Using expression data to fine map QTL associated with fertility in dairy cattle.

BACKGROUND: Female fertility is an important trait in dairy cattle. Identifying putative causal variants associated with fertility may help to improve the accuracy of genomic prediction of fertility. Combining expression data (eQTL) of genes, exons, gene splicing and allele specific expression is a promising approach to fine map QTL to get closer to the causal mutations. Another approach is to identify genomic differences between cows selected for high and low fertility and a selection experiment in New Zealand has created exactly this resource. Our objective was to combine multiple types of expression data, fertility traits and allele frequency in high- (POS) and low-fertility (NEG) cows with a genome-wide association study (GWAS) on calving interval in Australian cows to fine-map QTL associated with fertility in both Australia and New Zealand dairy cattle populations. RESULTS: Variants that were significantly associated with calving interval (CI) were strongly enriched for variants associated with gene, exon, gene splicing and allele-specific expression, indicating that there is substantial overlap between QTL associated with CI and eQTL. We identified 671 genes with significant differential expression between POS and NEG cows, with the largest fold change detected for the CCDC196 gene on chromosome 10. Our results provide numerous candidate genes associated with female fertility in dairy cattle, including GYS2 and TIGAR on chromosome 5 and SYT3 and HSD17B14 on chromosome 18. Multiple QTL regions were located in regions with large numbers of copy number variants (CNV). To identify the causal mutations for these variants, long read sequencing may be useful. CONCLUSIONS: Variants that were significantly associated with CI were highly enriched for eQTL. We detected 671 genes that were differentially expressed between POS and NEG cows. Several QTL detected for CI overlapped with eQTL, providing candidate genes for fertility in dairy cattle.

SWATH-MS Analysis of Blood Plasma and Circulating Small Extracellular Vesicles Enables Detection of Putative Protein Biomarkers of Fertility in Young and Aged Dairy Cows.

The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin; thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal; n = 30) and Aged (Primiparous; n = 20) dairy cows (Bos taurus) of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV; p value = 3.302e-07) and 0.95 (plasma; p value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (*p = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (***p = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle.

Kiss1 expression in the hypothalamic arcuate nucleus is lower in dairy cows of reduced fertility†.

We tested the hypothesis that divergent genetic merit for fertility of dairy cows is due to aberrant reproductive neuroendocrine function. The kisspeptin status of non-pregnant cows of either positive (POS) or negative (NEG) breeding values (BVs) for fertility was studied in three groups (n = 8), based on their previous post-partum period: POS cows, which had spontaneous ovarian cycles (POS-CYC) and NEG cows, which either cycled (NEG-CYC) or did not cycle (NEG-NONCYC). Ovarian cycles were synchronized, blood samples were taken to define endocrine status, and the animals were slaughtered in an artificial follicular phase. The brains and the pituitary glands were collected for quantitative polymerase chain reaction (qPCR) and in situ hybridization of hypothalamic GNRH1, Kiss1, TAC3, and PDYN and pituitary expression of LHB and FSHB. Gonadotropin releasing hormone (GnRH) and kisspeptin levels were quantified in snap frozen median eminence (ME). GNRH1 expression and GnRH levels in the ME were similar across groups. Kiss1 expression in the preoptic area of the hypothalamus was also similar across groups, but Kiss1 in the arcuate nucleus was almost 2-fold higher in POS-CYC cows than in NEG groups. TAC3 expression was higher in POS-CYC cows. The number of pituitary gonadotropes and the level of expression of LHB and FSHB were similar across groups. We conclude that the lower levels of Kiss1 and TAC3 in NEG cows with low fertility status and may lead to deficient GnRH and gonadotropin secretion.

Site-Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover.

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio-orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence-based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse-chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.

A microfluidic flow-focusing device for low sample consumption serial synchrotron crystallography experiments in liquid flow.

Serial synchrotron crystallography allows low X-ray dose, room-temperature crystal structures of proteins to be determined from a population of microcrystals. Protein production and crystallization is a non-trivial procedure and it is essential to have X-ray-compatible sample environments that keep sample consumption low and the crystals in their native environment. This article presents a fast and optimized manufacturing route to metal-polyimide microfluidic flow-focusing devices which allow for the collection of X-ray diffraction data in flow. The flow-focusing conditions allow for sample consumption to be significantly decreased, while also opening up the possibility of more complex experiments such as rapid mixing for time-resolved serial crystallography. This high-repetition-rate experiment allows for full datasets to be obtained quickly (∼1 h) from crystal slurries in liquid flow. The X-ray compatible microfluidic chips are easily manufacturable, reliable and durable and require sample-flow rates on the order of only 30 µl h-1.

Proteome profiling of exosomes derived from plasma of heifers with divergent genetic merit for fertility.

The current study evaluated exosomes isolated from plasma of heifers bred to have high or low fertility through developing extreme diversity in fertility breeding values, however, key animal traits (e.g., body weight, milk production, and percentage of North American genetics) remained similar between the 2 groups. The exosomes were isolated by a combined ultracentrifugation and size exclusion chromatography approach and characterized by their size distribution (nanoparticle tracking analysis), morphology (transmission electron microscopy), and presence of exosomal markers (immunoblotting). In addition, a targeted mass spectrometry approach was used to confirm the presence of 2 exosomal markers, tumor susceptibility gene 101 and flotillin 1. The number of exosomes from plasma of high fertility heifers was greater compared with low fertility heifers. Interestingly, the exosomal proteomic profile, evaluated using mass spectrometry, identified 89 and 116 proteins in the high and low fertility heifers respectively, of which 4 and 31 were unique, respectively. These include proteins associated with specific biological processes and molecular functions of fertility. Most notably, the tetratricopeptide repeat protein 41-related, glycodelin, and kelch-like protein 8 were identified in plasma exosomes unique to the low fertility heifers. These proteins are suggested to play a role in reproduction; however, the role of these proteins in dairy cow reproduction remains to be elucidated. Their identification underscores the potential for proteins within exosomes to provide information on the fertility status and physiological condition of the cow. This may potentially lead to the development of prognostic tools and interventions to improving dairy cow fertility.

Prepartum feeding level and body condition score affect immunological performance in grazing dairy cows during the transition period.

Precalving feeding level affects dry matter intake, postcalving energy balance, the risk of hepatic lipidosis and metabolic disease, and gene expression in liver and adipose tissue. These coincide with a higher risk of disease postpartum and, very likely, a failure to reach optimum production as well as reproductive targets. Current interpretation of the available evidence suggest that metabolic stressors affect the immune system of transition dairy cows and lead to reduced immunocompetence. The objective of the current study was to investigate the effect of precalving body condition score (BCS) and level of feeding on immunocompetence during the peripartum period. Twenty-three weeks before calving, 78 cows were allocated randomly to 1 of 6 treatment groups (n=13) in a 2 × 3 factorial arrangement: 2 precalving BCS categories (4.0 and 5.0, based on a 10-point scale) and 3 levels of energy intake during the 3 wk preceding calving (75, 100, and 125% of estimated requirements). Blood was sampled precalving and at 1, 2 and 4 wk after calving. Cells were analyzed by flow cytometry and quantitative real-time PCR. The numbers of T helper lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), natural killer cells (CD335+), and γδ T lymphocytes (WC1+) as well as their activation status [IL-2 receptor (CD25)+ cells] were highly variable between animals, but there was no evident effect of BCS, feeding level, or time. All groups presented with an increase in expression of cytokines in unstimulated blood cells in the week after calving, although this was significant only for IFNG in the BCS 4.0 group. Analysis of in vitro-stimulated cells allowed 2 general observations: (1) cows with high energy intake precalving (125%) had increased cytokine expression precalving, and (2) all cows had increased cytokine expression in the week after calving. The present study provides evidence that prepartum feed management can affect immunocompetence during the transition period. Considering the current results, optimally conditioned animals might benefit from a restricted precalving diet, whereas underconditioned cows can be fed to requirements.

Modulation of the immune system during postpartum uterine inflammation.

Postpartum uterine inflammation (endometritis) in the dairy cow is associated with lower fertility at both the time of infection and after the inflammation has resolved. We hypothesized that aberrant DNA methylation may be involved in the subfertility associated with uterine inflammation. The objective of this study was to characterize genome-wide DNA methylation and gene expression in the endometrium of dairy cows with subclinical endometritis (SCE). Endometrial tissues were obtained at 29 days postpartum (n = 12), and microarrays were used to characterize transcription and DNA methylation. Analyses revealed 1,856 probes differentially expressed in animals with SCE (n = 6) compared with controls (CON, n = 6, P < 0.05, Storey Multiple testing correction) and 2,976 probes with significant correlation between gene expression and bacteriology score. No significant associations among DNA methylation and gene expression were detected. Analysis of transcription data using the Database for Annotation, Visualization, and Integrated Discovery and Gene Set Enrichment Analysis identified several pathways and processes enriched in SCE cows, with the majority related to the immune response. Furthermore, the top ontology terms enriched in genes that had expression data correlated to bacteriology score were: Defense response, inflammatory response, and innate immune response. Gene expression profiles in cows with subclinical endometritis in this study indicate that the immune response is activated, potentially resulting in a local proinflammatory environment in the uterus. If this period of inflammation is prolonged it could result in tissue damage or failure to complete involution of the uterus, which may create a suboptimal environment for future pregnancy.

Grazing dairy cows had decreased interferon-γ, tumor necrosis factor, and interleukin-17, and increased expression of interleukin-10 during the first week after calving.

Peripartum, and especially during the transition period, dairy cows undergo dramatic physiological changes. These coincide with an increased risk of disease during the first 2 wk after calving and have been linked to dairy cows failing to achieve production as well as reproductive targets. Previous evidence suggests that these physiological changes affect the immune system and that transition dairy cows experience some form of reduced immunocompetence. However, almost all of these studies were undertaken in high-production, housed dairy cows. Grazing cows have much lower levels of production and this study aimed to provide clarity whether or not the dysfunctional attributes of the peripartum immune system reported in high production housed cows are evident in these animals. Therefore, cell culture techniques, flow cytometry, and quantitative PCR were applied to analyze the cellular composition of peripheral blood mononuclear cells from transition dairy cows as well as the performance of these cells in an in vitro assay. First, a combination of in vitro stimulation and quantitative PCR for cytokines was validated as a quantifiable immunocompetence assay in 29 cattle and a correlation of quantitative PCR and ELISA demonstrated. Second, the relative number of T helper cells, cytotoxic T cells, B cells, γδ T cells, natural killer cells, and monocytes in peripheral blood was measured, of which B cells and natural killer cells increased in number postcalving (n=29) compared with precalving. Third, following in vitro stimulation cytokine profiles indicated decreased expression of IFNγ, tumor necrosis factor, and IL-17 and increased expression of IL-10 wk 1 after calving, which later all returned to precalving values (n=39). Additionally, treatment of transition cows with a nonsteroidal anti-inflammatory drug (i.e., carprofen) administered on d 1, 3, and 5 postcalving (n=19; untreated control n=20) did not affect the cytokine expression at any time point. In conclusion, an immunocompetence assay has been developed that highlights a characteristic expression pattern for IFNγ, tumor necrosis factor, IL-17, and IL-10 that reflects a state of reduced immunocompetence in moderate-yielding pasture-based transition cows after calving, which is similar to that described for higher-yielding housed cows.

DNA methylation is correlated with gene expression during early pregnancy in Bos taurus.

Coordinated regulation of endometrial gene expression is essential for successful pregnancy establishment. A nonreceptive uterine environment may be a key contributor to pregnancy loss, as the majority of pregnancy losses occur prior to embryo implantation. DNA methylation has been highlighted as a potential contributor in regulating early pregnancy events in the uterus. It was hypothesized that DNA methylation regulates expression of key genes in the uterus during pregnancy. The correlation between DNA methylation and gene expression was tested. Endometrial samples from fertile and subfertile dairy cow strains were obtained at day 17 of pregnancy or the reproductive cycle. Microarrays were used to characterize genome-wide DNA methylation profiles and data compared with previously published transcription profiles. 39% of DNA methylation probes assayed mapped to RefSeq genes with transcription measurements. Correlations among gene expression and DNA methylation were assessed, and the 1,000 most significant correlations used for subsequent analysis. Of these, 52% percent were negatively correlated with gene expression. When this gene list was compared with previously reported gene expression studies on the same tissues, 42% were differentially expressed when pregnant and cycling animals were compared, and 11% were differentially expressed when pregnant fertile and subfertile animals were compared. DNA methylation status was correlated with gene expression in several pathways implicated in early pregnancy events. Although these data do not provide direct evidence of a causative association between DNA methylation and gene expression, this study provides critical support for an effect of DNA methylation in early pregnancy events and highlights candidate genes for future studies.

Recombinant production, crystallization and preliminary structural characterization of Schistosoma japonicum profilin.

Helminthic parasites of the genus Schistosoma contain a tegumental membrane, which is of crucial importance for modulation of the host immune response and parasite survival. The actin cytoskeleton plays an important role in the function of the tegument. Profilins are among the most important proteins regulating actin dynamics. Schistosoma japonicum possesses one profilin-like protein, which has been characterized as a potential vaccine candidate. Notably, profilins are highly immunogenic molecules in many organisms. Here, the profilin from S. japonicum was expressed, purified and crystallized. A native data set to 1.91 Šresolution and a single-wavelength anomalous diffraction (SAD) data set to a resolution of 2.2 Šwere collected. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 31.82, b = 52.17, c = 59.79 Šand a = 35.29, b = 52.15, c = 59.82 Å, respectively.

Genetic ancestry modifies fatty acid concentrations in different adipose tissue depots and milk fat.

The objective of this study was to determine the effect of cow genetic strain on fatty acid (FA) profiles in adipose tissue and milk. Adipose samples from two subcutaneous (shoulder and tail-head) and three visceral (kidney channel, mesenteric and omental) depots were obtained post mortem from New Zealand (NZ; n = 8) and North American (NA; n = 8) Holstein-Friesian cows. At the time of slaughter cows were in similar body condition (NZ: 4.0 ± 0.03, NA: 4.0 ± 0.02; ± SD) and stage of lactation (NZ: 90 ± 11.2 d; NA: 83 ± 4.3 d; ± SD). Milk was collected during the a.m. milking prior to slaughter and milk fat was extracted. Adipose and milk fat FA were quantified using gas chromatography. NZ cows had a lower proportion of saturated FA in shoulder, tail-head and omental adipose tissue and a greater proportion of mono-unsaturated FA and an elevated Δ9-desaturase index in shoulder and tail-head adipose tissue. The proportions of individual FA differed between adipose depots, with proportions of de-novo FA greater in subcutaneous compared with visceral adipose depots. Milk from NZ cows contained greater concentrations of short chain FA (C8 : 0-12 : 0) and CLA, and less cis-9 18 : 1 than milk from NA cows. Regression analysis identified moderate associations between milk FA and shoulder adipose tissue FA for 18 : 2 (R(2) = 0.24), 18 : 3 n - 3 (R(2) = 0.39), and polyunsaturated fatty acids (R(2) = 0.38). Results from this study support the hypothesis that genetic strain dictates FA profiles in adipose tissue and milk and may alter the metabolic status of the various adipose depots differently. The data further support the premise that genetic strain affects the metabolic status of the various adipose depots differently. Elucidating the mechanisms that regulate the different adipose depots in the NZ and NA strains will increase our understanding of tissue mobilization and replenishment.

Comparison of methods for deriving phenotypes from incomplete observation data with an application to age at puberty in dairy cattle.

BACKGROUND: Many phenotypes in animal breeding are derived from incomplete measures, especially if they are challenging or expensive to measure precisely. Examples include time-dependent traits such as reproductive status, or lifespan. Incomplete measures for these traits result in phenotypes that are subject to left-, interval- and right-censoring, where phenotypes are only known to fall below an upper bound, between a lower and upper bound, or above a lower bound respectively. Here we compare three methods for deriving phenotypes from incomplete data using age at first elevation (> 1 ng/mL) in blood plasma progesterone (AGEP4), which generally coincides with onset of puberty, as an example trait. METHODS: We produced AGEP4 phenotypes from three blood samples collected at about 30-day intervals from approximately 5,000 Holstein-Friesian or Holstein-Friesian × Jersey cross-bred dairy heifers managed in 54 seasonal-calving, pasture-based herds in New Zealand. We used these actual data to simulate 7 different visit scenarios, increasing the extent of censoring by disregarding data from one or two of the three visits. Three methods for deriving phenotypes from these data were explored: 1) ordinal categorical variables which were analysed using categorical threshold analysis; 2) continuous variables, with a penalty of 31 d assigned to right-censored phenotypes; and 3) continuous variables, sampled from within a lower and upper bound using a data augmentation approach. RESULTS: Credibility intervals for heritability estimations overlapped across all methods and visit scenarios, but estimated heritabilities tended to be higher when left censoring was reduced. For sires with at least 5 daughters, the correlations between estimated breeding values (EBVs) from our three-visit scenario and each reduced data scenario varied by method, ranging from 0.65 to 0.95. The estimated breed effects also varied by method, but breed differences were smaller as phenotype censoring increased. CONCLUSION: Our results indicate that using some methods, phenotypes derived from one observation per offspring for a time-dependent trait such as AGEP4 may provide comparable sire rankings to three observations per offspring. This has implications for the design of large-scale phenotyping initiatives where animal breeders aim to estimate variance parameters and estimated breeding values (EBVs) for phenotypes that are challenging to measure or prohibitively expensive.

Endometrial gene expression during early pregnancy differs between fertile and subfertile dairy cow strains.

A receptive uterine environment is a key component in determining a successful reproductive outcome. We tested the hypothesis that endometrial gene expression patterns differ in fertile and subfertile dairy cow strains. Twelve lactating dairy cattle of strains characterized as having fertile (n = 6) and subfertile (n = 6) phenotypes underwent embryo transfer on day 7 of the reproductive cycle. Caruncular and intercaruncular endometrial tissue was obtained at day 17 of pregnancy, and microarrays used to characterize transcriptional profiles. Statistical analysis of microarray data at day 17 of pregnancy revealed 482 and 1,021 differentially expressed transcripts (P value < 0.05) between fertile and subfertile dairy cow strains in intercaruncular and caruncular tissue, respectively. Functional analysis revealed enrichment for several pathways involved in key reproductive processes, including the immune response to pregnancy, luteolysis, and support of embryo growth and development, and in particular, regulation of histotroph composition. Genes implicated in the process of immune tolerance to the embryo were downregulated in subfertile cows, as were genes involved in preventing luteolysis and genes that promote embryo growth and development. This study provides strong evidence that the endometrial gene expression profile may contribute to the inferior reproductive performance of the subfertile dairy cow strain.

Modification of endometrial fatty acid concentrations by the pre-implantation conceptus in pasture-fed dairy cows.

The current study determined whether the pre-implantation conceptus modified endometrial fatty acid concentrations. Oestrus was synchronized in 14 mature lactating cows and embryos were transferred on day 7. Cows were slaughtered 10 d later, with each uterine horn flushed, the pre-implantation conceptus located, and inter-caruncular endometrial tissue collected from the gravid horn (containing the pre-implantation conceptus) and non-gravid horn. Endometrial fatty acid concentrations in the gravid and non-gravid horn were compared using linear models in restricted maxiumum likelihood. Investigations of the correlations among selected fatty acids and trophoblast weight or uterine fluid interferon-tau (IFN-τ) concentrations were also undertaken. The presence of the pre-implantation conceptus had relatively minor effects on endometrial fatty acid concentrations, but the ω6:ω3 ratio was greater and concentrations of stearic and oleic acid were slightly increased in the gravid horn. In the gravid horn, a negative linear relationship between the concentration of arachidonic acid and conceptus weight and IFN-τ concentration in the uterine luminal fluid were observed. In contrast, there was a positive relationship between concentrations of dihomo-γ-linolenic acid in the non-gravid horn and conceptus weight. In conclusion, the presence of the pre-implantation conceptus appears to modulate endometrial fatty acids, as indicated by the differences in endometrial fatty acid concentrations in the gravid and non-gravid uterine horns. The physiological implication of these local effects of the pre-implantation conceptus, on reproductive success requires further investigation.

Ovarian activity in Fleckvieh, Brown Swiss and two strains of Holstein-Friesian cows in pasture-based, seasonal calving dairy systems.

The objectives of the study were to compare the ovarian activity of Holstein-Friesian (CH HF), Fleckvieh (CH FV) and Brown Swiss (CH BS) dairy cows of Swiss origin with that of Holstein-Friesian (NZ HF) dairy cows of New Zealand origin, the latter being used as a reference for reproductive performance in pasture-based seasonal calving systems. Fifty, second-lactation NZ HF cows were each paired with a second-lactation Swiss cow (17, 15 and 18 CH HF, CH FV and CH BS respectively) in 13 pasture-based, seasonal-calving commercial dairy farms in Switzerland. Ovarian activity was monitored by progesterone profiling from calving to first breeding service. CH BS cows produced less energy-corrected milk (mean 22·8 kg/d) than the other breeds (26·0-26·5 kg/d) during the first 100 d of lactation. CH HF cows had the lowest body condition score (BCS) at calving and the greatest BCS loss from calving to 30 d post partum. Commencement of luteal activity (CLA) was later for NZ HF than for CH FV (51·5 v. 29·2 d; P <0·01), with CH HF and CH BS intermediate (43 d). On average, NZ HF and CH HF cows had one oestrous cycle before the onset of the seasonal breeding period; this was less (P<0·01) than either CH FV (1·7) or CH BS (1·6). There was a low prevalence of luteal persistency (3%) among the studied cows. First and second oestrous cycle inter-ovulatory intervals did not differ between breeds (20·5-22·6 d). The luteal phase length of CH BS during the second cycle was shorter (10·6 d) than that of the other breeds (13·8-16·0 d), but the inter-luteal interval was longer (9·8 d v. 7·0-8·0 d). The results suggest that the Swiss breeds investigated have a shorter interval from calving to CLA than NZ HF cows.

Homogeneous batch micro-crystallization of proteins from ammonium sulfate.

The emergence of X-ray free-electron lasers has led to the development of serial macromolecular crystallography techniques, making it possible to study smaller and more challenging crystal systems and to perform time-resolved studies on fast time scales. For most of these studies the desired crystal size is limited to a few micrometres, and the generation of large amounts of nanocrystals or microcrystals of defined size has become a bottleneck for the wider implementation of these techniques. Despite this, methods to reliably generate microcrystals and fine-tune their size have been poorly explored. Working with three different enzymes, L-aspartate α-decarboxylase, copper nitrite reductase and copper amine oxidase, the precipitating properties of ammonium sulfate were exploited to quickly transition from known vapour-diffusion conditions to reproducible, large-scale batch crystallization, circumventing the tedious determination of phase diagrams. Furthermore, the specific ammonium sulfate concentration was used to fine-tune the crystal size and size distribution. Ammonium sulfate is a common precipitant in protein crystallography, making these findings applicable to many crystallization systems to facilitate the production of large amounts of microcrystals for serial macromolecular crystallography experiments.

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Modulation of the maternal immune system by the pre-implantation embryo.

BACKGROUND: A large proportion of pregnancy losses occur during the pre-implantation period, when the developing embryo is elongating rapidly and signalling its presence to the maternal system. The molecular mechanisms that prevent luteolysis and support embryo survival within the maternal environment are not well understood. To gain a more complete picture of these molecular events, genome-wide transcriptional profiles of reproductive day 17 endometrial tissue were determined in pregnant and cyclic Holstein-Friesian dairy cattle. RESULTS: Microarray analyses revealed 1,839 and 1,189 differentially expressed transcripts between pregnant and cyclic animals (with > or = 1.5 fold change in expression; P-value < 0.05, MTC Benjamini-Hochberg) in caruncular and intercaruncular endometrium respectively. Gene ontology and biological pathway analysis of differentially expressed genes revealed enrichment for genes involved in interferon signalling and modulation of the immune response in pregnant animals. CONCLUSION: The maternal immune system actively surveys the uterine environment during early pregnancy. The embryo modulates this response inducing the expression of endometrial molecules that suppress the immune response and promote maternal tolerance to the embryo. During this period of local immune suppression, genes of the innate immune response (in particular, antimicrobial genes) may function to protect the uterus against infection.

Activation of microglial poly(ADP-ribose)-polymerase-1 by cholesterol breakdown products during neuroinflammation: a link between demyelination and neuronal damage.

Multiple sclerosis (MS) is a chronic demyelinating disease in which it has only recently been suggested that damage to neuronal structures plays a key role. Here, we uncovered a link between the release of lipid breakdown products, found in the brain and cerebrospinal fluid (CSF) of MS patients as well as in experimental autoimmune encephalomyelitis, and neuronal damage mediated by microglial activation. The concentrations of the breakdown product 7-ketocholesterol detected in the CSF of MS patients were capable of inducing neuronal damage via the activation and migration of microglial cells in living brain tissue. 7-ketocholesterol rapidly entered the nucleus and activated poly(ADP-ribose)-polymerase (PARP)-1, followed by the expression of migration-regulating integrins CD11a and intercellular adhesion molecule 1. These findings reveal a novel mechanism linking demyelination and progressive neuronal damage, which might represent an underlying insidious process driving disease beyond a primary white matter phenomenon and rendering the microglial PARP-1 a possible antiinflammatory therapeutic target.