Introduced and extinct: neglected archival specimens shed new light on the historical biogeography of an iconic avian species in the Mediterranean.

Collection specimens provide valuable and often overlooked biological material that enables addressing relevant, long-unanswered questions in conservation biology, historical biogeography, and other research fields. Here, we use preserved specimens to analyze the historical distribution of the black francolin (Francolinus francolinus, Phasianidae), a case that has recently aroused the interest of archeozoologists and evolutionary biologists. The black francolin currently ranges from the Eastern Mediterranean and the Middle East to the Indian subcontinent, but, at least since the Middle Ages, it also had a circum-Mediterranean distribution. The species could have persisted in Greece and the Maghreb until the 19th century, even though this possibility had been questioned due to the absence of museum specimens and scant literary evidence. Nevertheless, we identified four 200-year-old stuffed black francolins-presumably the only ones still existing-from these areas and sequenced their mitochondrial DNA control region. Based on the comparison with conspecifics (n = 396) spanning the entirety of the historic and current species range, we found that the new samples pertain to previously identified genetic groups from either the Near East or the Indian subcontinent. While disproving the former occurrence of an allegedly native westernmost subspecies, these results point toward the role of the Crown of Aragon in the circum-Mediterranean expansion of the black francolin, including the Maghreb and Greece. Genetic evidence hints at the long-distance transport of these birds along the Silk Road, probably to be traded in the commerce centers of the Eastern Mediterranean.

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

Historic samples reveal loss of wild genotype through domestic chicken introgression during the Anthropocene.

Human activities have precipitated a rise in the levels of introgressive gene flow among animals. The investigation of conspecific populations at different time points may shed light on the magnitude of human-mediated introgression. We used the red junglefowl Gallus gallus, the wild ancestral form of the chicken, as our study system. As wild junglefowl and domestic chickens readily admix, conservationists fear that domestic introgression into junglefowl may compromise their wild genotype. By contrasting the whole genomes of 51 chickens with 63 junglefowl from across their natural range, we found evidence of a loss of the wild genotype across the Anthropocene. When comparing against the genomes of junglefowl from approximately a century ago using rigorous ancient-DNA protocols, we discovered that levels of domestic introgression are not equal among and within modern wild populations, with the percentage of domestic ancestry around 20-50%. We identified a number of domestication markers in which chickens are deeply differentiated from historic junglefowl regardless of breed and/or geographic provenance, with eight genes under selection. The latter are involved in pathways dealing with development, reproduction and vision. The wild genotype is an allelic reservoir that holds most of the genetic diversity of G. gallus, a species which is immensely important to human society. Our study provides fundamental genomic infrastructure to assist in efforts to prevent a further loss of the wild genotype through introgression of domestic alleles.

Characterization of tongue dorsum microbiome in wine tasters.

Taste plays a paramount role in food and beverage choice, with recent studies pointing to a potential influence of the microorganisms from the tongue dorsum - particularly bacteria - on flavor perception. Thus, the association between tongue dorsum biofilm and taste is a fundamental prerequisite for a better understanding of the role played by these bacteria in wine tasting. To study this impact, we have analyzed the microbiomes from 58 samples of the tongue dorsum surface from professional wine tasters and 30 samples from non professional wine tasters. The microbiome of each sample was characterized through metagenome sequencing of the 16S rRNA gene for taxonomic discrimination of bacteria. A total of 497 taxa were identified in the tongue dorsum, and significant differences in diversity were observed between the wine taster and the control group. The comparison of bacterial diversity between samples collected before and after wine tasting along with the presence of new bacterial taxa indicates a direct effect of wine on the microbiome of frequent wine tasters, particularly in those tasting sparkling wines.

Gut Microbiome Studies in Livestock: Achievements, Challenges, and Perspectives.

The variety and makeup of the gut microbiome are frequently regarded as the primary determinants of health and production performances in domestic animals. High-throughput DNA/RNA sequencing techniques (NGS) have recently gained popularity and permitted previously unheard-of advancements in the study of gut microbiota, particularly for determining the taxonomic composition of such complex communities. Here, we summarize the existing body of knowledge on livestock gut microbiome, discuss the state-of-the-art in sequencing techniques, and offer predictions for next research. We found that the enormous volumes of available data are biased toward a small number of globally distributed and carefully chosen varieties, while local breeds (or populations) are frequently overlooked despite their demonstrated resistance to harsh environmental circumstances. Furthermore, the bulk of this research has mostly focused on bacteria, whereas other microbial components such as protists, fungi, and viruses have received far less attention. The majority of these data were gathered utilizing traditional metabarcoding techniques that taxonomically identify the gut microbiota by analyzing small portions of their genome (less than 1000 base pairs). However, to extend the coverage of microbial genomes for a more precise and thorough characterization of microbial communities, a variety of increasingly practical and economical shotgun techniques are currently available.

Ferroptosis regulation by the NGLY1/NFE2L1 pathway.

SignificanceFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2/NRF2 can both regulate oxidative stress pathways but are each regulated in a distinct manner, and whether these two transcription factors can regulate ferroptosis independent of one another is unclear. We find that NFE2L1 can promote ferroptosis resistance, independent of NFE2L2, by maintaining the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation. NFE2L2 can also promote ferroptosis resistance but does so through a distinct mechanism that appears independent of GPX4 protein expression. These results suggest that NFE2L1 and NFE2L2 independently regulate ferroptosis.

Characterization of a small molecule inhibitor of disulfide reductases that induces oxidative stress and lethality in lung cancer cells.

Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.

Spatial genetic structure and Ovis haplogroup as a tool for an adaptive conservation management of the endangered Cyprus mouflon.

The mouflon (Ovis gmelini ophion) is the largest wild terrestrial mammal of Cyprus. Regarded as the local flagship species, its population range is limited to the mountainous Paphos Forest and adjacent areas including part of Troodos National Forest Park (> 700 Km2). This species, protected by both national and international legislation, is classified as Endangered by the IUCN, with livestock intrusion, poaching and habitat loss as the main threatening factors. We determined the spatial genetic structure by investigating sexes separately and shed further light on the Cyprus mouflon placement among Ovis haplogroups (HGs) to enforce its protection within an adaptive conservation framework. We genotyped 108 samples collected across the entire species' range at both14 loci of the microsatellite DNA and the mitochondrial DNA Control Region (CR). Microsatellite genotyping indicated that the wild population retained low genetic variability, which, however, was not associated with a level of inbreeding raising particular concern (FIS = 0.12). An overall weak spatial genetic structure was disclosed, consistently with the limited extension of the mouflon range, the lack of significant physical barriers to dispersal and the intense gene flow mostly occurring along a northeast-southwest axis across the forest. Nevertheless, evidences of structure were found for females in compliance with their philopatric behaviour. The disclosure of unique features compared to Mediterranean and Near East conspecifics, such as the occurrence of a six-fold 76 bp-long repeated motif in the Cyprus mouflon CR, along with the outcome of a phylogenetic reconstruction (based on a far higher number of Ovis GenBank records and Cyprus haplotypes than in previous studies) inspired the proposal of a new haplogroup (HGC2) which included the Anatolian mouflon (O. g. anatolica, former HGX) as sister taxon. While both high habitat heterogeneity and low human disturbance were the main drivers in determining the overall spatial structure, future conservation efforts to preserve this valuable genetic resource should focus on avoiding possible introgressive hybridisation with co-grazing livestock to the edge of its range.

Novel genome reveals susceptibility of popular gamebird, the red-legged partridge (Alectoris rufa, Phasianidae), to climate change.

We produced a high-quality de novo genome assembly of the red-legged partridge A. rufa, the first reference genome of its genus, by utilising novel 10× Chromium technology. The estimated genome size was 1.19 Gb with an overall genome heterozygosity of 0.0022; no runs of homozygosity were observed. In total, 21,589 protein coding genes were identified and assigned to 16,772 orthologs. Of these, 201 emerged as unique to Alectoris and were enriched for positive regulation of epithelial cell migration, viral genome integration and maturation. Using PSMC analysis, we inferred a major demographic decline commencing ~140,000 years ago, consistent with forest expansion and reduction of open habitats during the Eemian interglacial. Present-day populations exhibit the historically lowest genetic diversity. Besides implications for management and conservation, this genome also promises key insights into the physiology of these birds with a view to improving poultry husbandry practices.

Genome-wide markers redeem the lost identity of a heavily managed gamebird.

Heavily managed wildlife may suffer from genetic homogenization and reshuffling of locally adapted genotypes with non-native ones. This phenomenon often affects natural populations by reducing their evolutionary potential and speeding up the ongoing biodiversity crisis. For decades, the red-legged partridge (Alectoris rufa), an intensively managed gamebird of conservation concern and considerable socio-economic importance, has been subjected to extensive releases of farm-reared hybrids with the chukar partridge (Alectoris chukar) and translocations irrespective of subspecific affinity. These practices have led to serious concerns that the genetic integrity and biogeographic structure of most red-legged partridge populations are irreversibly affected, as suggested by previous studies based on few genetic markers. Using over 168 000 genome-wide loci and a sampling across the entire A. rufa range, we detected unexpectedly limited and spatially uneven chukar introgression as well as significant intraspecific structure. We demonstrate that species widely feared to have irretrievably lost their genetic identity are likely to be much less affected by unsuitable management practices than previously assumed. Our results spell the need for a radical re-think on animal conservation, possibly restoring native status to populations long treated as compromised. Our study exemplifies how the application of innovative conservation-genomic methods is key to solving wildlife management problems dealing with introgressive hybridization worldwide.

A compendium of kinetic modulatory profiles identifies ferroptosis regulators.

Cell death can be executed by regulated apoptotic and nonapoptotic pathways, including the iron-dependent process of ferroptosis. Small molecules are essential tools for studying the regulation of cell death. Using time-lapse imaging and a library of 1,833 bioactive compounds, we assembled a large compendium of kinetic cell death modulatory profiles for inducers of apoptosis and ferroptosis. From this dataset we identify dozens of ferroptosis suppressors, including numerous compounds that appear to act via cryptic off-target antioxidant or iron chelating activities. We show that the FDA-approved drug bazedoxifene acts as a potent radical trapping antioxidant inhibitor of ferroptosis both in vitro and in vivo. ATP-competitive mechanistic target of rapamycin (mTOR) inhibitors, by contrast, are on-target ferroptosis inhibitors. Further investigation revealed both mTOR-dependent and mTOR-independent mechanisms that link amino acid metabolism to ferroptosis sensitivity. These results highlight kinetic modulatory profiling as a useful tool to investigate cell death regulation.

Markers for genetic change.

BACKGROUND AND AIMS: Wildlife conservation has focused primarily on species for the last decades. Recently, popular perception and laws have begun to recognize the central importance of genetic diversity in the conservation of biodiversity. How to incorporate genetic diversity in ongoing monitoring and management of wildlife is still an open question. METHODS: We tested a panel of multiplexed, high-throughput sequenced introns in the small mammal communities of two UNESCO World Heritage Sites on different continents to assess their viability for large-scale monitoring of genetic variability in a spectrum of diverse species. To enhance applicability across other systems, the bioinformatic pipeline for primer design was outlined. RESULTS: The number of loci amplified and amplification evenness decreased as phylogenetic distance increased from the reference taxa, yet several loci were still variable across multiple mammal orders. CONCLUSIONS: Genetic variability found is informative for population genetic analyses and for addressing phylogeographic and phylogenetic questions, illustrated by small mammal examples here.

Historic and modern genomes unveil a domestic introgression gradient in a wild red junglefowl population.

The red junglefowl Gallus gallus is the ancestor of the domestic chicken and arguably the most important bird species on Earth. Continual gene flow between domestic and wild populations has compromised its gene pool, especially since the last century when human encroachment and habitat loss would have led to increased contact opportunities. We present the first combined genomic and morphological admixture assessment of a native population of red junglefowl, sampled from recolonized parts of its former range in Singapore, partly using whole genomes resequenced from dozens of individuals. Crucially, this population was genomically anchored to museum samples from adjacent Peninsular Malaysia collected ~110-150 years ago to infer the magnitude of modern domestic introgression across individuals. We detected a strong feral-wild genomic continuum with varying levels of domestic introgression in different subpopulations across Singapore. Using a trait scoring scheme, we determined morphological thresholds that can be used by conservation managers to successfully identify individuals with low levels of domestic introgression, and selected traits that were particularly useful for predicting domesticity in genomic profiles. Our study underscores the utility of combined genomic and morphological approaches in population management and suggests a way forward to safeguard the allelic integrity of wild red junglefowl in perpetuity.

Kinetic Heterogeneity of Cancer Cell Fractional Killing.

Lethal drugs can induce incomplete cell death in a population of cancer cells, a phenomenon referred to as fractional killing. Here, we show that high-throughput population-level time-lapse imaging can be used to quantify fractional killing in response to hundreds of different drug treatments in parallel. We find that stable intermediate levels of fractional killing are uncommon, with many drug treatments resulting in complete or near-complete eradication of all cells, if given enough time. The kinetics of fractional killing over time vary substantially as a function of drug, drug dose, and genetic background. At the molecular level, the antiapoptotic protein MCL1 is an important determinant of the kinetics of fractional killing in response to MAPK pathway inhibitors but not other lethal stimuli. These studies suggest that fractional killing is governed by diverse lethal stimulus-specific mechanisms.

Non-native and hybrid in a changing environment: conservation perspectives for the last Italian red-legged partridge (Alectoris rufa) population with long natural history.

The ever-increasing biotic homogenization - especially when associated with introgressive hybridization - raises concern for the reduction of the spatial component of diversity in wildlife worldwide. Nonetheless, there is a growing attention to the potential conservation value of hybridization in fastening the adaptive evolutionary responses to rapidly changing selective pressures. Under these premises, we investigated the genetic affinity of the red-legged partridge (Alectoris rufa) population inhabiting Elba Island (Italy) in the context of the overall species phylogeography and particularly of the nominate subspecies it is traditionally ascribed to. Although notoriously hybrid with the congeneric A. chukar, this island population is of undisputedly value because of its long natural history and self-sustainability. As such, its adaptive conservation management calls for a comprehensive knowledge including the assessment of its geographic origin. For this purpose, 110 fecal samples were collected across Elba, genotyped at their joint Cytochrome-b and Control Region genes (2,249 characters), and compared with 149 conspecifics from all over the species distribution range. We confirmed a widespread A. chukar mitochondrial DNA introgression in Elba partridges, whereas their expected formal assignment to the nominate subspecies from Italy and France was rejected, since these turned out to be closely related to conspecifics from the Iberian Peninsula. This counterintuitive result found support in a large variety of literary sources and compelling evidences from personal testimonies revealing recent intense management with farm-reared birds of Spanish origin. Although the nativeness of Elba partridges was disproved, we advise local authorities to keep warranting the ongoing conservation efforts - and especially restore the connectivity between the western and eastern island sub-populations - as this resource may still be conceived as the ultimate repository for part of the otherwise extinct Italian A. r. rufa genome. Indeed, the admixture with conspecifics from the Iberian Peninsula does not necessarily mean that the entirety of the native nuclear genome of Elba partridges has been wiped out. Furthermore, these latter represent an interesting case study in conservation biology to investigate the possible role played by introgressive hybridization in the adaptation to recent land use and vegetation cover changes associated with rural abandonment in an insular yet heavily anthropized context.

From groups to communities in western lowland gorillas.

Social networks are the result of interactions between individuals at different temporal scales. Thus, sporadic intergroup encounters and individual forays play a central role in defining the dynamics of populations in social species. We assessed the rate of intergroup encounters for three western lowland gorilla ( Gorilla gorilla gorilla) groups with daily observations over 5 years, and non-invasively genotyped a larger population over four months. Both approaches revealed a social system much more dynamic than anticipated, with non-aggressive intergroup encounters that involved social play by immature individuals, exchanges of members between groups likely modulated by kinship, and absence of infanticide evidenced by infants not fathered by the silverback of the group where they were found. This resulted in a community composed of groups that interacted frequently and not-aggressively, contrasting with the more fragmented and aggressive mountain gorilla ( G. beringei beringei) societies. Such extended sociality can promote the sharing of behavioural and cultural traits, but might also increase the susceptibility of western lowland gorillas to infectious diseases that have decimated their populations in recent times.

GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis.

Oxygen is necessary for aerobic metabolism but can cause the harmful oxidation of lipids and other macromolecules. Oxidation of cholesterol and phospholipids containing polyunsaturated fatty acyl chains can lead to lipid peroxidation, membrane damage, and cell death. Lipid hydroperoxides are key intermediates in the process of lipid peroxidation. The lipid hydroperoxidase glutathione peroxidase 4 (GPX4) converts lipid hydroperoxides to lipid alcohols, and this process prevents the iron (Fe2+ )-dependent formation of toxic lipid reactive oxygen species (ROS). Inhibition of GPX4 function leads to lipid peroxidation and can result in the induction of ferroptosis, an iron-dependent, non-apoptotic form of cell death. This review describes the formation of reactive lipid species, the function of GPX4 in preventing oxidative lipid damage, and the link between GPX4 dysfunction, lipid oxidation, and the induction of ferroptosis.

Exogenous Monounsaturated Fatty Acids Promote a Ferroptosis-Resistant Cell State.

The initiation and execution of cell death can be regulated by various lipids. How the levels of environmental (exogenous) lipids impact cell death sensitivity is not well understood. We find that exogenous monounsaturated fatty acids (MUFAs) potently inhibit the non-apoptotic, iron-dependent, oxidative cell death process of ferroptosis. This protective effect is associated with the suppression of lipid reactive oxygen species (ROS) accumulation at the plasma membrane and decreased levels of phospholipids containing oxidizable polyunsaturated fatty acids. Treatment with exogenous MUFAs reduces the sensitivity of plasma membrane lipids to oxidation over several hours. This effect requires MUFA activation by acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) and is independent of lipid droplet formation. Exogenous MUFAs also protect cells from apoptotic lipotoxicity caused by the accumulation of saturated fatty acids, but in an ACSL3-independent manner. Our work demonstrates that ACSL3-dependent MUFA activation promotes a ferroptosis-resistant cell state.

Human-modified biogeographic patterns and conservation in game birds: The dilemma of the black francolin (Francolinus francolinus, Phasianidae) in Pakistan.

The ever-increasing human-mediated wildlife reshuffling is raising concern for the conservation of biodiversity. The loss of biological distinctiveness among regions lessens the genetic diversity and consequently the evolutionary potential of local biotas to tackle present-day global change and human disturbance. This process may be sometimes cryptic unless investigated by means of a molecular approach. In this respect, game birds are a paradigmatic case. The black francolin (Francolinus francolinus, Phasianidae) is a medium-sized galliform whose distribution range stretches from Cyprus to the Gulf of Bengal. Six morphologic subspecies are known, with three of which occurring in Pakistan, where the species is heavily hunted and used as pet for chirping competitions. We genotyped 98 samples (feathers) at both the entire mitochondrial DNA Control Region gene and nine microsatellite loci to get a deeper insight into the genetic diversity of the black francolin in Pakistan in order to offer cogent recommendations for its conservation management. We identified several mtDNA lineages that were consistent with the currently described subspecies/taxonomy whose pattern of co-occurrence is compatible with the geological history and the faunal movement routes of the region under study. However, the biparentally inherited microsatellites returned a quite discordant picture of an extensive, sex-biased genetic mixing due to the intensive relocations of already overharvested male individuals for chirping competitions. Our results indicated that the genetic integrity of the black francolin in Pakistan could be seriously at risk and call for monitoring and limiting its trade other than enhancing the public awareness of the importance of local biodiversity resources.

Systematic Quantification of Population Cell Death Kinetics in Mammalian Cells.

Cytotoxic compounds are important drugs and research tools. Here, we introduce a method, scalable time-lapse analysis of cell death kinetics (STACK), to quantify the kinetics of compound-induced cell death in mammalian cells at the population level. STACK uses live and dead cell markers, high-throughput time-lapse imaging, and mathematical modeling to determine the kinetics of population cell death over time. We used STACK to profile the effects of over 1,800 bioactive compounds on cell death in two human cancer cell lines, resulting in a large and freely available dataset. 79 potent lethal compounds common to both cell lines caused cell death with widely divergent kinetics. 13 compounds triggered cell death within hours, including the metallophore zinc pyrithione. Mechanistic studies demonstrated that this rapid onset lethal phenotype was caused in human cancer cells by metabolic disruption and ATP depletion. These results provide the first comprehensive survey of cell death kinetics and analysis of rapid-onset lethal compounds.