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The types of immune cells that populate the trachea after ILTV vaccination and infection have not been assessed. The objective of this study was to quantify CD4+, CD8α+, CD8β+, TCRγδ+, and MRC1LB+ cells that infiltrate the trachea after vaccination with chicken embryo origin (CEO), tissue culture origin (TCO), and recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccines, and after challenge of vaccinated and non-vaccinated chickens with a virulent ILTV strain. Eye-drop vaccination with CEO, or TCO, or in ovo vaccination with rHVT-LT did not alter the number of CD4+, CD8α+, CD8β+, TCRγδ+, and MRC1LB+ cells in the trachea. After challenge, the CEO vaccinated group of chickens showed swift clearance of the challenge virus, the mucosa epithelium of the trachea remained intact, and a limited number of CD4+, CD8α+, and CD8β+ cells were detected in the upper trachea mucosa. The TCO and rHVT-LT vaccinated groups of chickens showed narrow viral clearance with moderate disruption of the trachea epithelial integrity, and a significant increase in CD4+, CD8α+, CD8β+, and TCRγδ+ cells infiltrated the upper trachea mucosa. Non-vaccinated challenged chickens showed high levels of viral replication, the epithelial organization of the upper trachea mucosa was heavily disrupted, and the predominant infiltrates were CD4+, TCRγδ+, and MRC1LB+ cells. Hence, the very robust protection provided by CEO vaccination was characterized by minimal immune cell infiltration to the trachea mucosa. In contrast, partial protection induced by the TCO and rHVT-LT vaccines requires a prolonged period of T cell expansion to overcome the established infection in the trachea mucosa.

Caspases are a family of enzymes that play roles in cell death and inflammation. It has been suggested that in the execution phase of the apoptotic pathway, caspase-3, -6 and -7 are involved. The substrate specificities of two proteases (caspases 3 and 7) are highly similar, which complicates the design of compounds that selectively interact with a single enzyme exclusively. The recognition of residues other than Asp in the P1 position of the substrate by caspase-3/-7 has been reported, promoting interest in the effects of phosphorylation of amino acids in the direct vicinity of the scissile bond. To evaluate conflicting reports on this subject, we synthesized a series of known caspase-3 and -7 substrates and phosphorylated analogs, performed enzyme kinetic assays and mapped the peptide cleavage sites using internally quenched fluorescent peptide substrates. Caspases 3 and 7 will tolerate pSer at the P1 position but only poorly at the P2′ position. Our investigation demonstrates the importance of peptide length and composition in interpreting sequence/activity relationships. Based on the results, we conclude that the relationship between caspase-3/-7 and their substrates containing phosphorylated amino acids might depend on the steric conditions and not be directly connected with ionic interactions. Thus, the precise effect of phospho-amino acid residues located in the vicinity of the cleaved bond on the regulation of the substrate specificity of caspases remains difficult to predict. Our observations allow to predict that natural phosphorylated proteins may be cleaved by caspases, but only when extended substrate binding site interactions are satisfied.

We measured gonad size (diameter and circumference) by ultrasound and used it as a metric to assign stage of maturity in Burbot Lota lota from Lake Roosevelt, Washington. We collected paired gonad tissue and ultrasound measurements monthly from November 2017 to March 2018 and processed gonad tissue for histological analysis to confirm stage of maturity. We measured gonad diameter and circumference by ultrasound. We also measured excised gonad diameter (i.e., true gonad diameter) by digital calipers and excised gonad circumference (i.e., true gonad circumference) by a measuring tape. All late vitellogenic (stage 6) ovaries measured by ultrasound had a diameter greater than 3.90 cm, suggesting a value of 3.90 cm or greater may be used to characterize females capable of spawning in the current reproductive cycle. One mid-spermatogenic (stage 3) and all ripe (stage 4) testes were too large to be measured and were assigned a diameter of 5.11 cm, the maximum value capable of being measured by our ultrasound transducer. A value of 5.11 cm or greater may be used to characterize males capable of spawning in the current reproductive cycle. Testis circumference measured by ultrasound is not reported because some testes were wider than the ultrasound transducer and could not be measured. Measurements of testis diameter did not differ between measurement methods (ultrasound versus true), but ultrasound measurements of ovary diameter and circumference were higher than true measurements. We attributed the difference between measurement methods to flattening of the ovary while applying the ultrasound transducer. Gonad diameter and circumference measured by ultrasound were highly correlated with gonadosomatic index and ovarian follicle diameter, indicating gonad size measured by ultrasound is an appropriate index of gonad development in Burbot.

The hominin fossil record of Island Southeast Asia (ISEA) indicates that at least two endemic 'super-archaic' species-Homo luzonensis and H. floresiensis-were present around the time anatomically modern humans arrived in the region >50,000 years ago. Intriguingly, contemporary human populations across ISEA carry distinct genomic traces of ancient interbreeding events with Denisovans-a separate hominin lineage that currently lacks a fossil record in ISEA. To query this apparent disparity between fossil and genetic evidence, we performed a comprehensive search for super-archaic introgression in >400 modern human genomes, including >200 from ISEA. Our results corroborate widespread Denisovan ancestry in ISEA populations, but fail to detect any substantial super-archaic admixture signals compatible with the endemic fossil record of ISEA. We discuss the implications of our findings for the understanding of hominin history in ISEA, including future research directions that might help to unlock more details about the prehistory of the enigmatic Denisovans.

No-take reserves (NTRs) have been effective at conserving fish assemblages in tropical systems such as coral reefs, but have rarely been evaluated in turbid tropical estuaries. The present study evaluated the effect of a mangrove NTR on the conservation of juvenile fish abundance, commercial fish biomass and biodiversity at the assemblage level, and the abundance of juveniles, target and non-target adults at the family level. The evaluation incorporated one aspect of seascape connectivity, namely proximity to the sea, or in this case, the Gulf of Paria. Linear mixed models showed that the NTR had a positive effect only on species richness at the assemblage level. However, juvenile fish abundance, commercial fish biomass, taxonomic distinctness and functional diversity were not enhanced in the NTR. The inclusion of connectivity in these models still failed to identify any positive effects of the NTR at the assemblage level. Yet, there were significant benefits to juvenile fish abundance for 5 of 7 families, and for 1 family of non-target adults. Possible explanations for the limited success of the NTR for fish assemblages include failing to account for the ecology of fish species in NTR design, the drawbacks of ‘inside-outside’ (of the NTR) experimental designs and the fact that fishing does not always impact non-target species. It is important to recognise that mangrove NTRs do not necessarily benefit fish assemblages as a whole, but that finer-scale assessments of specific families may reveal some of the proclaimed benefits of NTRs in tropical estuaries.

The subset of the proteome that contains enzymes in their catalytically active form can be interrogated by using probes targeted toward individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, multiplex analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry giving us the ability to monitor the activity of multiple proteases in parallel. Using these probes, we were able to identify the distribution of four proteases with different active site geometries in three cell lines and peripheral blood mononuclear cells. This provides a framework for the use of mass cytometry for multiplexed enzyme activity detection.

Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1β (pro-IL18/pro-IL1β). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1′–P4′, the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1′–P4′ region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1′–P4′ region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1′–P4′-based inhibitors could be exploited to specifically target inflammatory caspases.

Excessive fishing pressure can induce population declines or complete collapse of fisheries. Unless commercial and recreational fisheries for K‐selected fishes, or those with slow growth and late maturation, are carefully managed, declines in abundance or fishery collapse is probable. Paddlefish Polyodon spathula,are a K‐selected species that experienced historical declines in abundance as a result of habitat degradation and overfishing. Mark‐recapture studies are well‐suited for long‐lived fishes by providing information on population density and vital rates. For sustainable commercial or recreational fisheries targeting species such as the paddlefish, managers require accurate estimates of population vital rates including survival, abundance, and exploitation. We used a Montana Fish, Wildlife & Parks (MFWP) mark‐recapture dataset and modified Jolly‐Seber (POPAN) models to estimate survival, recapture, probability of entry, and abundance of 8,518 tagged paddlefish over a 25‐year period. With many supporting estimates including stable survival (0.92 for females, mean of 0.82 for males), low exploitation rates (means of 2.6% for females and 2.9% for males), and stable abundance estimates (25‐year mean of 12,309 individuals for both sexes), the Fort Peck paddlefish population appears to be stable and well‐managed over the past 25 years. Presently, this is the only study focused on paddlefish in North America that has estimated survival and abundance for both male and female paddlefish using contemporary analyses. This research provided a unique opportunity to highlight that the effort exerted by management agencies to collect long‐term field data is extremely useful to our understanding of fish populations and management.

Yellowstone Lake in Yellowstone National Park, USA, has the longest ongoing suppression program for non-native lake trout (Salvelinus namaycush) in the western USA. Harvest data from the suppression program, along with data from an assessment program initiated in 2011, was used to estimate lake trout abundance and mortality rates. Abundance and biomass estimates were used to estimate stock–recruitment dynamics, which were inputs to a simulation model forecasting responses to continued suppression. Abundance increased during 1998–2012 when total annual mortality exceeded 0.59 and declined thereafter. The fishing mortality rate required to reduce abundance was 67% greater than predicted by models that used prerecruit survival estimates from the lake trout’s native range. Prerecruit survival in Yellowstone Lake was estimated at four to six times greater than native range survival rates. Simulated abundance continued to decline if recent suppression efforts were maintained. High prerecruit survival in Yellowstone Lake likely illustrates ecological release for an invasive species in an ecosystem containing few predators or competitors and demonstrates the potential pitfalls of assuming equal demographic rates for native and non-native populations.

Significance Genome-wide association studies have identified genetic variation at the ARMS2/HTRA1 locus as a risk factor for the development and progression of age-related macular degeneration (AMD). We have developed a potent anti-HtrA1 Fab inhibitor of HtrA1 proteolytic activity in the retina as a potential therapeutic for treating AMD. A set of proteomic analytical tools was established to characterize HtrA1 activity and discover in vivo HtrA1 substrates. These efforts led to the identification of an eye-specific and clinically applicable pharmacodynamic biomarker of anti-HtrA1 Fab activity. Analysis of HtrA1-mediated cleavage of Dickkopf-related protein 3 in the aqueous humor of patients with geographic atrophy provided evidence of anti-HtrA1 Fab activity and information on duration of activity in a phase 1 study., Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.

Summary Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1β. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora., Graphical abstract, Highlights • Carnivorans lack key NLRs and express a unique caspase-1/-4 hybrid protein • This protein is defective in mediating activation of common inflammasome pathways • What little activity occurs is driven by caspase-8, rather than caspase-1/-4, Species of the order Carnivora have evolutionarily acquired the expression of a unique caspase-1/-4 hybrid protein. Digby et al. show that this protein is a poor mediator of NLRP3- and caspase-4-dependent inflammasome activation. This downregulation in inflammasome pathways could impair pathogen detection and facilitate transmission of zoonotic infections.

Successful recruitment of endangered pallid sturgeon has not been documented in the upper Missouri River basin for decades, and research on the reproductive ecology of pallid sturgeon has been hindered by low sample size. A conservation propagation program was initiated in the 1990s, and the oldest age class of hatchery‐origin pallid sturgeon are becoming sexually mature increasing the number of reproductively‐active fish in the system. However, it is currently unknown how the reproductive ecology of hatchery‐origin pallid sturgeon relates to the few remaining wild fish. Following spring reproductive assessments, weekly relocations were recorded for each individual from late‐May to mid‐July to facilitate comparisons of spawning season movements among reproductive classifications and between spring hydrographs (2015 and 2016) for male pallid sturgeon. Mean total movement distances (±SE) were 104.5 km (18.9) for reproductively‐active wild males, 116.0 km (18.1) for reproductively‐active 1997‐year class males, and 20.6 km (3.0) for non‐reproductively‐active fish of unconfirmed sex. Movement characteristics of reproductively‐active males did not differ between 2015 and 2016 despite a difference of eight days in the timing of peak discharge and a difference of 79 m³/s (16.7%) in magnitude. Male aggregations were observed on the descending limb of the hydrograph in 2016 during temperatures suitable for spawning, but female pallid sturgeon underwent follicular atresia, similar to the other years of the study. Hatchery‐origin pallid sturgeon from the conservation propagation program appear to have retained reproductive characteristics from the wild broodstock, a key finding for a population where local extirpation of the wild stock is imminent.

Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult Schistosoma mansoni were incubated with 1 μM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the S. mansoni proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (β1), trypsin-type (β2), and chymotrypsin-type (β5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue 17 was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the β5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S β2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 μM 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.

Malaria is an important disease causing many death in several countries of Africa and Asia. In these continents, some plants such as Garcinia cola are used to fight against this disease because they contain xanthone derivatives which present antiplasmodial activity. The present theoretical study aims to establish a relationship between the electronic structure and the antiplasmodial activity of some xanthone derivatives, and more specifically to build a 2D-pharmacophore model in order to predict the biological activity of xanthone derivatives. The calculations are performed within the density functional theory (DFT) using the B3LYP/6-31G(d,p) level of theory. The developed approach quantitative structure-activity relationship (QSAR) follows the Klopman-Peradejordi-Gomez (KPG) methodology. We obtain a statistically significant equation relating the variation of the logarithm of half maximal inhibitory concentration (log(IC50)) with the variation of the numerical values of a set of eight local atomic reactivity descriptors (R = 0.98, R2 = 0.97, adj-R2 = 0.95, F(8.13) = 48.63, p

Contemporary group (CG) estimates of different phenotypes have not been widely explored for pigs. The objective of this study was to extend the traits used to derive environmental descriptors of the growing pig, to include CG estimates of early growth between birth and start of feed intake test (EADG), growth during feed intake test (TADG), lifetime growth (ADG), daily feed intake (DFI), backfat (BF) and muscle depth (MD). Pedigree and performance records (n = 7,746) from a commercial Australian piggery were used to derive environmental descriptors based on CG estimates of these six traits. The CG estimates of growth traits described different aspects of the environment from the CG estimates of carcass traits (r

Innovative chemotherapeutic agents that are active against Mycobacterium tuberculosis (Mtb) are urgently required to control the tuberculosis (TB) epidemic. The Mtb cell envelope has distinct (lipo)polysaccharides and glycolipids that play a critical role in Mtb survival and pathogenesis and disruption of pathways involved in the assembly of the Mtb cell envelope are the primary target of anti-tubercular agents. Here we introduce a previously unexplored approach whereby chemical agents directly target the extracellular glycans within the unique Mtb cell envelope, rather than the intracellular biosynthetic machinery. We designed and synthesised multimeric boronic acids that are selectivity lethal to Mtb and function by targeting these structurally unique and essential Mtb cell envelope glycans. By tuning the number of, and distance between, boronic acid units high selectivity to Mtb, low cytotoxicity against mammalian cells and no observable resistance was achieved. This non-conventional approach may prevent the development of drug-resistance and will act as a platform for the design of improved, pathogen-specific, next generation antibiotics.

Dimeric benzoboroxoles that are covalently linked by a short scaffold enhance selective anti-tubercular activity. These multimeric benzoboroxole compounds are capable of engaging the specific extracellular Mycobacterium tuberculosis glycans, do not lead to the evolution of resistance and bypass the need to cross the impermeable mycobacterial cell envelope barrier.

Lake charr Salvelinus namaycush life history and population dynamics metrics were reviewed to evaluate populations inside (n = 462) and outside (n = 24) the native range. Our goals were to create a database of metrics useful for evaluating population status and to test for large-scale patterns between metrics and latitude and lake size. An average lake charr grew from a 69-mm length at age-0 (L0) at 89 mm/year early growth rate (ω) to 50% maturity at 420 mm (L50) at age 8 (t50), and then continued to grow toward a 717-mm asymptotic length (L∞). L50 was positively correlated to ω, whereas t50 was inversely correlated to ω. Lake charr grew slower toward larger size and older age in northern latitudes and larger lakes than in southern latitudes and smaller lakes. Population density (number/ha) and yield density (kg/ha) decreased with lake size, and yield and total annual mortality (A) decreased with latitude. Native populations grew slower (ω), were heavier at 500 mm (W500), matured at shorter L50, grew to a shorter L∞, and suffered lower annual mortality A than non-native populations. Our review and database should be useful to managers and researchers for quantifying lake charr population status across the species range.

Zoonotic infections, such as COVID-19, reside in animal hosts before jumping species to humans. The Carnivora, like mink, carry many zoonoses yet how diversity in host immune genes across species impact upon pathogen carriage are poorly understood. Here we describe a progressive evolutionary downregulation of pathogen sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerisation domain leucine rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing lytic cell death and the formation of an NLRP3-caspase-8 containing inflammasome that inefficiently processes interleukin-1β (IL-1β). Inflammasomes regulate gut immunity, but the carnivorous diet is antimicrobial suggesting a tolerance to the loss of these immune pathways. The consequences of systemic inflammasome downregulation, however, can reduce the host sensing of specific pathogens such that they can reside undetected in the Carnivora.

The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATPbinding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substratespecific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools., This work was supported by a Sir Henry Dale Fellowship to E. F. jointly funded by the Wellcome Trust and Royal Society (104193/Z/14/Z and 104193/Z/14/B), a research grant from the Royal Society (RG120405), a research grant from the Leverhulme Trust (RPG-2019-087), the BBSRC for a studentship to H. P. and A. R. (BB/M01116X/1) and the MRC for a studentship for CMB (MR/N014294/1). We acknowledge equipment access, training, and support made available by the Research Technology Facility (managed by Dr Sarah Bennett) of the Warwick Integrative Synthetic Biology center (WISB), which received funding from EPSRC and BBSRC (BB/M017982/1). J. A. acknowledges support from the BBSRC through a New Investigator grant (BB/P010660/1) and the Universidad de Sevilla (Acciones Especiales del VI Plan Propio de Investigación y Transferencia). We are also grateful for the use of the University of East Anglia (UEA) Faculty of Science NMR facility. This project made use of time on ARCHER and JADE granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://hecbiosim.ac.uk), supported by the EPSRC (EP/R029407/1), Athena, at HPC Midlands+ funded by the EPSRC (EP/P020232/1) and used the University of Warwick Scientific Computing Research Technology Platform for computational access.

Lack of diversity in human genomics limits our understanding of the genetic underpinnings of complex traits, hinders precision medicine, and contributes to health disparities. To map genetic effects on gene regulation in the underrepresented Indonesian population, we have integrated genotype, gene expression, and CpG methylation data from 115 participants across three island populations that capture the major sources of genomic diversity on the region. In a comparison with a European dataset, we identify 166 uniquely Indonesia-specific eQTLs, highlighting the benefits of performing association studies on non-European populations. By combining local ancestry and archaic introgression inference eQTLs and methylQTLs, we identify regulatory loci driven by modern Papuan ancestry as well as introgressed Denisovan and Neanderthal variation. GWAS colocalization connects QTLs detected here to hematological traits. Our findings illustrate how local ancestry and archaic introgression drive variation in gene regulation across genetically distinct and in admixed populations.

Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.

The hominin fossil record of Island Southeast Asia (ISEA) indicates that at least two endemic ‘super-archaic’ species – Homo luzonensis and H. floresiensis – were present around the time anatomically modern humans (AMH) arrived in the region >50,000 years ago. Contemporary human populations carry signals consistent with interbreeding events with Denisovans in ISEA – a species that is thought to be more closely related to AMH than the super-archaic endemic ISEA hominins. To query this disparity between fossil and genetic evidence, we performed a comprehensive search for super-archaic introgression in >400 modern human genomes. Our results corroborate widespread Denisovan ancestry in ISEA populations but fail to detect any super-archaic admixture signals. By highlighting local megafaunal survival east of the Wallace Line as a potential signature of deep, pre-H. sapiens hominin-faunal interaction, we propose that this understudied region may hold the key to unlocking significant chapters in Denisovan prehistory.

Transmissible viral proventriculitis (TVP) is a disease of chickens, mostly in broilers of 2-8 wk of age. Chicken proventricular necrosis virus (CPNV), a birnavirus, is the etiologic agent. Characteristic gross lesions are enlargement, atony, and pallor of the proventriculus. Cases diagnosed in California between 2000 and 2018 (n = 477), originating from 93 different farms representing all major companies in the region, were analyzed. Frequency of cases varied widely between years, with no recognizable seasonality. The flocks were between 6 and 61 days of age; the average age was 34.0 days, and the median age was 35 days. In 166 cases, between 6.3% and 100% of the submitted birds had gross lesions in the proventriculus. The most common findings were enlarged or dilated proventriculi, thickened walls, and pale or mottled serosal appearance. Histopathologically, inflammation of the glands was the most frequent finding. Other lesions included necrosis, hyperplasia, or both conditions of the glandular epithelium; dilated glands; and occasionally fibrin deposition, fibrosis, and hemorrhages. Twenty-three proventriculi from six cases were tested by immunohistochemistry for the presence of CPNV antigen; 21 stained positive. In 209 cases, birds also had lesions in the bursa fabricii attributed to infectious bursal disease, but with no significant difference in the mean percentage of birds with gross lesions in the proventriculus between cases with or without lesions in the bursa fabricii. The results show that TVP is a common disease of broiler flocks in California and confirms that CPNV is the likely causative agent.

The uptake and metabolism of the disaccharide trehalose by Mycobacterium tuberculosis is essential for the virulence of this pathogen. Here we describe the chemoenzymatic synthesis of new azido-functionalised asymmetric trehalose probes that resist degradation by mycobacterial enzymes and are used to probe trehalose processing pathways in mycobacteria.\ud \ud

Mice with mutant ATG16L develop spontaneous, severe Alzheimer’s-like disease with age, despite normal autophagy., Noncanonical functions of autophagy proteins have been implicated in neurodegenerative conditions, including Alzheimer’s disease (AD). The WD domain of the autophagy protein Atg16L is dispensable for canonical autophagy but required for its noncanonical functions. Two-year-old mice lacking this domain presented with robust β-amyloid (Aβ) pathology, tau hyperphosphorylation, reactive microgliosis, pervasive neurodegeneration, and severe behavioral and memory deficiencies, consistent with human disease. Mechanistically, we found this WD domain was required for the recycling of Aβ receptors in primary microglia. Pharmacologic suppression of neuroinflammation reversed established memory impairment and markers of disease pathology in this novel AD model. Therefore, loss of the Atg16L WD domain drives spontaneous AD in mice, and inhibition of neuroinflammation is a potential therapeutic approach for treating neurodegeneration and memory loss. A decline in expression of ATG16L in the brains of human patients with AD suggests the possibility that a similar mechanism may contribute in human disease.

The Indian cheetah was hunted to extinction by the mid-20th century. While analysis of 139 bp of mitochondrial DNA (mtDNA) has confirmed that the Indian cheetah was part of the Asiatic subspecies (Acinonyx jubatus venaticus), the detailed relationships between cheetah populations remains unclear due to limited genetic data. We clarify these relationships by studying larger fragments of cheetah mtDNA, both from an Indian cheetah museum specimen and two African cheetah, one modern and one historic, imported into India at different times. Our results suggest that the most recent common ancestor of cheetah mtDNA is approximately twice as ancient as currently recognised. The Indian and Southeast African (Acinonyx jubatus jubatus) cheetah mtDNA diverged approximately 72 kya, while the Southeast and Northeast African (Acinonyx jubatus soemmeringii) cheetah mtDNA diverged around 139 kya. Additionally, the historic African cheetah sampled from India proved to have an A. j. jubatus haplotype, suggesting a hitherto unrecognised South African route of cheetah importation into India in the 19th century. Together, our results provide a deeper understanding of the relationships between cheetah subspecies, and have important implications for the conservation of A. j. venaticus and potential reintroduction of cheetahs into India.

The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, and yolk). The developing embryo and its membranes provide a diversity of cell types that allow for the successful replication of a wide variety of different viruses. Within the family Coronaviridae the embryonated egg has been used as a host system primarily for two avian coronaviruses within the genus Gammacoronavirus, infectious bronchitis virus (IBV) and turkey coronavirus (TCoV). IBV replicates well in the embryonated chicken egg, regardless of inoculation route; however, the allantoic route is favored as the virus replicates well in epithelium lining the chorioallantoic membrane, with high virus titers found in these membranes and associated allantoic fluids. TCoV replicates only in epithelium lining the embryo intestines and bursa of Fabricius; thus, amniotic inoculation is required for isolation and propagation of this virus. Embryonated eggs also provide a potential host system for detection, propagation, and characterization of other, novel coronaviruses.

Kakadu National Park and its wetlands are World Heritage and Ramsar listed and are at risk from invasive grasses. However, it appears that not all habitats and native vegetation are equally at risk. We conduct a spatial risk assessment for para grass (Urochloa mutica) invasion across seasonally inundated habitats of the 258 km2 Magela Creek floodplain within Kakadu National Park using Landsat 5 TM time-series imagery. Two maps, representing water depth and fire history, were derived from the imagery using object-based image analyses. Depth was modelled using a linear regression relationship established between 254 known water depth locations and the multi-date spectral index values of segmented image objects at corresponding locations (R2= 0.67; p < 0.0001). Binary fire-scar maps were then produced for each year of a 10-year period using visual interpretation and nearest neighbour classification. A map of the incidence of annual fire over this period was then calculated from the sum of the maps, overlaid. The maps were integrated in a GIS with an existing Landsat vegetation map to measure spatial inter-relationships between para grass, native vegetation, depth and fire. With a highly clustered distribution pattern, para grass occupied 1388 ha or 6% of the total floodplain area. However, its optimal depth habitat, estimated to be from 1.1 to 1.4 m, occurred over a much larger area (7180 ha) or 30% of the floodplain. Only 2% of this optimal area was actually occupied by para grass. Together the low occupancy of 'optimal-depth' habitat and a highly clustered distribution of para grass strongly suggested that, if left uncontrolled, it has capacity to spread further and eventually occupy much larger areas of this floodplain at high density. Landsat provided spatial information of suitable scale and accuracy to understand the landscape ecology of para grass; and from which to design and conduct further research, or trial management interventions to protect wetland vegetation at risk to weed invasion.

Despite many studies on the cytotoxic protease granzyme B, key aspects of its function remain unexplored due to the lack of selective probes for its activity. In this study, we fully mapped the substrate preferences of GrB using a set of unnatural amino acids, demonstrating previously unknown GrB substrate preferences that we then used to design novel substrate-based inhibitors and a GrB-activatable activity-based probe. We showed that our GrB probes react poorly with caspases, making them ideal for the in-depth analysis of GrB localization and function in cells. With our quenched fluorescence substrate, we determined GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with the GrB attack MDA-MB-231 target cells, and active GrB influences its target cell killing efficiency.

Members of the SCY1-like (SCYL) family of protein kinases are evolutionarily conserved and ubiquitously expressed proteins characterized by an N-terminal pseudokinase domain, centrally located Huntingtin, elongation factor 3, protein phosphatase 2A, yeast kinase TOR1 repeats, and an overall disorganized C-terminal segment. In mammals, three family members encoded by genesScyl1,Scyl2, andScyl3have been described. Studies have pointed to a role for SCYL1 and SCYL2 in regulating neuronal function and viability in mice and humans, but little is known about the biological function of SCYL3. Here, we show that the biochemical and cell biological properties of SCYL3 are similar to those of SCYL1 and both proteins work in conjunction to maintain motor neuron viability. Specifically, although lack ofScyl3in mice has no apparent effect on embryogenesis and postnatal life, it accelerates the onset of the motor neuron disorder caused byScyl1deficiency. Growth abnormalities, motor dysfunction, hindlimb paralysis, muscle wasting, neurogenic atrophy, motor neuron degeneration, and loss of large-caliber axons in peripheral nerves occurred at an earlier age inScyl1/Scyl3double-deficient mice than inScyl1-deficient mice. Disease onset also correlated with the mislocalization of TDP-43 in spinal motor neurons, suggesting that SCYL1 and SCYL3 regulate TDP-43 proteostasis. Together, our results demonstrate an overlapping role for SCYL1 and SCYL3in vivoand highlight the importance the SCYL family of proteins in regulating neuronal function and survival. Only male mice were used in this study.SIGNIFICANCE STATEMENTSCYL1 and SCYL2, members of the SCY1-like family of pseudokinases, have well established roles in neuronal function. Herein, we uncover the role of SCYL3 in maintaining motor neuron viability. Although targeted disruption ofScyl3in mice had little or no effect on embryonic development and postnatal life, it accelerated disease onset associated with the loss ofScyl1, a novel motor neuron disease gene in humans.Scyl1andScyl3double-deficient mice had neuronal defects characteristic of amyotrophic lateral sclerosis, including TDP-43 pathology, at an earlier age than didScyl1-deficient mice. Thus, we show that SCYL1 and SCYL3 play overlapping roles in maintaining motor neuronal viabilityin vivoand confirm that SCYL family members are critical regulators of neuronal function and survival.

For the αβ or γδTCR chains to integrate extracellular stimuli into the appropriate intracellular cellular response, they must use the 10 ITAMs found within the CD3 subunits (CD3γε, CD3δε, and ζζ) of the TCR signaling complex. However, it remains unclear whether each specific ITAM sequence of the individual subunit (γεδζ) is required for thymocyte development or whether any particular CD3 ITAM motif is sufficient. In this article, we show that mice utilizing a single ITAM sequence (γ, ε, δ, ζa, ζb, or ζc) at each of the 10 ITAM locations exhibit a substantial reduction in thymic cellularity and limited CD4−CD8− (double-negative) to CD4+CD8+ (double-positive) maturation because of low TCR expression and signaling. Together, the data suggest that ITAM sequence diversity is required for optimal TCR signal transduction and subsequent T cell maturation.

Mountain Whitefish Prosopium williamsoni were historically common throughout much of the U.S. Intermountain West. However, within the last decade Mountain Whitefish have exhibited population-level declines in some rivers. In the Madison River, Montana, anecdotal evidence indicates Mountain Whitefish abundance has declined and the population is skewed toward larger individuals, which is typically symptomatic of recruitment problems. Describing reproductive development, spawning behavior, and juvenile distribution will form a foundation for investigating mechanisms influencing recruitment. We collected otoliths and gonadal samples from fish of all size-classes to characterize fecundity, age at maturity, and spawning periodicity. We implanted radio tags in mature Mountain Whitefish and relocated tagged fish in autumn 2012–2014. Timing of spawning was determined from spawning status of captured females and from density of eggs collected on egg mats. In spring 2014, we seined backwater and channel site...

The activation mechanism of ZBP1/DAI to regulate virus-induced programmed cell death is not known. Kesavardhana et al. show that ZBP1 senses viral ribonucleoproteins to induce cell death upon influenza A virus infection. Apical activation of RIG-I–IFNAR signaling to upregulate ZBP1 and influenza-induced ZBP1 ubiquitination are critical events for ZBP1 activation., Innate sensing of influenza virus infection induces activation of programmed cell death pathways. We have recently identified Z-DNA–binding protein 1 (ZBP1) as an innate sensor of influenza A virus (IAV). ZBP1-mediated IAV sensing is critical for triggering programmed cell death in the infected lungs. Surprisingly, little is known about the mechanisms regulating ZBP1 activation to induce programmed cell death. Here, we report that the sensing of IAV RNA by retinoic acid inducible gene I (RIG-I) initiates ZBP1-mediated cell death via the RIG-I–MAVS–IFN-β signaling axis. IAV infection induces ubiquitination of ZBP1, suggesting potential regulation of ZBP1 function through posttranslational modifications. We further demonstrate that ZBP1 senses viral ribonucleoprotein (vRNP) complexes of IAV to trigger cell death. These findings collectively indicate that ZBP1 activation requires RIG-I signaling, ubiquitination, and vRNP sensing to trigger activation of programmed cell death pathways during IAV infection. The mechanism of ZBP1 activation described here may have broader implications in the context of virus-induced cell death.

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is complex and can be divided into two phases: early-MDV-IS associated with cytolytic infection in the lymphoid organs in chickens lacking maternal antibodies against MDV (MAbs) and late-MDV-IS that appears later in the pathogenesis and occurs even in chickens bearing MAbs. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model evaluates late-MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccines against challenge with ILT virus. In the present study, we have used this model to investigate the role of two factors (MDV pathotype and host sex) on the development of late-MDV-IS. Five MDV strains representing three different pathotypes: virulent (vMDV; 617A, GA), very virulent (vvMDV; Md5), and very virulent plus (vv+MDV; 648A, 686), were evaluated. Only vv+ strains were able to induce late-MDV-IS. An immunosuppression rank (IS-rank) was established based on the ability of MDV to reduce the efficacy of chicken embryo origin vaccine (values go from 0 to 100, with 100 being the highest immunosuppressive ability). The IS-rank of the evaluated MDV strains ranged from 5.97 (GA) to 20.8 (617A) in the vMDV strains, 5.97 to 16.24 in the vvMDV strain Md5, and 39.08 to 68.2 in the vv+ strains 648A and 686. In this study both male and female chickens were equally susceptible to MDV-IS by vv+MDV 686. Our findings suggest that late-MDV-IS is a unique feature of vv+ strains.

Background and purpose: Tuberculosis (TB) remains a major global health threat and is now the leading cause of death from a single infectious agent worldwide. The current TB drug regimen is inadequate and new anti-tubercular agents are urgently required to be able to successfully combat the increasing prevalence of drug-resistant TB. The purpose of this study was to investigate a piperidinol compound derivative that is highly active against the Mycobacterium tuberculosis bacillus. Experimental approach: The antibacterial properties of the piperidinol compound and its corresponding bis-Mannich base analogue were evaluated against Mycobacterium smegmatis and Gram-negative organisms. Cytotoxicity studies were undertaken in order to determine the selectivity index for these compounds. Spontaneous resistant mutants of Mycobacterium smegmatis were generated against the piperidinol and corresponding bis-Mannich base lead derivatives and whole genome sequencing employed to determine genetic modifications as result to selection pressure in the presence of these compounds. Key results: The piperidinol and the bis-Mannich base analogue were found to be selective for mycobacteria and rapidly kill this organism with a cytotoxicity selectivity index for mycobacteria of >30 fold. Whole genome sequencing of M. smegmatis strains resistant to the lead compounds led to the identification of a number of single nucleotide polymorphisms (SNPs) indicating multiple-targets. Conclusion and implications: Our results indicate that the piperidinol moiety represents an attractive compound class in the pursuit of novel anti-tubercular agents.

Conservation of sport fisheries and populations of several native fishes in the western United States is dependent on sustained success of removal programs targeting invasive Lake Trout Salvelinus namaycush. Gill-netting of spawning adults is one strategy used to decrease spawning success; however, additional complementary methods are needed to disrupt Lake Trout reproduction where bycatch in gill nets is unacceptable. We developed and tested two portable electrode arrays designed to increase Lake Trout embryo mortality in known spawning areas. Both arrays were powered by existing commercial electrofishing equipment. However, one array was moved across the substrate to simulate being towed behind a boat (i.e., towed array), while the other array was lowered from a boat and energized when sedentary (i.e., sedentary array). The arrays were tested on embryos placed within substrates of known spawning areas. Both arrays increased mortality of embryos (>90%) at the surface of substrates, but only the s...

Fisheries managers have implemented suppression programmes to control non-native lake trout, Salvelinus namaycush (Walbaum), in several lakes throughout the western United States. This study determined the feasibility of experimentally suppressing lake trout using gillnets in an isolated backcountry lake in Glacier National Park, Montana, USA, for the conservation of threatened bull trout, Salvelinus confluentus (Suckley). The demographics of the lake trout population during suppression (2009–2013) were described, and those data were used to assess the effects of suppression scenarios on population growth rate (λ) using an age-structured population model. Model simulations indicated that the population was growing exponentially (λ = 1.23, 95% CI: 1.16–1.28) prior to suppression. However, suppression resulted in declining λ (0.61–0.79) for lake trout, which was concomitant with stable bull trout adult abundances. Continued suppression at or above observed exploitation levels is needed to ensure continued population declines.