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BACKGROUND: While several studies have observed that solid organ transplant recipients experience diminished antibody responses to SARS-CoV-2 mRNA vaccination, data specific to heart and lung transplant (HT/LT) recipients remains sparse. METHODS: US adult HT and LT recipients completed their vaccine series between January 7 and April 10, 2021. Reactogencity and SARS-CoV-2 anti-spike antibody were assessed after a priming dose (D1) and booster dose (D2). Modified Poisson regression with robust variance estimator was used to evaluate associations between participant characteristics and antibody development. RESULTS: Of 134 heart recipients, there were 38% non-responders (D1-/D2-), 48% booster responders (D1-/D2+), and 14% priming dose responders (D1+/D2+). Of 103 lung recipients, 64% were non-responders, 27% were booster responders, and 9% were priming dose responders. Lung recipients were less likely to develop antibodies (p < .001). Priming dose antibody response was associated with younger recipient age (p = .04), transplant-to-vaccination time ≥6 years (p < .01), and lack of anti-metabolite maintenance immunosuppression (p < .001). Pain at injection site was the most commonly reported reaction (85% after D1, 76% after D2). Serious reactions were rare, the most common being fatigue (2% after D1 and 3% after D2). No serious adverse events were reported. CONCLUSIONS: HT and LT recipients experienced diminished antibody response following vaccination; reactogenicity was comparable to that of the general population. LT recipients may exhibit a more impaired antibody response than HT recipients. While current recommendations are to vaccinate eligible candidates and recipients, further studies characterizing the cell-mediated immune response and clinical efficacy of these vaccines in this population are needed.

Background The subfamily Phascolostrongylinae (Superfamily Strongyloidea) comprises nematodes that are parasitic in the gastrointestinal tracts of macropodid (Family Macropodidae) and vombatid (Family Vombatidae) marsupials. Currently, nine genera and 20 species have been attributed to the subfamily Phascolostrongylinae. Previous studies using sequence data sets for the internal transcribed spacers (ITS) of nuclear ribosomal DNA showed conflicting topologies between the Phascolostrongylinae and related subfamilies. Therefore, the aim of this study was to validate the phylogenetic relationships within the Phascolostrongylinae and its relationship with the families Chabertiidae and Strongylidae using mitochondrial amino acid sequences. Methods The sequences of all 12 mitochondrial protein-coding genes were obtained by next-generation sequencing of individual adult nematodes (n = 8) representing members of the Phascolostrongylinae. These sequences were conceptually translated and the phylogenetic relationships within the Phascolostrongylinae and its relationship with the families Chabertiidae and Strongylidae were inferred from aligned, concatenated amino acid sequence data sets. Results Within the Phascolostrongylinae, the wombat-specific genera grouped separately from the genera occurring in macropods. Two of the phascolostrongyline tribes were monophyletic, including Phascolostrongylinea and Hypodontinea, whereas the tribe Macropostrongyloidinea was paraphyletic. The tribe Phascolostrongylinea occurring in wombats was closely related to Oesophagostomum spp., also from the family Chabertiidae, which formed a sister relationship with the Phascolostrongylinae. Conclusion The current phylogenetic relationship within the subfamily Phascolostrongylinae supports findings from a previous study based on ITS sequence data. This study contributes also to the understanding of the phylogenetic position of the subfamily Phascolostrongylinae within the Chabertiidae. Future studies investigating the relationships between the Phascolostrongylinae and Cloacininae from macropodid marsupials may advance our knowledge of the phylogeny of strongyloid nematodes in marsupials. Graphical Abstract

Immune therapies have had limited efficacy in high-grade serous ovarian cancer (HGSC), as the cellular targets and mechanism(s) of action of these agents in HGSC are unknown. Here we performed immune functional and single-cell RNA sequencing transcriptional profiling on novel HGSC organoid/immune cell co-cultures treated with a unique bispecific anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) antibody compared with monospecific anti-PD-1 or anti-PD-L1 controls. Comparing the functions of these agents across all immune cell types in real time identified key immune checkpoint blockade (ICB) targets that have eluded currently available monospecific therapies. The bispecific antibody induced superior cellular state changes in both T and natural killer (NK) cells. It uniquely induced NK cells to transition from inert to more active and cytotoxic phenotypes, implicating NK cells as a key missing component of the current ICB-induced immune response in HGSC. It also induced a subset of CD8 T cells to transition from naïve to more active and cytotoxic progenitor-exhausted phenotypes post-treatment, revealing the small, previously uncharacterized population of CD8 T cells responding to ICB in HGSC. These state changes were driven partially through bispecific antibody-induced downregulation of the bromodomain-containing protein BRD1. Small-molecule inhibition of BRD1 induced similar state changes in vitro and demonstrated efficacy in vivo, validating the co-culture results. Our results demonstrate that state changes in both NK and a subset of T cells may be critical in inducing an effective anti-tumor immune response and suggest that immune therapies able to induce such cellular state changes, such as BRD1 inhibitors, may have increased efficacy in HGSC. Significance: This study indicates that increased efficacy of immune therapies in ovarian cancer is driven by state changes of NK and small subsets of CD8 T cells into active and cytotoxic states.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-β expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

MYB–bHLH–WDR (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat development including mucilage and tannin biosynthesis. The R2R3 MYBs MYB5, MYB23 and TRANSPARENT TESTA2 (TT2) participate in the MBW complexes with the WD-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). These complexes regulate GLABRA2 (GL2) and TTG2 expression in developing seeds. Microarray transcriptome analysis of ttg1-1- and wild-type (Ler) developing seeds identified 246 TTG1-regulated genes, which include all known metabolic genes of the tannin biosynthetic pathway. The first detailed TTG1-dependent metabolic pathways could be proposed for the biosynthesis of mucilage, jasmonic acid (JA) and cuticle including wax ester in developing seeds. We also assigned many known and previously uncharacterized genes to the activation/inactivation of hormones, plant immunity and nutrient transport. The promoters of six cuticle pathway genes were active in developing seeds. Expression of 11 genes was determined in the developing seeds of the combinatorial mutants of MYB5, MYB23 and TT2, and in the combinatorial mutants of GL2, HOMEODOMAIN GLABROUS2 (HDG2) and TTG2. These six TFs positively co-regulated the expression of four repressor genes while three of the six TFs repressed the wax biosynthesis genes examined, suggesting that the three TFs upregulate the expression of these repressor genes, which, in turn, repress the wax biosynthesis genes. Chromatin immunoprecipitation analysis identified 21 genes directly regulated by MYB5 including GL2, HDG2, TTG2, four repressor genes and various metabolic genes. We propose a multi-tiered regulatory mechanism by which MBWs regulate tannin, mucilage, JA and cuticle biosynthetic pathways.

Antibody-based therapeutics and vaccines are essential to combat COVID-19 morbidity and mortality after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Multiple mutations in SARS-CoV-2 that could impair antibody defenses propagated in human-to-human transmission and spillover or spillback events between humans and animals. To develop prevention and therapeutic strategies, we formed an international consortium to map the epitope landscape on the SARS-CoV-2 spike protein, defining and structurally illustrating seven receptor binding domain (RBD)–directed antibody communities with distinct footprints and competition profiles. Pseudovirion-based neutralization assays reveal spike mutations, individually and clustered together in variants, that affect antibody function among the communities. Key classes of RBD-targeted antibodies maintain neutralization activity against these emerging SARS-CoV-2 variants. These results provide a framework for selecting antibody treatment cocktails and understanding how viral variants might affect antibody therapeutic efficacy.

Increasing access to next‐generation sequencing (NGS) technologies is revolutionizing the life sciences. In disease ecology, NGS‐based methods have the potential to provide higher‐resolution data on communities of parasites found in individual hosts as well as host populations. Here, we demonstrate how a novel analytical method, utilizing high‐throughput sequencing of PCR amplicons, can be used to explore variation in blood‐borne parasite (Theileria—Apicomplexa: Piroplasmida) communities of African buffalo at higher resolutions than has been obtained with conventional molecular tools. Results reveal temporal patterns of synchronized and opposite fluctuations of prevalence and relative abundance of Theileria spp. within the host population, suggesting heterogeneous transmission across taxa. Furthermore, we show that the community composition of Theileria spp. and their subtypes varies considerably between buffalo, with differences in composition reflected in mean and variance of overall parasitemia, thereby showing potential to elucidate previously unexplained contrasts in infection outcomes for host individuals. Importantly, our methods are generalizable as they can be utilized to describe blood‐borne parasite communities in any host species. Furthermore, our methodological framework can be adapted to any parasite system given the appropriate genetic marker. The findings of this study demonstrate how a novel NGS‐based analytical approach can provide fine‐scale, quantitative data, unlocking opportunities for discovery in disease ecology.

Background The parasitic flatworm Clonorchis sinensis inhabits the biliary tree of humans and other piscivorous mammals. This parasite can survive and thrive in the bile duct, despite exposure to bile constituents and host immune attack. Although the precise biological mechanisms underlying this adaptation are unknown, previous work indicated that Niemann-pick type C2 (NPC2)-like sterol-binding proteins might be integral in the host-parasite interplay. Expansions of this family in some invertebrates, such as arthropods, have shown functional diversification, including novel forms of chemoreception. Thus, here we curated the NPC2-like protein gene complement in C. sinensis, and predicted their conserved and/or divergent functional roles. Methods We used an established comparative genomic-bioinformatic approach to curate NPC2-like proteins encoded in published genomes of Korean and Chinese isolates of C. sinensis. Protein sequence and structural homology, presence of conserved domains and phylogeny were used to group and functionally classify NPC2-like proteins. Furthermore, transcription levels of NPC2-like protein-encoding genes were explored in different developmental stages and tissues. Results Totals of 35 and 32 C. sinensis NPC2-like proteins were predicted to be encoded in the genomes of the Korean and Chinese isolates, respectively. Overall, these proteins had low sequence homology and high variability of sequence alignment coverage when compared with curated NPC2s. Most C. sinensis proteins were predicted to retain a conserved ML domain and a conserved fold conformation, with a large cavity within the protein. Only one protein sequence retained the conserved amino acid residues required in bovine NPC2 to bind cholesterol. Non-canonical C. sinensis NPC2-like protein-coding domains clustered into four distinct phylogenetic groups with members of a group frequently encoded on the same genome scaffolds. Interestingly, NPC2-like protein-encoding genes were predicted to be variably transcribed in different developmental stages and adult tissues, with most being transcribed in the metacercarial stage. Conclusions The results of the present investigation confirms an expansion of NPC2-like proteins in C. sinensis, suggesting a diverse array of functions beyond sterol binding and transport. Functional explorations of this protein family should elucidate the mechanisms enabling the establishment and survival of C. sinensis and related flukes in the biliary systems of mammalian hosts.

There is increasing interest in the potential of single-pass and timed electrofishing to assess status and trends in fish populations. However, where capture probability varies over time, there is a risk that uncalibrated electrofishing data could fail to detect, or provide biased estimates of trends. This study analysed a long-term electrofishing dataset collected over 50 years in an intensively studied catchment where egg deposition and emigrant production declined by c. 82% and 35% over the same time. The electrofishing data were used to illustrate the effects of changing capture probability on estimated trends in juvenile Atlantic salmon Salmo salar abundance. Temporal variability in capture probability was modelled. Trends in abundance were then estimated from uncalibrated single-pass electrofishing count data and compared with estimates from data calibrated for capture probability. The calibrated data revealed significant declines in S. salar fry (age 0) and parr (age ≥ 1) abundance. However, the trend estimates from the uncalibrated data were positively biased and not significant. Exploration of alternative (realistic) scenarios with different trends in true abundance and capture probability suggests that uncalibrated electrofishing data can provide very misleading estimates of trends. The problem is exacerbated in data where capture probability is low. It is recommended that single-pass and timed electrofishing methods should not be used to assess trends in fish populations without regular (annual) calibration.

Peatlandsare valuable but threatenedecosystems. Intervention to tackle direct threats is often necessary, but should be informed by scientific evidence to ensure it is effective and efficient. Herewe discuss a recent synthesis of evidence for the effects of interventions to conserve peatland vegetation -a fundamental component of healthy, functioning peatland ecosystems. The synthesis is unique in its broad scope (global evidence for a comprehensive list of 125 interventions) and practitioner-focused outputs (short narrative summaries in plain English, integrated into a searchable online database). Systematicliteraturesearches, supplemented by recommendationsfrom an international advisory board, identified162 publications containing 296 distinct tests of 66 of the interventions. Most of the articles studiedopen bogs or fens in Europe or North America. Only 36 interventions weresupported by sufficient evidence to assess their overall effectiveness. Mostof these interventions(85%) hadpositiveeffects, overall,on peatland vegetation-although this figure is likelyto have beeninflated by publication bias. We discuss how to use the synthesis, critically,to informconservation decisions.Reflecting on the content of the synthesiswe make suggestions for the future of peatland conservation,from monitoring overappropriate timeframes to routinely publishing resultsto build up the evidence base., MAVA, Arcadia

We have previously reported that the mouse gut microbiome contributes to pulmonary responses to ozone, a common asthma trigger, and that short-chain fatty acids, end products of bacterial fermentation, likely contribute to this role of the microbiome. A growing body of evidence indicates that there are sex-related differences in gut microbiota and these differences can have important functional consequences. The purpose of this study was to determine whether there are sex-related differences in the impact of the gut microbiota on pulmonary responses to ozone. After acute exposure to ozone, male mice developed greater airway hyperresponsiveness than female mice. This difference was abolished after antibiotic ablation of the gut microbiome. Moreover, weanling female pups housed in cages conditioned by adult male mice developed greater ozone-induced airway hyperresponsiveness than weanling female pups raised in cages conditioned by adult females. Finally, ad libitum oral administration via drinking water of the short-chain fatty acid propionate resulted in augmented ozone-induced airway hyperresponsiveness in male, but not female, mice. Overall, these data are consistent with the hypothesis that the microbiome contributes to sex differences in ozone-induced airway hyperresponsiveness, likely as a result of sex differences in the response to short-chain fatty acids.

Uterine carcinoma (UC) is the most common gynecologic malignancy in the United States. TP53 mutant UCs cause a disproportionate number of deaths due to limited therapies for these tumors and the lack of mechanistic understanding of their fundamental vulnerabilities. Here we sought to understand the functional and therapeutic relevance of TP53 mutations in UC. We functionally profiled targetable TP53 dependent DNA damage repair and cell cycle control pathways in a panel of TP53 mutant UC cell lines and patient-derived organoids. There were no consistent defects in DNA damage repair pathways. Rather, most models demonstrated dependence on defective G2/M cell cycle checkpoints and subsequent upregulation of Aurora kinase-LKB1-p53-AKT signaling in the setting of baseline mitotic defects. This combination makes them sensitive to Aurora kinase inhibition. Resistant lines demonstrated an intact G2/M checkpoint, and combining Aurora kinase and WEE1 inhibitors, which then push these cells through mitosis with Aurora kinase inhibitor-induced spindle defects, led to apoptosis in these cases. Overall, this work presents Aurora kinase inhibitors alone or in combination with WEE1 inhibitors as relevant mechanism driven therapies for TP53 mutant UCs. Context specific functional assessment of the G2/M checkpoint may serve as a biomarker in identifying Aurora kinase inhibitor sensitive tumors.

Information from Magnetic Resonance Imaging (MRI) is useful for diagnosis and treatment management of human neurological patients. MRI monitoring might also prove useful for non-human animals involved in neuroscience research provided that MRI is available and feasible and that there are no MRI contra-indications precluding scanning. However, MRI monitoring is not established in macaques and a resource is urgently needed that could grow with scientific community contributions. Here we show the utility and potential benefits of MRI-based monitoring in a few diverse cases with macaque monkeys. We also establish a PRIMatE MRI Monitoring (PRIME-MRM) resource within the PRIMatE Data Exchange (PRIME-DE) and quantitatively compare the cases to normative information drawn from MRI data from typical macaques in PRIME-DE. In the cases, the monkeys presented with no or mild/moderate clinical signs, were well otherwise and MRI scanning did not present a significant increase in welfare impact. Therefore, they were identified as suitable candidates for clinical investigation, MRI-based monitoring and treatment. For each case, we show MRI quantification of internal controls in relation to treatment steps and comparisons with normative data in typical monkeys drawn from PRIME-DE. We found that MRI assists in precise and early diagnosis of cerebral events and can be useful for visualising, treating and quantifying treatment response. The scientific community could now grow the PRIME-MRM resource with other cases and larger samples to further assess and increase the evidence base on the benefits of MRI monitoring of primates, complementing the animals' clinical monitoring and treatment regime.

Summary In sensory systems, representational features of increasing complexity emerge at successive stages of processing. In the mammalian auditory pathway, the clearest change from brainstem to cortex is defined by what is lost, not by what is gained, in that high-fidelity temporal coding becomes increasingly restricted to slower acoustic modulation rates. 1 , 2 Here, we explore the idea that sluggish temporal processing is more than just an inability for fast processing, but instead reflects an emergent specialization for encoding sound features that unfold on very slow timescales. 3 , 4 We performed simultaneous single unit ensemble recordings from three hierarchical stages of auditory processing in awake mice – the inferior colliculus (IC), medial geniculate body of the thalamus (MGB) and primary auditory cortex (A1). As expected, temporal coding of brief local intervals (0.001 – 0.1 s) separating consecutive noise bursts was robust in the IC and declined across MGB and A1. By contrast, slowly developing (∼1 s period) global rhythmic patterns of inter-burst interval sequences strongly modulated A1 spiking, were weakly captured by MGB neurons, and not at all by IC neurons. Shifts in stimulus regularity were not represented by changes in A1 spike rates, but rather in how the spikes were arranged in time. These findings show that low-level auditory neurons with fast timescales encode isolated sound features but not the longer gestalt, while the extended timescales in higher-level areas can facilitate sensitivity to slower contextual changes in the sensory environment.

Despite advances in high-throughput sequencing and bioinformatics, molecular investigations of snail intermediate hosts that transmit parasitic trematodes are scant. Here, we report the first transcriptome for Bulinus truncatus – a key intermediate host of Schistosoma haematobium – a blood fluke that causes urogenital schistosomiasis in humans. We assembled this transcriptome from short- and long-read RNA-sequence data. From this transcriptome, we predicted 12,998 proteins, 58% of which had orthologs in Biomphalaria glabrata – an intermediate host of Schistosoma mansoni – a blood fluke that causes hepato-intestinal schistosomiasis. We predicted that select protein groups are involved in signal transduction, cell growth and death, the immune system, environmental adaptation and/or the excretory/secretory system, suggesting roles in immune responses, pathogen defence and/or parasite-host interactions. The transcriptome of Bu. truncatus provides a useful resource to underpin future molecular investigations of this and related snail species, and its interactions with pathogens including S. haematobium. The present resource should enable comparative investigations of other molluscan hosts of socioeconomically important parasites in the future.

Background: There are few studies that have examined whether dysbiosis occurs in kidney donors and transplant recipients following kidney transplant surgery. Aim: To ascertain whether changes occur in the gastrointestinal microbiota of the kidney donor and recipient following kidney transplantation. Methods: Kidney transplant recipients and their donors were prospectively enrolled in a pilot study to collect one faecal sample prior to, and another faecal sample between four to eight weeks following surgery. Gastrointestinal microbiota richness, Shannon diversity measures and functional assessments of kidney donors and recipients were analysed via metagenomic sequencing. Results: The study included 12 donors (median age 56 years, 6 females) and 12 recipients (median age 51 years, 3 females). Donor microbiota showed no significant changes in gastrointestinal microbiota richness, Shannon diversity, or functional assessments before and after nephrectomy. Recipient microbiota was altered post-transplant, reflected in reductions of the mean (±SD) richness values (156 ± 46.5 to 116 ± 38.6, p = 0.002), and Shannon diversity (3.57 ± 0.49 to 3.14 ± 0.52, p = 0.007), and a dramatic increase in Roseburia spp. abundance post-transplant (26-fold increase from 0.16 ± 0.0091 to 4.6 ± 0.3; p = 0.006; FDR = 0.12). Functionally, the post-transplant microbial community shifted towards those taxa using the glycolysis pathway (1.2-fold increase; p = 0.02; FDR = 0.26) for energy metabolism, while those functions involved with reactive oxygen species degradation decreased (2.6-fold; p = 0.006; FDR = 0.14). Conclusion: Live donor kidney transplantation and standard care post-transplant result in significant alterations in gut microbiota richness, diversity, composition and functional parameters in kidney transplant recipients but not in their kidney donors.

Made available in DSpace on 2022-05-01T05:29:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 To investigate if differences in imprinting at tropho-microRNA (miRNA) genomic clusters can distinguish between pre-gestational trophoblastic neoplasia cases (pre-GTN) and benign complete hydatidiform mole (CHM) cases at the time of initial uterine evacuation. miRNA sequencing was performed on frozen tissue from 39 CHM cases including 9 GTN cases. DIO3, DLK1, RTL1, and MEG 3 mRNA levels were assessed by qRT-PCR. Protein abundance was assessed by Western blot for DIO3, DLK1, and RTL1. qRT-PCR and Western blot were performed for selenoproteins and markers of oxidative stress. Immunohistochemistry (IHC) was performed for DIO3 on an independent validation set of clinical samples (n = 42) and compared to normal placenta controls across gestational ages. Relative expression of the 14q32 miRNA cluster was lower in pre-GTN cases. There were no differences in protein abundance of DLK1 or RTL1. Notably, there was lower protein expression of DIO3 in pre-GTN cases (5-fold, p < 0.03). There were no differences in mRNA levels of DIO3, DLK1, RTL1 or MEG 3. mRNA levels were higher in all CHM cases compared to normal placenta. IHC showed syncytiotrophoblast-specific DIO3 immunostaining in benign CHM cases and normal placenta, while pre-GTN cases of CHM lacked DIO3 expression. We describe two new biomarkers of pre-GTN CHM cases: decreased 14q32 miRNA expression and loss of DIO3 expression by IHC. Differences in imprinting between benign CHM and pre-GTN cases may provide insight into the fundamental development of CHM. Division of Gynecologic Oncology Department of Obstetrics and Gynecology Brigham and Women’s Hospital, 75 Francis St. New England Trophoblastic Disease Center Division of Gynecologic Oncology Department of Obstetrics Gynecology and Reproductive Biology Brigham and Women’s Hospital Dana-Farber Cancer Institute Harvard Medical School, 75 Francis St University of Sao Paulo Trophoblastic Disease Center University of Sao Paulo Medical School Department of Pathology New York University Langone Health Department of Obstetrics and Gynecology University of Texas Southwestern Medical Center Botucatu Trophoblastic Disease Center Clinical Hospital of Botucatu Medical School Sao Paulo State University Sao Paulo State University-UNESP, Botucatu Botucatu Trophoblastic Disease Center Clinical Hospital of Botucatu Medical School Sao Paulo State University Sao Paulo State University-UNESP, Botucatu

Nematodes of the genus Macropostrongyloides inhabit the large intestines or stomachs of macropodid (kangaroos and wallabies) and vombatid (wombats) marsupials. This study established the relationships of seven species of Macropostrongyloides using mitochondrial (mt) protein amino acid sequence data sets. Phylogenetic analyses revealed that species of Macropostrongyloides (M. lasiorhini, M. baylisi, M. yamagutii, M. spearei, M. mawsonae and M. woodi) from the large intestines of their hosts formed a monophyletic assemblage with strong nodal support to the exclusion of M. dissimilis from the stomach of the swamp wallaby. Furthermore, the mitochondrial protein-coding genes provided greater insights into the diversity and phylogeny of the genus Macropostrongyloides, such data sets could potentially be used to elucidate the relationships among other parasitic nematodes of Australian marsupials.

Background Larvae of the Australian sheep blowfly, Lucilia cuprina, parasitise sheep by feeding on skin excretions, dermal tissue and blood, causing severe damage known as flystrike or myiasis. Recent advances in -omic technologies and bioinformatic data analyses have led to a greater understanding of blowfly biology and should allow the identification of protein families involved in host-parasite interactions and disease. Current literature suggests that proteins of the SCP (Sperm-Coating Protein)/TAPS (Tpx-1/Ag5/PR-1/Sc7) (SCP/TAPS) superfamily play key roles in immune modulation, cross-talk between parasite and host as well as developmental and reproductive processes in parasites. Methods Here, we employed a bioinformatics workflow to curate the SCP/TAPS protein gene family in L. cuprina. Protein sequence, the presence and number of conserved CAP-domains and phylogeny were used to group identified SCP/TAPS proteins; these were compared to those found in Drosophila melanogaster to make functional predictions. In addition, transcription levels of SCP/TAPS protein-encoding genes were explored in different developmental stages. Results A total of 27 genes were identified as belonging to the SCP/TAPS gene family: encoding 26 single-domain proteins each with a single CAP domain and a solitary double-domain protein containing two conserved cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domains. Surprisingly, 16 SCP/TAPS predicted proteins formed an extended tandem array spanning a 53 kb region of one genomic region, which was confirmed by MinION long-read sequencing. RNA-seq data indicated that these 16 genes are highly transcribed in all developmental stages (excluding the embryo). Conclusions Future work should assess the potential of selected SCP/TAPS proteins as novel targets for the control of L. cuprina and related parasitic flies of major socioeconomic importance. Graphical Abstract

In mice, both androgens and the gut microbiota modify pulmonary responses to ozone. We hypothesized that androgens affect gut microbiota and thereby impact pulmonary responses to ozone. To address this hypothesis, we transferred cecal microbiota from male castrated or sham castrated C57BL/6J mice into female germ‐free recipient C57BL/6J mice. Four weeks later mice were exposed to ozone (2 ppm) or room air for 3 hr. The gut microbiomes of castrated versus sham castrated donors differed, as did those of recipients of microbiota from castrated versus sham castrated donors. In recipients, ozone‐induced airway hyperresponsiveness was not affected by donor castration status. However, compared to mice receiving microbiota from sham castrated donors, mice receiving microbiota from castrated donors had elevated numbers of bronchoalveolar lavage (BAL) neutrophils despite evidence of reduced lung injury as measured by BAL protein. Serum concentrations of IL‐17A and G‐CSF were significantly greater in recipients of castrated versus sham castrated microbiota. Furthermore, BAL neutrophils correlated with both serum IL‐17A and serum G‐CSF. Our data indicate that androgen‐mediated effects on the gut microbiota modulate pulmonary inflammatory responses to ozone and suggest a role for circulating IL‐17A and G‐CSF in these events., While sex differences in ozone‐induced airway hyperresponsiveness (AHR) are apparent in mouse models, the role of androgens in ozone‐induced AHR have not been examined. Our data confirm previous reports that gut microbiota have the potential to impact host inflammatory responses to ozone and indicate that effects of these microbiota on systemic inflammation may contribute to responses to ozone. A better understanding of the mechanisms by which sex hormones manipulate the gut microbiome may ultimately lead to improved treatment of lung diseases such as asthma.

Oriental theileriosis is an economically important tickborne disease of bovines, caused by some members of the Theileria orientalis complex. Currently, 11 distinct operational taxonomic units (OTUs), or genotypes, are recognized based on their major piroplasm surface protein (MPSP) gene sequences. Two of these genotypes (i.e., chitose and ikeda) are recognized as pathogenic in cattle, causing significant disease in countries of the Asia-Pacific region. However, the true extent of genetic variation and associated virulence/pathogenicity within this complex is unknown. Here, we undertook a proof-of-principle study of a small panel of genomic DNAs (n = 13) from blood samples originating from individual cattle known to harbor T. orientalis, in order to assess the performance of a targeted &rdquo, next-generation&rdquo, sequencing-informatic approach to identify genotypes. Five genotypes (chitose, ikeda, buffeli, type 4, and type 5) were defined, multiple genotypes were found within individual samples, with dominant and minor sequence types representing most genotypes. This study indicates that this sequencing-informatic workflow could be useful to assess the nature and extent of genetic variation within and among populations of T. orientalis on a large scale, and to potentially employ panels of distinct gene markers for expanded molecular epidemiological investigations of socioeconomically important protistan pathogens more generally.

SUMMARYOptogenetic activation of Ntsr1+ layer 6 corticothalamic (L6 CT) neurons modulates thalamocortical sensory processing and perception for hundreds of milliseconds following laser offset. Naturally occurring sources of extrasensory inputs that could recruit L6 CTs prior to upcoming sensory stimuli have not been identified. Here, we found that 100% of L6 CTs in mouse primary auditory cortex (A1) expressed FoxP2, a protein marker found in brain areas that coordinate sensory inputs with movement. To test the idea that motor preparatory inputs could be a natural extrasensory activator of L6 CTs, we combined quantitative videography, optogenetically targeted single unit recordings, and two-photon imaging during self-initiated behavior. We found that A1 L6 CTs were activated hundreds of milliseconds prior to orofacial movements, but not whole-body movements associated with locomotion. These findings identify new local circuit arrangements for routing motor corollary discharge into A1 and suggest new roles for CT neurons in active sensing.

Low molecular weight polycyclic aromatic hydrocarbons (LMW PAHs

Electrofishing data are commonly collected to assess the status of salmonid populations. However, their interpretation can be challenging without a benchmark measure of abundance against which they can be compared, leading some practitioners to question the value of these data. Juvenile density models that relate salmonid abundance to habitat characteristics offer one approach for developing spatially explicit benchmark abundances. This study collated and analysed an electrofishing dataset for Atlantic salmon (Salmo salar) collected across Scotland between 1997 and 2015. Habitat was characterised using landscape proxies, derived from large scale spatial datasets. A two stage modelling approach related (1) capture probability to landscape and other covariates (2) fish density to landscape and other covariates, having adjusted for differences in capture probability. Capture probability varied with monitoring organisation, year and region, responded modally to day of the year, and decreased with upstream catchment area, river distance to sea and gradient. Salmon density increased non-linearly with upstream catchment area and river distance to sea and decreased with the percentage of the riparian zone containing conifer trees. There was a south-north gradient in density, with higher densities in the north. The density model was used to develop benchmark salmon densities (reference conditions) that would be expected in river catchments that are relatively un-impacted by anthropogenic pressures and are associated with high spawner densities, resulting in near-saturation of available freshwater habitat. The benchmark densities were based on the fixed effects from the density model, excluding the effects of riparian conifer woodland and the south-north gradient, adjusted for the site-wise mean observed density in the dataset. Comparing catchment scale predictions of juvenile production against this benchmark (or some percentage of it) could provide a valuable assessment tool.

Clonorchiasis is a neglected tropical disease caused by the Chinese liver fluke, Clonorchis sinensis, and is often associated with a malignant form of bile duct cancer (cholangiocarcinoma). Although some aspects of the epidemiology of clonorchiasis are understood, little is known about the genetics of C. sinensis populations. Here, we conducted a comprehensive genetic exploration of C. sinensis from endemic geographic regions using complete mitochondrial protein gene sets. Genomic DNA samples from C. sinensis individuals (n = 183) collected from cats and dogs in China (provinces of Guangdong, Guangxi, Hunan, Heilongjiang and Jilin) as well as from rats infected with metacercariae from cyprinid fish from the Russian Far East (Primorsky Krai region) were deep sequenced using the BGISEQ-500 platform. Informatic analyses of mitochondrial protein gene data sets revealed marked genetic variation within C. sinensis; significant variation was identified within and among individual worms from distinct geographical locations. No clear affiliation with a particular location or host species was evident, suggesting a high rate of dispersal of the parasite across endemic regions. The present work provides a foundation for future biological, epidemiological and ecological studies using mitochondrial protein gene data sets, which could aid in elucidating associations between particular C. sinensis genotypes/haplotypes and the pathogenesis or severity of clonorchiasis and its complications (including cholangiocarcinoma) in humans., Author summary Clonorchiasis is an important neglected tropical disease caused by the Chinese liver fluke, Clonorchis sinensis, which can induce malignant bile duct cancer (cholangiocarcinoma). Little precise information is available on the biology, epidemiology and population genetics of C. sinensis. For this reason, we explored here the genetic composition of C. sinensis populations in distinct endemic areas in China and Russia. Using a deep sequencing-informatic approach, we revealed marked mitochondrial genetic variation within and between individuals and populations of C. sinensis, with no particular affiliation with geographic or host origin. These molecular findings and the methodology established should underpin future genetic studies of C. sinensis causing human clonorchiasis and associated complications (cancer) as well as transmission patterns in endemic regions.

There are increasing calls for “conservation stocking” to counter declines in Atlantic salmon (Salmo salar) populations, the assumption being that stocking can bypass population bottlenecks and increase recruitment over natural processes. However, there are too few quantitative studies with sufficient data to assess the efficacy of conservation stocking. The Girnock Burn is a unique long-term monitoring site where adult and juvenile salmon numbers have been monitored for over 50 years, including 11 years with conservation stocking. Adults were monitored at a fixed trap and juveniles were monitored by electrofishing. In stocked years, ova were incubated in surface water to reduce density-independent over-winter mortality. In eight years, eyed ova were stocked at uniform densities to reduce local density-dependence. In three years, stocking replicated natural spatial variability in ova deposition removing any potential benefits of reduced local density-dependence. Juvenile production was estimated by summing the product of reach-scale density estimates and river area obtained from a novel spatial statistical river network model that incorporated the effects of capture probability, habitat and stock level. Capture probability varied with life-stage (age 0+ fry or ≥1+ parr), electrofishing pass and day of the year, but importantly also exhibited a positive temporal trend across years. Survival from ova to fry was density-independent and higher under uniform stocking than natural spawning or simulated natural spawning. Under uniform stocking, fry densities varied smoothly with altitude, while under natural spawning and simulated natural spawning, fry exhibited a more patchy distribution. Increased fry production did not translate to increased parr production, which was strongly density-dependent. This likely reflected the inability of fry to move between stocked locations and suitable overwintering habitat, decreasing survival between fry and parr life-stages. Consequently, there was no overall benefit of stocking. The modelling framework used in this study provides a valuable approach for interpreting long-term datasets where site locations, equipment and staffing vary over time. The long-term Girnock dataset was valuable in separating management action from natural population regulation and permitting understanding of ecological processes. The study indicates that conservation stocking can be ineffective, even where implemented to best scientific standards. It is therefore recommended that a detailed understanding of local population dynamics is obtained, and a realistic appraisal of the expected benefits of stocking is undertaken, before management actions are considered.

In this study, we explored the molecular alterations in the developmental switch from the L3 to the exsheathed L3 (xL3) and to the L4 stage of Haemonchus contortus in vitro using an integrated transcriptomic, proteomic and bioinformatic approach. Totals of 9,754 mRNAs, 88 microRNAs (miRNAs) and 1,591 proteins were identified, and 6,686 miRNA-mRNA pairs inferred in all larval stages studied. Approximately 16% of transcripts in the combined transcriptome (representing all three larval stages) were expressed as proteins, and there were positive correlations (r = 0.39-0.44) between mRNA transcription and protein expression in the three distinct developmental stages of the parasite. Of the predicted targets, 1,019 (27.0%) mRNA transcripts were expressed as proteins, and there was a negative correlation (r = -0.60 to -0.50) in the differential mRNA transcription and protein expression between developmental stages upon pairwise comparison. The changes in transcription (mRNA and miRNA) and protein expression from the free-living to the parasitic life cycle phase of H. contortus related to enrichments in biological pathways associated with metabolism (e.g., carbohydrate and lipid degradation, and amino acid metabolism), environmental information processing (e.g., signal transduction, signalling molecules and interactions) and/or genetic information processing (e.g., transcription and translation). Specifically, fatty acid degradation, steroid hormone biosynthesis and the Rap1 signalling pathway were suppressed, whereas transcription, translation and protein processing in the endoplasmic reticulum were upregulated during the transition from the free-living L3 to the parasitic xL3 and L4 stages of the nematode in vitro. Dominant post-transcriptional regulation was inferred to elicit these changes, and particular miRNAs (e.g., hco-miR-34 and hco-miR-252) appear to play roles in stress responses and/or environmental adaptations during developmental transitions of H. contortus. Taken together, these integrated results provide a comprehensive insight into the developmental biology of this important parasite at the molecular level in vitro. The approach applied here to H. contortus can be readily applied to other parasitic nematodes.

Layer 5 (L5) cortical projection neurons innervate far-ranging brain areas to coordinate integrative sensory processing and adaptive behaviors. Here, we characterize a plasticity in L5 auditory cortex (ACtx) neurons that innervate the inferior colliculus (IC), thalamus, lateral amygdala and striatum. We track daily changes in sound processing using chronic widefield calcium imaging of L5 axon terminals on the dorsal cap of the IC in awake, adult mice. Sound level growth functions at the level of the auditory nerve and corticocollicular axon terminals are both strongly depressed hours after noise-induced damage of cochlear afferent synapses. Corticocollicular response gain rebounded above baseline levels by the following day and remained elevated for several weeks despite a persistent reduction in auditory nerve input. Sustained potentiation of excitatory ACtx projection neurons that innervate multiple limbic and subcortical auditory centers may underlie hyperexcitability and aberrant functional coupling of distributed brain networks in tinnitus and hyperacusis., Deep layer auditory cortex neurons project to a number of limbic and subcortical auditory structures. Here, the authors show how these corticofugal projections adjust response gain following noise-induced cochlear damage.

A 36-year-old with metastatic and refractory choriocarcinoma following single- and multi-agent chemotherapy and surgical metastectomy experienced a durable remission after receiving therapy with an anti-endoglin monoclonal antibody and bevacizumab. Treatment options and scientific advances in this disease are highlighted.

Plasmodium falciparum parasites are purine auxotrophs that rely exclusively on the salvage of preformed purines from their human hosts to supply the requirement for purine nucleotides. Hypoxanthine-guanine-xanthine phosphor-ibosyltransferase (HGXPRT) catalyzes the freely reversible Mg2+-dependent conversion of 6-oxopurine bases to their respective nucleotides and inorganic pyrophosphate. The phosphoribosyl group is derived from 5-phospho-α-D-ribosyl 1-pyrophosphate (PRPP). The enzyme from malaria parasites (PfHGXPRT) is essential as hypoxanthine is the major precursor in purine metabolism. We used specific heavy atom labels in PRPP and hypoxanthine to measure primary (1-14C and 9-15N) and secondary (1-3H and 7-15N) intrinsic kinetic isotope effect (KIE) values for PfHGXPRT. Intrinsic isotope effects contain information for understanding enzymatic transition state properties. The transition state of PfHGXPRT was explored by matching KIE values predicted from quantum mechanical calculations to the intrinsic values determined experimentally. This approach provides information about PfHGXPRT transition state bond lengths, geometry, and atomic charge distribution. The transition state structure of PfHGXPRT was determined in the physiological direction of addition of ribose 5-phosphate to hypoxanthine by overcoming the chemical instability of PRPP. The transition state for PfHGXPRT forms nucleotides through a well-developed and near-symmetrical DN*AN, SN1-like transition state.

Human papillomavirus-negative keratinizing vulvar cancers typically harbor TP53 mutations as do their precursors, differentiated vulvar intraepithelial neoplasia. However, atypical verruciform proliferations are also associated with these malignancies and their pathogenesis is poorly understood. This study compared 11 atypical verruciform lesions, including atypical verruciform hyperplasia, vulvar acanthosis with altered differentiation, and verruciform lichen simplex chronicus, with 14 human papillomavirus-negative keratinizing squamous cell carcinomas. Extracted tissue DNA was subjected to targeted massively parallel sequencing of the exonic regions of 300 genes. Eight (73%) and six (55%) of eleven atypical verruciform lesions contained mutations in PIK3CA and ARID2, respectively. No TP53 mutations were identified. Eleven (79%) and five (36%) of fourteen keratinizing squamous cell carcinomas tested contained TP53 and CDKN2A mutations, respectively. Keratinizing squamous cell carcinomas displayed the majority of copy number variations with some variations (7p gain and 8p loss) shared by some cases in both groups. One patient developed atypical verruciform lesions with PIK3CA mutations followed by a keratinizing carcinoma with mutations in both PIK3CA and TP53. This study, for the first time segregates atypical verruciform lesions by virtue of a unique genotype (PIK3CA mutant/TP53 wild type) illustrating an example of progression to a TP53-mutated keratinizing carcinoma. The findings indicate that although PIK3CA mutations are found in

Ozone causes airway hyperresponsiveness, a defining feature of asthma. We have reported that the gut microbiome contributes to sex differences in ozone-induced airway hyperresponsiveness. Altering dietary fiber affects the gut microbiome. The purpose of this study was to determine the effects of dietary fiber on pulmonary responses to ozone and whether these effects differ by sex. We fed male and female mice fiber-free diets or diets enriched in one of two types of dietary fiber, cellulose and pectin, for 3 days before ozone exposure. Compared with control diets or pectin-enriched diets, cellulose-enriched diets attenuated ozone-induced airway hyperresponsiveness in male but not female mice. In contrast, fiber-free diets augmented responses to ozone in female but not male mice. Analysis of 16S rRNA sequencing of fecal DNA also indicated sex differences in the impact of dietary fiber on the gut microbiome and identified bacterial taxa that were associated with ozone-induced airway hyperresponsiveness. Our data suggest that microbiome-based therapies such as prebiotics may provide an alternative therapeutic strategy for air pollution-triggered asthma, but they indicate that such therapeutics may need to be tailored differently for males and females.

Objectives Cellular immunity against BK polyomavirus (BKV)‐encoded antigens plays a crucial role in long‐term protection against virus‐associated pathogenesis in transplant recipients. However, in‐depth understanding on dynamics of these cellular immune responses is required to develop better immune monitoring and immunotherapeutic strategies. Methods Here, we have conducted a proteome‐wide analysis of BKV‐specific T‐cell responses in a cohort of 53 healthy individuals and 26 kidney transplant recipients to delineate the functional and transcriptional profile of these effector cells and compared these characteristics to T cells directed against cytomegalovirus, which is also known to cause significant morbidity in transplant recipients. Results Profiling of BKV‐specific CD4+ and CD8+ T cells revealed that kidney transplant recipients with high levels of circulating viraemia showed significantly reduced T‐cell reactivity against large T and/or small T antigens when compared to healthy donors. Interestingly, T cells specific for these antigens showed strong cross‐recognition to orthologous JC virus (JCV) peptides, including those exhibiting varying degrees of sequence identity. Ex vivo functional and phenotypic characterisation revealed that the majority of BKV‐specific T cells from renal transplant recipients expressed low levels of the key transcriptional regulators T‐bet and eomesodermin, which was coincident with undetectable expression of granzyme B and perforin. However, in vitro stimulation of T cells with BKV epitopes selectively enhanced the expression of T‐bet, granzyme B and cellular trafficking molecules (CCR4, CD49d and CD103) with minimal change in eomesodermin and perforin. Conclusions These observations provide an important platform for the future development of immune monitoring and adoptive T‐cell therapy strategies for BKV‐associated diseases in transplant recipients, which may also be exploited for similar therapeutic value in JCV‐associated clinical complications., This manuscript provides comprehensive profiling of BK polyomavirus T‐cell responses in healthy virus carriers and kidney transplant recipients.

The incidence of infectious complications, compared with the general population and the pre-transplant status of the recipient, increases substantially following kidney transplantation, causing significant morbidity and mortality. The potent immunosuppressive therapy given to prevent graft rejection in kidney transplant recipients results in an increased susceptibility to a wide range of opportunistic infections including bacterial, viral and fungal infections. Over the last five years, several advances have occurred that may have changed the burden of infectious complications in kidney transplant recipients. Due to the availability of direct-acting antivirals to manage donor-derived hepatitis C infection, this has opened the way for donors with hepatitis C infection to be considered in the donation process. In addition, there have been the development of medications targeting the growing burden of resistant cytomegalovirus, as well as the discovery of the potentially important role of the gastrointestinal microbiota in the pathogenesis of post-transplant infection. In this narrative review, we will discuss these three advances and their potential implications for clinical practice.

Background: Human natural killer (NK) cells are key functional players in kidney transplant rejection. However, the respective contributions of the two functionally distinct human NK cell subsets (CD56bright cytokine-producing vs. CD56dim cytotoxic effector) in episodes of allograft rejection remain uncertain, with current immunohistochemical methods unable to differentiate these discrete populations. We report the outcomes of an innovative multi-color flow cytometric-based approach to unequivocally define and evaluate NK cell subsets in human kidney allograft rejection. Methods: We extracted renal lymphocytes from human kidney transplant biopsies. NK cell subsets were identified, enumerated, and phenotyped by multi-color flow cytometry. Dissociation supernatants were harvested and levels of soluble proteins were determined using a multiplex bead-based assay. Results were correlated with the histopathological patterns in biopsies—no rejection, borderline cellular rejection, T cell-mediated rejection (TCMR), and antibody-mediated rejection (AMR). Results: Absolute numbers of only CD56bright NK cells were significantly elevated in TCMR biopsies. In contrast, both CD56bright and CD56dim NK cell numbers were significantly increased in biopsies with histopathological evidence of AMR. Notably, expression of the activation marker CD69 was only significantly elevated on CD56dim NK cells in AMR biopsies compared with no rejection biopsies, indicative of a pathogenic phenotype for this cytotoxic NK cell subset. In line with this, we detected significantly elevated levels of cytotoxic effector molecules (perforin, granzyme A, and granulysin) in the dissociation supernatants of biopsies with a histopathological pattern of AMR. Conclusions: Our results indicate that human NK cell subsets are differentially recruited and activated during distinct types of rejection, suggestive of specialized functional roles.

Oestrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, regulates breast cancer cell proliferation and promotes motility and invasion. To determine the mechanisms of LRH-1 action in breast cancer, we performed gene expression microarray analysis following RNA interference for LRH-1. Interestingly, gene ontology (GO) category enrichment analysis of LRH-1–regulated genes identified oestrogen-responsive genes as the most highly enriched GO categories. Remarkably, chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1 showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα−regulated genes by LRH-1 through binding to oestrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 overexpression stimulated ERα recruitment, while LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at oestrogen response elements controls the expression of oestrogen-responsive genes.

Cortical layers (L) 5 and 6 are populated by intermingled cell-types with distinct inputs and downstream targets. Here, we made optogenetically guided recordings from L5 corticofugal (CF) and L6 corticothalamic (CT) neurons in the auditory cortex of awake mice to discern differences in sensory processing and underlying patterns of functional connectivity. Whereas L5 CF neurons showed broad stimulus selectivity with sluggish response latencies and extended temporal non-linearities, L6 CTs exhibited sparse selectivity and rapid temporal processing. L5 CF spikes lagged behind neighboring units and imposed weak feedforward excitation within the local column. By contrast, L6 CT spikes drove robust and sustained activity, particularly in local fast-spiking interneurons. Our findings underscore a duality among sub-cortical projection neurons, where L5 CF units are canonical broadcast neurons that integrate sensory inputs for transmission to distributed downstream targets, while L6 CT neurons are positioned to regulate thalamocortical response gain and selectivity.

Parasitic worms cause very significant diseases in animals and humans worldwide, and their control is critical to enhance health, well-being and productivity. Due to widespread drug resistance in many parasitic worms of animals globally, there is a major, continuing demand for the discovery and development of anthelmintic drugs for use to control these worms. Here, we established a practical, cost-effective and semi-automated high throughput screening (HTS) assay, which relies on the measurement of motility of larvae of the barber’s pole worm (Haemonchus contortus) using infrared light-interference. Using this assay, we screened 80,500 small molecules and achieved a hit rate of 0.05%. We identified three small molecules that reproducibly inhibited larval motility and/or development (IC50 values of ~4 to 41 µM). Future work will critically assess the potential of selected hits as candidates for subsequent optimisation or repurposing against parasitic nematodes. This HTS assay has a major advantage over most previous assays in that it achieves a ≥ 10-times higher throughput (i.e., 10,000 compounds per week), and is thus suited to the screening of libraries of tens of thousands to hundreds of thousands of compounds for subsequent hit-to-lead optimisation or effective repurposing and development. The current assay should be adaptable to many socioeconomically important parasitic nematodes, including those that cause neglected tropical diseases (NTDs). This aspect is of relevance, given the goals of the World Health Organization (WHO) Roadmap for NTDs 2021–2030, to develop more effective drugs and drug combinations to improve patient outcomes and circumvent the ineffectiveness of some current anthelmintic drugs and possible drug resistance.

Background The development of intrinsic and acquired resistance to antineoplastic agents is a major obstacle to successful chemotherapy in ovarian cancers. Identification and characterisation of chemoresponse-associated biomarkers are of paramount importance for novel therapeutic development. Methods Global RNA expression profiles were obtained by high-throughput microarray analysis. Cell cycle, proliferation rate, and paclitaxel sensitivity of ovarian cancer cells harbouring cyclin A1-inducible expression construct were compared with and without tetracycline induction, as well as when the cyclin A1 expression was suppressed by short inhibiting RNA (siRNA). Cellular senescence was evaluated by β-galactosidase activity staining. Results Global RNA expression profiling and subsequent correlation studies of gene expression level and drug response has identified that elevated expression of cyclin A1 (CCNA1) was significantly associated with cellular resistance to paclitaxel, doxorubicin and 5-fluorouracil. The role of cyclin A1 in paclitaxel resistance was confirmed in ovarian cancer cells that harbour an inducible cyclin A1 expression construct, which showed reduced paclitaxel-mediated growth inhibition and apoptosis when cyclin A1 expression was induced, whereas downregulation of cyclin A1 expression in the same cell lines using cyclin A1-specific siRNAs sensitised the cells to paclitaxel toxicity. However, ovarian cancer cells with ectopic expression of cyclin A1 demonstrated slowdown of proliferation and senescence-associated β-galactosidase activity. Conclusions Our profiling and correlation studies have identified cyclin A1 as one chemoresistance-associated biomarker in ovarian cancer. The results of the characterisation studies suggest that cyclin A1 functions as an oncogene that controls proliferative and survival activities in tumourigenesis and chemoresistance of ovarian cancer.

// Yuanyuan Hua 1, 2 , Pui-Wah Choi 2 , Alexander J. Trachtenberg 3 , Allen C. Ng 2 , Winston P. Kuo 3, 4 , Shu-Kay Ng 5 , Daniela M. Dinulescu 6 , Martin M. Matzuk 7 , Ross S. Berkowitz 2 , Shu-Wing Ng 2 1 Department of Obstetrics & Gynecology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, PR China 2 Department of Obstetrics/Gynecology and Reproductive Biology, Brigham and Women’s Hospital, Boston, Massachusetts, USA 3 Harvard Catalyst Laboratory for Innovative Translational Technologies, Harvard Medical School, Boston, Massachusetts, USA 4 Predicine, Inc., Hayward, California, USA 5 School of Medicine and Menzies Health Institute Queensland, Griffith University, Nathan, Australia 6 Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA 7 Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, Texas, USA Correspondence to: Shu-Wing Ng, email: sng@partners.org Keywords: ovarian cancer, fallopian tube Received: May 11, 2016 Accepted: August 13, 2016 Published: September 01, 2016 ABSTRACT Epithelial ovarian carcinoma accounts for 90% of all ovarian cancer and is the most deadly gynecologic malignancy. Recent studies have suggested that fallopian tube fimbriae can be the origin of cells for high-grade serous subtype of epithelial ovarian carcinoma (HGSOC). A mouse HGSOC model with conditional Dicer-Pten double knockout ( Dicer-Pten DKO) developed primary tumors, intriguingly, from the fallopian tube stroma. We examined the growth and epithelial phenotypes of the Dicer-Pten DKO mouse tumor cells contributable by each gene knockout. Unlike human ovarian epithelial cancer cells that expressed full-length E-cadherin, the Dicer-Pten DKO stromal tumor cells expressed cleaved E-cadherin fragments and metalloproteinase 2, a mixture of epithelial and mesenchymal markers. Although the Dicer-Pten DKO tumor cells lost the expression of mature microRNAs as expected, they showed high levels of tRNA fragment expression and enhanced AKT activation due to the loss of PTEN function. Introduction of a Dicer1- expressing construct into the DKO mouse tumor cells significantly reduced DNA synthesis and the cell growth rate, with concurrent diminished adhesion and ZO1 epithelial staining. Hence, it is likely that the loss of Dicer promoted mesenchymal-epithelial transition in fallopian tube stromal cells, and in conjunction with Pten loss, further promoted cell proliferation and epithelial-like tumorigenesis.

1. Wildlife disease surveillance is the first line of defence against infectious disease. Fluctuations in host populations and disease prevalence are a known feature of wildlife disease systems. However, the impact of such heterogeneities on the performance of surveillance is currently poorly understood. 2. We present the first systematic exploration of the effects of fluctuations prevalence and host population size on the efficacy of wildlife disease surveillance systems. In this study efficacy is measured in terms of ability to estimate long term prevalence and detect disease risk. 3. Our results suggest that for many wildlife disease systems fluctuations in population size and disease lead to bias in surveillance-based estimates of prevalence and over-confidence in assessments of both the precision of prevalence estimates and the power to detect disease. 4. Neglecting such ecological effects may lead to poorly designed surveillance and ultimately to incorrect assessments of the risks posed by disease in wildlife. This will be most problematic in systems where prevalence fluctuations are large and disease fade-outs occur. Such fluctuations are determined by the interaction of demography and disease dynamics and although particularly likely in highly fluctuating populations typical of fecund short lived hosts, can’t be ruled out in more stable populations of longer lived hosts. 5. Synthesis and Applications: Fluctuations in population size and disease prevalence should be considered in the design and implementation of wildlife disease surveillance and the framework presented here provides a template for conducting suitable power calculations. Ultimately understanding the impact of fluctuations in demographic and epidemiological processes will enable improvements to wildlife disease surveillance systems leading to better characterisation of, and protection against endemic, emerging and re-emerging disease threats.

// Yanli Zhang 1 , Jamie Sui-Lam Kwok 2 , Pui-Wah Choi 1 , Minghua Liu 2 , Junzheng Yang 1 , Margit Singh 1 , Shu-Kay Ng 4 , William R. Welch 5 , Michael G. Muto 1 , Stephen KW Tsui 2 , Stephen P. Sugrue 3 , Ross S. Berkowitz 1 , Shu-Wing Ng 1 1 Laboratory of Gynecologic Oncology, Division of Gynecologic Oncology, Department of Obstetrics, Gynecology and Reproductive Biology, Harvard Medical School, Boston, MA, USA 2 School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong 3 Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL, USA 4 School of Medicine and Menzies Health Institute Queensland, Griffith University, Meadowbrook, Australia 5 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Correspondence to: Shu-Wing Ng, e-mail: sng@partners.org Keywords: ovarian cancer, tumorigenesis, RNA metabolism, cell adhesion, RNA sequencing Received: October 21, 2015 Accepted: January 24, 2016 Published: February 08, 2016 ABSTRACT Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

Herbicide-resistant weeds are an increasing global problem in crop production systems. To lessen the incidence of herbicide resistance and to prevent the spread of herbicide-resistant weeds many farmers in Australia have adopted weed seed control measures at grain harvest. One new option is known as the Harrington Seed Destructor (HSD). It is a machine that intercepts crop residue from the harvester and then mechanically destroys embedded weed seeds. In this study, the RIM (Ryegrass Integrated Management) model was used to investigate the economic worth of the HSD within integrated weed management strategies applicable to different weed environments, rotations, sizes of cropping programmes and crop yields. Use of the HSD generated increased returns compared to many other weed management strategies in several scenarios, but especially when non-selective herbicide resistance occurred and large areas of high-yielding crops were grown. Emerging trends in grain farming that include larger areas sown to crops, a greater incidence of herbicide-resistant weeds and higher crop yields, when combined with further manufacturing improvement of the HSD, will only further favour the use of the HSD as a key component of integrated weed management.

Corticothalamic (CT) neurons comprise the largest component of the descending sensory corticofugal pathway, but their contributions to brain function and behavior remain an unsolved mystery. To address the hypothesis that layer 6 (L6) CTs may be activated by extra-sensory inputs prior to anticipated sounds, we performed optogenetically targeted single-unit recordings and two-photon imaging of Ntsr1-Cre+ L6 CT neurons in the primary auditory cortex (A1) while mice were engaged in an active listening task. We found that L6 CTs and other L6 units began spiking hundreds of milliseconds prior to orofacial movements linked to sound presentation and reward, but not to other movements such as locomotion, which were not linked to an explicit behavioral task. Rabies tracing of monosynaptic inputs to A1 L6 CT neurons revealed a narrow strip of cholinergic and non-cholinergic projection neurons in the external globus pallidus, suggesting a potential source of motor-related input. These findings identify new pathways and local circuits for motor modulation of sound processing and suggest a new role for CT neurons in active sensing.

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

Genomic analyses indicate that 50% of high-grade serous ovarian cancers (HGSC) harbor a genomic alteration in a DNA damage repair gene that may lead to functional defects. Using functional assays on patient-derived HGSC organoid cultures to test the capacity of the tumor cells to repair double-strand DNA breaks and to protect stalled replication forks, we have found that many HGSCs have stalled fork protection defects regardless of the genomic background of the tumor and that these defects correlate with sensitivity to replication stress inducing therapeutic agents. We hypothesized that gaining a better understanding of the mechanisms of replication fork instability and stability in HGSC organoid cultures would help to better understand the mechanisms of therapeutic sensitivity of the tumor cells. The purpose of this work is to understand how replication fork stabilization either in the primary tumor or through selection post-treatment leads to alterations in tumor cell biology, including therapeutic sensitivity and interaction of the tumor cells with the surrounding microenvironment. We utilized bulk RNA sequencing analysis of HGSC organoid cultures with varied replication fork protection capacity, some matched pairs of untreated and post-neoadjuvant tumors, to stratify differences in functional profiles in fork stable versus unstable tumors; we then used basic molecular biology techniques to understand the mechanisms of fork stabilization and how this stabilization affects the therapeutic sensitivity of the cells. We also developed and utilized multiple functional assays to assess the interaction of HGSC organoids of varying fork protection capacity with their immune microenvironment in different drug exposure settings. We identified multiple proteins that through either up- or downregulation lead to stabilization of replication forks in the tumor cells and found that the mechanisms of stabilization can occur at both the level of the replication fork and the overall transcriptional level of the cell and can alter the therapeutic sensitivity of the cells. We have determined that replication fork stability leads to increased mesenchymal characteristics in tumors and to decreased activation of the antitumor immune response within the cultures after treatment with DNA damage repair and immuno-oncologic (IO) agents. Overall, these results indicate that replication fork stabilization in HGSC through multiple different mechanisms can lead to altered interactions of the tumor cells with their microenvironment and altered therapeutic sensitivity. This abstract is also being presented as Poster B10. Citation Format: Sarah J. Hill, Patrick Lizotte, Nikolas Kesten, Neil S. Horowitz, Michael G. Muto, Michael J. Worley, Colleen M. Feltmate, Ross S. Berkowitz, Henry Long, Ursula A. Matulonis, Christopher P. Crum, Myles Brown, Alan D. D'Andrea. Dissecting mechanisms of replication fork stabilization in patient-derived high-grade serous organoid cultures and their impact on therapeutic sensitivity and the immune-tumor interaction [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR06.

The prevalent porcine helminth, Ascaris suum, compromises pig health and reduces farm productivity worldwide. The closely related human parasite, A. lumbricoides, infects more than 800 million people representing a disease burden of 1.31 million disability-adjusted life years. The infections are often chronic in nature, and the parasites have a profound ability to modulate their hosts' immune responses. This study provides the first in-depth characterisation of extracellular vesicles (EVs) from different developmental stages and body parts of A. suum and proposes the role of these vesicles in the host-parasite interplay. The release of EVs from the third- (L3) and fourth-stage (L4) larvae and adults was demonstrated by transmission electron microscopy (TEM), and sequencing of EV-derived RNA identified a number of microRNAs (miRNAs) and transcripts of potential host immune targets, such as IL-13, IL-25 and IL-33, were identified. Furthermore, proteomics of EVs identified several proteins with immunomodulatory properties and other proteins previously shown to be associated with parasite EVs. Taken together, these results suggest that A. suum EVs and their cargo may play a role in host-parasite interactions. This knowledge may pave the way to novel strategies for helminth infection control and knowledge of their immune modulatory potential.