Biology subjects

Graf, J.

Publications and source records attributed to Graf, J..

27 records · Page 2Linked to original sources

bioRxiv2021

Stratification Of Chemotherapy-Treated Stage III Colorectal Cancer Patients Using Multiplexed Imaging And Single Cell Analysis Of T Cell Populations

Colorectal cancer (CRC) has one of the highest cancer incidences and mortality rates. In stage III, postoperative chemotherapy benefits <20% of patients, while more than 50% will develop distant metastases. Predictive biomarkers for identification of patients with increased risk for disease recurrence are currently lacking, with progress in biomarker discovery hindered by the diseases inherent heterogeneity. The immune profile of colorectal tumors has previously been found to have prognostic value. The aims of this study were to evaluate immune signatures in the tumor microenvironment (TME) using an in situ multiplexed immunofluorescence imaging and single cell analysis technology (Cell DIVE). Tissue microarrays (TMAs) with up to three 1mm diameter cores per patient were prepared from 117 stage III CRC patients treated with adjuvant fluoropyrimidine/oxaliplatin chemotherapy. Single sections underwent multilplexed immunofluorescence with Cy3- and Cy5-conjugated antibodies for immune cell markers (CD45, CD3, CD4, CD8, FOXP3, PD1) and cell segmentation markers (DAPI, pan-cytokeratin, AE1, NaKATPase and S6). We applied a probabilistic multi-class, multi-label classification algorithm based on multi-parametric models to build statistical models of protein expression to classify immune cells. Expert annotations of immune cell markers were made on a range of images, and Support Vector Machines (SVM) were used to derive a statistical model for cell classification. Images were also manually scored independently by a Pathologist as high, moderate or low, for stromal and total immune cell content. Excellent agreement was found between manual and total automated scores (p<0.0001). Higher levels of multi-marker classified regulatory T cells (CD3+CD4+FOXP3+PD1-) were significantly associated with disease-free survival (DFS) and overall-survival (OS) (p=0.049 and 0.032), compared to FOXP3 alone. Our results also showed that PD1- Tregs rather than PD1+ Tregs were associated with improved survival. Overall, compared to single markers, multi-marker classification provided more accurate quantitation of immune cells with greater potential for predicting patient outcomes.

cancer biology↗

bioRxiv2020

Multicohort Analysis of Publicly-available Monocyte Expression Data Identifies Gene Signatures to Accurately Monitor Subset-specific Changes in Human Diseases

Monocytes and monocyte-derived cells play important roles in the regulation of inflammation, both as precursors as well as effector cells. Monocytes are heterogeneous and characterized by three distinct subsets in humans. Classical and non-classical monocytes represent the most abundant subsets, each carrying out distinct biological functions. Consequently, altered frequencies of different subsets have been associated with inflammatory conditions, such as infections and autoimmune disorders including lupus, rheumatoid arthritis, inflammatory bowel disease, and, more recently, COVID-19. Dissecting the contribution of different monocyte subsets to disease is currently limited by samples and cohorts, often resulting in underpowered studies and, consequently, poor reproducibility. Public transcriptomes provide an alternative source of data characterized by high statistical power and real world heterogeneity. However, most transcriptome datasets profile bulk blood or tissue samples, requiring the use of in silico approaches to quantify changes in the levels of specific cell types. Here, we integrated 853 publicly available microarray expression profiles of sorted human monocyte subsets from 45 independent studies to identify robust and parsimonious gene expression signatures, consisting of 10 genes specific to each subset. These signatures, although derived using only datasets profiling healthy individuals, maintain their accuracy independent of the disease state in an independent cohort profiled by RNA-sequencing (AUC = 1.0). Furthermore, we demonstrate that our signatures are specific to monocyte subsets compared to other immune cells such as B, T, dendritic cells (DCs) and natural killer (NK) cells (AUC = 0.87~0.88, p<2.2e-16). This increased specificity results in estimated monocyte subset levels that are strongly correlated with cytometry-based quantification of cellular subsets (r = 0.69, p = 6.7e-4). Consequently, we show that these monocyte subset-specific signatures can be used to quantify changes in monocyte subsets levels in expression profiles from patients in clinical trials. Finally, we show that proteins encoded by our signature genes can be used in cytometry-based assays to specifically sort monocyte subsets. Our results demonstrate the robustness, versatility, and utility of our computational approach and provide a framework for the discovery of new cellular markers.

systems biology↗

bioRxiv2020

Simultaneous Single-Cell Genome and Transcriptome Sequencing of Termite Hindgut Protists Reveals Metabolic and Evolutionary Traits of Their Endosymbionts

Different protist species which colonize the hindguts of wood feeding Reticulitermes termites are associated with endosymbiotic bacteria belonging to the genus Endomicrobium. In this study, we focused on the endosymbionts of three protist species from Reticulitermes flavipes, which included Pyrsonympha vertens, Trichonympha agilis, and Dinenympha species II. Since these protist hosts represented members of difference taxa which colonize different niches within the hindguts of their termite hosts, we investigated if these differences translated to differential gene content and expression in their endosymbionts. Following assembly and comparative genome and transcriptome analyses, we discovered that these endosymbionts differed with respect to possible niche specific traits such carbon metabolism. Our analyses supported that genes related to carbon metabolism were acquired by horizontal gene transfer (HGT) from donor taxa which are present in termites hindgut community. In addition, our analyses supported that these endosymbionts have retained and expressed genes related to natural transformation (competence) and recombination. Taken together, the presence of genes acquired by HGT and a putative competence pathway supported that these endosymbionts are not cut-off from gene flow and that competence may be a mechanism by which members of the Endomicrobium can acquire new traits. ImportanceThe composition and structure of wood, which contains cellulose, hemicellulose and lignin, prevents most organisms from using this common food source. Termites are a rare exception among animals, and they rely on a complex microbiome housed in their hindguts to use wood as a source of food. The lower termite R. flavipes houses a variety of protist and prokaryotes that are the key players the disassembly of lignocellulose. In this paper we describe the genomes and the gene expression profiles of five Endomicrobium endosymbionts living inside three different protist species from R. flavipes. Data from these genomes suggest that these Endomicrobium species have different mechanisms for using both carbon and nitrogen. In addition, they harbor genes that may be used to import free DNA from their environment. This process of DNA-uptake may cntribute to the high levels of horizontal gene transfer often seen in the Endomicrobium species.

microbiology↗

bioRxiv2020

Systematic Detection of Large-Scale Multi-Gene Horizontal Transfer in Prokaryotes

Horizontal gene transfer (HGT) is central to prokaryotic evolution. However, little is known about the "scale" of individual HGT events. In this work, we introduce the first computational framework to help answer the following fundamental question: How often does more than one gene get horizontally transferred in a single HGT event? Our method, called HoMer, uses phylogenetic reconciliation to infer single-gene HGT events across a given set of species/strains, employs several techniques to account for inference error and uncertainty, combines that information with gene order information from extant genomes, and uses statistical analysis to identify candidate horizontal multi-gene transfers (HMGTs) in both extant and ancestral species/strains. HoMer is highly scalable and can be easily used to infer HMGTs across hundreds of genomes. We apply HoMer to a genome-scale dataset of over 22000 gene families from 103 Aeromonas genomes and identify a large number of plausible HMGTs of various scales at both small and large phylogenetic distances. Analysis of these HMGTs reveals interesting relationships between gene function, phylogenetic distance, and frequency of multi-gene transfer. Among other insights, we find that (i) the relative frequency of HMGT increases as divergence between genomes increases, (ii) HMGTs often have conserved gene functions, and (iii) rare genes are frequently acquired through HMGT. We also analyze in detail HMGTs involving the zonula occludens toxin and type III secretion systems. By enabling the systematic inference of HMGTs on a large scale, HoMer will facilitate a more accurate and more complete understanding of HGT and microbial evolution.

evolutionary biology↗

bioRxiv2020

Intraneuronal chloride accumulation via NKCC1 is not essential for hippocampal network development in vivo

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in-vitro excitatory GABA actions and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics and behavioral performance are subtle. Our data reveal a neural network function of depolarizing GABA in the hippocampus in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

neuroscience↗

bioRxiv2020

Effects of host species identity and diet on biodiversity of oral and rectal microbiomes of Puerto Rican bats

Microbiomes perform vital functions for their mammalian hosts, making them potential drivers of host evolution. Understanding effects of environmental factors and host characteristics on the composition and biodiversity of microbiomes may provide novel insights into the origin and maintenance of these symbiotic relationships. Our goals were to (1) characterize biodiversity of oral and rectal microbiomes of bats in Puerto Rico; and (2) determine the effects of geographic location and host characteristics on that biodiversity. We collected bats and their microbiomes from 3 sites, and used 4 metrics (species richness, Shannon diversity, Camargo evenness, Berger-Parker dominance) to characterize biodiversity. We evaluated the relative importance of site, host sex, host species identity, and host foraging guild on microbiome biodiversity. Microbiome biodiversity was highly variable among conspecifics. Geographical location exhibited consistent effects, whereas host sex did not do so. Within each host guild, host species exhibited consistent differences in oral and rectal microbiome biodiversity. Oral microbiome biodiversity was indistinguishable between guilds, whereas rectal microbiome biodiversity was significantly greater in carnivores than in herbivores. The high intraspecific and spatial variation in microbiome biodiversity necessitate a large number of samples to isolate the effects of environmental or host characteristics on microbiomes. Species-specific biodiversity of oral microbiomes suggests these communities are structured by direct interactions with the host immune system via epithelial receptors. In contrast, the number of microbial taxa that a host gut supports may be contingent on the number and kinds of functions a host requires of its microbiome.

ecology↗

bioRxiv2019

Macrobdella decora: Old World Leech Gut Microbial Community Structure Conserved in a New World Leech

Leeches are found in terrestrial, aquatic, and marine habitats on all continents. Sanguivorous leeches have been used in medicine for millennia. Modern scientific uses include studies of neurons, anticoagulants, and gut microbial symbioses. Hirudo verbana, the European medicinal leech, maintains a gut community dominated by two bacterial symbionts, Aeromonas veronii and Mucinivorans hirudinis, which sometimes account for as much as 97% of the total crop microbiota. The highly simplified gut anatomy and microbiome of H. verbana make it an excellent model organism for studying gut microbial dynamics. The North American medicinal leech, Macrobdella decora, is a hirudinid leech native to Canada and the northern U.S.A. In this study we show that M. decora symbiont communities are very similar to those in H. verbana. This similarity allowed for an extensive study in which wild caught animals were sampled to determine effects of geographic separation, time of collection, and feeding on the microbiome. Through 16S V4 rRNA deep sequencing we show that: i) the M. decora gut and bladder microbial communities are distinct, ii) the M. decora gut community is affected by feeding and long periods of starvation, and iii) geographic separation does not appear to affect the overall gut microbial community structure. We propose that M. decora is a replacement for H. verbana for studies of wild-caught animals and offer evidence for the conservation of annelid symbionts. Successful culturing and comparison of dominant symbionts from M. decora and H. verbena will provide the ability to assess host-symbiont co-evolution in future work.\n\nIMPORTANCEBuilding evidence implicates the gut microbiome in regulating animal digestion, nutritional acquisition, immune regulation, development, and even mood regulation. Because of the difficulty of assigning causative relationships in complex gut microbiomes a simplified model for testing hypotheses is necessary. Previous research in Hirudo verbana has suggested this animal as a highly simplified and tractable animal model of gut symbioses. Our data show that Macrobdella decora may work just as well as H. verbana without the drawback of being an endangered organism and with the added convenience of easy access to field-caught specimens. The similarity of the microbial community structure of species from two different continents reveals the highly-conserved nature of the microbial symbionts in sanguivorous leeches and confirms the medicinal leech as a highly simplified, natural animal model in which to study gut symbioses.

microbiology↗

bioRxiv2019

Identification and characterization of putative Aeromonas spp. T3SS effectors

The genetic determinants of bacterial pathogenicity are highly variable between species and strains. However, a factor that is commonly associated with virulent Gram-negative bacteria, including many Aeromonas spp., is the type 3 secretion system (T3SS), which is used to inject effector proteins into target eukaryotic cells. In this study, we developed a bioinformatics pipeline to identify T3SS effector proteins, applied this approach to the genomes of 105 Aeromonas strains isolated from environmental, mutualistic, or pathogenic contexts and evaluated the cytotoxicity of the identified effectors through their heterologous expression in yeast. The developed pipeline uses a two-step approach, where candidate families are initially selected using HMM profiles with minimal similarity scores against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. Using our approach, we identified 21 Aeromonas T3SS likely effector groups, of which 8 represented known or characterized effectors, while the remaining 13 had not previously been described in Aeromonas. We experimentally validated our in silico findings by assessing the cytotoxicity of representative effectors in Saccharomyces cerevisiae BY4741, with 15 out of 21 assayed proteins eliciting a cytotoxic effect in yeast. The results of this study demonstrate the utility of our approach, combining a novel in silico search method with in vivo experimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera.

microbiology↗

bioRxiv2018

Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing

BackgroundShort-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated.\n\nResultsWe sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions.\n\nConclusionsGenome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes and enabled the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing is cost effective and dramatically advances studies of microbial evolution and genome-driven drug discovery.

genomics↗