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An accurate T-cell quantification is prognostically and therapeutically relevant in various malignancies. We previously developed a digital PCR-based approach offering a precise T-cell enumeration in small amounts of DNA. However, it may be challenging to apply this method in malignant specimens, as copy number instability can disturb the underlying mathematical model. For example, approximately 24% of the tumors from The Cancer Genome Atlas pan-cancer data set carried a copy number alteration affecting our TRB gene T-cell marker, which would cause an underestimation or overestimation of the T-cell fraction. In this study, we introduce a multiplex digital PCR experimental setup to quantify T cells in copy number unstable DNA samples. By implementing a so-called regional corrector, genetic alterations involving the T-cell marker locus can be recognized and corrected for. This novel setup is evaluated mathematically in silico and validated in vitro by measuring T-cell presence in various samples with a known T-cell fraction. The utility of the approach is further demonstrated in copy number altered cutaneous melanomas. Our novel multiplex setup provides a simple, but accurate, DNA-based T-cell quantification in both copy number stable and unstable specimens. This approach has potential clinical and diagnostic applications, as it does not depend on availability of T-cell epitopes, has low requirements for sample quantity and quality, and can be performed in a relatively easy experiment.

Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.

Background COVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed. Methods We evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays’ performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10–40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence. Results Combined IgG + IgM sensitivities ranged from 33.9 to 94.6%, while combined specificities ranged from 92.6 to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG + IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG + IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 μg/mL), followed by a similar LOD of 1.5 μg/mL for CareHealth, Cellex, KHB, and Vivachek. Conclusion We provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Canine African Trypanosomosis (CAT) is a rarely described disease with frequently lethal outcome. A 5-year-old female mongrel dog weighing 22 kg was presented in Austria due to unilateral uveitis, pancytopenia, and anorexia 4 months after return from a trip through Western Africa. Trypanosoma spp. flagellates were detected in a blood smear and identified as Trypanosoma congolense forest type by PCR. Initial treatment with imidocarb and miltefosine led to clinical improvement but only isometamidium chloride hydrochloride applied intramuscularly led to complete eradication of the pathogen from the dog’s blood 4 months later.

Zusammenfassung Hintergrund und Ziele Alpha-2-Makroglobulin (A2 M) ist ein Plasmaprotein mit proteolytischer Wirkung auf viele Proteasen. Bei Patienten mit einem angeborenen Alpha-1-Antitrypsinmangel (AATM) liegt ein funktioneller Protease-Überschuss vor. Ziel dieser Studie war es, die Hypothese zu prüfen, dass bei Patienten mit AATM ein erhöhter A2M-Spiegel nachweisbar ist. Methoden Patienten mit AATM (Genpolymorphismus Pi*ZZ, Pi*SZ, Pi*MZ und seltene Genvarianten) wie auch Probanden mit einem normwertigen AAT-Spiegel (gesunde Probanden, Pi*MM) wurden der A2M- und AAT-Testung unterzogen. Die Serumkonzentrationen der Proteine wurden mittels Nephelometrie bestimmt. Die Polymorphismen Pi*Z und Pi*S wurden durch Polymerase-Kettenreaktion (PCR) nachgewiesen, seltene Genvarianten wurden durch Sequenzierung ermittelt. Ergebnisse In unsere Studie wurden insgesamt 291 Individuen eingeschlossen. Es konnte gezeigt werden, dass es bei Vorliegen eines Genpolymorphismus (Pi*ZZ) und einem Alpha-1-Antitrypsin-Serumspiegel Schlussfolgerungen In dieser Studie konnte eine inverse Korrelation zwischen den Serumspiegeln von AAT und A2M bei Vorliegen eines Genpolymorphismus (Pi*ZZ) nachgewiesen werden. Weitere Studien sind erforderlich, um zu erfassen, welche klinische Bedeutung erhöhte A2M-Serumpiegel bei Patienten mit einem schweren AAT-Mangel Pi*ZZ und seltenen Genvarianten, deren AAT-Serumspiegel unter 50 mg/dl liegt, haben könnten.

Background The Blastomyces antigen concentration in urine (BACU) test is used to diagnose blastomycosis and monitor treatment in dogs. It is unknown if a higher BACU is associated with shorter survival. Objectives To determine if the magnitude of BACU before treatment is associated with survival in dogs with blastomycosis. Animals Fifty‐two dogs with blastomycosis. Methods Retrospective case review. BACU, radiographic lung severity (RLS) score (0‐4 scale), and survival time up to 1 year after diagnosis were obtained through medical record review of dogs with Blastomyces dermatitidis. Results The overall survival was: discharge, 87%; 1 week, 85%; 2 months, 74%; and 6 months, 69%. BACU correlated with RLS score (r s = 0.33, P = .02). BACU and RLS scores were lower in survivors to 2 months than nonsurvivors (average BACU difference of 2.5 ng/mL, 95% confidence interval [CI]: 0.2‐4.8 ng/mL, P = .04; median RLS difference of 2; range, 0‐4, P = .02). Dogs with BACU 5 ng/mL (P = .03) and dogs with severe (3‐4) RLS scores (P = .04). All dogs with a BACU

The Athabasca Oil Sands Region (AOSR) in northeast Alberta contains Canada’s largest reserve of hydrocarbons, and the third largest in the world. Extraction and processing operations generate contaminants and nutrients that are aerially deposited across the region. However, structured environmental monitoring efforts were only initiated decades following the establishment (1967) and escalation (~ 1980) of commercial operations. We examined whether diatom assemblages preserved in 210Pb-dated sediment cores retrieved from strategically selected lakes have responded to aerial deposition of contaminants. The relative amount of contaminant inputs was tracked using sedimentary dibenzothiophene (DBT) concentrations and DBT enrichment factors, which are established proxies for AOSR activities. We observed no relationship between diatom assemblage changes and DBT enrichment. The nature of the diatom changes differed among lakes, regardless of DBT enrichment, suggesting that diatom responses were related to site-specific conditions. Moreover, diatom assemblage changes at several sites tracked trends in whole-lake primary production. Collectively, these records indicate that regional warming is likely the primary driver of recent diatom assemblage changes in these shallow, closed-basin lakes.

Tree diversity exerts a strong influence on consumer communities, but most work has involved single time point measurements over short time periods. Describing temporal variation associated with diversity effects over longer time periods is necessary to fully understand the effects of tree diversity on ecological function. We conducted a year-long study in an experimental system in southern Mexico assessing the effects of tree diversity on the abundance and diversity of foraging birds. To this end, we recorded bird visitation patterns in 32 tree plots (21 × 21 m; 12 tree species monocultures, 20 four-species polycultures) every 45 days (n = 8 surveys) and for each plot estimated bird abundance, richness, functional diversity (FD) and phylogenetic diversity (PD). In each case, we reported temporal (intra-annual) variation in the magnitude of tree diversity effects, and calculated the temporal stability of these bird responses. Across surveys, tree diversity noticeably affected bird responses, demonstrated by significantly higher abundance (43%), richness (32%), PD (25%) and FD (25%) of birds visiting polyculture plots compared to monoculture plots, as well as a distinct species composition between plot types. We also found intra-annual variation in tree diversity effects on these response variables, ranging from surveys for which the diversity effect was not significant to surveys where a significant 80% increase (e.g. for bird FD and PD) was observed in polyculture relative to monoculture plots. Notably, tree diversity increased the stability of all bird responses, with polycultures having a greater stability abundance (18%), richness (38%), PD (32%), and FD (35%) of birds visiting tree species polycultures compared to monocultures. These results show that tree diversity not only increases bird visitation to plots, but also stabilizes bird habitat usage over time in ways that could implicate insurance-related mechanisms. Such findings are highly relevant for understanding the long-term effects of plant diversity on vertebrates and the persistence of bird-related ecosystem functions. More work is needed to unveil the ecological mechanisms behind temporal variation in vertebrate responses to tree diversity and their consequences for community structure and function.

High-throughput sequencing (HTS) is central to the study of population genomics and has an increasingly important role in constructing phylogenies. Choices in research design for sequencing projects can include a wide range of factors, such as sequencing platform, depth of coverage and bioinformatic tools. Simulating HTS data better informs these decisions, as users can validate software by comparing output to the known simulation parameters. However, current standalone HTS simulators cannot generate variant haplotypes under even somewhat complex evolutionary scenarios, such as recombination or demographic change. This greatly reduces their usefulness for fields such as population genomics and phylogenomics. Here I present the R package jackalope that simply and efficiently simulates (i) sets of variant haplotypes from a reference genome and (ii) reads from both Illumina and Pacific Biosciences platforms. Haplotypes can be simulated using phylogenies, gene trees, coalescent-simulation output, population-genomic summary statistics, and Variant Call Format (VCF) files. jackalope can simulate single, paired-end or mate-pair Illumina reads, as well as reads from Pacific Biosciences. These simulations include sequencing errors, mapping qualities, multiplexing and optical/PCR duplicates. It can read reference genomes from fasta files and can simulate new ones, and all outputs can be written to standard file formats. jackalope is available for Mac, Windows and Linux systems.

Many countries in sub-Saharan Africa have experienced lower COVID-19 caseloads and fewer deaths than countries in other regions worldwide. Under-reporting of cases and a younger population could partly account for these differences, but pre-existing immunity to coronaviruses is another potential factor. Blood samples from Sierra Leonean Lassa fever and Ebola survivors and their contacts collected before the first reported COVID-19 cases were assessed using enzyme-linked immunosorbent assays for the presence of antibodies binding to proteins of coronaviruses that infect humans. Results were compared to COVID-19 subjects and healthy blood donors from the United States. Prior to the pandemic, Sierra Leoneans had more frequent exposures than Americans to coronaviruses with epitopes that cross-react with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), SARS-CoV, and Middle Eastern respiratory syndrome coronavirus (MERS-CoV). The percentage of Sierra Leoneans with antibodies reacting to seasonal coronaviruses was also higher than for American blood donors. Serological responses to coronaviruses by Sierra Leoneans did not differ by age or sex. Approximately a quarter of Sierra Leonian pre-pandemic blood samples had neutralizing antibodies against SARS-CoV-2 pseudovirus, while about a third neutralized MERS-CoV pseudovirus. Prior exposures to coronaviruses that induce cross-protective immunity may contribute to reduced COVID-19 cases and deaths in Sierra Leone.

Background: More than 90% of the global 400 000 annual malaria deaths occur in Africa. The current SARS-CoV-2 pandemic has resulted in more than 830 000 deaths in its first 10 months. Case presentation: This case describes a patient who had travelled from Mozambique to Cape Town, presented with a mild febrile illness, and was diagnosed with both COVID-19 and uncomplicated Plasmodium falciparum malaria infection. She responded well to malaria treatment and had an uneventful COVID-19 admission. Her blood smear showed a low malaria parasitaemia and a relatively high gametocyte load. Conclusion: We postulate that her clinical course and abnormal smear could well be due to reciprocal disease-modifying effects of the infections. The presenting symptoms of COVID-19 may mimic endemic infectious diseases including malaria, tuberculosis, pneumocystis pneumonia and influenza thus there is a need for clinical vigilance to identify and treat such co-infections. The full article is available at https://doi.org/10.36303/JMLSTSA.2020.2.2.57

A series of fifteen acetylcholinesterase inhibitors were designed and synthesised based upon the previously identified lead compound 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl 1-benzylpiperidine-4-carboxylate (5) which showed good inhibitory activity (IC50 0.03 ± 0.07 μM) against acetylcholinesterase. A series of compounds were prepared wherein the ester linker in the original lead compound was exchanged for a more metabolically stable amide linker and the indanone moiety was exchanged for a range of aryl and aromatic heterocycles. The two most active analogues 1-benzyl-N-(5,6-dimethoxy-8H-indeno[1,2-d]thiazol-2-yl)piperidine-4-carboxamide (28) and 1-benzyl-N-(1-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) piperidine-4-carboxamide (20) afforded in vitro IC50 values of 0.41 ± 1.25 and 5.94 ± 1.08 μM, respectively. In silico screening predicts that 20 will be a blood brain-barrier permeant, and molecular dynamic simulations are indicative of a close correlation between the binding of 20 and the Food and Drug Administration-approved cholinesterase inhibitor donepezil (1).

10 páginas, 2 tablas y 3 figuras., Terpenoids are secondary metabolites produced in most plant tissues and are often considered toxic or repellent to plant enemies. Previous work has typically reported on intra-specific variation in terpene profiles, but the effects of plant sex, an important axis of genetic variation, have been less studied for chemical defences in general, and terpenes in particular. In a prior study, we found strong genetic variation (but not sexual dimorphism) in terpene amounts in leaves of the dioecious shrub Baccharis salicifolia. Here we build on these findings and provide a more in-depth analysis of terpene chemistry on these same plants from an experiment consisting of a common garden with male (N = 19) and female (N = 20) genotypes sourced from a single population. Our goal in the present study was to investigate quantitative and qualitative differences in terpene profiles associated with plant sex and genotypic variation. For this, we quantified leaf mono- and sesquiterpene amount, richness, and diversity (quantitative profile), as well as the composition of compounds (qualitative profile). We found no evidence of sexual dimorphism in monoterpene or sesquiterpene profiles. We did, however, find significant genotypic variation in amount, diversity, and composition of monoterpenes, but no effects on sesquiterpenes. These findings indicated that genotypic variation in terpene profiles largely surpassed variation due to sexual dimorphism for the studied population of this species., This research was funded by National Science Foundation grants DEB-1354734, DEB-1457029 and DEB-1120794 to KAM. XM was supported by a Spanish National Research Grant (AGL2015-70748-R), a grant from the Regional Government of Galicia (IN607D 2016/001) and the Ramon y Cajal Research Programme (RYC-2013-13230).

Feline iris melanoma, the most common feline intraocular tumour, has a reported metastatic rate of 19-63%. However, there is a lack of knowledge about its molecular biology. Previous studies have reported that feline iris melanomas do not harbour mutations comparable to common mutations found in their human counterpart. Nevertheless, there are differences in the gene expression patterns. The aim of this study was to investigate the protein expression of B-RAF oncogene serine/threonine kinase (BRAF), G protein subunit alpha q (GNAQ) and 11 (GNA11), KIT proto-oncogene receptor tyrosine kinase (KIT), and Ras association family member 1 (RASSF1) in feline iris melanomas. Fifty-seven formalin-fixed paraffin embedded (FFPE) iris melanomas and 25 FFPE eyes without ocular abnormalities were stained with antibodies against the respective proteins using immunofluorescence. Averaged pixel intensities/μm2 and percentage of stained area from total tissue area were measured and the results were compared. Compared to the control group, iris melanomas showed overexpression of BRAF, GNAQ, GNA11 and KIT. The higher expression of BRAF, GNAQ, GNA11 and KIT in feline iris melanomas suggest that these proteins may play a key role in the development of feline iris melanomas and KIT may present a possible target for future therapies in cats with feline iris melanomas.

Although rollout of combined antiretroviral treatment (cART) has blunted human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) onset, there is increased development of cardiovascular diseases (CVDs) in HIV-infected individuals. While most HIV-infected individuals on cART achieve viral suppression, this may not necessarily result in complete immunological recovery. This study therefore evaluated T-cell-mediated changes and coagulation markers in HIV-positive individuals to ascertain their potential to increase CVD risk. Eighty participants were recruited (Worcester, South Africa), and fasted blood was collected to evaluate: 1) immune activation (CD38 expression on CD4+and CD8+T cells) and thrombus formation [tissue factor (CD142)] on CD4+and CD8+T cells; 2) monocyte subpopulations (nonclassical, intermediate, and classical); and 3) classical regulatory T (Treg) cells with activation markers [glycoprotein A repetitions predominant (GARP) and special AT-rich sequence-binding protein 1 (SATB-1)]. High- and low-density lipoprotein subclasses (Lipoprint) were also determined. This study revealed four key findings for HIV-positive patients: 1) coexpression of the CD142 coagulation marker together with immune activation on both CD4+and CD8+T cells during chronic infection stages; 2) Treg cell activation and upregulated GARP and SATB-1 contributing to Treg dysfunction in chronic HIV; 3) proatherogenic monocyte subset expansion with significant correlation between T-cell activation and macrophage activation (marker: CD163); and 4) significant correlation between immune activation and lipid subclasses, revealing crucial changes that can be missed by traditional lipid marker assessments (LDL and HDL). These data also implicate lipopolysaccharide-binding protein as a crucial link between immune activation, lipid alterations, and increased CVD risk.NEW & NOTEWORTHY With combined antiretroviral treatment rollout, HIV-AIDS patients are increasingly associated with cardiovascular diseases onset. This study demonstrated the significant interplay between adaptive immune cell activation and monocyte/macrophage markers in especially HIV-positive individuals with virological failure and on second line treatment. Our data also show a unique link between immune activation and lipid subclass alterations, revealing important changes that can be missed by traditional lipid marker assessments (e.g., LDL and HDL).

Severe COVID‐19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS‐CoV‐2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco‐2, Calu‐3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon‐induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS‐CoV‐2 emerging variants display enhanced syncytia formation., Spike protein mutations expressed by emerging SARS‐CoV‐2 variants‐of‐concern differentially affect host cell‐to‐cell fusion, ACE2 receptor binding, and antibody escape.

Here, we discuss the presence and roles of heterogeneity in the development of uveal melanoma. Both genetic and cellular heterogeneity are considered, as their presence became undeniable due to single cell approaches that have recently been used in uveal melanoma analysis. However, the presence of precursor clones and immune infiltrate in uveal melanoma have been described as being part of the tumour already decades ago. Since uveal melanoma grow in the corpus vitreous, they present a unique tumour model because every cell present in the tumour tissue is actually part of the tumour and possibly plays a role. For an effective treatment of uveal melanoma metastasis, it should be clear whether precursor clones and normal cells play an active role in progression and metastasis. We propagate analysis of bulk tissue that allows analysis of tumour heterogeneity in a clinical setting.

There is an urgent need for accurate, scalable and cost-efficient models of the tumor microenvironment. Here, we detail how to fabricate and use the metabolic microenvironment chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is accessibility to the blood stream. Whereas perivascular tumor cells have direct access to oxygen and nutrients, cells further from the vasculature must survive under progressively more ischemic environments. The MEMIC simulates this differential access to nutrients, allow co-culturing any number of cell types, and it is optimized for live imaging and other microscopy-based analyses. Owing to a modular design and full experimental control, the MEMIC provides insights into the tumor microenvironment that would be difficult to obtain via other methods. As proof of principle, we show that cells sense gradual changes in metabolite concentration leading to predictable molecular and cellular spatial patterns. We propose the MEMIC as a complement to standard in vitro and in vivo experiments, diversifying the tools available to accurately model, perturb and monitor the tumor microenvironment., Editor's choice: We present how to fabricate the MEMIC, an experimental model of the tumor microenvironment, describing proof-of-principle experiments and providing image analysis tools that are helpful when using this system.

International audience; Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA in situ hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion, and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

Life history variation is thought to be mainly a result of energetic trade-offs among fitness components; however, detecting these trade-offs in natural populations has yielded mixed results. Individual quality and environmental variation may mask expected relationships among fitness components because some higher quality individuals may be able to acquire more resources and invest more in all functions. Thus, life history variation may be more affected by variation in individual quality than varying strategies to resolve energetic trade-offs, e.g. costs of reproduction. Here, we investigated whether variation in female quality or costs of reproduction is a larger factor in shaping differences in life history trajectories by assessing the relationship between survival and individual reproductive performance using a 32-year longitudinal data set of repeated reproductive measurements from 273 individually marked, known-aged female grey seals (Halichoerus grypus) from the Sable Island breeding colony. We defined individual reproductive performance using two traits: reproductive frequency (a female’s probability of breeding) and provisioning performance (provisions given to young measured by offspring mass), computed using mixed effects models separately for (1) all reproductive events, and (2) an age-class specific reproductive investment. Individual differences contributed a large portion of the variance in reproductive traits, with individuals displaying a range in individual reproductive frequencies from 0.45 to 0.94, and a range of average pup weaning masses from 34.9 kg to 61.8 kg across their lifetime. We used a Cormack-Jolly-Seber open-population model to estimate the effect of these reproductive performance traits on adult survival probability. Our approach estimated a positive relationship between reproductive performance and survival, where individuals that consistently invest well in their offspring survive longer. The best supported model estimated survival as a function of age-class specific provisioning performance, where late-life performance was quite variable and had the greatest impact on survival, possibly indicating individual variation in senescence. There was no evidence to support a trade-off in reproductive performance and survival at the individual level. These results suggest that in grey seals, individual quality is a stronger driver in life history variation than varying strategies to mitigate trade-offs among fitness components.

Euphorbia usambarica is a traditional medicine used for gynecologic, endocrine, and urogenital illnesses in East Africa, however, its constituents and bioactivities have not been investigated. A variety of compounds isolated from Euphorbia species have been shown to have activity against latent HIV-1, the major source of HIV-1 persistence despite antiretroviral therapy. We performed bioactivity-guided isolation to identify 15 new diterpenoids (1–9, 14–17, 19, and 20) along with 16 known compounds from E. usambarica with HIV-1 latency reversal activity. Euphordraculoate C (1) exhibits a rare 6/6/3-fused ring system with a 2-methyl-2-cyclopentenone moiety. Usambariphanes A (2) and B (3) display an unusual lactone ring constructed between C-17 and C-2 in the jatrophane structure. 4β-Crotignoid K (14) revealed a 250-fold improvement in latency reversal activity compared to crotignoid K (13), identifying that configuration at the C-4 of tigliane diterpenoids is critical to HIV-1 latency reversal activity. The primary mechanism of the active diterpenoids 12–14 and 21 for the HIV-1 latency reversal activity was activation of PKC, while lignans 26 and 27 that did not increase CD69 expression, suggesting a non-PKC mechanism. Accordingly, natural constituents from E. usambarica have the potential to contribute to the development of HIV-1 eradication strategies.

Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighbouring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha and Beta spread and fusion in cell cultures. Alpha and Beta replicated similarly to D614G reference strain in Vero, Caco-2, Calu-3 and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Alpha, Beta and D614G fusion was similarly inhibited by interferon induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes differentially modified fusogenicity, binding to ACE2 and recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.SynopsisThe Spike protein of the novel SARS-CoV-2 variants are comparative more fusogenic than the earlier strains. The mutations in the variant spike protein differential modulate syncytia formation, ACE2 binding, and antibody escape.The spike protein of Alpha, Beta and Delta, in the absence of other viral proteins, induce more syncytia than D614GThe ACE2 affinity of the variant spike proteins correlates to their fusogenicityVariant associated mutations P681H, D1118H, and D215G augment cell-cell fusion, while antibody escape mutation E484K, K417N and Δ242-244 hamper it.Variant spike-mediated syncytia formation is effectively restricted by IFITMs

The lack of effective treatments for autism spectrum disorder (ASD) and congenital hydrocephalus (CH) reflects the limited understanding of the biology underlying these common neurodevelopmental disorders. Although ASD and CH have been extensively studied as independent entities, recent human genomic and pre-clinical animal studies have uncovered shared molecular pathophysiology. Here, we review and discuss phenotypic, genomic, and molecular similarities between ASD and CH, and identify the PTEN-PI3K-mTOR (phosphatase and tensin homolog-phosphoinositide 3-kinase-mammalian target of rapamycin) pathway as a common underlying mechanism that holds diagnostic, prognostic, and therapeutic promise for individuals with ASD and CH.

International audience; We present the largest exome sequencing study of autism spectrum disorder (ASD) to date (n = 35,584 total samples, 11,986 with ASD). Using an enhanced analytical framework to integrate de novo and case-control rare variation, we identify 102 risk genes at a false discovery rate of 0.1 or less. Of these genes, 49 show higher frequencies of disruptive de novo variants in individuals ascertained to have severe neurodevelopmental delay, whereas 53 show higher frequencies in individuals ascertained to have ASD; comparing ASD cases with mutations in these groups reveals phenotypic differences. Expressed early in brain development, most risk genes have roles in regulation of gene expression or neuronal communication (i.e., mutations effect neurodevelopmental and neurophysiological changes), and 13 fall within loci recurrently hit by copy number variants. In cells from the human cortex, expression of risk genes is enriched in excitatory and inhibitory neuronal lineages, consistent with multiple paths to an excitatory-inhibitory imbalance underlying ASD.

The mitochondrial antiviral signaling protein (MAVS) is part of the cell’s innate immune mechanism of defense. MAVS mRNA is bicistronic and can give rise to a full length-MAVS and a shorter isoform termed miniMAVS. In response to viral infections, viral RNA can be sensed by the cytosolic RNA sensors retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation-associated protein 5 (MDA5) and activate NF-κB through interaction with MAVS. MAVS can also sense cellular stress and activate an anti-oxidative stress (AOS) response through the activation of NF-κB. Because NF-κB is a main cellular transcription factor for HIV-1, we wanted to address what role MAVS plays in HIV-1 reactivation from latency in CD4 T cells. Our results indicate that RIG-I agonists required full length-MAVS whereas the AOS response induced by Dynasore through its catechol group can reactivate latent HIV-1 in a MAVS dependent manner through miniMAVS isoform. Furthermore, we uncover that PKC agonists, a class of latency-reversing agents, induce an AOS response in CD4 T cells and require miniMAVS to fully reactivate latent HIV-1. Our results indicate that the AOS response, through miniMAVS, can induce HIV-1 transcription in response to cellular stress and targeting this pathway adds to the repertoire of approaches to reactivate latent HIV-1 in ‘shock-and-kill’ strategies.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that specifically target bacterial metabolites but are also identified as innate-like sensors of viral infection. Individuals with chronic HIV-1 infection have lower numbers of circulating MAIT cells compared with healthy individuals, yet the features of the MAIT TCR repertoire are not well known. We isolated and stimulated human PBMCs from healthy non-HIV–infected donors (HD), HIV-infected progressors on antiretroviral therapy, and HIV-infected elite controllers (EC). We sorted MAIT cells using flow cytometry and used a high-throughput sequencing method with bar coding to link the expression of TCRα, TCRβ, and functional genes of interest at the single-cell level. We show differential patterns of MAIT TCR usage among the groups. We observed expansions of certain dominant MAIT clones in HIV-infected individuals upon Escherichia coli stimulation, which was not observed in clones of HD. We also found different patterns of CDR3 amino acid distributions among the three groups. Furthermore, we found blunted expression of phenotypic genes in HIV individuals; most notably, HD mounted a robust IFNG response to stimulation, whereas both HIV-infected progressors and EC did not. In conclusion, our study describes the diverse MAIT TCR repertoire of persons with chronic HIV-1 infection and suggest that MAIT clones of HIV-infected persons may be primed for expansion more than that of noninfected persons. Further studies are needed to examine the functional significance of unique MAIT cell TCR usage in EC.

Background Alveolar echinococcosis (AE) is caused by metacestode larva of the tapeworm Echinococcus multilocularis. AE diagnostics currently rely on imaging techniques supported by serology, but unequivocal detection of AE is difficult. Although polymerase chain reaction (PCR)-based methods to detect tapeworm DNA in biopsies have been suggested for several species, no validated protocol adhering to accepted guidelines has so far been presented for AE diagnostics. We herein established a PCR protocol for metacestode biopsies and technically evaluated the method using isolated parasite DNA and cells, biopsies of clinically relevant material, and formalin fixed paraffin-embedded (FFPE) human tissue blocks. We compared the results with an immunochemical (IHC) approach using the monoclonal antibody Em2G11 specific for the antigen Em2 of E. mulitlocularis. Methodology/Principal findings Based on tapeworm 12S rDNA sequences we established and validated a PCR protocol for robust detection of as little as 50 parasite cells per specimen and report 127 cases of positive identification of Echinococcus species in samples from humans and animals. For further validation, we analyzed 45 liver, heart, brain, and soft tissue samples as well as cytological probes of aspirates of FFPE-material from 18 patients with clinically confirmed AE. Of each patient we analyzed (i) fully viable lesions with laminated layer; (ii) tissue with mAbEm2G11-positive small particles of E. multilocularis (spems); (iii) mAbEm2G11-negative tissue adjacent to the main lesion; and (iv) lymph node tissue with mAbEm2G11-positive spems. To identify the areas for the PCR-based approach, we performed IHC-staining with the monoclonal antibody Em2G11. Micro-dissected tissue of these areas was then used for PCR-analysis. 9 of 15 analyzed samples with viable E. multilocularis lesions with laminated layer were positive by PCR. Of this group, all samples preserved for less than 6 years (6/6) were tested positive. 11 of 15 samples of spems and 7 of 9 samples of the control group mAbEm2G11-negative tissue were negative by PCR. We further show that all probes from lymph nodes with spems are PCR negative. Conclusions/Significance We present a sensitive PCR method for the detection of E. multilocularis in human tissue, particularly in fresh biopsy material and tissue blocks stored for less than 5 years. While the diagnostic sensitivity of material containing only spems was higher using IHC, PCR detection was possible in IHC negative liver tissue and in patients with negative serology. Our results support the view that spems do not contain parasitic DNA or viable cells of the parasite. spems thus most probably do not directly contribute to metastasis formation during AE., Author summary Alveolar echinococcosis (AE) is a potentially lethal zoonosis during which metacestode larval tissue of the tapeworm Echinococcus multilocularis grows like a malignant tumor, infiltrating the human liver. Current AE diagnosis, which mainly relies on imaging techniques (ultrasound, computed tomography, magnetic resonance tomography) supported by serology, is not always suitable to unequivocally identify an infection. In this work the authors present a PCR-based approach to detect the parasite in biopsy material taken from patients. The method was first validated for routine laboratory use employing isolated parasite DNA as well as defined numbers of parasite cells in clinically relevant settings. The method was then verified under clinically relevant settings using pathology samples from patients with defined AE. Particularly in cases when these samples had been stored for less than 6 years, the PCR methodology was highly suitable to identify the infection, even when serology was negative. The authors show that small particles of E. multilocularis (spems), containing surface material of the parasite and often located in the draining adjacent lymph nodes, are PCR negative and, thus, most probably do not contain viable parasite cells. The authors conclude that in cases of doubtful serology and imaging, a combination of immunochemical staining methods with PCR are reliable means to unequivocally detect the infection.

There is an urgent need for accurate, scalable, and cost-efficient models of the complexity and heterogeneity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC can provide novel insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells can sense gradual changes in metabolite concentration, and tune intracellular cell signaling to form multicellular spatial patterns of cell proliferation. We also show that ischemic macrophages reduce epithelial features in neighboring tumor cells highlighting the power of this system to study cell-cell interactions and non-cell autonomous effects of the metabolic microenvironment. We propose that the MEMIC can be easily adapted to study early development, ischemic stroke, and other systems where multiple cell types interact within heterogeneous environments.

BackgroundCOVID-19 has resulted in significant morbidity and mortality worldwide. Lateral flow assays can detect anti-Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) antibodies to monitor transmission. However, standardized evaluation of their accuracy and tools to aid in interpreting results are needed.MethodsWe evaluated 20 IgG and IgM assays selected from available tests in April 2020. We evaluated the assays’ performance using 56 pre-pandemic negative and 56 SARS-CoV-2-positive plasma samples, collected 10-40 days after symptom onset, confirmed by a molecular test and analyzed by an ultra-sensitive immunoassay. Finally, we developed a user-friendly web app to extrapolate the positive predictive values based on their accuracy and local prevalence.ResultsCombined IgG+IgM sensitivities ranged from 33.9% to 94.6%, while combined specificities ranged from 92.6% to 100%. The highest sensitivities were detected in Lumiquick for IgG (98.2%), BioHit for both IgM (96.4%), and combined IgG+IgM sensitivity (94.6%). Furthermore, 11 LFAs and 8 LFAs showed perfect specificity for IgG and IgM, respectively, with 15 LFAs showing perfect combined IgG+IgM specificity. Lumiquick had the lowest estimated limit-of-detection (LOD) (0.1 μg/mL), followed by a similar LOD of 1.5 μg/mL for CareHealth, Cellex, KHB, and Vivachek.ConclusionWe provide a public resource of the accuracy of select lateral flow assays with potential for home testing. The cost-effectiveness, scalable manufacturing process, and suitability for self-testing makes LFAs an attractive option for monitoring disease prevalence and assessing vaccine responsiveness. Our web tool provides an easy-to-use interface to demonstrate the impact of prevalence and test accuracy on the positive predictive values.

Projection neurons in the mammalian olfactory bulb (OB) not only receive direct input from the nose but also directly project to diverse cortical and subcortical areas. While morphological and physiological studies have highlighted functional heterogeneity, no molecular markers have been described that could reliably delineate subtypes of projection neurons. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich OB neuronal populations for projection neurons. We then performed high-throughput single nucleus and bulk RNA deep sequencing of such enriched populations. Transcriptome analysis together with RNA in situ hybridization allowed us to identify three distinct mitral and five distinct tufted cell populations with characteristic cell adhesion and cell excitability-related gene expression patterns. Using a novel computational approach to integrate bulk and snRNA-seq data we propose that different mitral cell populations selectively project to different regions of olfactory cortex. Together, we have identified potential molecular and gene regulatory mechanisms underlying projection neuron diversity and provide critical new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

Neuronal lesions in Parkinson’s disease (PD) are commonly associated with α-synuclein (α-Syn)-induced cell damage that are present both in the central and peripheral nervous systems of patients, with the enteric nervous system also being especially vulnerable. Here we bring together evidence that the development and presence of PD depends on specific sets of interlinking factors that include neuro-inflammation, systemic inflammation, α-Syn-induced cell damage, vascular dysfunction, iron dysregulation, gut and periodontal dysbiosis. We argue that there is significant evidence that bacterial inflammagens fuel this systemic inflammation, and might be central to the development of PD. We also discuss the processes whereby lipopolysaccharides may be involved in causing nucleation of proteins, including of α-Syn. Lastly, we review evidence that pre-and probiotics, as well as antibiotics and faecal transplant treatment might be valuable treatments in PD. A most important consideration, however, is that these therapeutic options need to be validated and tested in randomized controlled clinical trials. However, targeting underlying mechanisms of PD, including gut dysbiosis and iron toxicity, have potentially opened up possibilities of a wide variety of novel treatments which may relieve the characteristic non-motor deficits of PD, and may even slow the progression and/or accompanying gut-related conditions of the disease.

Background Single-cell RNA sequencing is a powerful technology to discover new cell types and study biological processes in complex biological samples. A current challenge is to predict transcription factor (TF) regulation from single-cell RNA data. Results Here, we propose a novel approach for predicting gene expression at the single-cell level using cis-regulatory motifs, as well as epigenetic features. We designed a tree-guided multi-task learning framework that considers each cell as a task. Through this framework we were able to explain the single-cell gene expression values using either TF binding affinities or TF ChIP-seq data measured at specific genomic regions. TFs identified using these models could be validated by the literature. Conclusion Our proposed method allows us to identify distinct TFs that show cell type–specific regulation. This approach is not limited to TFs but can use any type of data that can potentially be used in explaining gene expression at the single-cell level to study factors that drive differentiation or show abnormal regulation in disease. The implementation of our workflow can be accessed under an MIT license via https://github.com/SchulzLab/Triangulate.

BackgroundActivating Gαq signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT2R) forms a rare alternative. The role of wild-type CysLT2R in uveal melanoma remains unknown.MethodsWe performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study wild-type and mutant CYSLTR2.Results1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was subclonal in the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in 2/3 CYSLTR2 mutant melanomas from the TCGA cohort. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 originated from melanoma cells and correlated to tumour inflammation.ConclusionsOur findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT2R an attractive therapeutic target in uveal melanoma. In GNAQ, GNA11 and PLCB4 mutant melanomas, expression of wild-type CYSLTR2 possibly facilitates an interaction with immune cells in the microenvironment and may also have therapeutic potential.

Alveolar echinococcosis (AE) is caused by the intermediate stage of Echinococcus multilocularis. We aimed to correlate computed tomography (CT) data with histology to identify distinct characteristics for different lesion types. We classified 45 samples into five types with the Echinococcus multilocularis Ulm Classification for Computed Tomography (EMUC-CT). The various CT lesions exhibited significantly different histological parameters, which led us to propose a progression model. The initial lesion fit the CT type IV classification, which comprises a single necrotic area with the central located laminated layer, a larger distance between laminated layer and border zone, a small fibrotic peripheral zone, and few small particles of Echinococcus multilocularis (spems). Lesions could progress through CT types I, II, and III, characterized by shorter distances between laminated layer and border zone, more spems inside and surrounding the lesion, and a pronounced fibrotic rim (mostly in type III). Alternatively, lesions could converge to a highly calcified, regressive state (type V). Our results suggest that the CT types mark sequential stages of the infection, which progress over time. These distinct histological patterns advance the understanding of interactions between AE and human host; moreover, they might become prognostically and therapeutically relevant., publishedVersion

Objective The morphology of the corneal epithelium in two age groups of horses is described. Distribution patterns of proliferation‐, differentiation‐, stem cell‐associated markers and cell junction proteins were assessed. Methods Corneal samples from 12 horses (six foals and six adult horses) were analyzed after H&E staining and immunohistochemistry using the following antibodies: E‐cadherin, β‐catenin, Connexin 43 (Cx43), tight junction protein 1 (TJP1), cytokeratin (CK) 14, CK 19, CK 3, CK 10, vimentin, Ki67, p63, nerve growth factor (NGF), ABCG2, and epithelial growth factor receptor. Semiquantitative analysis of crypt, limbal, peripheral, and central zone was performed. Semithin and ultrathin sections were used for ultrastructural evaluation of the epithelium. Results The height of the epithelium varied between age groups and crypts were consistently present. In the peripheral and central epithelium, three types of basal cells resembling a pseudostratified epithelium were characterized. Potential stem cell markers (CK 14, p63, NGF, and ABCG2) were present in all zones with decreasing frequency toward the center. Cornea‐specific differentiation marker CK 3 was not expressed in the most basal cell layer of the limbal epithelium. E‐cadherin, β‐catenin, and Cx43 revealed a similar apico‐lateral signal pattern throughout the entire epithelium; only TJP1 was additionally seen at the basal surface. Conclusions This study presents a systematic semiquantitative evaluation of the equine corneal epithelium, showing the presence of crypts as potential stem cell niche with CK 14, p63, NGF, and ABCG2 as relevant markers for cells with regenerative capacity. The pseudostratified arrangement of the basal layer was a unique finding.

Background: Among HIV-positive individuals, increased levels of inflammation and immune activation persist even in the setting of effective antiretroviral therapy (ART) and are associated with greater rates of non-AIDS events. The etiology of this persistent inflammation is incompletely understood.Methods: Using a well-characterized cohort of 322 HIV-infected individuals on suppressive ART, we conducted a case-control study. Cytomegalovirus (CMV) immunoglobulin G (IgG) levels, plasma biomarkers, and T-cell phenotypes were measured/characterized from samples collected 1 year after ART initiation. Conditional logistic regression for matched case-control studies analyzed the associations of year 1 CMV-specific IgG level with the subsequent occurrence of any non-AIDS event. Correlations between continuous CMV IgG antibody levels and soluble and cellular markers were assessed.Results: We found that higher levels of CMV IgG were associated with increased risk of non-AIDS events (OR = 1.58 per IQR [95% CI: 1.12, 2.24], P = 0.01) and with elevated soluble and cellular markers of inflammation.Conclusions: The magnitude of the host immune response to CMV may play a role in the persistent inflammation and resultant morbid events observed in the HIV-positive population.

Background Alveolar (AE) and cystic echinococcosis (CE) in humans are caused by the metacestode of the tapeworms Echinococcus multilocularis and Echinococcus granulosus sensu lato (s.l.). Immunohistochemistry with the monoclonal antibodies (mAb) Em2G11, specific for AE, and the mAb EmG3, specific for AE and CE, is an important pillar of the histological diagnosis of these two infections. Our aim was to further evaluate mAb EmG3 in a diagnostic setting and to analyze in detail the localization, distribution, and impact of small particles of Echinococcus multilocularis (spems) and small particles of Echinococcus granulosus s.l. (spegs) on lymph nodes. Methodology/principal findings We evaluated the mAb EmG3 in a cohort of formalin-fixed, paraffin embedded (FFPE) specimens of AE (n = 360) and CE (n = 178). These samples originated from 156 AE-patients and 77 CE-patients. mAb EmG3 showed a specific staining of the metacestode stadium of E. multilocularis and E. granulosus s.l. and had a higher sensitivity for spems than mAb Em2G11. Furthermore, we detected spegs in the surrounding host tissue and in almost all tested lymph nodes (39/41) of infected patients. 38/47 lymph nodes of AE showed a positive reaction for spems with mAb EmG3, whereas 29/47 tested positive when stained with mAb Em2G11. Spegs were detected in the germinal centers, co-located with CD23-positive follicular dendritic cells, and were present in the sinuses. Likewise, lymph nodes with spems and spegs in AE and CE were significantly enlarged in size in comparison to the control group. Conclusions/significance mAb EmG3 is specific for AE and CE and is a valuable tool in the histological diagnosis of echinococcosis. Based on the observed staining patterns, we hypothesize that the interaction between parasite and host is not restricted to the main lesion since spegs are detected in lymph nodes. Moreover, in AE the number of spems-affected lymph nodes is higher than previously assumed. The enlargement of lymph nodes with spems and spegs points to an immunological interaction with the small immunogenic particles (spems and spegs) of Echinococcus spp., Author summary Echinococcosis is a life-threatening disease in humans that is caused by the larval stages of the tapeworms Echinococcus multilocularis and Echinococcus granulosus s.l.. For the histological diagnosis of alveolar echinococcosis (AE) and cystic echinococcosis (CE) the two monoclonal antibodies (mAb) Em2G11, specific for AE, and the mAb EmG3, specific for AE and CE are available. We have analyzed a large cohort of formalin-fixed, paraffin embedded human tissue (FFPE) specimens of AE (n = 360) and CE (n = 178) using these two antibodies. We show that the mAb EmG3 has a specific staining pattern of the metacestode stadium of E. multilocularis and E. granulosus s.l. while staining of the larval state of E. multilocularis is limited to the mAb Em2G11. We further identified so called “small particles of Echinococcus multilocularis” (spems) and “small particles of Echinococcus granulosus” (spegs) not only in the main lesion of AE and CE, but also in the adjacent tissue and in the vast majority of lymph nodes analyzed draining the main lesion. The lymph nodes with these microparticles are enlarged as compared to a control group. We conclude that immunochemistry with these two antibodies is a valuable adjunct for the final histological diagnosis of AE und CE. The frequent detection of microparticles of E. multilocularis and E. granulosus in lymph nodes of patients with AE and CE argue for a larger interface of interaction of the parasite with the host`s immune system than previously assumed and point to further mechanisms of these infections in humans which may be the basis for immunological reactions in the host.

“Shock and kill” therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity., “Shock and kill” therapeutic strategies toward HIV eradication are based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) and the consequent killing of the reactivated cell by either the cytopathic effect of HIV or an arm of the immune system. We have recently found several benzotriazole and benzotriazine analogues that have the ability to reactivate latent HIV by inhibiting signal transducer and activator of transcription 5 (STAT5) SUMOylation and promoting STAT5 binding to the HIV long terminal repeat and increasing its transcriptional activity. To understand the essential structural groups required for biological activity of these molecules, we performed a systematic analysis of >40 analogues. First, we characterized the essential motifs within these molecules that are required for their biological activity. Second, we identified three benzotriazine analogues with similar activity. We demonstrated that these three compounds are able to increase STAT5 phosphorylation and transcriptional activity. All active analogues reactivate latent HIV in a primary cell model of latency and enhance the ability of interleukin-15 to reactivate latent HIV in cells isolated from aviremic participants. Third, this family of compounds also promote immune effector functions in vitro in the absence of toxicity or global immune activation. Finally, initial studies in mice suggest lack of acute toxicity in vivo. A better understanding of the biological activity of these compounds will help in the design of improved LRAs that work via inhibition of STAT5 SUMOylation.

Food poisoning in humans caused by Salmonella enterica remains a significant global public health concern, with the majority of infections associated with the consumption of contaminated eggs or poultry products. The safety and efficacy of a novel inactivated trivalent Salmonella enterica vaccine containing in addition to Salmonella serovars Enteritidis (O:9, serogroup D) and Typhimurium (O:4, serogroup B) also serovar Infantis (O:7, serogroup C1) formulated with an aluminium hydroxide-gel adjuvant was evaluated under field conditions. A total of 10,229 broiler breeder pullets, housed under commercial conditions, were vaccinated at 10 and 17 weeks of age by the intramuscular route in the breast muscle. The vaccine was safe with no local or systemic reactions or adverse effects on bird performance related to the vaccine detected. Vaccination resulted in notable increases in serovar specific antibodies that were maintained until at least 56 weeks of age. Vaccinated birds subjected to homologous challenges around onset of lay showed significantly reduced faecal shedding and organ invasion. Following heterologous challenge with S. Hadar (O:8, serogroup C2) faecal shedding was significantly reduced. These results demonstrate that this novel vaccine could play a significant role in a comprehensive Salmonella control programme intended to reduce both the incidence of food poisoning in humans and the use of antibiotics during poultry production.

Background A strong correlation exists between type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD), with CVD and the presence of atherosclerosis being the prevailing cause of morbidity and mortality in diabetic populations. T2DM is accompanied by various coagulopathies, including anomalous clot formation or amyloid fibrin(ogen), the presence of dysregulated inflammatory molecules. Platelets are intimately involved in thrombus formation and particularly vulnerable to inflammatory cytokines. Methods The aim of this current study was therefore to assess whole blood (hyper)coagulability, platelet ultrastructure and receptor expression, as well as the levels of IL-1β, IL-6, IL-8 and sP-selectin in healthy and diabetic individuals. Platelet morphology was assessed through scanning electron microscopy (SEM), while assessment of GPIIb/IIIa receptor expression was performed with confocal microscopy and flow cytometry with the addition of FITC-PAC-1 and CD41-PE antibodies. IL-1β, IL-6 and IL-8 and sP-selectin levels were assessed using a multiplex assay. Results In T2DM there is significant upregulation of circulating inflammatory markers, hypercoagulation and platelet activation, with increased GPIIb/IIIa receptor expression, as seen with flow cytometry and confocal microscopy. Analyses showed that these receptors were additionally shed onto microparticles, which was confirmed with SEM. Conclusions Cumulatively, this provides mechanistic evidence that pathological states of platelets together with amyloid fibrin(ogen) in T2DM, might underpin an increased risk for cardiovascular events.

IntroductionLatency reversal agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-κB and, in turn, induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, and rapamycin, an mTOR inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 viral reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and can synergize with other LRAs to bring dormant proviruses out of latency. In this study we investigated whether a broad panel of epigenetic modifiers, including HDACi, could effectively dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal.MethodsWe screened an epigenetic modifier library to identify compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce a broad panel of pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo.ResultsWe identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-α, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when used in combination with Ingenol-3,20-dibenzoate.ConclusionThe addition of SBHA to Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when used in combination with Ingenol-3,20-dibenzoate suggests that SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.