Patent Docs

Peach Genome Dissected to Provide Insights into Ecological Influences

By Kevin E. Noonan --

The humble peach has been the inspiration for pies, album titles, independent movies, and a fictional woman's baseball team, but is also an important food species, yielding 24.5 million tons globally in 2018.  Like all plant species, their sessile nature makes them a good target for environmental studies because, unlike most animals, they must respond to changes in their environment by adaptation rather than flight.  They have a diverse temperate host range from their origins in southwestern China, being found in temperate and subtropical regions, wet and dry climates, and in harsh environments (high altitudes, severe cold, and drought conditions).  This range suggests that the peach is a good species for studying climate effects on their evolution as illustrated by genetic changes, particularly in view of the climate change the globe has been experiencing for the past ~40 years that is expected to continue.

An international group of researchers* recently reported the results of their taking this suggestion, in a paper entitled "Genomic analyses provide insights into peach local adaptation and responses to climate change" in the journal Genome Research.  The basis for the study was 263 diverse accessions that consisted of 52 wild relatives and 211 landraces ("a domesticated, locally adapted, traditional variety of a species of animal or plant that has developed over time, through adaptation to its natural and cultural environment of agriculture and pastoralism, and due to isolation from other populations of the species").  The 263 peach accessions could be divided into seven major groups:  YG (Yun-gui plateau), NW (Northwest China), NP (North Plain China), YT (Yangtze River Middle and Backward), NE (Northeast China), TB (Tibet plateau), and ST (South China Subtropical) groups.  Placing their genome assay results in context, these researchers reported on the effects of glaciation, which had caused a sharp decline of effective population size during the two largest Pleistocene glaciations:  the Xixiabangma glaciation (that occurred 1.17–0.8 million years ago, mya) and the Naynayxungla glaciation (occurring 0.78–0.50 mya); there was also a smaller decline in population size during the "last glacial maximum" (LGM) about 20,000 years ago.  Also reported was a slight expansion of peach populations from the Tibetan plateau (TB) after the Naynayxungla glaciation about 300,000 years ago.

In total, 342.7 Gb of paired-end sequences were assayed, with a median depth of 5.3-fold corresponding to 91.7% of a reference peach genome ("Lovell"); the peach genome is relatively small (227.4 Mb) compared with other fruit species (e.g., apple, 650 Mb; and strawberry, 805 Mb).  These sequencing studies resulted in the identification of 4,611,842 single-nucleotide polymorphisms (SNPs), with 1,931,310 residing in introns (∼11.33%) and another 848,638 (∼4.98%) in protein-coding exons.  These researches also identified 1,049,266 small insertions and deletions (indels) (< 6 bp) and 106,388 larger structural variations (>30 bp).  These variations were used as genetic markers for mapping through time genomic changes and crossbreeding between the different geographically defined groups.  The results of these genetic analyses showed a pattern of extensive admixture and possible gene flow among landrace groups.

Genetic analyses identified specific loci involved in adaptation to local conditions.  These include a total of 2092 genomic regions found for all seven groups (comprising 19.1 Mb, ∼8.4%) and "189, 387, 301, 235, 280, 339, and 378 regions for the YG, NW, NP, YT, NE, TB, and ST groups, respectively."  These loci were termed candidate selection regions (CSRs) by these researchers, and together harbored 3742 genes, corresponding to 396, 966, 635, 403, 573, 743, and 680 genes for the YG, NW, NP, YT, NE, TB, and ST groups.  In addition, some of these genes were shared among different groups, "suggesting the unique adaptive patterns for each group and that different climates may shape distinct genomic regions" according to the authors.  Assessment of these genes identified several related to response to different types of stimuli and stress that were overrepresented; included were temperature, radiation, salt, DNA damage, osmotic, toxin, and biotic stimulus.  These results suggested to these researchers that these genes were involved in adaptive evolution in response to these stresses.

Turning to specific forms of environmental stress, the researchers found that peach specimens from high-salt soils in northwestern China were enriched for two cation/H+ exchanger family genes.  Additional genes found from to be enriched in these specimens were genes encoding proteins having a leucine-rich repeat (LRR) domain, comprising 121 of 612 members (∼19.8%).  These researchers noted that such genes were "considered to be one of the most important domains involved in plant resistance."  Pattern recognition receptor (PPR) proteins, which "form one of the largest protein families in land plants that are related to environmental responses" and are present with 286 members in the peach genome, were found in 79 (∼27.6%) of the identified candidate selection regions (CSRs).

Specific genetic characteristics were reported for certain groups.  For the YG group (which is found on the Yun-gui plateau (Southwest China), a low-latitude (∼23.3–26.6° N) and high-altitude (∼2000 m) region with acidic soil (pH 4.5∼5.5)), genes (107) related to "metal ion (including potassium, iron, and zinc) binding and transport, cell membrane function, and response to toxins" were found to be overrepresented.  These scientists found that expression of a phenotype encoded by these genes would be "consistent with functions in overcoming cation deficiency and aluminum toxicity that are common in acidic soils."  For the YT group, characterized by high temperature and high humidity areas in middle and lower regions of the Yangtze river, the researchers found "high enrichments of the LRR domain (24) genes," as well as other genes related to stress responses (40 genes), in comparison to other groups.

The paper also reported on genes correlated with altitude and adaptations to altitude.

These include 2755 association SNPs, involving 2408 genes, of which 1670 association SNPs were unique, and ∼51.9% of the associations were shared across different types of environmental variable (EVs), which suggested to them that different EVs may shape the same genomic regions.  These "hotspots" were located at "the top and bottom of Chromosome 2," wherein the top was enriched with genes encoding NBS-LRR proteins and the bottom was highly enriched with genes associated with responses to a series of stresses, as well as at the top of Chromosome 4, which was "highly enriched with genes associated encoding LRR domain-containing proteins."  Overall, a "total of 75 genomic loci associated with more than five EVs were identified and these loci were highly enriched with genes in stress-related pathways, such as plant–pathogen interaction, MAPK signaling pathway, response to stress, and defense response."

Related to specific stresses, the researchers found that "genes involved in ion transport were highly enriched in those associated with soil pH . . ., as soil pH affects absorption of metal ions in plants."  Also enriched were genes associated with "programmed cell death (PCD), innate immune response, plant–pathogen interaction, and DNA repair," as well as "secondary metabolism, including flavonoid metabolic process, jasmonic acid (JA) biosynthesis, and plant hormone signal transduction."  These researchers also identified "temperature-associated SNPs [including] 10 association hotspots on Chromosomes 1, 2, 3, 4, 5, 6, and 7 for more than eight EVs related to temperature and altitude."  These findings were significant, according to the authors, because "[t]olerance to low temperature in winter is a major factor that restricts the spread of peach to extremely cold regions."  One particular SNP identified in this way was "located in the gene PpAHP5" which is part of a gene cluster encoding six histidine phosphotransfer proteins (AHP); these have been "reported to be involved in mediating cold signaling" and was found to be "up-regulated by cold," and cold-resistant cultivars expressed the gene at higher levels than cold-sensitive ones.  Similarly, the researchers reported precipitation-related "hotspots"; one of these showed enrichment of several stress response related genes.

Of economic importance, the level of fruit produced by cultivars adapted to different environmental conditions was determined, and the researchers reported finding differential expression of genes resulting in higher soluble sugar content.  Environmental factors associated with this phenotype include aridity, high diurnal temperature variation, and long sunshine duration, according to these scientists, leading them to conclude that "higher soluble sugar content in accessions from northwestern China represents an adaptive trait driven by the local drought environment."

Other phenotypes include fruit flesh color, which the researchers found "strong geographic patterns, with ∼80% of yellow-fleshed peach landraces originating from northwestern China" and to be related to carotenoid metabolism (particularly overexpression of genes associated with carotenoid synthesis and loss-of-function mutations leading to carotenoid accumulation).  Studies of genetic variants associated with high altitude (Tibet) adapted cultivars were also reported, including specific patterns of gene expression, enrichment and loss (339 genomic regions, harboring 743 genes for high altitude cultivars, including ones involved in flavonoid production and UVB protection, and morphological changes in stomata thought to be involved in resistance to high-altitude hypoxia).

The depth of genetic information and environmental factor correlations (much more extensive than set forth in this synopsis) led these researcher to conclude:

In summary, this study provides new insights into peach adaptation to its habitat and how climate has shaped the genome of a perennial tree through natural selection.  These results also provide a new resource for studies of peach evolutionary biology and breeding, especially with regard to enhancing stress resistance.

* Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences; National Horticulture Germplasm Resources Center, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences; Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture & Forestry Sciences, Huazhong Agricultural University;  Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Sciences;  IRTA–Centre de Recerca en Agrigenòmica (CSIC-IRTA-UAB-UB); Boyce Thompson Institute for Plant Research, Cornell University; U.S. Department of Agriculture– Agricultural Research Service, Robert W. Holley Center for Agriculture and Health

Giraffe Genome Reveals Relevant Adaptations

By Kevin E. Noonan --

The giraffe is the tallest extant terrestrial animal, and its iconic long neck (6 feet) provides advantages for foraging for food and detecting predators on the veldt over long distances.  As a consequence, however, the giraffe has a blood pressure two-fold higher than other ruminant animals needed to bring blood to the brain that is so far away from the animal's heart.

An earlier (2016) study of giraffe genome and genome of the related okapi (Okapia johnstoni) was not optimal, being restricted to 17,210 genes identified by comparison to cattle (Bos taurus) genome.  Last Wednesday, a team of Chinese and Norse scientists published a paper entitled "A towering genome: Experimentally validated adaptations to high blood pressure and extreme stature in the giraffe," in Science Advances.  In this paper, the authors described their work on Rothschild's giraffe (Giraffa camelopardalis rothschildi), which provided a higher "completeness" for this genome than previous studies.  The analysis provided the sequence of a 2.44 Gb assembly covering about 98% of giraffe genomic DNA.  Using comparisons with genomic DNA of cattle, goat, and okapi (with sperm whale as "outgroup"), the authors reported an assemblage of a putative common ancestor between giraffe and cattle; this resulted in a prediction of an evolutionary history of 4 chromosome fissions and 17 fusions that has resulted in the 15 haploid chromosome complement of modern giraffes (albeit admitting the need for further analysis to understand the significance of this result).

In this chromosomal complement these researchers discerned 101 genes under positive selection and 359 undergoing "rapid evolution," related to growth and development, nervous and visual systems, circadian rhythm, and blood pressure regulation.  One gene in particular was the focus this research:  the giraffe fibroblast growth factor receptor-like protein 1 (FGFRL1) gene was found to have seven non-synonymous mutations affecting the FGF binding domain, the most in any genetic sequence comparison to other ruminant mammals.  This gene was known to be involved in bone mineral density and hypertension resistance (thought to permit blood flow to the heights the giraffe's neck requires).  To investigate the relevance of these mutations to giraffe physiology, the researchers used CRISPR-Cas9 to introduce these mutations into the mouse FGFRL1 gene.  The resulting phenotype in these mice was found to be resistant to treatment with a high blood pressure-inducing drug (angiotensin II) while showing no developmental changes in cardiac structure; wildtype mice showed "significantly increased blood pressure" as a result of drug treatment.  These genetically altered mice also displayed significantly higher bone density as adults, with skeletal hypoplasia immediately postnatally.  Otherwise no deleterious effects were noted in these mice.  Together these results suggested these mutations could be responsible, at least in part, for the giraffe's characteristic long-necked phenotype.

This genetic research also detected differences in genes involving cardiac development, blood vessel characteristics and increased glomerular filtration rate in giraffe kidney, as well as genes involved in platelet function (including the phosphatidylinositol metabolism genes PIP4K2A, ISYNA1, MTMR3, CDS1, and INPP1) and ion transport related to cardiac contraction.

With regard to genetic adaptation of sensory genes (important for an herbivorous ungulate subject to predation by, inter alia, lions), these researchers found differences in genes related to eye development, vision, hearing, and balance.  Like other ruminants, giraffes have only two opsin genes, suggesting the absence of trichromatic color vision.  Perhaps curiously, the giraffe genome has lost 53 olfactory-related genes compared with the opaki, due to segmental deletion of sets of genes spatially clustered in the opaki genome, which these researchers speculated was a sensory "tradeoff" with their increased visual acuity.

Giraffes are known to have sleep durations among the lowest of all mammals.  Consistent with this phenotype circadian rhythm genes are altered in giraffes, particularly having a translation stop codon in the PER1 gene involved in circadian rhythm maintenance.

These researchers conclude their paper saying:

Overall, these results show that pleiotropy is a plausible mechanism for contributing to the suite of co-adaptations necessary in the evolution of the giraffe's towering stature.  However, because of the complexity of cardiovascular and sensory systems, more research on the functional consequences of giraffe-specific genetic variants is needed.

* School of Ecology and Environment, Northwestern Polytechnical University, Xi'an, China; Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi'an, China; College of Animal Science and Technology, Jilin Agricultural University, Changchun, China. BGI-Qingdao, BGI-Shenzhen, Qingdao, China; Shaanxi Key Laboratory for Animal Conservation, College of Life Sciences, Xi'an, China; Jiaxing SynBioLab. Co. Ltd., Jiaxing, China;  Research Center of Traditional Chinese Medicine, The Affiliated Hospital to Changchun University of Chinese Medicine, Changchun, China; Center for Evolutionary Hologenomics, GLOBE Institute, University of Copenhagen, Øster Voldgade, Copenhagen, Denmark; Norwegian University of Science and Technology, University Museum, 7491 Trondheim, Norway; Section for Computational and RNA Biology, Department of Biology, University of Copenhagen, Copenhagen, Denmark; Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, China.

Image of Rothschild's giraffe (Giraffa camelopardalis rothschildi) at Murchison Falls NP, UGANDA by Bernard DUPONT, from the Wikimedia Commons under the Creative Commons Attribution-ShareAlike 2.0 Generic license.

IP Organizations Support Continued Opposition to Waiver Proposal

By Donald Zuhn --

Late last month, a group of intellectual property organizations sent a letter to members of Congress and officials at the Patent and Copyright Offices to express their support for the United States' continued opposition to the TRIPS waiver proposal being discussed at the World Trade Organization (WTO).

The letter's signatories included the American Intellectual Property Law Association (AIPLA), Intellectual Property Owners Association (IPO), Licensing Executives Society (USA and Canada), Inc. (LES USA-Canada), and New York Intellectual Property Law Association (NYIPLA).  The group's letter was addressed to Sen. Patrick Leahy (D-VT), Chairman of the Senate Subcommittee on Intellectual Property; Sen. Thom Tillis (R-NC), Ranking Member of the Subcommittee on Intellectual Property; Rep. Henry C. "Hank" Johnson (D-GA), Chair of the House Subcommittee on the Courts, Intellectual Property, and the Internet; Rep. Darrell Issa (R-CA), Ranking Member of the Subcommittee on the Courts, Intellectual Property, and the Internet; Drew Hirshfeld, Performing the functions and duties of the Under Secretary of Commerce for Intellectual Property and Director of the U.S. Patent and Trademark Office; and Shira Perlmutter, Register of Copyrights and Director of the U.S. Copyright Office.

The organizations begin their letter by declaring that they "strongly support equitable, widespread, and successful distribution of vaccines, medicines, diagnostics, personal protective equipment, and other measures necessary to meet the challenges of COVID-19," and by noting that the intellectual property rights of their members "have fueled the innovation that has allowed us to combat COVID-19."  The letter also points out that IP system "will continue to fuel the next generation of solutions."

With respect to the WTO waiver proposal (which we discussed here), the group argues that the proposal "incorrectly portrays IP as a barrier to rapid innovation, R&D collaboration, and ample manufacturing of COVID-19 technologies," which the organizations state is contrary to the experience of their members.  The organizations also state that they "know of no data to suggest that patents and other IP rights are hindering vaccine development or delivery."  With regard to IP rights related to testing, treatments, and personal protective equipment, the group reiterates that "[w]e are not aware of any examples where IP has been used to limit access to COVID-related technology ‒ rather innovator companies have partnered and shared IP to create tools to address this pandemic."

The letter also notes that the manufacturing of COVID-19 vaccines is a complicated process, and cautions that "[p]oor quality vaccines being produced by underqualified manufacturers could have extreme negative consequences."

The organizations conclude their letter by arguing that "should the proposed TRIPS waiver be implemented, it would have an immediate chilling effect on their continued research and collaboration needed to overcome, for example, new variants of the virus, to create vaccines for children, and to develop better delivery mechanisms."  The group therefore "urge[s] the U.S. to continue its opposition to the TRIPS waiver proposal."

For additional information regarding this topic, please see:

• "Industry Coalition Supports Continued Efforts to Oppose Waiver Proposal," March 29, 2021
• "BIO and PhRMA Urge Biden Administration to Oppose Proposed WTO TRIPS Waiver," March 11, 2021

Evolution of SARS-CoV-2 from Bat to Human Pathogen

By Kevin E. Noonan --

A recent COVID post elicited the comment that we were just a "ray of sunshine."  Following in this tradition, this post concerns a recent report, in PLOS Biology, that the evolution of the SARS-CoV-2 virus (which causes COVID-19) evinced the development of a "highly efficient human pathogen" (to quote Billie Eilish, "Duh!").

The paper, entitled "Natural selection in the evolution of SARS-CoV-2 in bats created a generalist virus and highly capable human pathogen," was reported by an international team* of researchers.  It was known prior to this work that SARS-CoV-2 and the virus that caused the SARS outbreak in 2002-2003 (which these authors term "SARS-CoV-1" to avoid confusion) arose in bats, and that SARS-CoV-2 is particularly infective in humans, inter alia, for having a furin protease cleavage site (Arg-X-X-Arg/ Arg-X-Arg/Lys-Arg) in its Spike protein, which facilitates protease-related binding to human angiotensin-converting enzyme 2 (ACE2) because furin is expressed in human lung tissue.  The interesting conclusions drawn by these authors is that mutational adaptations that facilitate human infection by the virus occurred prior to the jump from bats to humans, which accounts (at least in part) for the rapid dissemination/infectivity in the pandemic because the virus did not require long-term incubation to achieve the species switch typical of other zoonotic viruses.  Specifically, the authors report:

[U]nlike most other RNA viruses which acquire adaptations after switching to a new host species for efficient replication and spreading as successfully as exhibited by SARS-CoV-2, the Sarbecoviruses [a closely related bat viral species]—which already transmit frequently among bat species—can exploit the generalist properties of their ACE2 binding ability, facilitating successful infection of non-bat species, including humans.

The paper reports these researchers' comparison between signs of positive selection during the pandemic with historic selection in related bat viruses.  They assessed 133,741 SARS-CoV-2 virus samples collected in the first 11 months of the pandemic with 69 samples of related (Sarbecovirus) virus genomes for the frequencies of nonsynonymous (dN) mutations (which result in amino acid sequence differences that can be subject to selective pressures) with synonymous (dS) mutations (which change at the nucleic acid but not amino acid sequence).  Nonsynonymous mutations were expected to arise (i.e., be detected) more slowly in the viral population, because while both synonymous and nonsynonymous changes arise "randomly" there is no basis for selection of synonymous ones.  As expected, dN mutations arose at 4% the rate of dS when comparing SARS-CoV-2 with related bat Sarbecovirus RaTG13.  The authors further report that "[t]he vast majority of 20,687 observed mutations occur at very low frequency, with 79% of mutations observed in 10 or fewer of the 133,741 SARS-CoV-2 genome sequences analysed."  They conclude from these data that "SARS-CoV-2 is evolving relatively slowly with no dramatic increases in selective pressures occurring over the sampling period [from] December 2019 to October 2020."

Perhaps surprisingly, the D614G mutation, putatively associated with higher rates of infection (see "The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity"), was detected in samples collected early in the pandemic, whereas other mutated sites occurred later, suggesting that the D614G variant has a higher capacity for positive selection in unvaccinated/uninfected populations because it has had a longer time to spread through the virus populations.  In addition, these researchers found that most of the other mutations were only transiently present in the viral population.

In comparisons with other bat viruses, "diversifying" selection, associated with the ability of the virus to "jump" species, was found in the earliest branches of virus evolution from the bat virus.  These researchers further report that there was "no evidence of selection in the terminal branch leading to SARS-CoV-2[, c]onsistent with the nonhuman progenitor of SARS-CoV-2 requiring little or no novel adaptation to successfully infect humans" (although they also caution that "no model can detect all signatures of historic genomic adaptation, and mutations which may enable SARS-CoV-2 to infect humans could have arisen by genetic drift in the reservoir host before human exposure").

Also found was evidence of a related bat virus, RmYN02, that has a sequence in comparison with SARS-CoV-2 that indicates recombination with prototype of SARS-CoV-2 as early as 1976, evidence of how long this virus was "brewing" in bat populations over time and consistent with "direct" bat-to-human transmission; indeed, the researchers found no evidence that related viruses are found in pangolins.

Further (and having the character of improvident destiny or the propensity for God to play dice contrary to Einstein's preferences), the sequence comparisons with bat viruses are consistent not with sequence changes related to a change in host species (from bat to human) but more consistent with a history of sequence changes related to the bat host(s) (such as immune avoidance or tissue preferences) having arisen that "preselected" the virus for effective human infection.  As these authors write:  "[t]hese results suggest that the majority of adaptive changes which generated SARS-CoV-2 took place prior to its emergence in the human population."

Exceptions are the mutations related to UK (B.1.1.7) or South African (B.1.351) mutations, which seem associated with "host immunity due to previous exposure and/or chronic infections of probably immunocompromised individuals," i.e., mutations more in line with conventional stories of how zoonotic viruses show sequence changes related to adaptation to a new host.

There has been evidence supporting these conclusions (outside the depth of the genetic analyses presented here; see "Sequence Comparisons Illustrate Susceptibility to Coronavirus Infection") regarding the biology of the observed ease of transmission to other hosts due to evolution of a "generalist" virus that arose in bats.  In this regard, the authors state "[t]he apparent 'success' of these bat viruses to transmit to multiple other mammals and spread with few to no significant genomic changes further supports the hypothesis that the SARS-CoV-2 progenitor is from a viral lineage with a relatively generalist nature."  This is consistent with a mechanism wherein this "generalist" virus arose in bat populations over the past ~100 years as a function of host switching or tissue tropism within bat species, perhaps due to resistance in bats (see "How Bats Are Different").

Finally, and consistent with these ideas, the authors present a histogram of the various related coronavirus species showing the genetic distance between SARS-CoV-1 and SARS-CoV-2 and illustrating that the genetic changes characterizing the increased human infectivity of SARS-CoV-2 arose late in the evolution of this species.

The authors conclude with a cautionary note:

An overarching point stemming from these observations is the lack of sampling and knowledge of the diversity in this viral subgenus.  In particular, the closest known bat viruses to SARS-CoV-2 are relatively divergent in time[], and the apparent generalist nature of these viruses suggests that there are species of wild mammals, yet to be sampled, infected with nCoV-like viruses.  Serological studies of communities in China that come into contact with bats indicate that incidental and dead-end spillover of SARS-like viruses into humans do occur[].  Due to the high diversity and generalist nature of these Sarbecoviruses, a future spillover, potentially coupled with a recombination event with SARS-CoV-2, is possible, and such a "SARS-CoV-3" emergence could be sufficiently divergent to evade either natural or vaccine-acquired immunity, as demonstrated for SARS-CoV-1 versus SARS-CoV-2.  We must therefore dramatically ramp up surveillance for Sarbecoviruses at the human–animal interface and monitor carefully for future SARS-CoV emergence in the human population.

* MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom; Temple University, Institute for Genomics and Evolutionary Medicine, Philadelphia, Pennsylvania; Center for Infectious Disease Dynamics, Department of Biology, Pennsylvania State University, University Park, Pennsylvania; Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium

Industry Coalition Supports Continued Efforts to Oppose Waiver Proposal

By Donald Zuhn --

Earlier today, a group of fifteen industry and trade organizations sent a letter to five members of the Biden Administration, to express their strong support for the Administration's work to leverage international mechanisms to help address COVID-19 and also for the Administration's continued efforts to oppose "a problematic proposal at the World Trade Organization to waive IP global protections," which would "remov[e] patent, industrial designs, copyright, and trade secret protection for any products and services so long as they can be tied to COVID-19."

The letter's signatories included the Advanced Medical Technology Association (AdvaMed), Biotechnology Innovation Organization (BIO), Incubate Coalition, Latino Coalition, National Association of Manufacturers (NAM), National Foreign Trade Council (NFTC), National LGBT Chamber of Commerce (NGLCC), National Puerto Rican Chamber of Commerce, Pharmaceutical Research and Manufacturers of America (PhRMA), Small Business & Entrepreneurship Council (SBE), Software and Information Industry Association (SIIA), Telecommunications Industry Association (TIA), U.S. Chamber of Commerce, U.S. Council for International Business (USCIB), and U.S. Pan Asian American Chamber of Commerce (USPAACC).  The coalition's letter was addressed to Gina M. Raimondo, Secretary of Commerce; Katherine C. Tai, U.S. Trade Representative; Brian Deese, Assistant to the President for Economic Policy; Jeffrey Zients, Coordinator of the COVID-19 Response and Counselor to the President; and Jacob Sullivan, Assistant to the President for National Security Affairs.

The letter begins by noting that "[n]o country will be safe, until the virus is controlled everywhere, including at home and abroad, in developed and developing countries," and acknowledging "robust U.S. leadership" in global efforts to ramp-up the development and manufacturing of COVID-19 supplies and treatments to defeat the pandemic.  The coalition states that the U.S. "has led the world in promoting policies that advance scientific innovation, improve global health, and foster economic growth," and points out that "[t]hese policies have created R&D-intensive industries that contribute $6 trillion towards the U.S. gross domestic product and support approximately 45 million U.S. jobs in all 50 states."

The letter also commends the country's innovative companies for responding to the COVID-19 pandemic by "leverage[ing] their extraordinary R&D capacity to launch the unprecedented development and delivery of diagnostics, medical equipment, treatments, vaccines, digital tools, and information sharing faster than ever before."  The coalition suggests, however, that this response would not have been possible without a robust IP environment in the U.S.  The coalition also expresses support for one of the Administration's most recent COVID-19 vaccine global initiatives -- joining with Australia, India, and Japan to expand manufacturing and distribution of COVID-19 vaccines in the Indo-Pacific region.

Turning to the WTO waiver proposal (which we discussed here), the coalition writes that:

Unfortunately, some countries have chosen this moment to pursue their longstanding goals to weaken IP rights, including through a problematic proposal at the World Trade Organization to waive IP global protections.  This waiver is as vague as it is broad, removing patent, industrial designs, copyright, and trade secret protection for any products and services so long as they can be tied to COVID-19.  Proponents of the waiver have claimed, without evidence, that it would advance public health.  In reality, the waiver would undermine the global response to COVID-19 and would not achieve its stated goal to rapidly expand vaccines production.

The letter adds that the WTO waiver proposal distracts from addressing the manufacturing and logistical issues that constitute "[t]he greatest barriers to faster global access to vaccines and therapies."

The coalition concludes its letter by expressing support for "the Administration's global leadership and collaboration with key global vaccine mechanisms, consistent focus on efforts to identify and eliminate the real barriers to end the pandemic and ensure faster global vaccination, and continued efforts to oppose this waiver."  The letter also encourages the Administration to continue to oppose the WTO waiver proposal, and to continue to work with Japan, the European Union, the United Kingdom, Switzerland, and Brazil "to foster a more productive, comprehensive conversation" with WTO Director-General Ngozi Okonjo-Iweala "about ensuring equitable access to COVID-19 products by tackling trade barriers."

Neanderthal Ancestors Can Be Human Guardian Angels for COVID Infection, Too

By Kevin E. Noonan --

When it comes to SARS-CoV-2 infection (and resulting COVID-19), it seems our Neanderthal ancestors giveth and taketh away.  Genetic material inherited from interbreeding between Neanderthals and early humans has been shown to increase the risk of serious COVID infection (see "Inherited Neanderthal Gene Encodes Genetic Risk for COVID-19").  Recently, further explication of correlations between Neanderthal remnants in genomic DNA of modern humans has shown that a sequence on chromosome 12 is associated with milder COVID disease.

These results were reported by the same researchers who reported the earlier, more sinister connection between Neanderthal DNA and COVID-19, which included Svante Pääbo, who almost single-handedly created the science of detecting Neanderthal DNA in archeological samples (and living humans; see Neanderthal Man: In Search of Lost Genomes).  In this more recent study, published in the Proceedings of the National Academy of Sciences USA, and entitled "A genomic region associated with protection against severe COVID-19 is inherited from Neandertals," it is a region of chromosome 12 that contains the relevant, seemingly protective genetic locus.  Humans bearing this haplotype have a 22% reduction in risk of severe COVID infection.  It is present "at substantial frequencies in all regions of the world outside Africa," a hallmark of Neanderthal DNA which arose after humans migrated from the place of our initial evolutionary development.  Unlike the earlier, deleterious locus on chromosome 3, for which there is no known function, the region of chromosome 12 associated with milder COVID infection is known to encode proteins that are expressed "during infections with RNA viruses" according to these authors.  Another unique inheritance pattern is that this locus appears to have increased in frequency between 20,000 and 10,000 years ago and again approximately 1,000 years ago, which suggests selection that could have been very effective at times when effective medical intervention would have been practically non-existent.

With regard to the function of this locus in Neanderthals themselves, the authors posit that these early humans would have needed to adapt to environments outside Africa which undoubtedly would have included new pathogens that cause infectious disease.  Such a pattern of genetic variants that affect genes involved in innate immunity have been shown to have been introduced into Homo sapiens sapiens ancestors from Neanderthal and Denisovan populations, including for example genes that reduce susceptibility to H. pylori infection.

The current study involved genetic assessment of 2,244 terminally ill COVID 19 patients as part of the Genetic of Mortality in Critical Care (GenOMICC) consortium.  In addition to the two loci that have been discussed previously and herein, genetic loci relevant to COVID-19 infection have also been found on chromosomes 6, 19, and 21.  Specifically with regard to the chromosome 12 locus, these researchers reported association of single nucleotide polymorphisms (SNPs) with this relevant chromosomal loci; this locus satisfied the further criterion of being absent from the genomes of 108 Yoruba individuals (the loci on chromosome 6, 19, and 21 failed this characterization test).  The chromosome 12 SNPs were found to be in linkage disequilibrium (i.e., are separated at meiosis at lower than expected frequencies) and formed a ~75kb haplotype.  The researchers interpreted this inheritance pattern to be consistent with introduction of these sequences into the modern human gene pool by interbreeding with Neanderthals.  Further genetic analyses informed these researchers that this region was not a remnant of DNA inherited by both Neanderthals and modern humans from the time (~550,000 years ago) they diverged but rather were more consistent with mixing when the two populations co-habited beginning ~100,000 years ago.  This locus is also present in the three authentic Neanderthal genomes available in current databases.

The geographic distribution of this locus in modern human populations is illustrated by this figure,

which shows the veritable absence in Africa and variable frequencies (25-30%) around most of the rest of the world.

The researchers found three genes (OAS1, OAS2, and OAS3) that encode oligoadenylate synthases present at this Neanderthal-derived locus.  These genes in modern humans are induced by interferon produced by RNA virus infection and which produce a product (short-chain polyadenylates) that activate a double-stranded RNA specific nuclease.  Other antiviral responses are induced as well as a consequence of this generic induction, suggesting a mechanism for resistance to RNA virus infection.  Finer scale genetic mapping of SNPs and COVID severity protection implicated OAS3 (although these authors state that this association is "tenuous").  Also of interest is a SNP in the OAS1 gene which alters splicing of the encoded mRNA transcript and produces several isoforms of the encoded protein in its modern human counterpart. Compared with these isoforms, the researchers reported that the ancestral Neanderthal protein has higher enzymatic activity.  This isoform is also present in African populations outside this locus, which these authors posit was an ancestral form lost in humans during the time they left Africa.  Functionally, this splice site variant has been associated in earlier studies with protection against West Nile Virus.  The Neanderthal-derived locus also has missense variants in the OAS1 and OAS2 genes, and two synonymous variants in the OAS3 gene, but these are a mixture of variants present in ancestral African DNA and later acquired in Neanderthal DNA.  The missense variant in the OAS1 gene has been previously associated with moderate-to-strong protection against SARS-CoV infection, although these researchers could not rule out the possibility that the responsible genetic factor was merely closely linked to this gene.  Finally, this Neanderthal locus on chromosome 12 also seems to provide increased resistance to Hepatitis C infection.

The authors conclude by noting the suggestion that this Neanderthal-derived DNA, particularly the OAS loci contained therein, had undergone positive selection over the past 1,000 years and their hope that "[f]uture studies of human remains from historical times will clarify whether, and when, this occurred."

BIO and PhRMA Urge Biden Administration to Oppose Proposed WTO TRIPS Waiver

By Donald Zuhn --

One year ago today, World Health Organization Director-General Tedros Adhanom declared the COVID-19 outbreak to be a pandemic.  At the time of the announcement, the WHO noted that there were 118,000 cases reported globally, but in its most recent situation report, the WHO indicated that as of March 7, 2021 there have been 116,166,652 cases globally.

The response to the COVID-19 pandemic by the global biopharmaceutical industry over the past fourteen months has been unprecedented.  From December 30, 2019, when SARS coronavirus was first detected in a patient sample, and January 10, 2020, when the complete genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was released, multiple treatments and vaccines have been developed and approved for use in combating COVID-19.  And the rapid development of these treatments and vaccines has, as the Pharmaceutical Research and Manufacturers of America (PhRMA) recently put it, been "nothing short of incredible."  For example, the U.S. Food and Drug Administration granted Emergency Use Authorization (EUA) for Gilead Sciences' remdesivir 123 days after the virus was first detected in a patient sample, granted EUA to Eli Lilly's antibody treatment 315 days after the virus was first detected, and granted EUA for the Pfizer-BioNTech vaccine 347 days after the virus was first detected.  And since the pandemic was declared a year ago, the FDA has also granted EUA for Regeneron's antibody treatment, as well as for Moderna's and Johnson & Johnson's vaccines, with other vaccines expected to be granted EUA by the FDA shortly.

However, despite the speed with which the above treatments and vaccines have been developed, India and South Africa have been seeking since October to have the Council for Trade-Related Aspects of Intellectual Property Rights (TRIPS) of the World Trade Organization (WTO) recommend "a waiver from the implementation, application and enforcement of Sections 1, 4, 5, and 7 of Part II of the TRIPS Agreement in relation to prevention, containment or treatment of COVID-19" to the General Council of the WTO.  The two countries also recommended that "[t]he waiver should continue until widespread vaccination is in place globally."  According to their October 2, 2020 letter, the waiver is necessary because of "several reports about intellectual property rights hindering or potentially hindering timely provisioning of affordable medical products to the patients," as well as "urgent legal amendments to . . . national patent laws" being introduced by some WTO members "to expedite the process of issuing compulsory/government use licenses."

Last week, the Biotechnology Innovation Organization (BIO) and PhRMA sent separate letters to the Biden Administration to provide their thoughts on the Indian and South African proposal.  In the PhRMA letter*, the organization congratulated President Biden for the more than 80 million vaccine doses that have been administered to Americans to date, adding that "[a]s we continue to work to vaccinate Americans, we must also acknowledge that COVID-19 anywhere in the world means our collective fight against this virus is not done."  With respect to the importance of IP protections in combating the pandemic, PhRMA stated that:

Intellectual property protections have been essential not only to speed the research and development of new treatments and vaccines, but also to facilitate sharing of technology and information to scale up vaccine manufacturing to meet global needs.  Eliminating those protections would undermine the global response to the pandemic, including ongoing effort to tackle new variants, create confusion that could potentially undermine public confidence in vaccine safety, and create a barrier to information sharing.  Most importantly, eliminating protections would not speed up production.

Turning to the waiver proposal, PhRMA noted that "[i]n requesting the waiver, India and South Africa argued without evidence that intellectual property is hindering the global response to the pandemic and that the waiver would help scale up research, development, manufacturing and supply of needed products."  PhRMA urged the Biden Administration to join the European Union, United Kingdom, Japan, Canada, Switzerland, Brazil, and Norway, as the United States did last year, in opposing the waiver, suggesting that "[p]erhaps more than any other time in history, society is seeing and benefiting from the innovation supported by intellectual property."  PhRMA also pointed out that current estimates indicate that COVID-19 vaccine manufacturers will supply approximately 10 billion doses by the end of 2021, which would be sufficient to vaccinate the entire current global vaccine eligible population.

In the BIO letter, the organization began by noting that "[c]learly, the global pandemic can only be ended with global efforts that reach everywhere this deadly virus is prevalent," adding that the group "supports strong, efficient, and effective efforts to see that successful COVID products get to patients everywhere in the world that need them."  The letter points out that within months following the outbreak, more than 800 global development projects had been initiated to create vaccines, treatments, and diagnostics.

However, like PhRMA, BIO noted that it had "strong concerns about some proposals being made that would set back and complicate such efforts," pointing to the waiver proposal, which BIO said was "both a wrong-headed and ineffective means of spurring further efforts at access."  The letter argues that "the scientific and regulatory barriers required before one can safely and efficiently produce these advanced technologies are simply too high to be accomplished by anything other than cooperative, collaborative partnerships," asserting that:

The TRIPS waiver by contrast could spur a spate of confusing, mutually inconsistent and heavy-handed "compulsory" demands by governments all over the world for supply and technology transfer, which would distract from the need for coordinated international efforts and the cooperative work that currently is being advanced, and thus ultimately undermine the very goals of quick, safe access that it seeks to promote.

BIO concluded its letter by stating that "the world needs to be working together to advance the coordinated efforts that have been organized," while also declaring that "[t]he TRIPS waiver would be a divisive and ineffective detour from that critical work."

* The PhRMA letter was signed by the CEO's of thirty biopharmaceutical companies:  Eli Lilly and Company; Takeda Pharmaceuticals U.S.A., Inc.; Novartis; AbbVie; Bayer AG; Amgen Inc.; Biogen, Inc.; Astellas Pharma US Inc.; BioMarin Pharmaceutical Inc.; AstraZeneca Pharmaceuticals LP; Boehringer Ingelheim USA Corporation; Bristol Myers Squibb Company; GlaxoSmithKline; CSL Behring; Incyte Corporation; Eisai Inc.; Ipsen Biopharmaceuticals, Inc.; Genentech, Inc.; Johnson & Johnson; Gilead Sciences, Inc.; Lundbeck LLC; Merck and Co., Inc.; Sanofi; Novo Nordisk Inc.; Sunovion Pharmaceuticals, Inc.; Otsuka North America Pharmaceutical Business; Teva Pharmaceutical Industries, Ltd.; Pfizer Inc.; UCB; and Sage Therapeutics.

DNA from Pleistocene Era Mammoth Lineages Elucidated

By Kevin E. Noonan --

The wooly mammoth (Mammuthus primigenius) is an iconic animal, like the saber tooth tiger or dire wolf, from a time in human history when our position at the top of the global food chain was decidedly not assured (and being something's prey was not limited to just other humans).  Perhaps this is a reason that resurrection of mammoths using Jurassic Park-like technology has some currency and appeal (but see How to Clone a Mammoth for reasons why this may not be such a good idea).  Perhaps paradoxically, the mammoth arose in Africa 5 million years ago and like its (very) distant Homo sapiens relatives migrated to colonize the Northern Hemisphere, with most of the evolutionary adaptations to new habitats (and speciation that accompanied them) rising in the Pleistocene Era (from about 2.6 million to 12 years ago).  From these speciation events arose the Columbian mammoth in North America about 1.5 million years ago and the "classic" wooly mammoth in northeastern Siberia about 700,000 years ago.

Recently an international team* elucidated some genetic relationships between mammoths using DNA extracted from specimens from the Early and Middle Pleistocene, in a paper entitled "Million-year-old DNA sheds light on the genomic history of mammoths," in the journal Nature.  These results, termed "deep-time paleogenomics" by the authors, were obtained from three specimens, two of which were more than 1 million years old.  The specimens represented two distinct lineages present in Eastern Siberia in early Pleistocene, one of which resulted in wooly mammoth populations that survived to become contemporaneous with humans.  The third lineage gave rise to first mammoths in North America (Mammuthus columbi), resulting from a hybrid between these two lineages in Siberia in the Middle Pleistocene.  And perhaps not surprisingly, these researchers found most of the adaptation to cold seen in these animals were present in the samples from one million years ago.

As reported by these researchers, DNA was extracted from the three specimens using now established techniques for highly degraded DNA samples and libraries prepared.  The libraries were sequenced and these sequences compared to genomic DNA sequences from the African savannah elephant (Loxodonta africana) and mitochondrial DNA from the Asian elephant (Elephas maximus).  Perhaps not unexpectedly, the specimen DNA was "considerably more fragmented and had higher levels of cytosine deamination than DNA from permafrost-preserved samples dating to the Late Pleistocene subepoch," which required modifications to the sequence analysis protocols.  Nevertheless, these researchers reported that they obtained complete mitochondrial DNA genomes from the three samples and 49 million (from the oldest sample, named Krestovka), 884 million (the Adycha sample)  and 3,671 million base pairs (the Chukochya sample) of nuclear genomic data from the three samples (with the highest DNA recovery coming from the most recent specimen, which from tooth morphology they thought to be an early wooly mammoth specimen).

The ages of these specimens were then determined using the mitochondrial DNA sequences, ranging from 1.65 Ma (2.08–1.25 Ma; Krestovka), 1.34 Ma (1.69–1.06 Ma; Adycha) and 0.87 Ma (1.07–0.68 Ma; Chukochya). Genomic DNA was used to verify the two specimens having the highest amount of gDNA sequence obtained, against estimates of the highest number of changes from the latest Africa savannah element ancestor, which yielded 1.28 Ma (1.64–0.92 Ma) and 0.62 Ma (1.00–0.24 Ma), respectively.  These results were similar to the dates derived from the generally more intact mitochondrial DNA, but the authors cautioned that "this analysis is based on low-coverage data and the confidence intervals are wide."  Nevertheless, the authors also note that "[t]he DNA-based age estimates for the [the two genomic DNA] specimens are consistent with the geological age inferences that were independently derived from biostratigraphy and palaeomagnetism."

The genetic analyses indicated that the samples were divergent from known mammoth species from the Late Pleistocene, and were consistent with the younger two specimens (Adycha and Chukochya) being ancestral to the wooly mammoths, while the oldest specimen (Krestovka) apparently diverged prior to the species split between the wooly mammoth and the Columbian (North American) mammoths.  The oldest specimen was estimated by these authors to have split from the later mammoth lineages between 2.66 and 1.78 million years ago based on both mitochondrial and genomic DNA data and had fewer derived alleles that differed from the Asian elephant than the younger two (indicating a closer evolutionary relationship).  Due to the relative closeness in time for the eldest and one of the younger specimens, these results are consistent with these two lineages together inhabiting eastern Siberia in the Early Pleistocene, with the earliest having diverged from all later mammoth species before mammoths appeared in North America.

Regarding the Columbian (North American) mammoth, the genetic analyses also indicated that these mammoths shared a higher proportion of it ancestry from the earlier-diverging Krestovka species, which showed "excess derived allele-sharing between the Columbian mammoth and the Krestovka specimen."  These statistics were further consistent with one migration of n admixture of mammoth populations of the species from the Krestovka specimen (38-43% ancestry) and the wooly mammoth (57-62% ancestry).  Further analysis of later Columbian mammoth specimens indicated a second admixture with North American wooly mammoth species.

The researchers then assessed the genetic adaptations expected to be found in animals descendent from African elephants living in Siberia, notably cold-tolerance and open-habitat adaptations.  These analyses were performed using protein-coding portions of the genetic information derived from these samples in comparison with known adaptations from Late Pleistocene wooly mammoths (as further compared with African and Asian elephants, who carried the "ancestral" alleles).  As reported in the paper, "85.2% (782 out of 918) and 88.7% (2,578 out of 2,906) of the mammoth-specific protein-coding changes were already present in the genomes of Adycha . . . and Chukochya [specimens], respectively."  These specimens showed no specific genetic changes arising in these populations in response to known changes in climate and mammoth morphology arising in the Middle Pleistocene.  These researchers also interrogated the DNA samples for 91 changes known to have arisen in the mammoth lineage.  These included genes thought to be involved in "hair growth, circadian rhythm, thermal sensation and white and brown fat deposits," the researchers finding "the vast majority of coding changes were present in both the Adycha (87%) and Chukochya (89%) genomes."  In contrast, another gene ("TRPV3, which encodes a temperature-sensitive transient receptor channel that is potentially involved in thermal sensation and hair growth") possessed only 2 of 4 amino acid sequence changes found in Late Pleistocene wooly mammoths, which indicated to these authors that these genetic alterations "occurred over several hundreds of thousands of years, rather than during a single brief burst of adaptive evolution."

The authors conclude by a discussion of their results suggesting an admixture of an earlier-arising mammoth ancestor with wooly mammoths to produce the Columbian (North American) mammoth through a "hybrid speciation event" resulting in an almost 50:50 mix of ancestor species DNA.  Mitochondrial DNA assessments suggested that the most recent common female ancestor of all Late Pleistocene Columbian mammoths lived about 420 thousand years ago, which these authors use as a minimum date for the admixture event.  Because it is known that mammoths existed in North America 1.5 million years ago, these authors posit initial North American colonization by the Krestovka ancestral species.  They conclude their report by stating:

The retrieval of DNA that is more than one million years old confirms previous theoretical predictions that the ancient genetic record can be extended beyond what has been previously shown.  We anticipate that the additional recovery and analysis of Early and Middle Pleistocene genomes will further improve our understanding of the complex nature of evolutionary change and speciation.  Our results highlight the value of perennially frozen environments for extending the temporal limits of DNA recovery, and hint at a future deep-time chapter of ancient DNA research in which specimens from high latitudes will have an important role.

* From Centre for Palaeogenetics, Stockholm, Sweden; Department of Bioinformatics and Genetics, Swedish Museum of Natural History, Stockholm, Sweden; Department of Cell and Molecular Biology, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; Department of Zoology, Stockholm University, Stockholm, Sweden; Section for Computational and RNA Biology, Department of Biology, University of Copenhagen, Copenhagen, Denmark; The Francis Crick Institute, London, UK; Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA; Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany; Department of Biological Sciences, Middle East Technical University, Ankara, Turkey; Department of Earth Sciences, Natural History Museum, London, UK; College of Plant Protection, China Agricultural University, Beijing, China; The Arctic University Museum of Norway, UiT – The Arctic University of Norway, Tromsø, Norway; Geological Institute, Russian Academy of Sciences, Moscow, Russia; Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA; Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, CA; and Department of Archaeology and Classical Studies, Stockholm University, Stockholm, Sweden.

Image of Woolly mammoth model Royal BC Museum in Victoria by Thomas Quine, from the Wikimedia Commons under the Creative Commons Attribution 2.0 Generic license.

Do mRNA-based COVID Vaccines Have an Achilles Heel?

By Kevin E. Noonan --

The COVID-19 pandemic has spread throughout the globe, infecting more than 90 million people and causing almost two million deaths (see "Tracking coronavirus' global spread").  SARS-CoV-2 infection is the cause of the COVID-19 pandemic; this virus is recognized as the latest viral infection in humans of zoonotic origins, in this case bats first arising in Wuhan, China.

But the world's hope against the pandemic has been raised by the development of effective vaccines against virus infection in record time, using technology based on the mRNA encoding a particular viral protein called Spike.  The rapidity of vaccine development is one of the advantages of using modern RNA technology, and this is a direct consequence of two aspects of this technology.  First, RNA sequencing technology has permitted quick determination of the genetic sequence of the viral RNA.  In particular, this technology identified the sequence of the viral Spike protein, which is major antigenic determinant and required for virus entry into infected cells, attaching to the angiotensin 1 converting enzyme 2 (ACE-2) receptor.  Second, because this protein is necessary for infectivity, immunological responses directed at the encoded protein were believed to be (and turned out to be) particularly effective in provoking immunity to infection.  And because just this specific protein can be produced in the laboratory (and then in industrial quantities) for producing the vaccine used to raise immunity, many of the deleterious effects, both biological and temporal, of using "whole" viruses can be avoided.  It is a remarkable demonstration of applying technology developed over the past 40 years to solve a critical problem in global health.  And it would have been almost impossible to have achieved these vaccines "the old fashioned way," i.e., using virus samples and either genetically attenuating them or inactivating them with heat, chemicals, and like methods.

But the capacity of the virus to mutate and change portions of the sequence of the Spike protein has raised concerns that the current vaccines, which were all raised with specific Spike protein-encoded RNAs, might be ineffective for such variants.  This potential deficiency raises the possibility if not the likelihood that vaccination against the known ("original") Spike protein might in fact select for the variants, which could spread even in an inoculated population.  The only way to forestall if not prevent such an outcome would be a massive inoculation effort that could prevent sufficient replication of the virus in naïve members of the population to not give the virus replication and mutation opportunities to outrun inoculation efforts.

But such a strategy is impractical in a global epidemic, if only due to the logistics of inoculating a large fraction of the world's population when transmission across national boundaries, oceans, and continents is facilitated by global travel.  And the most draconian lockdown, quarantine, and travel ban regimes are unlikely to be effective unless aided by population and geography, for example, in New Zealand which is almost literally at the end of the world.

The variant problem is one that arises precisely because of the way the vaccine has been made.  Viruses like all living things are genetically heterogenous, and the SARS-CoV-2 virus "in the wild" is likely comprised of thousands if not millions of slightly different genetic sequences, some of which are found in the Spike protein.  In addition, the biologically active portion of the Spike protein, i.e., the portion that interacts with the ACE-2 receptor and facilitates virus entry into infected cells, is not necessarily the most antigenic portion of the protein, albeit because the vaccines are "neutralizing" antibodies (which means just what it sounds like) antibodies produced by the vaccine must interfere with such binding.  When vaccines are prepared from populations of viruses they can be expected to comprise a somewhat representative population of these slightly variable proteins; for SARS-CoV-2 these would be not only the Spike protein but any protein antigenically available to the immune system.

As a result, any vaccines produced using mRNA technology cannot have the plurality of viral proteins and the populations of variant viral proteins that are provided by more conventional immunological methods.  The immunity thereby provided is specific but its very specificity renders the immunological protection narrow compared with more traditional vaccines.  This narrowness provides the virus with an opportunity for immune avoidance and thus infection even in individuals immunized against the current Spike protein.  And paradoxically, the vaccine may in fact select (in the Darwin, "survival of the fittest" meaning) for such variants, which can propagate through the human population in spite of even robust vaccination efforts.

Fortunately, there may be two silver linings in this viral cloud.  First, the very rapidness of the production of these first mRNA vaccines may permit equally (or perhaps even faster) development of mRNA vaccines specific for these variants, and the provision of immunological "cocktails" of vaccines directed against the most prevalent or infectious variants.  Second, many variants may not be capable of immune evasion, so despite the genetic changes created during infection some portion of the new variants will not be capable of infecting vaccinated individuals.  Third, there is a balance between such variants and their effectiveness in infection that could select for less infectious or perhaps less deleterious strains of the virus; the thought in classical immunology was that pathogens that were limited to human infections over time attenuated because the more virulent strains burned themselves out while the milder forms permitted their hosts to live long enough to infect more, other individuals.  This is one explanation for why influenza infection is constant, because those viruses can be passed through fowl (ducks and chickens) and swine (pigs and hogs) as well as humans, and thus selection in these animal populations can produce virus populations more virulent in people.

There may be a grandeur in this biological view of life, but when it comes to pandemic infection a perhaps more apt meme is that nature truly is red in tooth and claw (and Spike proteins), and pathological infections are almost impossible to avoid in the long run.  In the short term, however, the hope is that they can be tamped down enough to put out the fire this time, and for us to learn the lesson of the importance of eternal vigilance and preparedness against the next potential pandemic.

Dire Wolf DNA Shows Distinct New World Lineage

By Kevin E. Noonan --

When I awoke, the dire wolf
Six hundred pounds of sin
Was grinning at my window
All I said was, "Come on in"

But don't murder me, I beg of you
Don't murder me, please, don't murder me

"Dire Wolf," Grateful Dead, Workingman's Dead

The dire wolf (Canis dirus), prototype of the various wolves that were important members of the House Stark family of characters in Game of Thrones, was found uniquely in North America until its extinction in the late Pleistocene (~13,000 years ago).  The relationship between this species and the indigenous gray wolf (Canis lupus), the coyote (Canis latrans), and the Asiatic wild dog or dhole (Cuon alpinus) is uncertain, however, based purely on shared morphologic characteristics.  Perhaps this "lone wolf" property, and its great size (~68kg/150lbs, despite artistic exaggeration), contributed to its iconic stature in popular culture as diverse as George R.R. Martin and the Grateful Dead, but "mythic" is not too exaggerated a description of this mighty beast in popular culture.

Recently, a diverse and international group of researchers* have explored the relationships between these North American canid species, using both morphological characteristics and genetic comparisons between modern wolves and dire wolf fossils, from both mitochondria and, in a more limited extent genomic DNA (albeit focusing on comparisons of only one gene, COL1).  Sites where the more than 700 fossil dire wolf specimens used in these studies were obtained (and their associated academic institutions) were Natural Trap Cave, Wyoming (University of Kansas); Gigantobison Bay, Idaho (Idaho Museum Natural History); Sheridan Pit, Ohio (Cincinnati Museum Center); Guy Wilson Cave, Tennessee (University of Tennessee); American Falls Reservoir, Idaho (Idaho Museum Natural History); and Rancho La Brea Tar Seeps, California (La Brea Tar Pits and Museum), the latter being the predominant site for obtaining dire wolf fossils (100-fold more than gray wolf fossils from this site).

The paper begins by debunking (somewhat) the idea that dire wolves were "sister-species" or conspecific with gray wolves.  Although morphology is highly similar between these species as evaluated by various comparative criteria, these authors screened 46 fossil dire wolf specimens dating between 12,900 and 50,000 years ago for mitochondrial DNA (mtDNA) and, at lower complexity, genomic DNA (which is much less well preserved).  One of the La Brea Tar Pit specimens provided DNA from type-1 collagen gene (COL1) of sufficient quality for comparison.  These experiments suggested that dire wolves were not closely related to either gray wolves, coyotes, African wolves (Canis lupaster) or dogs (Canis familiaris).  mtDNA analyses supported these results, that dire wolves formed their own group evolutionarily highly divergent from grey wolves and coyotes, as illustrated by this phylogenetic tree:

Further, these researchers analyzed nuclear genomic data of the dire wolves together with previously published genomic data from eight extant canids:  grey wolf, coyote, African wolf, dhole, Ethiopian wolf (Canis simensis), African wild dog (Lycaon pictus), Andean fox (Lycalopex culpaeus) and grey fox (Urocyon cinereoargenteus—an outgroup).  Only the grey wolves, coyotes, dholes, and grey foxes had geographical ranges that overlapped the dire wolf during the Pleistocene prior to its extinction.  These analyses confirmed what these scientists characterized as a "distant evolutionary relationship" between the dire wolf and other canid species.  Further genomic comparisons indicated that the dire wolf was one branch of the canid lineage (the other two being African jackals and all other canid species), giving the surprising result that grey wolves are more closely related to African wild dogs, Ethiopian wolves, and dholes than they are to dire wolves.

Using these results to provide a "clock" to estimate when dire wolves diverged from other canid species, this paper reports that dire wolves shared a common ancestor with other canid species about 5.7 million years ago (the estimate having a range of 4.0-8.5 million years), with further divergence from African jackals about 5.1 million years ago (3.5-7.6 million years ago).  Their genetic analyses also showed no evidence of extensive interbreeding between dire wolves and any other extant North American canid species.  There was some evidence of genetic admixture between ancestors of dire wolves and ancestors of grey wolves, coyotes, and dholes about 3 million years ago.  These results are consistent with the ready genetic admixture that occurs, inter alia, between modern coyotes and grey wolves.  The authors state that, in this context, "our finding of no evidence for gene flow between dire wolves and grey wolves, coyotes or their common ancestor—despite substantial range overlap with dire wolves during the Late Pleistocene—suggests that the common ancestor of grey wolves and coyotes probably evolved in geographical isolation from members of the dire wolf lineage."  Given evolution of other canid species, and their status of immigration from Asia, these results suggest to these researchers that the dire wolf arose in North America and may be related to the also-extinct Armbruster's wolf (Canis armbrusteri).  Their genetic analyses also suggest to these researchers that dire wolves are properly classified as their own separate genus, Aenocyon, an idea first proposed (not on genetic comparison grounds) in 1918 (see Merriam, J. C., 1918, Note on the systematic position of the wolves of the Canis dirus group. Bull. Dept. Geol. Univ. California 10, 531–33).  This genetic and geographic isolation of the dire wolf from other canid species would explain the estimated age of the dire wolf lineage and the evolutionary distance of this species from other canids.

The paper ends with speculation regarding why dire wolves became extinct at the end of the Pleistocene (associated with megafaunal extinctions, i.e., die-offs of animals with weights greater than 100 pounds such as the American lion, the short-faced bear, mammoths, mastodons, ground sloths, and giant beavers) and other canids like coyotes and grey wolves in North America did not, in view of the "overall phenotypic similarities" between these beasts.  These speculations include greater morphological plasticity and dietary flexibility in canid species other than the dire wolf, or the ability to interbreed with other canids (which has been shown to permit acquired traits like coat color and enhanced immunity).  The researchers suggest that the inability of dire wolves to benefit from such phenotype acquisition through interbreeding might have prevented them from resisting diseases carried by Old World taxa arriving over the Bering land bridge.

While the information is limited (e.g., only five dire wolf genomic DNA samples were sufficiently intact to be assayed), the results from these studies are another example of the power of genetic analysis to "fill in the blanks" from the fossil record to illuminate animal (and human) migration in the Pleistocene era that led to population patterns of animals in the New World in the Holocene (current) Era.

* Department of Archaeology, Durham University, Durham, UK; Australian Centre for Ancient DNA, School of Biological Sciences, University of Adelaide, Adelaide, South Australia, Australia; Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA; School of Biological and Chemical Sciences, Queen Mary University of London, London, UK; Department of Archaeology, Classics and Egyptology, University of Liverpool, Liverpool, UK; School of Natural Sciences and Psychology, Liverpool John Moores University, Liverpool, UK; The Palaeogenomics & Bio-Archaeology Research Network, Research Laboratory for Archaeology and History of Art, The University of Oxford, Oxford, UK; Department of Anatomy, Des Moines University, Des Moines, IA; Department of Zoology, University of Oxford, Oxford, UK; Department of Anthropology, National Museum of Natural History, Smithsonian Institution, Washington, DC; Center of Excellence in Paleontology & Department of Geosciences, East Tennessee State University, Johnson City, TN; Department of Archaeology, University of Exeter, Exeter, UK; Institute of Archaeology, Russian Academy of Sciences, Moscow, Russia; ARAID Foundation, Instituto Universitario de Investigación en Ciencias Ambientales (IUCA) - Aragosaurus Group, Universidad de Zaragoza, Zaragoza, Spain; Department of Earth Sciences, Natural History Museum, London, UK; Section for Evolutionary Genomics, The GLOBE Institute, University of Copenhagen, Copenhagen, Denmark; Applied Paleoscience, Bothell, WA; Department of Archaeology, University of Sydney, Sydney, New South Wales, Australia; Department of Archaeology, University of Aberdeen, Aberdeen, UK; Department of Archaeology, Simon Fraser University, Burnaby, Canada; Institut des Sciences de l'Evolution - Montpellier, CNRS, Université de Montpellier, IRD, EPHE, Montpellier, France; Laboratoire Evolution & Diversité Biologique, UPS/CNRS/IRD, Université Paul Sabatier, Toulouse, France; Australian Museum Research Institute, Australian Museum, Sydney, New South Wales, Australia; Department of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz, CA; Institute of Plant and Animal Ecology, Urals Branch of the Russian Academy of Sciences, Yekaterinburg, Russia; Ural Federal University, Yekaterinburg, Russia; Department of Anthropology, Texas A&M University, College Station, TX; Center for Evolution and Medicine, Arizona State University, Tempe, AZ; School of Human Evolution and Social Change, Arizona State University, Tempe, AZ; Halmos College of Arts and Sciences, Nova Southeastern University, Fort Lauderdale, FL; Department of Archaeology, University of York, York, UK; Institute of Systematics and Ecology of Animals, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia; Idaho Museum of Natural History, Idaho State University, Pocatello, ID; Zoological Institute of the Russian Academy of Sciences, St Petersburg, Russia; Sobolev Institute of Geology and Mineralogy, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia; Tomsk State University, Tomsk, Russia; McDonald Institute for Archaeological Research, University of Cambridge, Cambridge, UK; Greenland Institute of Natural Resources, Nuuk, Greenland; NTNU University Museum, Trondheim, Norway; Institute of Human Origins, Arizona State University, Tempe, AZ; Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, CA; South Australian Museum, Adelaide, South Australia, Australia; Palaeogenomics Group, Department of Veterinary Sciences, Ludwig Maximilian University, Munich, Germany

Going from Bad to Worse: Evidence for Neuro-COVID Infections

By Kevin E. Noonan --

The COVID-19 pandemic has spread throughout the globe, infecting more than 90 million people and causing almost two million deaths (see "Tracking coronavirus’ global spread").  SARS-CoV-2 infection is the cause of the COVID-19 pandemic; this virus is recognized as the latest viral infection in humans of zoonotic origins, in this case bats first arising in Wuhan, China.  The infection is known to be introduced into the lungs, and infection due to the viral Spike protein binding to the angiotensin 1 converting enzyme 2 (ACE-2) receptor expressed in the lung.

Among the many consequences of SARS-CoV-2 infection are respiratory disease, as well as effects in the liver as well as neurological tissues.  The capacity of SARS-CoV-2 to infect brain is supported by finding viral RNA and proteins in brain tissue on autopsy, but the frequency of neurological infection remains unknown.  Regarding COVID-related disease in brain, there are a number of possible bases for the anecdotal reports of impaired function (including persistent headache and impaired consciousness and cognition), including inter alia reduced blood-borne oxygen due to impaired respiration.  This is a particular risk in patients with mild disease symptoms, because humans sense lack of oxygen indirectly by increased levels of carbon dioxide in the blood and thus brain and other neurological tissues can be oxygen-deprived unknowingly.  In addition, it has long been known that there can be psychological sequellae to severe, life-threatening, debilitating diseases and thus the effects of psychology rather than pathology is not easy to tweeze out of the clinical presentation of these effects.

This week, an international group of researchers* illuminated how SARS-CoV-2 can infect neurological tissue and provides a viral explanation of neurological COVID infection, in a paper entitled "Neuroinvasion of SARS-CoV-2 in human and mouse brain," in the Journal of Experimental Medicine.  The experiments these researchers performed are illustrated in the paper's "graphical abstract":

The first of these methods used human neural progenitor cells and human brain organoids produced therefrom, which the authors reference as having been used to study Zika virus infection (which had been associated with anencephaly in fetuses in utero).  These experiments showed infection of 2-week old human neural progenitor cells, wherein peak viral titers were found 12 hours after infection and were associated with increased cell death.  SARS-CoV-2 infection was found in 9-week old human brain organoids within 24 hours of infection and significant infection within 96 hours post-infection.  "[T]he majority of the SARS-CoV-2–infected cells were localized within MAP2-positive cellular fields of mature neurons," according to the authors, but infection was also observed in "SOX2-positive neural stem cells with bipolar morphology and cells localized around the neural tube–like structures."  Viral particles were observed by electron microscopy to be located intracellularly associated with endoplasmic reticulum, and were associated with "extensive neuronal cell death."  The authors conclude from these organoid studies that "SARS-CoV-2 can infect cells of neural origin and suggested that infected cells can promote death of nearby cells."

Turning to molecular studies, the researchers reported investigation into whether some cells are more susceptible to virus-induced cell death than others, using single-cell RNA profiling.  These experiments were performed using 60-day old organoids virus-infected or mock-infected, and 96,205 cells were assayed in 31 clusters comprising "neural progenitors/outer radial glia, intermediate progenitor/ interneurons, neurons, and cortical neurons."  The researchers reported these experiments showed "widespread infectivity of SARS-CoV-2 in neurons, radial glia, and neuronal progenitor cells."  When compared with the effects of Zika virus (ZIKV) infection on neural tissues, "SARS-CoV-2–infected brain organoid up-regulated pathways related to cell division, organelle fission, and metabolic processes, while ZIKV showed enrichment in type I IFN pathways."  These results were consistent, according to these researchers, with reports that "SARS-CoV-2 induces a moderate IFN-stimulated gene response in other tissues" as well as "previous reports of specific virus replication being controlled by alternative pathways by neurons."

When comparing SARS-CoV-2 infected cells with adjacent uninfected cells, the infected cells showed "enrichment of genes corresponding to viral transcription, along with enrichment for metabolic processes including electron transport–coupled proton transport, cytochrome c to oxygen, and NADH to ubiquinone" whereas the uninfected cells showed "a mitochondrial catabolic state with the up-regulation of alcohol metabolism, cholesterol synthesis, and regulation of cell death."  Consistent with the mechanism of infection in other tissues, experiments using ACE2 receptor-blocking antibodies showed that expression of ACE2 receptor was necessary for neural tissue infection, despite the low frequency of detecting ACE2 receptor mRNA expression in neural tissues.  In addition, IgG antibodies immunologically specific for the viral Spike protein were found in cerebrospinal fluid from patients with virus-associated acute encephalopathy, which antibodies were capable of blocking virus infection in human brain organoids.

The authors also produced a mouse model of SARS-CoV-2 infection using transgenic mice that expressed human ACE2 in brain tissue. Intranasal administration of the virus produced infection in neural cells in the forebrain; in contrast there was "a relatively low density of infected cells [in] the dentate gyrus, the globus pallidus, and cortical layer 4."  In perhaps the most disquieting result reported in this paper, mice induced to express human ACE2 receptor in either lung tissue or ventricular structures in brain by Adenovirus infection were shown in later SARS-CoV-2 infection to have "signs of lung pathology but no weight loss or death," but both weight loss and death caused by brain infection even at much lower (100-fold) administered virus dose, suggesting neurological infection has high "neuroreplicative potential and lethal consequences."

Finally, the paper reported results of three human autopsies of COVID-19 patients, all of which "had been admitted to the intensive care unit, . . . sedated and ventilated due to respiratory failure [within 3-18 days], and [wherein] their difficulty to be weaned from mechanical ventilation indicated the severity and highly pathogenic nature of the disease course."  The patterns of virus-specific immunostaining were somewhat idiosyncratic, but death was associated with "a temporal sequence of continued ischemic events."  Somewhat curiously, regions of SARS-CoV-2 infection were not associated with lymphocyte or leukocyte infiltration which indicates, according to these authors, that "although SARS-CoV-2 has neurotropic properties and can infect neurons in patients, it did not invoke an immune response typical of other neurotropic virus."

The paper concludes that "the brain is a site for high replicative potential for SARS-CoV-2," that "SARS-CoV-2 causes significant neuronal death in human brain organoids," that "ACE2 is expressed at the protein level and is functionally required for SARS-CoV-2 infection in human brain organoids," and that "there is robust antibody response against the virus within the CSF," which can cause "cascading downstream effects in causing and amplifying CNS inflammation."  The mouse model studies reported "for the first time that SARS-CoV-2 neuroinvasion in mice can have significant remodeling of brain vasculature."  Human autopsy results, according to this paper, showed that neural pathology in humans was a consequence of acute ischemic damage caused by the infection (although these researchers emphasized that the small sample size limited the conclusions that they could draw from these results).  Despite these caveats, the paper concludes with these researchers asserting that:

Altogether, our study provides clear demonstration that neurons can become a target of SARS-CoV-2 infection, with devastating consequences of localized ischemia in the brain and cell death, highlighting SARS-CoV-2 neurotropism and guiding rational approaches to treatment of patients with neuronal disorders.

While gratifying to the extent that these researchers have explicated the ability of SARS-CoV-2 to infect neural tissues and produce neurological disease, the paper suggests that any hope that 2021 will be a better year than 2020 may be unfounded, or at least too much to hope for, as well as both a cautionary tale of the dangers of this infection and the importance of achieving effective vaccination quickly and universally.

* From the Departments of Immunobiology, Genetics, Pathology, Internal Medicine, Comparative Medicine, and Neuroscience, Yale Medical School; Hôpital Pitié-Salpêtrière  and the Sorbonne, Paris.

Molecular Mechanism for (In)Famous Warburg Effect in Cancer Elucidated

By Kevin E. Noonan --

Cancer, the "Emperor of All Maladies" as it has been termed, has been studied for millennia.  President Nixon's "War on Cancer" resulted in slow but steady progress, aided by the biotechnology revolution, the development of monoclonal antibodies (see Herceptin®) and more recent developments in immunological interventions like CAR-T cells (see "Global CAR-T Cell Therapy Market (2021 to 2027) - Size, Forecasts, Trials and Trends -ResearchAndMarkets.com").

But the basis for many characteristics of cancer biology remain elusive, including what is classically termed the Warburg effect, i.e., the tendency of cancer cells to be dependent upon aerobic glycolysis instead of oxidative phosphorylation even in the presence of oxygen and thus not being an adaptation for low oxygen conditions as it is in normal cells.  (Those with long memories will remember the saga of the eminent Efraim Racker being fooled by a graduate student who presented falsified evidence of a protein kinase cascade that regulated glycolysis.)  This effect has been known since the 1920's (during the Golden Age of research into the biochemical basis of intermediary metabolism) when Otto Heinrich Warburg found that depriving tumor cells of oxygen and glucose resulted in cell death.  But the change has remained phenomenology perhaps until now.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) which is an inherited form of kidney cancer is characterized by deficiency in fumarate hydratase (FH), which catalyzes the reaction:

as part of the Krebs (TCA) cycle in the mitochondria.  Until now there has been no understanding of why or whether this metabolic deficiency results in tumors that rapidly grow and metastasize.  In a recent paper, a team of university and National Institutes' researchers* recently reported that the growth aggressive phenotype is associated with expression of the Warburg effect, in a paper entitled "Mitochondrial DNA alterations underlie an irreversible shift to aerobic glycolysis in fumarate hydratase-deficient renal cancer," in Science Signaling.  These researchers show that the enzymatic deficiency is associated with an accumulation of fumarate in the cells, and this results in increased mutation rates in mitochondrial DNA as a consequence of fumarate accumulation.  The authors draw the conclusion that these cancer cells are "rewired" to promote tumor progression and metastasis due to a loss of mitochondria and a "metabolic shift" to aerobic glycolysis.

FH is encoded by a nuclear-encoded gene but the enzyme is necessary for competent mitochondrially mediated metabolic function.  The basis for high fumarate's deleterious effects on mitochondria is the "nonenzymatic formation of an S-(2-succinyl)-cysteine (2SC) adduct that inactivates multiple proteins involved in tumor biology and metabolism."  The researchers found that there was a loss of respiratory-chain components encoded by mitochondrial DNA and with S-(2-succinyl)-cysteine (2SC) adduct-mediated inhibition of mitochondrial DNA replication.  This in turn was caused by inactivation of the sole mitochondrial DNA polymerase as well as mtDNA maintenance and repair proteins, leading to mitochondrial depletion in these cells.  The authors termed this mitochondrial depletion a "defining feature" of this tumor type and its aggressive growth and metastasis phenotype.

The researchers studied a cohort of 24 HLRCC patients aged 21-70 with the majority (75%) having advanced metastatic disease.  Assessment of proteins in the respiratory chain showed disruption associated with morphological defects on the mitochondrial inner membrane (where these proteins are located in normal cells).  As summarized by the authors, "[t]ogether, these data demonstrate an impairment in the assembly of respiratory CI, CIII, and CIV in HLRCC tumor mitochondria with a substantial loss of protein abundance of mitochondrially encoded subunits."

These results were consistent with the detected down-regulation of mitochondrial mRNA synthesis in these cells which was not seen in the expression of nuclear gene-encoded mRNAs members of Krebs cycle or respiratory chain proteins, as illustrated in this Figure:

As stated in the paper "[o]ur transcriptome analyses indicated that the underlying cause of respiratory chain dysfunction in HLRCC FH^−/− tumors was the selective loss of expression of mtDNA-encoded subunits" of the respiratory chain"; the authors were able to correlate this reduction in mtDNA gene expression with a reduction in the number of mitochondria per cell, from an average of 833 mtDNA molecules in normal renal cells to between 344 and 294 copies in primary and metastatic HLRCC tumors, respectively.

These changes were mirrored in HLRCC tumor-derived cell lines, albeit not entirely consistently.  To evaluate these inconsistencies the researchers restored normal FH levels by introducing heterologous FH-encoding constructs that showed "[r]obust FH activity."  Nevertheless, functional respiratory chain function was not established in these cells, consistent with the defect being associated with mitochondrial loss rather than merely FH disfunction:  "[o]ur observations indicate that loss of FH activity had a lasting impact on mitochondrial function even after FH activity was restored and fumarate levels were reduced in the tumor-derived cells."

These results led the researchers to evaluate the status of mtDNA in these cell lines by DNA sequencing.  These experiments showed multiple deactivating mutations in several mtDNA-encoded genes involved in respiration, including mutations not found in the Human Mitochondrial DNA database (HmtDB), which the authors assert suggested that "these mutations are not tolerated in normal human tissues."  When similar experiments were performed in the HLRCC tumor samples, almost all (22) showed one or more somatic changes in mtDNA, each sample having on average 2.27 (range, 1-7) mutations in the mtDNA, wherein more than half of these mutations resided in protein-encoding mtDNA.  Such patterns of mtDNA mutations were not found in other types of renal cell carcinoma not associated with FH deficiencies.

Finally, an analysis of the protein components of mtDNA, which encode proteins known to be involved in maintaining DNA integrity, were found by these researchers to be covalently modified by succinylation at cysteine (Cys) residues, resulting in their inactivation.  These results were consistent with the loss of mitochondria population in these cells.  These results were observed in both cells from HLRCC tumor cell lines and HLRCC patient samples.

These researchers summarized their results with this diagram:

and by noting that FH deficiency resulted in inactivation of core enzymes involved in mtDNA integrity, repair, and replication and irreversible mitochondrial loss.  Mitochondrial loss "obligates cells to compensate for energy loss by shifting to dependence on aerobic glycolysis as an energy source."

These changes were also associated with upregulation of enzymatic activity that promoted lipid synthesis, necessary for rapid tumor growth.  As summarized by the authors:

[T]he metabolic compensations dictated by loss of mtDNA may result in features of the aggressive growth phenotype that characterizes HLRCC tumors, including increased fatty acid biosynthesis contributing to formation of new cell membranes, generation of the intermediates of the citric acid cycle that are used in anaplerotic reactions, and synthesis of other precursors necessary for biosynthesis of amino acids and nucleotides that are needed for rapid growth and invasiveness of this type of cancer.

And:

In summary, mitochondrial dysfunction forces metabolic remodeling in HLRCC tumors that favors anabolic pathways offering crucial bioenergetic and biosynthetic advantages for tumor growth and metastasis.  Understanding that mtDNA mutations and deletions represent the fundamental cause for the shift to complete dependence on aerobic glycolysis in this aggressive form of inherited, genetically defined RCC will hopefully provide insights into the development of effective forms of therapy for patients with HLRCC and other related cancers.

* National Institutes of Health, National Cancer Institute, National Institute of Child Health and Human Development, Frederick National Laboratory for Cancer Research, and the University of Kentucky

Epigenetic Changes Implicated in Age-related Diminution in Vision and Its Possible Reversal

By Kevin E. Noonan --

The Fountain of Youth -- an enduring aspiration, particularly as the ravages of age reduce human faculties prior to leading inexorably to death.  Reduction in sight is the human faculty that can have the greatest effect on quality of life in the aged -- a faculty that begins to decline in the 4th or 5th decade of life and doesn't get better (when it does) without medical intervention.

But what if there were a way to rejuvenate sight?  That prospect is the tantalizing suggestion in a paper published on December 2nd entitled "Reprogramming to recover youthful epigenetic information and restore vision," Nature 588: 124-29*.  The basis of the report is the recognition that many of the age-related effects on vision are an example of gene expression differences associated with epigenetic changes in chromosomal DNA.  Epigenetics is a phenomenon of gene structure and expression involving small differences in nucleotide bases, typically methylation of cytosine residues at specific (CpG) sites.  These changes have been studied in normal development, where gene expression changes arise as different cell types properly differentiate and act as a molecular "clock" reflecting age.  The ability to turn back cellular time has been demonstrated by the development of induced pluripotent stem cells (iPSCs), wherein terminally differentiated somatic cells (typically fibroblasts) can be turned into pluripotent cells.  Pluripotent cells are capable of differentiating into cells of each embryonic germinal layer (ectoderm, mesoderm, endoderm), and iPSCs can be produced by expressing four specific genes: OCT4, SOX2, KLF4 and MYC.  All of these genes encode transcription factors capable of affecting (and effecting) developmentally relevant gene expression.  Consequent to this "de-differentiation" occasioned by expression of these genes is a "resetting" of the epigenetic patterns associated with development.  In this paper the researchers hypothesized that resetting these epigenetic patterns could also rejuvenate neuroretinal cells to reinvigorate and overcome the ocular nerve damages by glaucoma in an animal model.

Because one of these genes (MYC) is also associated with cancer development (i.e., it is an oncogene) the researchers developed an inducible expression construct that expressed only the  OCT4, SOX2, and KLF4 members of the quartet (OSK).  (This decision was also informed by the experience of other researchers that continuous expression of all four genes in animal models resulted in teratomas or was fatal within days of introduction.)  Their system used a polycistronic (i.e., all the genes in one linear array) construct of all three genes regulated by a tetracycline response element (TRE) promoter in a adeno-associated viral vector.  This construct was tested by introduction into fibroblasts from aged (20 month old) mice and gene expression related to aging (i.e., that showed differential expression with age) was evaluated.  These studies showed that OSK expression for 5 days resulted in a "youthful" mRNA expression pattern in these genes (without any effect on the terminal differentiation state of the fibroblasts).

The TRE promoter enabled selection for or against expression of the OSK gene cassette; as the authors explain "[t]he TRE promoter can be activated either by reverse tetracycline-controlled transactivator (rtTA) in the presence of the tetracycline derivative doxycycline (DOX) ('Tet-On') or by tetracycline-controlled transactivator (tTA) in the absence of DOX ('Tet-Off')."  Simply put, the presence of absence of DOX in the animal's drinking water determined whether the expression cassette is "on" or "off," as illustrated in this figure:

Long-term (10-18 months) expression of this cassette was achieved in both young (5 months-old) and aged mice with no tumorigenesis or other negative side effects being observed.

To test the ability of induced OSK expression to rejuvenate optical nerve cells the researchers examined retinal ganglion cells (RGC, which project axons away from the retina informing the optic nerve) in an optic nerve crush injury model (which mimics the effects of optic nerve injury due to inter alia glaucoma).  The construct was delivered by injection into the vitreous humor and resulted in about 37% of the RGCs taking up and expressing the OSK genes in response to DOX administration.  A separate cohort of mice were administered versions of the construct where DOX inhibited OSK expression.  In these experiments, "the greatest extent of axon regeneration and RGC survival occurred when all three genes were delivered and expressed as a polycistron within the same AAV particle" according to the researchers.  In contrast, inhibition of OSK expression in the "Tet-Off" mice showed no axonal growth.  Moreover, delivery of the OSK genes individually in separate viral vectors or in pairs also did not show axonal growth, indicating the need for these genes to be expressed together in proper relative amounts provided by the polycistronic construct.  The researchers also found OSK expression induced expression of Stat3, a gene know to encourage regeneration.  These results were obtained in using 12-month-old mice as well as 1- and 3-month-old mice, which indicated, as the authors note, that "ageing does not greatly diminish the ability of OSK transcription factors to induce axon regeneration."  Increased axonal growth from RGCs was found even after crush injury, an effect found with no other treatment modalities.

The researchers then determined whether these reinvigorated RGCs showed changes in DNA methylation patterns.  In the absence of DOX-induced OSK expression injury in this model caused an "accelerated" aging pattern, whereas in the presence of DOX-induced OSK expression counteracted this effect according to the results reported in this paper.  Interestingly, this preservation of a "youthful" pattern of DNA methylation was found to be enriched at genes "associated with light detection and synaptic transmission."  Having shown this association the researchers then investigated whether axonal regeneration required youthful changes in DNA methylation.  These experiments were performed by reducing expression of genes that caused DNA demethylation in RGCs (and whose expression was known to be increased in cells expressing OSK) and detecting that axonal regeneration did not occur in these mice even in the presence of DOX-induced OSK expression.

Whether these effects of OSK expression would also be seen in human neurons was investigated using differentiated human neurons in vitro.  Neurons harboring an OSK-encoding construct were treated with vincristine (a drug that occasions axon injury) and DOX-induced OSK expression was shown to "counteract[] axonal loss and the advancement of DNA methylation age," showing a 15-fold greater area of proliferation in OSK-expressing cells than control vincristine-treated neural cells.  These cells also showed the demethylation-dependent characteristics that were shown in RGCs in the mouse optic nerve crush injury model.

The most clinically significant result disclosed in this paper involved the effect of OSK expression in a glaucoma model in vivo.  Intraocular pressure was increased to pathological levels by injecting microbeads unilaterally into the anterior chamber of mouse eye for 21 days.  At 4 weeks, after these animals showed correspondingly unilateral decreases in axonal density and the number of RGCs present in the treated eye.  The viral vector encoding inducible OSK expression thereafter was introduced by intravitreal injection followed by DOX-induced OSK expression for 4 weeks.  Compared with control (introduction of saline or viral vectors not encoding OSK into the microbead-treated eyes) the OSK vector-treated eyes showed "restored axon density equivalent to that in the non-glaucomatous eyes, with no evidence of RGC proliferation."  These mice also showed a reversal of vision loss caused by the glaucomatous injury.  Together these results indicated that OSK expression could be a therapy for glaucoma in humans.

Finally, the paper reports efforts to determine whether OSK expression could improve age-related (as opposed to injury- or pathology-related) vision problems.  In these experiments, 3-and 11-month-old mice were treated by intravitreal injection of DOX-inducible OSK encoding constructs and OSK expression induced for 4 weeks.  Twelve-month-old mice showed age-related visual acuity and RGS electrical activity diminution which was reversed by DOX-induced OSK expression.  However, these phenotypic changes were not observed to be associated with an increased number of RGCs or axon density, which prompted these researchers to hypothesize that the effect were dependent on changes in gene expression ("transcriptomic changes" as these were termed in the paper).  RGCs from treated or untreated 12-month-old mice were isolated and compared with RGCs from 5-month-old mice and expression of 464 genes were found to be altered: expression of almost all (90%) of these genes were found to be restored to youthful levels in OSK-expressing RGCs.  The participation of DNA methylation changes in aged RGCs in producing a youthful pattern of gene expression was further assessed and validated using artificial intelligence/machine learning approaches.

The results reported in this paper suggest therapeutic interventions that could improve vision in the aged human population even in the absence of vision-impairing pathologies such as glaucoma.  Although cautious to mention that "we do not wish to imply that DNA methylation is the only epigenetic mark involved in this process" and "[i]t is likely to involve other transcription factors and epigenetic modifications," the authors are not blind to the implication that:

[W]e show that it is possible to safely reverse the age of a complex tissue and restore its biological function in vivo.  Using the eye as a model system, we present evidence that the ectopic expression of OSK transcription factors safely induces in vivo epigenetic restoration of aged CNS neurons, without causing a loss of cell identity or pluripotency.  Instead, OSK promotes a youthful epigenetic signature and gene-expression pattern that causes the neurons to function as though they were young again.  The requirement for active demethylation in this process supports the idea that changes in DNA methylation patterns are involved in the ageing process and its functional reversal.

* By researchers from Harvard Medical School, Yale University School of Medicine, Massachusetts General Hospital, UCLA Geffen School of Medicine, and The University of New South Wales Medical School.

Neanderthal-specific Variants in Sodium Ion Channel Gene Associated with Sensitivity to Pain in Modern Humans

By Kevin E. Noonan --

Over the past decade, genetic archeology has revealed two branches of the human family tree, one known since the 19th Century (the Neanderthals) and the other more recently discovered (the Denisovans, an Asian relative of the Neanderthal population).  These populations evolved without genetic intermingling with Homo sapiens sapiens for about 500,000 years, which resulted in the accumulation of genetic variants specific for these populations.  But the migration of modern human populations out of Africa and throughout the world resulted in interbreeding between humans and Neanderthals or Denisovans about 60,00-40,000 years ago, and as a result there are genetic sequences inherited from them in modern human DNA that can be distinguished on the basis of these population-specific genetic alterations (see, e.g., Inherited Neanderthal Gene Encodes Genetic Risk for COVID-19).

Recent work* has found a cluster of genetic alterations ultimately inherited by modern humans from Neanderthals in a paper entitled "A Neanderthal Sodium Channel Increases Pain Sensitivity in Present-Day Humans," published in Cell Current Biology 30: P3465-69.  These alterations were found in a Neanderthal version of a gene, the SCN9A gene, that encodes Nav1.7 voltage-gated sodium ion channel involved in nerve transmission in peripheral nerves and implicated in pain sensation.  In modern humans, mutations in this gene causing a "loss of function" phenotype (inactivation of this gene) are associated with pain insensitivity and anosmia, while "gain of function" mutations  (increased activity) are associated with idiopathic small-fiber neuropathy and increased sensitivity to pain.

The Neanderthal-specific alleles reported in this paper contained three amino acid variants (M932L, V991L, and D1908G) compared with the gene found in modern humans; alleles containing all three of these variant amino acids are not found in any living primate other than humans (see below).  These researchers performed a series of experiments to determine the physiological effects of these variants assessed alone, in pairs, and in the presence of all three variant amino acids.  These experiments involved synthesizing messenger RNAs (mRNAs) corresponding to each species of variant and introducing them into Xenopus laevis oocytes (which provided a more easily manipulated experimental platform for establish the effects) and then human cells.  The oocyte experiments, which involved co-expression with a human gene encoding a protein (termed the "β3 subunit") that forms a functional complex with Nav1.7 protein, evaluated the electrophysiological function of the channel and showed a shift compared to the human gene product indicating increased sensitivity.  Physiologically, the channel having the Neanderthal variants remained "open" for a longer time and, the researchers wrote, "is expected to lower the threshold for the generation of an action potential" (i.e., activate the physiological mediator of nerve activity that can be perceived as pain).

When tested individually and in combinations (M932L+V991L, V991L+D1908G, M932L+D1908G, and M932L, V991L, and D1908G), none of the single amino acid variant-containing species showed any differential effect on function.  When tested in pairs, two of the variants (M932L+D1908G and M932L+V991L) also showed no effect.  However the third combination (V991L+D1908G) showed the same type of shift detected in the triple variant.  When these amino acids were mapped to the protein (shown below) the V991L and D1908G residues were found on the intracellular portion of the protein while the M932L residue was found extracellularly, perhaps indicating a need for an interaction prevented by the intervening cell membrane for the M932L variant.  The D1908G variant (but not the other two) is also found in Denisovan DNA and can be inherited homozygously; as the authors speculate, "[t]his suggests that the D1908G substitution occurred in the common ancestor of Neanderthals and Denisovans but that it may have had a functional effect only in Neanderthals, where also the V991L substitution occurred."

Similar experiments were performed using human embryonic kidney cells, where similar results were obtained for these variant Nav1.7 protein species.

The authors next reported the results of their investigations of whether any of these variants are found in DNA from present-day humans, examining 2,535 genomes in the 1000 Genomes database.  None of these variants were found in DNA from African or European populations, but the M932L and V991L variants were found in Asian and American populations at low frequencies (0.9-7.8% and 0.5-23.8%, respectively) but in a pattern indicting co-inheritance (termed "linkage disequilibrium").  The D1908G variant was also detected in these populations, at frequencies of 0-17.1% in Asia and 0.5-52.9% in America; where present this variant is in linkage disequilibrium with the M932L and V991L variants.

These researchers also tested whether these variants were inherited from a common ancestor to Neanderthals, Denisovans, and modern humans or whether they arose in ancient Neanderthal and Denisovan populations and were introduced later by cross-breeding with Homo sapiens sapiens.  The genetic signal providing the assay was the size of the genetic element in which these variants are found, which were expected to be larger if the introduction was more recent than a common ancestor because the "genetic reshuffling" caused by meiotic recombination would not have had as long a time to disrupt co-inheritance.  Indeed this was the found to be the case, when a comparison was made between DNA fragments from Yoruba individuals and ones from Neanderthal and Denisovan samples containing alleles likely to have been co-inherited.  The researchers found one ~26 kilobasepair (kb) fragment containing the M932L and V991L variants and another ~110 kb fragment containing the D1908G variant whose inheritance patterns were consistent with later introgression of these variants from interbreeding between the different populations.

The M932L and V991L variants have also been associated in modern populations with increased sensitivity to pain, and small-fiber neuropathy, consistent with the experimental results reported in this paper.  A survey of 362,944 unrelated "individuals of British ancestry" revealed  none that while were homozygous for the three variants, 1,327 (0.4%) carried an allele bearing all three variants in heterozygous form.  And their genetic makeup correlated to their responses to questions about their sensitivity to pain, which was that they "had experienced one or more forms of pain more often than non-carriers."  However, there were no individuals in this dataset having both V991L and D1908G variants.  And while gender was not a factor, age seemed to be, with "an approximately linear and positive correlation with reported pain between the ages of 40 and 70."

The authors conclude with reasoned speculation on the significance of their findings:

[G]iven the electrophysiological effects of the [amino acid] substitutions, nociception, i.e., the input to the central nervous system from peripheral nerves in response to harmful or potentially harmful stimuli, is likely to have been higher in Neanderthals than in modern humans.  The translation of such input into the conscious perception of pain is modulated both at the level of the spinal cord and the brain.  Thus, it is not possible to conclude that Neanderthals necessarily experienced more pain than modern humans do.  Yet the input from Neanderthal peripheral nerve endings would have allowed Neanderthals to be more sensitive to stimuli, as suggested by the observations in present-day people heterozygous for the Neanderthal Nav1.7 variant.

*H. Zeberg, M. Dannemann, K. Sahlholm, K. Tsuo, T. Maricic, V. Wiebe, W. Hevers, H. Robinson, J. Kelso, and S. Pääbo