Similar Functional Profiles Research Articles

S. aureus Eap is a polyvalent inhibitor of neutrophil serine proteases

Staphylococcus aureus expresses three high-affinity neutrophil serine protease (NSP) inhibitors known as the extracellular adherence protein domain (EAPs) proteins. Whereas EapH1 and EapH2 are comprised of a single EAP domain, the modular extracellular adherence protein (Eap) from S. aureus strain Mu50 consists of four EAP domains. We recently reported that EapH2 can simultaneously bind and inhibit cathepsin-G (CG) and neutrophil elastase (NE), which are the two most abundant NSPs. This unusual property of EapH2 arises from independent CG and NE-binding sites that lie on opposing faces of its EAP domain. Here we used X-ray crystallography and enzyme assays to show that all four individual domains of Eap (i.e. Eap1, Eap2, Eap3, and Eap4) exhibit an EapH2-like ability to form ternary complexes with CG and NE that inhibit both enzymes simultaneously. We found that Eap1, Eap2, and Eap3 have similar functional profiles insofar as NSP inhibition is concerned, but that Eap4 displays an unexpected ability to inhibit two NE enzymes simultaneously. Using X-ray crystallography, we determined that this second NE-binding site in Eap4 arises through the same region of its EAP domain that also comprises its CG-binding site. Interestingly, small angle X-ray scattering data showed that stable tail-to-tail dimers of the NE/Eap4/NE ternary complex exist in solution. This arrangement is compatible with NSP-binding at all available sites in a two-domain fragment of Eap. Together, our work implies that Eap is a polyvalent inhibitor of NSPs. It also raises the possibility that higher-order structures of NSP-bound Eap may have unique functional properties.

Stored platelet hemostatic phenotype and function is not altered when donors are on testosterone replacement therapy.

Critical shortages in the national blood supply have led to a re-evaluation of previously overlooked donor sources for blood products. As a part of that effort, red blood cells collected from therapeutic phlebotomy of donors on testosterone replacement therapy (TRT) have been conditionally approved for transfusion. However, platelets from TRT donors are not currently approved for use due to limited data on effects of supraphysiologic testosterone on recipient safety and platelet function. The objective of this study was to provide a comprehensive profile of phenotype and function in platelets from TRT and control donors. Platelets in plasma were collected from TRT and control donors (N = 10 per group; age- and sex-matched) and stored at room temperature for 7 days. On storage Day 1 (D1) and Day 7 (D7), platelet products were analyzed for platelet count, metabolic parameters (i.e., glucose, lactate, mitochondrial function), surface receptor expression, aggregation, thrombin generation, and thrombus formation under physiological flow conditions. TRT donor platelets were not significantly different than control donor platelets in terms of count, surface phenotype, metabolic function, ability to aggregate, thrombin generation, or ability to form occlusive thrombus under arterial flow regimes. Both groups were similar to each other by D7, but had significantly lost hemostatic function compared to D1. Platelets derived from donors undergoing TRT have similar phenotypic and functional profiles compared to those derived from control donors. This suggests that therapeutic phlebotomy of TRT donors may provide a useful source for platelet products.

Investigation of the functional impact of CHED- and FECD4-associated SLC4A11 mutations in human corneal endothelial cells.

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.

Evaluation of multiple displacement amplification for metagenomic analysis of low biomass samples.

Combining multiple displacement amplification (MDA) with metagenomics enables the analysis of samples with extremely low DNA concentrations, making them suitable for high-throughput sequencing. Although amplification bias and nonspecific amplification have been reported from MDA-amplified samples, the impact of MDA on metagenomic datasets is not well understood. We compared three MDA methods (i.e. bulk MDA, emulsion MDA, and primase MDA) for metagenomic analysis of two DNA template concentrations (approx. 1 and 100pg) derived from a microbial community standard "mock community" and two low biomass environmental samples (i.e. borehole fluid and groundwater). We assessed the impact of MDA on metagenome-based community composition, assembly quality, functional profiles, and binning. We found amplification bias against high GC content genomes but relatively low nonspecific amplification such as chimeras, artifacts, or contamination for all MDA methods. We observed MDA-associated representational bias for microbial community profiles, especially for low-input DNA and with the primase MDA method. Nevertheless, similar taxa were represented in MDA-amplified libraries to those of unamplified samples. The MDA libraries were highly fragmented, but similar functional profiles to the unamplified libraries were obtained for bulk MDA and emulsion MDA at higher DNA input and across these MDA libraries for the groundwater sample. Medium to low-quality bins were possible for the high input bulk MDA metagenomes for the most simple microbial communities, borehole fluid, and mock community. Although MDA-based amplification should be avoided, it can still reveal meaningful taxonomic and functional information from samples with extremely low DNA concentration where direct metagenomics is otherwise impossible.

Endovascular treatment of acute ischemic stroke patients with tandem lesions: antegrade versus retrograde approach.

The optimal technique for treating tandem lesions (TLs) with endovascular therapy is debatable. The authors evaluated the functional, safety, and procedural outcomes of different approaches in a multicenter study. Anterior circulation TL patients treated from January 2015 to December 2020 were divided on the basis of antegrade versus retrograde approach and included. The evaluated outcomes were favorable modified Rankin Scale (mRS) score (mRS score 0-2) at 3 months, ordinal shift in mRS score, successful recanalization, excellent recanalization, first-pass effect (FPE), time from groin puncture to successful recanalization, symptomatic intracranial hemorrhage (sICH), and 90-day mortality. Among 691 patients treated at 16 centers, 286 patients (174 antegrade and 112 retrograde approach patients) with acute stenting were included in the final analysis. There were no significant differences in mRS score 0-2 at 90 days (52.2% vs 50.0%, adjusted odds ratio [aOR] 0.83, 95% CI 0.42-1.56, p = 0.54), favorable shift in 90-day mRS score (aOR 1.03, 95% CI 0.66-1.29, p = 0.11), sICH (4.0% vs 4.5%, aOR 0.64, 95% CI 0.24-1.51, p = 0.45), successful recanalization (89.4% vs 93%, aOR 0.49, 95% CI 0.19-1.28, p = 0.19), excellent recanalization (51.4% vs 58.9%, aOR 0.59, 95% CI 0.40-1.07, p = 0.09), FPE (58.3% vs 69.7%, aOR 0.62, 95% CI 0.44-1.15, p = 0.21), and mortality at 90 days (16.6% vs 14.0%, aOR 0.94, 95% CI 0.35-2.44, p = 0.81) between the groups. The median (interquartile range) groin puncture to recanalization time was significantly longer in the antegrade group (59 [43-90] minutes vs 49 [35-73] minutes, p = 0.036). The retrograde approach was associated with faster recanalization times with a similar functional and safety profile when compared with the antegrade approach in patients with acute ischemic stroke with TL.

Platelets from blood diversion pouches (DPs) are a suitable alternative for functional, bioenergetic, and metabolomic analyses.

The collection of the first blood flow into a diversion pouch (DP) has become widely adopted in blood donation systems to reduce whole-blood unit contamination from skin bacteria. The strict control of pre-analytical variables, such as blood collection and proper anticoagulant selection, is critical to diminish experimental variability when studying different aspects of platelet biology. We hypothesize that the functional, mitochondrial, and metabolomic profiles of platelets isolated from the DP are not different from the ones isolated from standard venipuncture (VP), thus representing a suitable collection method of platelets for experimental purposes. Whole blood from the blood DP or VP was collected. Platelets were subsequently isolated and washed following standard protocols. Platelet function was assessed by flow cytometry, light transmission aggregometry, clot retraction, and under flow conditions using the total thrombus formation analyzer (T-TAS). Mitochondrial function and the platelet metabolome profiles were determined by the Seahorse extracellular flux analyzer (Agilent, Santa Clara, CA, USA) and ultra-high-pressure liquid chromatography-mass spectrometry metabolomics, respectively. Platelets isolated from VP and the DP have similar functional, mitochondrial, and metabolic profiles with no significant differences between both groups at baseline and upon activation by any of the assays mentioned above. The findings of our study support the use of platelets from the DP for performing functional and metabolic studies on platelets from a wide range of blood donors. The DP may serve as an alternative blood collection method to standard VP, allowing the study of diverse aspects of platelet biology, such as age, sex, race, and ethnicity, in many eligible individuals for blood donation.

The tropical cookbook: Termite diet and phylogenetics-Over geographical origin-Drive the microbiome and functional genetic structure of nests.

Termites are key decomposers of dead plant material involved in the organic matter recycling process in warm terrestrial ecosystems. Due to their prominent role as urban pests of timber, research efforts have been directed toward biocontrol strategies aimed to use pathogens in their nest. However, one of the most fascinating aspects of termites is their defense strategies that prevent the growth of detrimental microbiological strains in their nests. One of the controlling factors is the nest allied microbiome. Understanding how allied microbial strains protect termites from pathogen load could provide us with an enhanced repertoire for fighting antimicrobial-resistant strains or mining for genes for bioremediation purposes. However, a necessary first step is to characterize these microbial communities. To gain a deeper understanding of the termite nest microbiome, we used a multi-omics approach for dissecting the nest microbiome in a wide range of termite species. These cover several feeding habits and three geographical locations on two tropical sides of the Atlantic Ocean known to host hyper-diverse communities. Our experimental approach included untargeted volatile metabolomics, targeted evaluation of volatile naphthalene, a taxonomical profile for bacteria and fungi through amplicon sequencing, and further diving into the genetic repertoire through a metagenomic sequencing approach. Naphthalene was present in species belonging to the genera Nasutitermes and Cubitermes. We investigated the apparent differences in terms of bacterial community structure and discovered that feeding habits and phylogenetic relatedness had a greater influence than geographical location. The phylogenetic relatedness among nests' hosts influences primarily bacterial communities, while diet influences fungi. Finally, our metagenomic analysis revealed that the gene content provided both soil-feeding genera with similar functional profiles, while the wood-feeding genus showed a different one. Our results indicate that the nest functional profile is largely influenced by diet and phylogenetic relatedness, irrespective of geographical location.

Apilactobacillus kunkeei releases RNA-associated membrane vesicles and proteinaceous nanoparticles.

Extracellularly released particles, including membrane vesicles, have increasingly been recognized as important for bacterial community functions and host-interaction processes, but their compositions and functional roles differ between species and also between strains of the same species. In this study, we have determined the composition of membrane vesicles and protein particles identified in the cell-free pellets of two strains of Apilactobacillus kunkeei, a defensive symbiont of honeybees. The membrane vesicles were separated from the extracellular particles using density gradient ultracentrifugation. The peaks of the RNA and protein distributions were separated from each other and the highest concentration of RNA was observed in the fractions that contained the membrane vesicles while the highest protein concentration coincided with the fractions that contained extracellular particles. A comparative proteomics analysis by LC-MS/MS showed that 37 proteins with type-I signal peptides were consistently identified across the fractionated samples obtained from the cell-free pellets, of which 29 were orthologs detected in both strains. Functional predictions of the extracellular proteins revealed the presence of glycoside hydrolases, glycosyltransferases, giant proteins and peptidases. The extracellular transcriptomes mapped to a broad set of genes with a similar functional profile as the whole cell transcriptome. This study provides insights into the composition of membrane vesicles and extracellular proteins of a bee-associated symbiont.

Biofilm dysbiosis and caries activity: a surface or an individual issue?

This study aimed to analyze the functional profile of supragingival biofilm from sound (CAs), active (CAa), and inactive (CAi) enamel caries lesions from caries-active individuals to provide insights into the diversity of biological processes regarding biofilm dysbiosis. A metatranscriptome analysis was performed in biofilm samples collected from five caries-active individuals. Total RNA was extracted, and the microbial cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2). Bioinformatics analysis of mRNAs revealed a similar functional profile related to all analyzed conditions (CAa, CAi, and CAs). However, active and inactive surfaces share up-regulated genes (gtsA; qrtT; tqsA; pimB; EPHX1) related to virulence traits that were not overrepresented in sound surfaces. From a functional perspective, what matters most is the individual carious status rather than the surface condition. Therefore, pooling samples from various sites can be carried out using naturally developed oral biofilms but should preferably include carious surfaces. Metatranscriptome data from subjects with caries activity have shown that biofilms from sound, arrested, and active lesions are similar in composition and function.

P12.07.B Getting to the core of microglia versus bone marrow-derived macrophages in glioblastoma

Abstract Background Microglia and bone marrow-derived macrophages (BMDMs) are two ontogenetically distinct myeloid populations present within glioblastoma that can comprise 30-50% of the tumour mass. Historically, these cell types have been conflated and studied as a single population of ‘tumour-associated macrophages’. Recent advances in single-cell omics have allowed population delineation, suggesting microglia and BMDMs may play different roles within the tumour and subsequently differentially affect tumour progression. Despite building evidence for the unique functions of these cells within glioblastoma, the inherent heterogeneity of the tumour landscape has complicated such studies. Indeed, macrophages exist as phenotypically and functionally diverse populations that are polarised in a context-dependent manner. Hence, to understand the differences between microglia and BMDMs within glioblastoma, both ontogeny and spatial location must be considered. Material and Methods To elucidate the functional roles of microglia and BMDMs across the tumour landscape, a publicly available RNAseq dataset was utilised to classify myeloid cells into four populations based on spatial location and ontogeny. These were tumour core BMDMs and microglia, or tumour periphery BMDMs and microglia. Differential gene analysis was then performed to identify significant differentially expressed genes (DEGs) between classified myeloid populations. Tumour core DEGs were then compared against the Ivy Glioblastoma Atlas to define their expression across anatomical tumour regions. Finally, myeloid DEGs were validated at the protein level on human glioblastoma tissue through immunohistochemistry. Results Microglia and BMDMs showed different spatial distributions across the tumour landscape and displayed distinct functional expression profiles. Microglia held a more chemotactic and pro-inflammatory profile, whereas BMDMs held a more pro-tumoural profile. However, a comparison of microglia between the tumour core and periphery revealed that tumour microglia upregulate many pro-tumoural genes, including multiple genes that have previously been defined as ‘BMDM-enriched’. Moreover, we found myeloid DEGs identified within the tumour core cluster to distinct spatial tumour regions such as the vascular or hypoxic niche. Immunohistochemical staining reflected these spatial expression profiles, identifying a distinct population of phagocytic macrophages within the hypoxic niche. Conclusion Although microglia and BMDMs represent two ontogenetically distinct myeloid populations within glioblastoma, both cell types can adopt similar functional expression profiles within the tumour core, particularly within tumour niches. This indicates that myeloid cell function is strongly influenced by the tumour microenvironment, rather than ontogeny alone.

Developmental profiles of children at risk for autism spectrum disorder at school entry.

Functional abilities in children with autism spectrum disorder (ASD) are highly heterogenous, and impairments can overlap with non‐ASD neurodevelopmental disorders. We compared the profiles of children assessed for ASD with and without an ASD diagnosis using a retrospective cohort study of 101,739 children born in British Columbia (2000–2008). The children were grouped into the following five comparison groups: (1) ASD− (n = 1131), (2) ASD+ (n = 1583), (3) Ministry of Education designated ASD+ (n = 654), (4) special need other than ASD (n = 11,663), and (5) typically developing (n = 86,708). Five developmental domains were assessed using the Early Development Instrument. ANCOVA was used to control for covariates, Tukey's HSD test for multiple comparisons, and Cohen's d for effect size. The ASD− group had slightly higher scores than the ASD+ group with small to medium effect sizes in all domains (d = 0.20–0.48). The ASD− group had slightly higher scores than the Ministry of Education ASD+ group in only three domains with small effect sizes (d = 0.21–0.25). The ASD− group had lower scores in all domains compared to the typically developing group with large effect sizes in all domains (d = 1.12–1.77). The ASD− group received less education funding at school entry than both ASD+ groups. Overall, only small to medium differences in development were detected between the ASD− and ASD+ groups. While these children differ diagnostically, they share similar functional profiles and have substantially more difficulties than typically developing children. Therefore, differences in levels of support at school entry raise critical questions of equity.Lay SummaryComparison of children in British Columbia who have been referred for an autism assessment, with or without a diagnosis, shows similarities in their functional and developmental profiles in kindergarten. Furthermore, both groups of children have more difficulties than typically developing children. However, children who have been referred for assessment without an autism diagnosis receive less financial support at school entry, raising important questions on equity.

Betacoronavirus-specific alternate splicing.

Viruses can subvert a number of cellular processes including splicing in order to block innate antiviral responses, and many viruses interact with cellular splicing machinery. SARS-CoV-2 infection was shown to suppress global mRNA splicing, and at least 10 SARS-CoV-2 proteins bind specifically to one or more human RNAs. Here, we investigate 17 published experimental and clinical datasets related to SARS-CoV-2 infection, datasets from the betacoronaviruses SARS-CoV and MERS, as well as Streptococcus pneumonia, HCV, Zika virus, Dengue virus, influenza H3N2, and RSV. We show that genes showing differential alternative splicing in SARS-CoV-2 have a similar functional profile to those of SARS-CoV and MERS and affect a diverse set of genes and biological functions, including many closely related to virus biology. Additionally, the differentially spliced transcripts of cells infected by coronaviruses were more likely to undergo intron-retention, contain a pseudouridine modification, and have a smaller number of exons as compared with differentially spliced transcripts in the control groups. Viral load in clinical COVID-19 samples was correlated with isoform distribution of differentially spliced genes. A significantly higher number of ribosomal genes are affected by differential alternative splicing and gene expression in betacoronavirus samples, and the betacoronavirus differentially spliced genes are depleted for binding sites of RNA-binding proteins. Our results demonstrate characteristic patterns of differential splicing in cells infected by SARS-CoV-2, SARS-CoV, and MERS. The alternative splicing changes observed in betacoronaviruses infection potentially modify a broad range of cellular functions, via changes in the functions of the products of a diverse set of genes involved in different biological processes.

Critical Assessment of MetaProteome Investigation (CAMPI): a multi-laboratory comparison of established workflows

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.

Factors Driving Bacterial Microbiota of Eggs from Commercial Hatcheries of European Seabass and Gilthead Seabream.

A comprehensive understanding of how bacterial community abundance changes in fishes during their lifecycle and the role of the microbiota on health and production is still lacking. From this perspective, the egg bacterial communities of two commercially farmed species, the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata), from different aquaculture sites were compared, and the potential effect of broodstock water microbiota and disinfectants on the egg microbiota was evaluated. Moreover, 16S ribosomal RNA gene sequencing was used to profile the bacterial communities of the eggs and broodstock water from three commercial hatcheries. Proteobacteria were the most common and dominant phyla across the samples (49.7% on average). Vibrio sp. was the most highly represented genus (7.1%), followed by Glaciecola (4.8%), Pseudoalteromonas (4.4%), and Colwellia (4.2%), in eggs and water across the sites. Routinely used iodine-based disinfectants slightly reduced the eggs’ bacterial load but did not significantly change their composition. Site, species, and type of sample (eggs or water) drove the microbial community structure and influenced microbiome functional profiles. The egg and seawater microbiome composition differed in abundance but shared similar functional profiles. The strong impact of site and species on egg bacterial communities indicates that disease management needs to be site-specific and highlights the need for species- and site-specific optimization of disinfection protocols.

A multiscale approach for bridging the gap between potency, efficacy, and safety of small molecules directed at membrane proteins

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting “undruggable” regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand–membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel multiscale platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

NASH limits anti-tumour surveillance in immunotherapy-treated HCC

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1–5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH–HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH–HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH–HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

LC\u2013MS based case-by-case analysis of the impact of acidic and basic charge variants of bevacizumab on stability and biological activity

The present study investigates the impact of charge variants on bevacizumab's structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product's composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab's therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.

Optimised generation of iPSC-derived macrophages and dendritic cells that are functionally and transcriptionally similar to their primary counterparts.

Induced pluripotent stem cells (iPSC) offer the possibility to generate diverse disease-relevant cell types, from any genetic background with the use of cellular reprogramming and directed differentiation. This provides a powerful platform for disease modeling, drug screening and cell therapeutics. The critical question is how the differentiated iPSC-derived cells translate to their primary counterparts. Our refinement of a published differentiation protocol produces a CD14+ monocytic lineage at a higher yield, in a smaller format and at a lower cost. These iPSC-derived monocytes can be further differentiated into macrophages or dendritic cells (DC), both with similar morphological and functional profiles as compared to their primary counterparts. Transcriptomic analysis of iPSC-derived cells at different stages of differentiation as well as comparison to their blood-derived counterparts demonstrates a complete switch of iPSCs to cells expressing a monocyte, macrophage or DC specific gene profile. iPSC-derived macrophages respond to LPS treatment by inducing expression of classic macrophage pro-inflammatory response markers. Interestingly, though iPSC-derived DC show similarities to monocyte derived DC, they are more similar transcriptionally to a newly described subpopulation of AXL+ DC. Thus, our study provides a detailed and accurate profile of iPSC-derived monocytic lineage cells.

Overlap of functional heartburn and reflux hypersensitivity with proven gastroesophageal reflux disease

AbstractBackgroundWhen esophageal acid exposure time (AET) is physiologic, ROME IV stratifies functional esophageal disorders into functional heartburn (FH, with negative reflux‐symptom association) and reflux hypersensitivity (RH, with positive reflux‐symptom association), and hypothesizes that FH/RH could overlap with proven gastroesophageal reflux disease (GERD). We compared conventional FH/RH diagnosed off antisecretory therapy with overlap FH/RH coexisting with proven GERD.MethodsAdult patients with heartburn underwent pH‐impedance testing on therapy (proven GERD) and off therapy (unproven GERD) and completed validated questionnaires defining symptoms (Reflux Diagnostic Questionnaire, RDQ), affective state (Hospital Anxiety and Depression Scale, HADS; Visceral Sensitivity Index, VSI), and functional diagnoses (irritable bowel syndrome, IBS; functional dyspepsia, FD on ROME diagnostic questionnaire). AET (pathologic: >6%, physiologic <4%) and mean nocturnal baseline impedance (MNBI) were calculated. Clinical, psychological, and functional profiles were compared between FH and RH and to a comparison cohort of non‐erosive reflux disease (NERD, AET > 6%).Key ResultsStudy patients included 19 with RH (10 with overlap RH), 60 with FH (31 with overlap FH), and 26 patients with NERD. Clinical, psychological, and functional profiles of conventional FH/RH were similar to overlap FH/RH. Compared with NERD, combined FH and RH cohorts had higher anxiety scores, and lower proportions with IBS. Compared with FH with low MNBI, FH defined using both normal AET and normal MNBI had higher depression scores (p = 0.008), and trended toward higher anxiety scores (p = 0.059).Conclusions and InferencesConventional and overlap FH and RH have similar clinical, psychological and functional profiles, thereby proving ROME IV hypotheses.

Metagenome assembled-genomes reveal similar functional profiles of CPR/Patescibacteria phyla in soils.

Soil microbiome is one of the most heterogeneous biological systems. State-of-the-art molecular approaches such as those based on single-amplified genomes (SAGs) and metagenome assembled-genomes (MAGs) are now improving our capacity for disentailing soil microbial-mediated processes. Here, we analysed publicly available datasets of soil microbial genomes and MAG's reconstructed from the Amazon's tropical soil (primary forest and pasture) and active layer of permafrost, aiming to evaluate their genome size. Our results suggest that the Candidate Phyla Radiation (CPR)/Patescibacteria phyla have genomes with an average size fourfold smaller than the mean identified in the RefSoil database, which lacks any representative of this phylum. Also, by analysing the potential metabolism of 888 soil microbial genomes, we show that CPR/Patescibacteria representatives share similar functional profiles, but different from other microbial phyla and are frequently neglected in the soil microbial surveys. Finally, we argue that the use of MAGs may be a better choice over SAGs to expand the soil microbial databases, like RefSoil.