Strong Selective Pressure Research Articles

Assessment of genetic diversity of freshwater genus Labeo through DNA barcoding and phylogenetic analysis inhabited in the River Indus

ObjectivesGenus Labeo of family Cyprinidae comprising of important group of fish species are known for ornamental purpose, food, extracting oil, medicinal value and for potential viable aquaculture. The present study was conducted to assess the morphometric and molecular identification of freshwater fishes of genus Labeo including Catla catla, Labeo rohita, Labeo calbasu, Labeo gnius, Labeo bata from the three fish resources of River Indus, Punjab, Pakistan. MethodsOut of 75, thirty fish specimen were amplified and sequenced with 650 bp of mitochondrial COI gene for the evaluation of fish biodiversity, management and conservation by using different bioinformatics techniques. ResultsMaximum and minimum genetic divergence between fishes of C. catla, L. calbasu, L. boga, L. gonius and L. rohita, were 0.052 to 0.105, respectively. Highest divergence was found between Labeo bata and Labeo rohita. Interspecific mean distance was found higher than intraspecific distance. In nucleotide base composition, AT contents was higher than GC contents indicated the clear pattern of anti-G bias. Transition rate was k1 = 8.84 for purines and transversional rate was k2 = 15.47 for pyrimidines with transition/transversion bias R = 5.62. Tajima D test and nucleotide diversity was measured D = 1.382883 and π = 0.049981 respectively. Positive value of D indicated a strong selective pressure and lack of population expansion. Thirty species were identified with e-value 0.0 and similar identity 99.85 %–100 % with maximum 600 bp length. Phylogenetic tree revealed two major clades with 95 % high bootstrap values identity showed closet relationship among these species. ConclusionsCurrent study supported the efficacy of the COI gene for the species identification in River Indus and develop the concept for establishing Pakistani fish gene bank which is still needed.

Genomic Analysis Points to Multiple Genetic Mechanisms for Non-Transformable Campylobacter jejuni ST-50.

Campylobacter jejuni and Campylobacter coli are well known for their natural competence, i.e., their capacity for the uptake of naked DNA with subsequent transformation. This study identifies non-transformable C. jejuni and C. coli strains from domestic animals and employs genomic analysis to investigate the strain genotypes and their associated genetic mechanisms. The results reveal genetic associations leading to a non-transformable state, including functional DNase genes from bacteriophages and mutations within the cts-encoded DNA-uptake system, which impact the initial steps of the DNA uptake during natural transformation. Interestingly, all 38 tested C. jejuni ST-50 strains from the United States exhibit a high prevalence of non-transformability, and the strains harbor a variety of these genetic markers. This research emphasizes the role of these genetic markers in hindering the transfer of antimicrobial resistance (AMR) determinants, providing valuable insights into the genetic diversity of Campylobacter. As ST-50 is a major clone of C. jejuni globally, we additionally determined the prevalence of the genetic markers for non-transformability among C. jejuni ST-50 from different regions of the world, revealing distinct patterns of evolution and a strong selective pressure on the loss of competence in ST-50 strains, particularly in the agricultural environment in the United States. Our findings contribute to a comprehensive understanding of genetic exchange mechanisms within Campylobacter strains, and their implications for antimicrobial resistance dissemination and evolutionary pathways within specific lineages.

Illegal dumping of oil and gas wastewater alters arid soil microbial communities.

The Permian Basin, underlying southeast New Mexico and west Texas, is one of the most productive oil and gas (OG) provinces in the United States. Oil and gas production yields large volumes of wastewater with complex chemistries, and the environmental health risks posed by these OG wastewaters on sensitive desert ecosystems are poorly understood. Starting in November 2017, 39 illegal dumps, as defined by federal and state regulations, of OG wastewater were identified in southeastern New Mexico, releasing ~600,000 L of fluid onto dryland soils. To evaluate the impacts of these releases, we analyzed changes in soil geochemistry and microbial community composition by comparing soils from within OG wastewater dump-affected samples to unaffected zones. We observed significant changes in soil geochemistry for all dump-affected compared with control samples, reflecting the residual salts and hydrocarbons from the OG-wastewater release (e.g., enriched in sodium, chloride, and bromide). Microbial community structure significantly (P < 0.01) differed between dump and control zones, with soils from dump areas having significantly (P < 0.01) lower alpha diversity and differences in phylogenetic composition. Dump-affected soil samples showed an increase in halophilic and halotolerant taxa, including members of the Marinobacteraceae, Halomonadaceae, and Halobacteroidaceae, suggesting that the high salinity of the dumped OG wastewater was exerting a strong selective pressure on microbial community structure. Taxa with high similarity to known hydrocarbon-degrading organisms were also detected in the dump-affected soil samples. Overall, this study demonstrates the potential for OG wastewater exposure to change the geochemistry and microbial community dynamics of arid soils.IMPORTANCEThe long-term environmental health impacts resulting from releases of oil and gas (OG) wastewater, typically brines with varying compositions of ions, hydrocarbons, and other constituents, are understudied. This is especially true for sensitive desert ecosystems, where soil microbes are key primary producers and drivers of nutrient cycling. We found that releases of OG wastewater can lead to shifts in microbial community composition and function toward salt- and hydrocarbon-tolerant taxa that are not typically found in desert soils, thus altering the impacted dryland soil ecosystem. Loss of key microbial taxa, such as those that catalyze organic carbon cycling, increase arid soil fertility, promote plant health, and affect soil moisture retention, could result in cascading effects across the sensitive desert ecosystem. By characterizing environmental changes due to releases of OG wastewater to soils overlying the Permian Basin, we gain further insights into how OG wastewater may alter dryland soil microbial functions and ecosystems.

Predictability and conceptual repeatability of the predator-associated burst speed ecophenotype in western mosquitofish (Gambusia affinis).

Predation exerts strong selection pressure on morphological traits and is often studied in freshwater fishes. A common morphological ecophenotype driven by predation from pursuit predators is the predator-associated burst speed (PABS) ecophenotype. This ecophenotype, characterized by a smaller head, smaller body, and larger caudal region, is commonly found in western mosquitofish (Gambusia affinis) residing in environments with sunfish (family Centrarchidae) predators. However, the repeatability and transferability of the PABS ecophenotype across populations have not been tested. The four objectives of this study were to (1) assess the repeatability of methods to confirm the presence of the PABS ecophenotype in independent populations, (2) test whether morphology is correlated with predator abundance, (3) assess the repeatability of utilizing a discriminant function analysis (DFA) to accurately classify fish to predator status, and (4) use a DFA to conduct a cross-validation test of the PABS model using previously studied populations of G. affinis to predict predator status of newly studied populations. There was consistency in the occurrence of the PABS ecophenotype among populations with predators. Permuted MANOVA (PMANOVA) models fit to each dataset revealed a significant effect of collection site nested within predator status (i.e., presence or absence of predator), suggesting location-specific variations of the PABS ecophenotype. In addition, a PMANOVA model fit to body shape as a function of predator abundance (i.e., 0, 0.1-1.0, or >1.0 predators per minute of electrofishing) revealed a significant effect of predation level in the newly studied populations, suggesting morphology differs among populations with varying predator abundances. Cross-study validation of the DFA revealed low between-study accuracy compared to within-study accuracy, but elongation of the caudal region in the presence of predators was consistent across studies. Our findings ultimately suggest that although the PABS ecophenotype at studied locations provides partial predictive capacity at unstudied locations, the nature and magnitude of the PABS ecophenotype depend on environmental settings, encounter histories with predators, level of abundance of predators, or other spatially structured mechanisms.

Whole-genome resequencing of major populations revealed domestication-related genes in yaks

BackgroundThe yak is a symbol of the Qinghai-Tibet Plateau and provides important basic resources for human life on the plateau. Domestic yaks have been subjected to strong artificial selection and environmental pressures over the long-term. Understanding the molecular mechanisms of phenotypic differences in yak populations can reveal key functional genes involved in the domestication process and improve genetic breeding.Material and methodHere, we re-sequenced 80 yaks (Maiwa, Yushu, and Huanhu populations) to identify single-nucleotide polymorphisms (SNPs) as genetic variants. After filtering and quality control, remaining SNPs were kept to identify the genome-wide regions of selective sweeps associated with domestic traits. The four methods (π, XPEHH, iHS, and XP-nSL) were used to detect the population genetic separation.ResultsBy comparing the differences in the population stratification, linkage disequilibrium decay rate, and characteristic selective sweep signals, we identified 203 putative selective regions of domestic traits, 45 of which were mapped to 27 known genes. They were clustered into 4 major GO biological process terms. All known genes were associated with seven major domestication traits, such as dwarfism (ANKRD28), milk (HECW1, HECW2, and OSBPL2), meat (SPATA5 and GRHL2), fertility (BTBD11 and ARFIP1), adaptation (NCKAP5, ANTXR1, LAMA5, OSBPL2, AOC2, and RYR2), growth (GRHL2, GRID2, SMARCAL1, and EPHB2), and the immune system (INPP5D and ADCYAP1R1).ConclusionsWe provided there is an obvious genetic different among domestic progress in these three yak populations. Our findings improve the understanding of the major genetic switches and domestic processes among yak populations.

Escherichia coli adapts metabolically to 6- and 7-fluoroindole, enabling proteome-wide fluorotryptophan substitution

Nature has scarcely evolved a biochemistry around fluorine. However, modern science has shown that fluorinated organic molecules are suitable building blocks for biopolymers, from peptides and proteins up to entire organisms. Here, we conducted adaptive laboratory evolution (ALE) experiments to introduce organofluorine into living microorganisms. By cultivating Escherichia coli with fluorinated indole analogs, we successfully evolved microbial cells capable of utilizing either 6-fluoroindole or 7-fluoroindole for growth. Our improved ALE protocols enabled us to overcome previous challenges and adaptation was achieved, enabling a former growth inhibiting unnatural molecule to become a substrate for the cell’s protein synthesis machinery to the extent that the entire proteome underwent Trp to F-Trp substitution. In the ALE experiments, we supplied fluoroindoles to Trp-auxotrophic E. coli bacteria, exerting strong selective pressure that led to microbial adaptation. Within the cells, these indoles were converted into the corresponding amino acids (6- and 7-fluorotryptophan) and globally incorporated into the proteome at tryptophan sites. This study is a first step and establishes a strong foundation for further exploration of the mechanisms underlying fluorine-based life and how a former antimetabolite can become a vital nutrient.

The early ontogenetic development of immune cells and organs in teleosts

Fully developed teleosts possess a highly developed immune system comprising both innate and adaptive elements, but when hatching from the egg, the yolksac larva is still at an ontogenetically incomplete stage with regard to physiological, including immunological, functions. The immune system in these young fish stages is far less developed when compared to the youngs appearing from reptile and avian eggs and from most mammals at parturition. In those vertebrate groups the early ontogenetic development of the fetus is highly protected. The lack of a fully developed immune system in yolksac larvae of fish is critical, because this stage encounters a potentially hostile and infectious aquatic environment. The strong selective pressure on the immune system of the yolksac larva and the youngest fry stages explains the existence of a multi-facetted innate system, which is protecting the young fish stages against viral, bacterial and parasitic infections. The sequential development of immune cells and organs depends on host species and its environmental setting. However, a strong armament comprising innate cells (neutrophilic granulocytes, macrophages) and molecules (receptors, lectins, complement, AMPs and constitutively expressed immunoglobulins) protect the earliest stages. The adaptive immune elements, including T-cells and B-cells, occur gradually in headkidney, spleen, thymus, tonsils, bursa equivalent (if present) and mucosa associated lymphoid cells. A functional protective response following immunization occur later.

Bacteria colonization in tumor microenvironment creates a favorable niche for immunogenic chemotherapy

The tumor microenvironment (TME) presents differential selective pressure (DSP) that favors the growth of cancer cells, and monovalent therapy is often inadequate in reversing the cancer cell dominance in the TME. In this work, we introduce bacteria as a foreign species to the TME and explore combinatorial treatment strategies to alter DSP for tumor eradication. We show that cancer-selective chemotherapeutic agents and fasting can provide a strong selection pressure against tumor growth in the presence of bacteria. Moreover, we show that an immunogenic drug (oxaliplatin), but not a non-immunogenic one (5-FU), synergizes with the bacteria to activate both the innate and adaptive immunity in the TME, resulting in complete tumor remission and a sustained anti-tumor immunological memory in mice. The combination of oxaliplatin and bacteria greatly enhances the co-stimulatory and antigen-presenting molecules on antigen-presenting cells, which in turn bridge the cytotoxic T cells for cancer-cell killing. Our findings indicate that rational combination of bacterial therapy and immunogenic chemotherapy can promote anticancer immunity against the immunosuppressive TME.

Chemical Camouflage Induced by Diet in a Pest Treehopper on Host Plants.

Ants patrol foliage and exert a strong selective pressure on herbivorous insects, being their primary predators. As ants are chemically oriented, some organisms that interact with them (myrmecophiles) use chemical strategies mediated by their cuticular hydrocarbons (CHCs) to deal with ants. Thus, a better understanding of the ecology and evolution of the mutualistic interactions between myrmecophiles and ants depends on the accurate recognition of these chemical strategies. Few studies have examined whether treehoppers may use an additional strategy called chemical camouflage to reduce ant aggression, and none considered highly polyphagous pest insects. We analyzed whether the chemical similarity of the CHC profiles of three host plants from three plant families (Fabaceae, Malvaceae, and Moraceae) and the facultative myrmecophilous honeydew-producing treehopper Aetalion reticulatum (Hemiptera: Aetalionidae), a pest of citrus plants, may play a role as a proximate mechanism serving as a protection against ant attacks on plants. We found a high similarity (>80%) between the CHCs of the treehoppers and two of their host plants. The treehoppers acquire CHCs through their diet, and the chemical similarity varies according to host plant. Chemical camouflage on host plants plays a role in the interaction of treehoppers with their ant mutualistic partners.

Strong selection signatures for Aleutian disease tolerance acting on novel candidate genes linked to immune and cellular responses in American mink (Neogale vison)

Aleutian disease (AD) is a multi-systemic infectious disease in American mink (Neogale vison) caused by Aleutian mink disease virus (AMDV). This study aimed to identify candidate regions and genes underlying selection for response against AMDV using whole-genome sequence (WGS) data. Three case–control selection signatures studies were conducted between animals (N = 85) producing high versus low antibody levels against AMDV, grouped by counter immunoelectrophoresis (CIEP) test and two enzyme-linked immunosorbent assays (ELISA). Within each study, selection signals were detected using fixation index (FST) and nucleotide diversity (θπ ratios), and validated by cross-population extended haplotype homozygosity (XP-EHH) test. Within- and between-studies overlapping results were then evaluated. Within-studies overlapping results indicated novel candidate genes related to immune and cellular responses (e.g., TAP2, RAB32), respiratory system function (e.g., SPEF2, R3HCC1L), and reproduction system function (e.g., HSF2, CFAP206) in other species. Between-studies overlapping results identified three large segments under strong selection pressure, including two on chromosome 1 (chr1:88,770–98,281 kb and chr1:114,133–120,473) and one on chromosome 6 (chr6:37,953–44,279 kb). Within regions with strong signals, we found novel candidate genes involved in immune and cellular responses (e.g., homologous MHC class II genes, ITPR3, VPS52) in other species. Our study brings new insights into candidate regions and genes controlling AD response.

Genome-scale identification and comparative analysis of transcription factors in thermophilic cyanobacteria

BackgroundThe transcription factors (TFs) in thermophilic cyanobacteria might represent a uniquely evolved gene repertoire in light of the strong selective pressure caused by hostile habitats. Understanding the molecular composition of the TF genes in thermophilic cyanobacteria will facilitate further studies regarding verifying their exact biochemical functions and genetic engineering. However, limited information is available on the TFs of thermophilic cyanobacteria. Herein, a thorough investigation and comparative analysis were performed to gain insights into the molecular composition of the TFs in 22 thermophilic cyanobacteria.ResultsThe results suggested a fascinating diversity of the TFs among these thermophiles. The abundance and type of TF genes were diversified in these genomes. The identified TFs are speculated to play various roles in biological regulations. Further comparative and evolutionary genomic analyses revealed that HGT may be associated with the genomic plasticity of TF genes in Thermostichus and Thermosynechococcus strains. Comparative analyses also indicated different pattern of TF composition between thermophiles and corresponding mesophilic reference cyanobacteria. Moreover, the identified unique TFs of thermophiles are putatively involved in various biological regulations, mainly as responses to ambient changes, may facilitating the thermophiles to survive in hot springs.ConclusionThe findings herein shed light on the TFs of thermophilic cyanobacteria and fundamental knowledge for further research regarding thermophilic cyanobacteria with a broad potential for transcription regulations in responses to environmental fluctuations.

Whole genome resequencing reveals significant genetic differentiation between Exserohilum turcicum populations from maize and sorghum and candidate effector genes related to host specificity (Retracted).

After the manuscript was accepted, inconsistencies in the analyses were detected. These inconsistencies affected the general conclusion of the manuscript. This article was retracted on 27 March 2024. A peer-reviewed revised version was subsequently accepted: https://doi.org/10.1094/PHYTO-05-24-0172-R. Exserohilum turcicum is a devastating fungal pathogen that infects both maize and sorghum, leading to severe leaf diseases of the two crops. According to host specificity, pathogenic isolates of E. turcicum are divided into two formae speciales, namely E. turcicum f. sp. zeae and E. turcicum f. sp. sorghi. To date, the molecular mechanism underlying the host specificity of E. turcicum is marginally known. In this study, the whole genomes of 60 E. turcicum isolates collected from both maize and sorghum were resequenced, which enabled identification of 147,847 high-quality SNPs in total. Based on the SNPs, all isolates were clustered into four genetic groups that had a close relationship with host source. This observation was validated by the result of principal component analysis. The analysis of population structure revealed that there was obvious genetic differentiation between maize and sorghum host populations. Further analysis showed that 5,431 SNPs, including 612 nonsynonymous SNPs, were completely co-segregated with host source. These nonsynonymous SNPs were located in 539 genes in which 18 genes were predicted to encode secretory proteins, including six putative effector genes. The sequence polymorphism analysis of the six effector genes in 60 isolates indicated that these genes were perfectly co-segregated with host source. All SNVs in the coding regions of these genes were non-synonymous substitutions, suggesting that these genes were subject to strong positive selection pressure. These findings provide new insights into the molecular basis of host specificity in E. turcicum.

Molecular characterization of canine parvovirus type 2 (CPV2) reveals a high prevalence of the CPV2c genotype among dogs suffering from diarrhea

AbstractCanine parvovirus 2 (CPV-2) is a highly contagious virus in dogs that typically causes hemorrhagic enteritis and a high mortality rate in unvaccinated puppies. The genetic variability and antigenic diversity of CPV-2 hinder its effective prevention of infection by vaccination. To investigate the epidemiology and genetic characteristics of CPV-2 in China, rectal swabs from affected dogs were collected from different animal clinics in Kunshan from 2022 to 2023. Preliminary detection and capsid gene sequencing of CPV-2 were performed using previously described primers and protocols. The overall detection rate for CPV-2 was 16.5% (33/200). A significant association was found between the CPV-2-positivity and clinical signs, age, breed and vaccination status. Sequence analysis revealed the presence of CPV-2c genotypes in all positive samples, which were genetically similar to other Asian CPV-2c strains. Notably, four key mutations (A5G, F267Y, Y324I and Q370R) were detected in all isolates, and one novel mutation (I447M) was detected in three CPV-2 isolates. These mutations in the CPV-2 strains could impact vaccine efficacy and the effectiveness of the virus immune evasion. Surprisingly, no recombination events were observed between the identified CPV-2c strains and reference strains from China. Our data revealed that amino acid residues 324, 426 and 440 of VP2 may under strong selection pressure. This pattern of genetic variation in the CPV-2 lineage warrants continuous laboratory-based surveillance programs in other parts of China to better understand the pattern of seasonal distribution and association between emerging genotypes and the intensity of disease severity.

Microbial diversity and potential functional dynamics within the rhizocompartments of Dendrobium huoshanense.

Understanding the microbial diversity and potential functional dynamics within the rhizocompartments of Dendrobium huoshanense is crucial for unraveling the plant-microbe interactions that influence its medicinal properties. This study is the first to characterize the microbiome associated with the rhizocompartments of D. huoshanense, including its cultivation medium, rhizosphere, rhizoplane, and root endosphere, using high-throughput sequencing and subsequent bioinformatic analysis. Bacterial phylogenetic diversity was significantly higher in the endosphere than in the rhizosphere, while fungal α-diversity significantly decreased from the cultivation medium to the endosphere. Both bacterial and fungal niche widths decreased from the cultivation medium to the endosphere. β-Diversity analysis revealed distinct spatial patterns in both bacterial and fungal communities across the rhizocompartments, with the most pronounced differences between the cultivation medium and the endosphere. Taxonomically, Proteobacteria and Ascomycota were predominant in the endosphere for bacterial and fungal communities, respectively. Functional predictions showed significant enrichment of pathways related to xenobiotics biodegradation, lipid metabolism, and nitrogen fixation in the endosphere, while functions associated with plant pathogens and saprotrophs were significantly reduced. The results indicate a shift from generalist to specialist microbes from the cultivation medium to the endosphere, suggesting that D. huoshanense exerts strong selective pressure for endophytic fungi. Interestingly, a high proportion of fungi with unknown functions were found in the endosphere, highlighting an area for further research regarding the medicinal efficacy of D. huoshanense. Overall, this study provides foundational data for understanding the adaptive evolution of these microbial communities in response to specific microhabitats.

Dall's sheep horn growth and harvest management in the Mackenzie Mountains, Northwest Territories, Canada

AbstractAcross most of their native North American range, the horns of mountain sheep (Ovis spp.) males are getting smaller, a pattern attributed to selective hunting pressure. We measured the horns of 755 Dall's sheep males (Ovis dalli dalli) in the southern Mackenzie Mountains, Northwest Territories, between 2002 and 2017. For each male, we measured the circumference and length of each annulus for the right horn and calculated horn volume for each year. We examined changes in horn size in 4 different outfitter areas, using age at harvest as a covariate. Hunting pressure across years in the study area was consistently low, and this population did not experience the decline in horn size observed in several other mountain sheep populations in Canada. Over the 16‐year period, the average horn volume of harvested males was stable and even increased in 1 outfitter area. Local management of Dall's sheep delivered independently by the guide outfitters in the Mackenzie Mountains appears to contribute to maintaining a population of males that has not been adversely affected by strong selective hunting pressure. The resilience of this management strategy may be challenged by environmental changes associated with rapid warming in northern mountain environments.

Tumour cells can escape antiproliferative pressure by interferon-β through immunoediting of interferon receptor expression

Type I interferons (IFNs) play a central role not only in innate immunity against viral infection, but also in the antitumour response, e.g. through a direct impact on cell proliferation. Particularly for cancer arising in the context of chronic inflammation, constant exposure to IFNs may constitute a strong selective pressure during tumour evolution. Expansion of neoplastic subclones resistant to the antiproliferative effects of IFNs may contribute to immunoediting of tumours, leading to more aggressive disease. Experimental evidence for this development of IFN-insensitivity has been scarce and its molecular mechanism is unclear. In this study we demonstrate that six weeks exposure of cells to IFN-β in vitro reduces their sensitivity to its antiproliferative effects, and that this phenotype was stable for up to four weeks. Furthermore, we observed substantial differences in cellular sensitivity to growth inhibition by IFN-β in a panel of ten different liver cancer cell lines, most prominently in a pair of highly dedifferentiated cell lines, and least in cells from well-differentiated tumours. In both, long-term IFN selection and in dedifferentiated tumour cell lines, we found IFNAR2 expression to be substantially reduced, suggesting the receptor complex to be a sensitive target amenable to immunoediting. Beyond new insights into possible molecular processes in tumour evolution, these findings might prove valuable for the development of biomarkers allowing to stratify tumours for their sensitivity to IFN treatment in the context of patient tailored therapies.

A plethora of rodents: Rattlesnake predators generate unanticipated patterns of venom resistance in a grassland ecosystem

Predation has the potential to impart strong selective pressures on organisms within their environments, resulting in adaptive changes in prey that minimize risk of predation. Pressures from venomous snakes present an exceptional challenge to prey, as venom represents a unique chemical arsenal evolutionarily tailored to incapacitate prey. In response, venom resistance has been detected in various snake prey species, and to varying degrees. This study analyzes venom resistance in an eastern Colorado grassland habitat, where the Prairie Rattlesnake (Crotalus viridis) and Desert Massasauga Rattlesnake (Sistrurus tergeminus edwardsii) co-occur with a suite of grassland rodents. We test for venom resistance across rodent and snake pairings using two geographically distant field sites to determine the role of 1) predation pressure and trophic ecology, and 2) sympatric and allopatric patterns of venom resistance. Resistance was measured using serum-based metalloproteinase inhibition assays to determine potential inhibition of proteolytic activity, augmented by median lethal dose (LD50) assays on rodent species to assess toxicity of crude venoms. Resistance is present in several rodent species, with strong resistance present in populations of Eastern Woodrat (Neotoma floridana), Ord's Kangaroo Rat (Dipodomys ordii), and Northern Grasshopper Mouse (Onychomys leucogaster). Resistance is less developed in other species, including the House Mouse (Mus musculus) and Plains Pocket Mouse (Perognathus flavescens). An unexpected differential is present, where Lincoln County Kangaroo Rats are highly resistant to venom of co-occurring Prairie Rattlesnakes yet are sensitive to an allopatric population of Prairie Rattlesnakes in Weld County. Lincoln Co. Northern Grasshopper Mice also demonstrate extremely elevated resistance to Weld Co. Prairie Rattlesnake venoms, and they may possess resistance mechanisms for myotoxin a, an abundant component of Weld Co. C. v viridis venoms. This study illustrates the complexity of venom resistance in biological communities that can exist when incorporating multiple species interactions. Future studies aimed at characterizing resistance mechanisms at the molecular level will provide a more detailed physiological context for understanding mechanisms by which resistance to venoms occurs.

Community assembly of the human piercing microbiome.

Predicting how biological communities respond to disturbance requires understanding the forces that govern their assembly. We propose using human skin piercings as a model system for studying community assembly after rapid environmental change. Local skin sterilization provides a 'clean slate' within the novel ecological niche created by the piercing. Stochastic assembly processes can dominate skin microbiomes due to the influence of environmental exposure on local dispersal, but deterministic processes might play a greater role within occluded skin piercings if piercing habitats impose strong selection pressures on colonizing species. Here we explore the human ear-piercing microbiome and demonstrate that community assembly is predominantly stochastic but becomes significantly more deterministic with time, producing increasingly diverse and ecologically complex communities. We also observed changes in two dominant and medically relevant antagonists (Cutibacterium acnes and Staphylococcus epidermidis), consistent with competitive exclusion induced by a transition from sebaceous to moist environments. By exploiting this common yet uniquely human practice, we show that skin piercings are not just culturally significant but also represent ecosystem engineering on the human body. The novel habitats and communities that skin piercings produce may provide general insights into biological responses to environmental disturbances with implications for both ecosystem and human health.

Identifying PTPRJ As a Novel Mediator of CEBPA-Mutated AML

10-20% of acute myeloid leukaemia (AML) cases in children and adults carry mutations in the transcription factor CCAAT/enhancer binding protein alpha (CEBPA). CEBPA is transcribed from a single exon and produces two isoforms, p30 and p42, with characteristic mutations, either frameshift mutations in the N-terminal portion ( CEBPA Nterm) leading to production of only the p30 isoform, or in frame mutations in the C-terminal portion ( CEBPA Cterm), disrupting DNA binding. The most frequent secondary driver mutation in CEBPA-mutated AML, is the second allele of CEBPA. Additionally, in rare germline CEBPA cases, the founding mutation is usually CEBPA Nterm, with allindividuals showing acquisition of a second hit mutation in the CEBPA Cterm on the alternate CEBPA allele at the time of AML diagnosis, which has near 100% penetrance in these families. This highlights a strong selective pressure for this very specific biallelic mutation pattern. We have developed zebrafish models with germline cebpa Nterm and cebpa Cterm mutations that faithfully recapitulates the disease, with all homozygous and double heterozygous mutants showing loss of mature myeloid cells, and impaired survival. All double or compound mutants die by 10 weeks of age, with haematopoietic stem and progenitor cell (HSPC) expansion, and leukaemia in around 30% of these animals. Our model also demonstrates distinct phenotypes for cebpa Nterm and cebpa Cterm mutations during developmental haematopoiesis prior to leukaemia onset. We observed that cebpa Nterm and cebpa Cterm mutations have opposite effects on the expression of c-myb, a transcription factor expressed by proliferating HSPC, from 3 days post fertilization. cebpa Nterm/Nterm mutants show an increase in c-myb while cebpa Cterm/Cterm show reduced c-myb expression during development. To uncover the mechanisms by which cebpa Nterm and cebpa Cterm effect HSPCs we undertook RNASeq of sorted Tg(cd41:eGFP) lo HSPCs from fish of all possible genotype combinations. We identified both shared and unique expression profiles between genotypes. We also identified 4 genes that that were differentially regulated in both cebpa Nterm/Nterm and cebpa Cterm/Cterm HSPC, but in opposing vectors (upregulated in cebpa Cterm/Cterm and downregulated in cebpa Nterm/Nterm) (Figure 1). We hypothesised that these 4 genes are candidate drivers of selective pressure for the common CEBPA Cterm/Ntermmutation combination in human AML, and thus the malignant phenotype it confers. One of these candidate genes, PTPRJ, has also recently been identified as a frequently mutated gene in CEBPA-mutated AML in children thereby validating the relevance of our dataset and our approach. To define the effects of these 4 candidate genes we then conducted an F0 CRISPR knock-out screen in all cebpa mutant combinations in our zebrafish model, using c-myb expression as a phenotypic readout. We identified ptprja as our primary candidate gene and showed that loss of ptprja alone and in combination with cebpa Cterm/+ reduced expression of c-myb but had no effect on expression of c-myb in cebpa Cterm/Cterm, indicating that ptprja loss reduces c- myb-expressing HSPC and that this is dependent on functional cebpa DNA binding. We then examined the impact of ptprja knockout in juvenile zebrafish. Homozygous and double heterozygous mutants develop a pre-leukaemia expansion of HSPC before they succumb to death. We showed that that loss of ptprja in cebpa Cterm/Cterm fish accelerates this pre-leukaemic expansion of Tg(cd41:eGFP) lo HSPCs in 4 week fish (Figure 2). We also showed an opposing effect in the cebpa Nterm/Nterm mutants at 2 weeks, where loss of ptprja resulted in decreased pre-leukemic expansion of HSPC. These data indicate a role for PTPRJ in mediating HSPC expansion in CEBPA-mutated AML and suggest that PTPRJ may mediate the mechanism of clonal selection in those with a CEBPA mutation that drives the acquisition of a second CEBPA mutation on the opposing allele, leading to the common CEBPA Cterm/Nterm in patients with CEBPA-mutated AML.

Early Generation Telomerase Deficient Mice to Investigate Clonal Hematopoiesis in Aging

Clonal hematopoiesis is an age-associated condition characterized by the expansion of hematopoietic stem cell (HSC) pools with somatic mutations, observed in 10-30% of individuals older than 70 years. This phenomenon is primarily driven by mutations in transcriptional regulators such as DNMT3A, TET2, and ASXL1. While the clonal expansion of mutated HSCs is ubiquitous with age, it appears largely benign in the absence of strong selective pressures. These benign clones might compensate for the loss of regenerative HSC capacity during aging, and skew differentiation towards the myeloid lineage. Telomere erosion plays a significant role in this process. Telomeres normally prevent the activation of the DNA damage response (DDR) machinery. However, with each cell division, telomeres shorten until they reach a critical length, eventually leading to apoptosis or senescence. Previous studies have suggested a potential involvement of telomere attrition in the development of clonal hematopoiesis (Hinds et al. Blood 2016; Zink et al. Blood 2017). Late generation telomerase deficient (Terc-/- G5) mice develop premature bone marrow failure and mortality, however earlier generation Terc-/- G3 (3 homozygous crosses) have shortened telomeres but appear relatively normal. Importantly, inhibitors of telomerase have recently entered clinical trials for haematological malignancies and therefore understanding the long-term effects of telomerase inhibition is important. By studying the interplay between telomere attrition, HSC aging, and the development of CHIP, we aim to gain insights into the mechanisms underlying age-associated hematopoietic changes and their potential clinical implications. We examined young (age 8-10 week) and aged (10-12 month) Terc-/- G3 mice. Telomere lengths were significantly reduced in bone marrow cells from young Terc G3 (15 kb) when compared to wild-type mice (30 kb) with mild reduction in BM cellularity. Absolute long-term HSC numbers were reduced, as were megakaryocyte erythroid progenitors, but there was preservation of granulocyte macrophage progenitors and common myeloid progenitors. Spleen progenitor numbers were not affected. In comparison, aged (10-12 months old) Terc G3 mice showed enhanced differentiation skewing in the peripheral blood with significantly increased myeloid (n = 8 wt; n = 12 Terc ko; p = 0.0004) and concomitantly reduced B cell (p = 0.0002) and T cell (p = 0.0320) populations. There was marked mobilization of bone marrow progenitors to the spleen, most notably GMP (p = 0.0004) but also MEP and CMP in aged Terc G3. Furthermore, aged Terc G3 ko now exhibited marked reduction in bone marrow CMP (p &amp;lt; 0.0001) and MEP (p &amp;lt; 0.0001), but skewing towards GMP (p&amp;lt;0.001) resulting in normal total GMP numbers, but markedly reduced CMP and MEP numbers (p &amp;lt; 0.0001). Compared to young Terc G3 mice, the aged Terc G3 mice had relative expansion of the BM LT-HSC population, and therefore normal numbers of BM LT-HSC compared to aged matched controls. Aged Terc G3 BM LTHSC have been isolated for gene expression and clonality analysis. Together, these findings are consistent with accelerated aging in G3 Terc-/- mice, providing a model for examining clonal hematopoiesis. In order to assess the cell intrinsic function of Terc G3 LTHSC, we next performed non-competitive bone marrow transplantation experiments from Terc ko or wild-type donors into lethally irradiated young wild-type recipients (n = 9 per group), and analysed these at 4 months post-transplant. There was reduced, but stable reconstitution by Terc G3 cells, consistent with previous competitive bone marrow transplants in Terc G3 (Bruedigam et al. Cell Stem Cell 2014). We observed a similar reduction in bone marrow cellularity in Terc ko transplanted recipients (p = 0.003). Lineage skewing was less pronounced but CMP percentages were significantly reduced in Terc ko compared to wild-type conditions (p = 0.03). In contrast to primary mice, mobilization of progenitors to the spleen was not observed, suggesting that this finding was ameliorated by normal bone marrow and splenic supporting cells. The aged G3 Terc ko model faithfully recapitulates the haematopoietic phenotype of aging in humans and we propose to utilise this model to further our understanding of the mechanisms and physiological consequences of clonal hematopoiesis, to eventually help identify strategies to mitigate its clinical consequences.