Somatic mutation patterns at Ig and Non-Ig Loci.

The reverse transcriptase (RT) model of immunoglobulin (Ig) somatic hypermutation (SHM) has received insufficient scientific attention. This is understandable given that DNA deamination mediated by activation-induced deaminase (AID), the initiating step of Ig SHM, has dominated experiments since 2002. We summarise some key history of the RT Ig SHM model dating to 1987. For example, it is now established that DNA polymerase η, the sole DNA repair polymerase involved in post-replication short-patch repair, is an efficient cellular RT. This implies that it is potentially able to initiate target site reverse transcription by RNA-directed DNA repair at AID-induced lesions. Recently, DNA polymerase θ has also been shown to be an efficient cellular RT. Since DNA polymerase θ plays no significant role in Ig SHM, it could serve a similar RNA-dependent DNA polymerase role as DNA polymerase η at non-Ig loci in the putative RNA-templated nucleotide excision repair of bulky adducts and other mutagenic lesions on the transcribed strand. A major yet still poorly recognised consequence of the proposed RT process in Ig SHM is the generation of significant and characteristic strand-biased mutation signatures at both deoxyadenosine/deoxythymidine and deoxyguanosine/deoxycytidine base pairs. In this historical perspective, we highlight how diagnostic strand-biased mutation signatures are detected in vivo during SHM at both Ig loci in germinal centre B lymphocytes and non-Ig loci in cancer genomes. These strand-biased signatures have been significantly obscured by technical issues created by improper use of the polymerase chain reaction technique. A heightened awareness of this fact should contribute to better data interpretation and somatic mutation pattern recognition both at Ig and non-Ig loci.

RNA-directed DNA repair and antibody somatic hypermutation.

Somatic hypermutation at antibody loci affects both deoxyadenosine-deoxythymidine (A/T) and deoxycytidine-deoxyguanosine (C/G) pairs. Deamination of C to deoxyuridine (U) by activation-induced deaminase (AID) explains how mutation at C/G pairs is potentiated. Mutation at A/T pairs is triggered during the initial stages of repair of AID-generated U lesions and occurs through an as yet unknown mechanism in which polymerase η has a major role. Recent evidence confirms that human polymerase η can act as a reverse transcriptase. Here, we compare the popular suggestion of mutation at A/T pairs through nucleotide mispairing (owing to polymerase error) during short-patch repair synthesis with the alternative proposal of mutation at A/T pairs through RNA editing and RNA-directed DNA repair.

Analysis of SARS-CoV-2 haplotypes and genomic sequences during 2020 in Victoria, Australia, in the context of putative deficits in innate immune deaminase anti-viral responses.

The SARS-CoV-2 epidemic infections in Australia during 2020 were small in number in epidemiological terms and are well described. The SARS-CoV-2 genomic sequence data of many infected patients have been largely curated in a number of publicly available databases, including the corresponding epidemiological data made available by the Victorian Department of Health and Human Services. We have critically analysed the available SARS-CoV-2 haplotypes and genomic sequences in the context of putative deficits in innate immune APOBEC and ADAR deaminase anti-viral responses. It is now known that immune impaired elderly co-morbid patients display clear deficits in interferon type 1 (α/ß) and III (λ) stimulated innate immune gene cascades, of which APOBEC and ADAR induced expression are part. These deficiencies may help explain some of the clear genetic patterns in SARS-CoV-2 genomes isolated in Victoria, Australia, during the 2nd Wave (June-September, 2020). We tested the hypothesis that predicted lowered innate immune APOBEC and ADAR anti-viral deaminase responses in a significant proportion of elderly patients would be consistent with/reflected in a low level of observed mutagenesis in many isolated SARS-CoV-2 genomes. Our findings are consistent with this expectation. The analysis also supports the conclusions of the Victorian government's Department of Health that essentially one variant or haplotype infected Victorian aged care facilities where the great majority (79%) of all 820 SARS-CoV-2 associated deaths occurred. The implications of our data analysis for other localized epidemics and efficient coronavirus vaccine design and delivery are discussed.

Nature of Acquired Immune Responses, Epitope Specificity and Resultant Protection from SARS-CoV-2.

The primary global response to the SARS-CoV-2 pandemic has been to bring to the clinic as rapidly as possible a number of vaccines that are predicted to enhance immunity to this viral infection. While the rapidity with which these vaccines have been developed and tested (at least for short-term efficacy and safety) is commendable, it should be acknowledged that this has occurred despite the lack of research into, and understanding of, the immune elements important for natural host protection against the virus, making this endeavor a somewhat unique one in medical history. In contrast, as pointed out in the review below, there were already important past observations that suggested that respiratory infections at mucosal surfaces were susceptible to immune clearance by mechanisms not typical of infections caused by systemic (blood-borne) pathogens. Accordingly, it was likely to be important to understand the role for both innate and acquired immunity in response to viral infection, as well as the optimum acquired immune resistance mechanisms for viral clearance (B cell or antibody-mediated, versus T cell mediated). This information was needed both to guide vaccine development and to monitor its success. We have known that many pathogens enter into a quasi-symbiotic relationship with the host, with each undergoing sequential change in response to alterations the other makes to its presence. The subsequent evolution of viral variants which has caused such widespread concern over the last 3-6 months as host immunity develops was an entirely predictable response. What is still not known is whether there will be other unexpected side-effects of the deployment of novel vaccines in humans which have yet to be characterized, and, if so, how and if these can be avoided. We conclude by remarking that to ignore a substantial body of well-attested immunological research in favour of expediency is a poor way to proceed.

Introduction-Panspermia, 2020.

This current volume is, in many ways, a 2020 update to the important 1999-2000 compendium by Sir Fred Hoyle and Professor N. Chandra Wickramasinghe's "Astronomical Origins of life: Steps towards Panspermia." The emerging new paradigm of biology that connects life on Earth with the wider cosmos is covered in considerable depth showing that terrestrial biological evolution is best understood as a cosmically derived habitat and an interconnected genetic system. The various chapters here discuss all aspects of this interconnectedness, particularly relevant now in this time of the coronavirus pandemic (COVID-19) as the human race reacts to the many microbes and viral pathogens that arrive regularly from space.

The efficient Lamarckian spread of life in the cosmos.

In this Chapter we discuss the various mechanisms that are available for the possible transfer of cosmic microbial living systems from one cosmic habitat to another. With the 100 or so habitable planets that are now known to exist in our galaxy alone transfers of cometary dust carrying life including fragments of icy planetoids/asteroids would be expected to occur on a routine basis. It is thus easy to view the galaxy as a single connected "biosphere" of which our planet Earth is a minor component. The Hoyle-Wickramasinghe Panspermia paradigm provides a cogent biological rationale for the actual widespread existence of Lamarckian modes of inheritance in terrestrial systems (which we review here). Thus the Panspermia paradigm provides the raison d'etre for Lamarckian Inheritance. Under a terrestrially confined neoDarwinian viewpoint such an association may have been thought spurious in the past. Our aim here is to outline the main evidence for rapid terrestrial-based Lamarckian-based evolutionary hypermutation processes dependent on reverse transcription-coupled mechanisms among others. Such rapid adaptation mechanisms would be consistent with the effective cosmic spread of living systems. For example, a viable, or cryo-preserved, living system traveling through space in a protective matrix will of necessity need to adapt rapidly and proliferate on landing in a new cosmic niche. Lamarckian mechanisms thus come to the fore and supersede the slow (blind and random) genetic processes expected under neoDarwinian Earth centred theories.

Experiments to prove continuing microbial ingress from Space to Earth.

A wide range of evidence for pointing to our cosmic origins is close to the point of being overwhelming. Yet the long-entrenched paradigm of Earth-centered biology appears to prevail in scientific culture. A matter of crucial importance is to carry out a decisive experiment that is long overdue-establishing empirically beyond any doubt that extraterrestrial microbiota reaches the surface of the Earth at the present day. Such an experiment may of course happen naturally by the appearance of pandemics of new disease as discussed in an earlier chapter.

The sociology of science and generality of the DNA/RNA/protein paradigm throughout the cosmos.

The theory of cometary panspermia argues that life cannot have originated on Earth in the time available. It must have an ultimate, but still undiscovered cosmological source. The origin of life remains an open question. Life on Earth was introduced by impacting comets, and its further evolution was driven by the subsequent acquisition of cosmically derived genes. Explicit predictions of this theory stating how the acquisition of new genes drives evolution, are compared with recent developments in relation to horizontal gene transfer, and the role of retroviruses in evolution. Precisely stated predictions of the theory of cometary panspermia are shown to have been verified.

Cometary panspermia and origin of life?

A range of astronomical observations are shown to be in accord with the theory of cometary panspermia. This theory posits that comets harbor a viable biological component in the form of bacteria and viruses that led to origin and evolution of life on Earth. The data includes (1) infrared, visual and ultraviolet spectra of interstellar dust, (2) infrared spectra of the dust released from comet Halley in 1986, (3) infrared spectra of comet Hale-Bopp in 1997, (4) near and mid-infrared spectra of comet Tempel I in 2005, (5) the discovery of an amino acid and degradation products attributable to biology in the material recovered from the Stardust Mission in 2009, (6) jets from comet Lovejoy showing both a sugar and Ethyl alcohol and finally, (7) a diverse set of data that has emerged from the Rosetta mission. The conjunction of all the available data points to cometary biology and interstellar panspermia as being inevitable.

Origin of new emergent Coronavirus and Candida fungal diseases-Terrestrial or cosmic?

The origins and global spread of two recent, yet quite different, pandemic diseases is discussed and reviewed in depth: Candida auris, a eukaryotic fungal disease, and COVID-19 (SARS-CoV-2), a positive strand RNA viral respiratory disease. Both these diseases display highly distinctive patterns of sudden emergence and global spread, which are not easy to understand by conventional epidemiological analysis based on simple infection-driven human- to-human spread of an infectious disease (assumed to jump suddenly and thus genetically, from an animal reservoir). Both these enigmatic diseases make sense however under a Panspermia in-fall model and the evidence consistent with such a model is critically reviewed.

Blood and saliva-derived exomes from healthy Caucasian subjects do not display overt evidence of somatic mosaicism.

Somatic mosaicism is a normal occurrence during development in the tissues and organs. As part of establishing a "healthy population "(HP) background or base-line, we investigated whether such mosaicism can be routinely detected in the circulating DNA secured from a rigorously designed healthy human liquid biopsy clinical trial (saliva, blood). We deployed next generation (NG) whole exome sequencing (WES) at median exome coverage rates of 97.2 % (-to-30x) and 70.0 % (-to-100x). We found that somatic mosaicism is not detectable by such standard bulk WES sequencing assays in saliva and blood DNA in 24 normal healthy Caucasians of both sexes from 18 to 60 years of age. We conclude that for circulating DNA using standard WES no novel somatic mutational variants can be detected in protein-coding regions of normal healthy subjects. This implies that the extent within normal tissues of somatic mosaicism must be at a lower level, below the detection threshold, for these circulating DNA WES read depths.

A proposed reverse transcription mechanism for (CAG)n and similar expandable repeats that cause neurological and other diseases.

The mechanism of (CAG)n repeat generation, and related expandable repeat diseases in non-dividing cells, is currently understood in terms of a DNA template-based DNA repair synthesis process involving hairpin stabilized slippage, local error-prone repair via MutSß (MSH2-MSH3) hairpin protective stabilization, then nascent strand extension by DNA polymerases-ß and -δ. We advance a very similar slipped hairpin-stabilized model involving MSH2-MSH3 with two key differences: the copying template may also be the nascent pre-mRNA with the repair pathway being mediated by the Y-family error-prone enzymes DNA polymerase-η and DNA polymerase-κ acting as reverse transcriptases. We argue that both DNA-based and RNA-based mechanisms could well be activated in affected non-dividing brain cells in vivo. Here, we compare the advantages of the RNA/RT-based model proposed by us as an adjunct to previously proposed models. In brief, our model depends upon dysregulated innate and adaptive immunity cascades involving AID/APOBEC and ADAR deaminases that are known to be involved in normal locus-specific immunoglobulin somatic hypermutation, cancer progression and somatic mutations at many off-target non-immunoglobulin sites across the genome: we explain how these processes could also play an active role in repeat expansion diseases at RNA polymerase II-transcribed genes.

Regulatory T cells and co-evolution of allele-specific MHC recognition by the TCR.

What is the evolutionary mechanism for the TCR-MHC-conserved interaction? We extend Dembic's model (Dembic Z. In, Scand J Immunol e12806, 2019) of thymus positive selection for high-avidity anti-self-MHC Tregs among double (CD4 + CD8+)-positive (DP) developing thymocytes. This model is based on competition for self-MHC (+ Pep) complexes presented on cortical epithelium. Such T cells exit as CD4 + CD25+FoxP3 + thymic-derived Tregs (tTregs). The other positively selected DP T cells are then negatively selected on medulla epithelium removing high-avidity anti-self-MHC + Pep as T cells commit to CD4 + or CD8 + lineages. The process is likened to the competitive selection and affinity maturation in Germinal Centre for the somatic hypermutation (SHM) of rearranged immunoglobulin (Ig) variable region (V[D]Js) of centrocytes bearing antigen-specific B cell receptors (BCR). We now argue that the same direct SHM processes for TCRs occur in post-antigenic Germinal Centres, but now occurring in peripheral pTregs. This model provides a potential solution to a long-standing problem previously recognized by Cohn and others (Cohn M, Anderson CC, Dembic Z. In, Scand J Immunol e12790, 2019) of how co-evolution occurs of species-specific MHC alleles with the repertoire of their germline TCR V counterparts. We suggest this is not by 'blind', slow, and random Darwinian natural selection events, but a rapid structured somatic selection vertical transmission process. The pTregs bearing somatic TCR V mutant genes then, on arrival in reproductive tissues, can donate their TCR V sequences via soma-to-germline feedback as discussed in this journal earlier. (Steele EJ, Lindley RA. In, Scand J Immunol e12670, 2018) The high-avidity tTregs also participate in the same process to maintain a biased, high-avidity anti-self-MHC germline V repertoire.

Lamarck and Panspermia - On the Efficient Spread of Living Systems Throughout the Cosmos.

We review the main lines of evidence (molecular, cellular and whole organism) published since the 1970s demonstrating Lamarckian Inheritance in animals, plants and microorganisms viz. the transgenerational inheritance of environmentally-induced acquired characteristics. The studies in animals demonstrate the genetic permeability of the soma-germline Weismann Barrier. The widespread nature of environmentally-directed inheritance phenomena reviewed here contradicts a key pillar of neo-Darwinism which affirms the rigidity of the Weismann Barrier. These developments suggest that neo-Darwinian evolutionary theory is in need of significant revision. We argue that Lamarckian inheritance strategies involving environmentally-induced rapid directional genetic adaptations make biological sense in the context of cosmic Panspermia allowing the efficient spread of living systems and genetic innovation throughout the Universe. The Hoyle-Wickramasinghe Panspermia paradigm also developed since the 1970s, unlike strictly geocentric neo-Darwinism provides a cogent biological rationale for the actual widespread existence of Lamarckian modes of inheritance - it provides its raison d'être. Under a terrestrially confined neo-Darwinian viewpoint such an association may have been thought spurious in the past. Our aim is to outline the conceptual links between rapid Lamarckian-based evolutionary hypermutation processes dependent on reverse transcription-coupled mechanisms among others and the effective cosmic spread of living systems. For example, a viable, or cryo-preserved, living system travelling through space in a protective matrix will need of necessity to rapidly adapt and proliferate on landing in a new cosmic niche. Lamarckian mechanisms thus come to the fore and supersede the slow (blind and random) genetic processes expected under a traditional neo-Darwinian evolutionary paradigm.

Molecular model linking Th2 polarized M2 tumour-associated macrophages with deaminase-mediated cancer progression mutation signatures.

A new and diverse range of somatic mutation signatures are observed in late-stage cancers, but the underlying reasons are not fully understood. We advance a "combinatorial association model" for deaminase binding domain (DBD) diversification to explain the generation of previously observed cancer-progression associated mutation signatures. We also propose that changes in the polarization of tumour-associated macrophages (TAMs) are accompanied by the expression of deaminases with a new and diverse range of DBDs, and thus accounting for the generation of new somatic mutation signatures. The mechanism proposed is molecularly reminiscent of combinatorial association of heavy (H) and light (L) protein chains following V(D)J recombination of immunoglobulin molecules (and similarly for protein chains in heterodimers α/ß and γ/δ of V(D)Js of T Cell Receptors) required for pathogen antigen recognition by B cells and T cells, respectively. We also discuss whether extracellular vesicles (EVs) emanating from tumour enhancing M2-polarized macrophages represent a likely source of the de novo deaminase DBDs. We conclude that M2-polarized macrophages extruding EVs loaded with deaminase proteins or deaminase-specific transcription/translation regulatory factors and like information may directly trigger deaminase diversification within cancer cells, and thus account for the many new somatic mutation signatures that are indicative of cancer progression. This hypothesis now has a plausible evidentiary base, and it is worth direct testing in future investigations. A long-term objective would be to identify molecular biomarkers predicting cancer progression (or metastatic disease) and to support the development of new drug targets before metastatic pathways are activated.

Cause of Cambrian Explosion - Terrestrial or Cosmic?

We review the salient evidence consistent with or predicted by the Hoyle-Wickramasinghe (H-W) thesis of Cometary (Cosmic) Biology. Much of this physical and biological evidence is multifactorial. One particular focus are the recent studies which date the emergence of the complex retroviruses of vertebrate lines at or just before the Cambrian Explosion of ∼500 Ma. Such viruses are known to be plausibly associated with major evolutionary genomic processes. We believe this coincidence is not fortuitous but is consistent with a key prediction of H-W theory whereby major extinction-diversification evolutionary boundaries coincide with virus-bearing cometary-bolide bombardment events. A second focus is the remarkable evolution of intelligent complexity (Cephalopods) culminating in the emergence of the Octopus. A third focus concerns the micro-organism fossil evidence contained within meteorites as well as the detection in the upper atmosphere of apparent incoming life-bearing particles from space. In our view the totality of the multifactorial data and critical analyses assembled by Fred Hoyle, Chandra Wickramasinghe and their many colleagues since the 1960s leads to a very plausible conclusion - life may have been seeded here on Earth by life-bearing comets as soon as conditions on Earth allowed it to flourish (about or just before 4.1 Billion years ago); and living organisms such as space-resistant and space-hardy bacteria, viruses, more complex eukaryotic cells, fertilised ova and seeds have been continuously delivered ever since to Earth so being one important driver of further terrestrial evolution which has resulted in considerable genetic diversity and which has led to the emergence of mankind.