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Abstract Background and aims Previously, we found that FK506 binding protein 51 (Fkbp51) knockout (KO) mice resist high fat diet-induced fatty liver and alcohol-induced liver injury. The aim of this research is to identify the mechanism of Fkbp51 in liver injury. Methods Carbon tetrachloride (CCl4)-induced liver injury was compared between Fkbp51 KO and wild type (WT) mice. Step-wise and in-depth analyses were applied, including liver histology, biochemistry, RNA-Seq, mitochondrial respiration, electron microscopy, and molecular assessments. The selective FKBP51 inhibitor (SAFit2) was tested as a potential treatment to ameliorate liver injury. Results Fkbp51 knockout mice exhibited protection against liver injury, as evidenced by liver histology, reduced fibrosis-associated markers and lower serum liver enzyme levels. RNA-seq identified differentially expressed genes and involved pathways, such as fibrogenesis, inflammation, mitochondria, and oxidative metabolism pathways and predicted the interaction of FKBP51, Parkin, and HSP90. Cellular studies supported co-localization of Parkin and FKBP51 in the mitochondrial network, and Parkin was shown to be expressed higher in the liver of KO mice at baseline and after liver injury relative to WT. Further functional analysis identified that KO mice exhibited increased ATP production and enhanced mitochondrial respiration. KO mice have increased mitochondrial size, increased autophagy/mitophagy and mitochondrial-derived vesicles (MDV), and reduced reactive oxygen species (ROS) production, which supports enhancement of mitochondrial quality control (MQC). Application of SAFit2, an FKBP51 inhibitor, reduced the effects of CCl4-induced liver injury and was associated with increased Parkin, pAKT, and ATP production. Conclusions Downregulation of FKBP51 represents a promising therapeutic target for liver disease treatment. Graphical Abstract

Abstract Subcutaneous patient-derived xenografts (PDXs) are an important tool for childhood cancer research. Here, we describe a resource of 68 early passage PDXs established from 65 pediatric solid tumor patients. Through genomic profiling of paired PDXs and patient tumors (PTs), we observe low mutational similarity in about 30% of the PT/PDX pairs. Clonal analysis in these pairs show an aggressive PT minor subclone seeds the major clone in the PDX. We show evidence that this subclone is more immunogenic and is likely suppressed by immune responses in the PT. These results suggest interplay between intratumoral heterogeneity and antitumor immunity may underlie the genetic disparity between PTs and PDXs. We further show that PDXs generally recapitulate PTs in copy number and transcriptomic profiles. Finally, we report a gene fusion LRPAP1-PDGFRA. In summary, we report a childhood cancer PDX resource and our study highlights the role of immune constraints on tumor evolution.

Abstract Mechanisms of sex-dependent orofacial pain are widely understudied. A significant gap in knowledge exists about comprehensive regulation of tissue-specific trigeminal sensory neurons in diseased state of both sexes. Using RNA sequencing of FACS sorted retro-labeled sensory neurons innervating tongue tissue, we determined changes in transcriptomic profiles in males and female mice under naïve as well as tongue-tumor bearing conditions Our data revealed the following interesting findings: (1) FACS sorting obtained higher number of neurons from female trigeminal ganglia (TG) compared to males; (2) Naïve female neurons innervating the tongue expressed immune cell markers such as Csf1R, C1qa and others, that weren’t expressed in males. This was validated by Immunohistochemistry. (3) Accordingly, immune cell markers such as Csf1 exclusively sensitized TRPV1 responses in female TG neurons. (4) Male neurons were more tightly regulated than female neurons upon tumor growth and very few differentially expressed genes (DEGs) overlapped between the sexes, (5) Male DEGs contained higher number of transcription factors whereas female DEGs contained higher number of enzymes, cytokines and chemokines. Collectively, this is the first study to characterize the effect of sex as well as of tongue-tumor on global gene expression, pathways and molecular function of tongue-innervating sensory neurons.

Summary: Patient-derived xenografts (PDX) remain valuable models for understanding the biology and for developing novel therapeutics. To expand current PDX models of childhood leukemia, we have developed new PDX models from Hispanic patients, a subgroup with a poorer overall outcome. Of 117 primary leukemia samples obtained, successful engraftment and serial passage in mice were achieved in 82 samples (70%). Hispanic patient samples engrafted at a rate (51/73, 70%) that was similar to non-Hispanic patient samples (31/45, 70%). With a new algorithm to remove mouse contamination in multi-omics datasets including methylation data, we found PDX models faithfully reflected somatic mutations, copy-number alterations, RNA expression, gene fusions, whole-genome methylation patterns, and immunophenotypes found in primary tumor (PT) samples in the first 50 reported here. This cohort of characterized PDX childhood leukemias represents a valuable resource in that germline DNA sequencing has allowed the unambiguous determination of somatic mutations in both PT and PDX.

Non-neuronal cells constitute 90%–95% of sensory ganglia. These cells, especially glial and immune cells, play critical roles in the modulation of sensory neurons. This study aimed to identify, profile, and summarize the types of trigeminal ganglion (TG) non-neuronal cells in naïve male mice using published and our own data generated by single-cell RNA sequencing, flow cytometry, and immunohistochemistry. TG has five types of non-neuronal cells, namely, glial, fibroblasts, smooth muscle, endothelial, and immune cells. There is an agreement among publications for glial, fibroblasts, smooth muscle, and endothelial cells. Based on gene profiles, glial cells were classified as myelinated and non-myelinated Schwann cells and satellite glial cells. Mpz has dominant expression in Schwann cells, and Fabp7 is specific for SCG. Two types of Col1a2+ fibroblasts located throughout TG were distinguished. TG smooth muscle and endothelial cells in the blood vessels were detected using well-defined markers. Our study reported three types of macrophages (Mph) and four types of neutrophils (Neu) in TG. Mph were located in the neuronal bodies and nerve fibers and were sub-grouped by unique transcriptomic profiles with Ccr2, Cx3cr1, and Iba1 as markers. A comparison of databases showed that type 1 Mph is similar to choroid plexus-low (CPlo) border-associated Mph (BAMs). Type 2 Mph has the highest prediction score with CPhi BAMs, while type 3 Mph is distinct. S100a8+ Neu were located in the dura surrounding TG and were sub-grouped by clustering and expressions of Csf3r, Ly6G, Ngp, Elane, and Mpo. Integrative analysis of published datasets indicated that Neu-1, Neu-2, and Neu-3 are similar to the brain Neu-1 group, while Neu-4 has a resemblance to the monocyte-derived cells. Overall, the generated and summarized datasets on non-neuronal TG cells showed a unique composition of myeloid cell types in TG and could provide essential and fundamental information for studies on cell plasticity, interactomic networks between neurons and non-neuronal cells, and function during a variety of pain conditions in the head and neck regions.

Obesity is an enormous global health problem, and obesity‐induced nonalcoholic steatohepatitis (NASH) is contributing to a rising incidence and mortality for hepatocellular carcinoma (HCC). Increase in de novo lipogenesis and decrease in fatty acid β‐oxidation (FAO) underlie hepatic lipid accumulation in NASH. Astrocyte‐elevated gene‐1/metadherin (AEG‐1) overexpression contributes to both NASH and HCC. AEG‐1 harbors an LXXLL motif through which it blocks activation of peroxisome proliferator activated receptor α (PPARα), a key regulator of FAO. To better understand the role of LXXLL motif in mediating AEG‐1 function, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, we generated a mouse model (AEG‐1‐L24K/L25H) in which the LXXLL motif in AEG‐1 was mutated to LXXKH. We observed increased activation of PPARα in AEG‐1‐L24K/L25H livers providing partial protection from high‐fat diet–induced steatosis. Interestingly, even with equal gene dosage levels, compared with AEG‐1–wild‐type livers, AEG‐1‐L24K/L25H livers exhibited increase in levels of lipogenic enzymes, mitogenic activity and inflammation, which are attributes observed when AEG‐1 is overexpressed. These findings indicate that while LXXLL motif favors steatotic activity of AEG‐1, it keeps in check inflammatory and oncogenic functions, thus maintaining a homeostasis in AEG‐1 function. AEG‐1 is being increasingly appreciated as a viable target for ameliorating NASH and NASH‐HCC, and as such, in‐depth understanding of the functions and molecular attributes of this molecule is essential. Conclusion: The present study unravels the unique role of the LXXLL motif in mediating the balance between the metabolic and oncogenic functions of AEG‐1.

Abstract Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3–L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3–L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Abstract Background Studies of the gut microbiome are becoming increasingly important. Such studies require stool collections that can be processed or frozen in a timely manner so as not to alter the microbial content. Due to the logistical difficulties of home-based stool collection, there has been a challenge in selecting the appropriate sample collection technique and comparing results from different microbiome studies. Thus, we compared stool collection and two alternative clinic-based fecal microbiome collection techniques, including a newer glove-based collection method. Results We prospectively enrolled 22 adult men from our prostate cancer screening cohort SABOR (San Antonio Biomarkers of Risk for prostate cancer) in San Antonio, TX, from 8/2018 to 4/2019. A rectal swab and glove tip sample were collected from each participant during a one-time visit to our clinics. A single stool sample was collected at the participant’s home. DNA was isolated from the fecal material and 16 s rRNA sequencing of the V1-V2 and V3-V4 regions was performed. We found the gut microbiome to be similar in richness and evenness, noting no differences in alpha diversity among the collection methods. The stool collection method, which remains the gold-standard method for the gut microbiome, proved to have different community composition compared to swab and glove tip techniques (p 0.05). Despite differences between home-based stool and office-based fecal collection methods, we noted that the distance metrics for the three methods cluster by participant indicating within-person similarities. Additionally, no taxa differed among the methods in a Linear Discriminant Analysis Effect Size (LEfSe) analysis comparing all-against-all sampling methods. Conclusion The glove tip method provides similar gut microbiome results as rectal swab and stool microbiome collection techniques. The addition of a new office-based collection technique could help easy and practical implementation of gut microbiome research studies and clinical practice.

Cells reprogram their transcriptome in response to stress, such as heat shock. In free-living bacteria, the transcriptomic reprogramming is mediated by increased DNA-binding activity of heat shock sigma factors and activation of genes normally repressed by heat-induced transcription factors. In this study, we performed transcriptomic analyses to investigate heat shock response in the obligate intracellular bacterium Chlamydia trachomatis, whose genome encodes only three sigma factors and a single heat-induced transcription factor. Nearly one-third of C. trachomatis genes showed statistically significant (≥1.5-fold) expression changes 30 min after shifting from 37 to 45°C. Notably, chromosomal genes encoding chaperones, energy metabolism enzymes, type III secretion proteins, as well as most plasmid-encoded genes, were differentially upregulated. In contrast, genes with functions in protein synthesis were disproportionately downregulated. These findings suggest that facilitating protein folding, increasing energy production, manipulating host activities, upregulating plasmid-encoded gene expression, and decreasing general protein synthesis helps facilitate C. trachomatis survival under stress. In addition to relieving negative regulation by the heat-inducible transcriptional repressor HrcA, heat shock upregulated the chlamydial primary sigma factor σ66 and an alternative sigma factor σ28. Interestingly, we show for the first time that heat shock downregulates the other alternative sigma factor σ54 in a bacterium. Downregulation of σ54 was accompanied by increased expression of the σ54 RNA polymerase activator AtoC, thus suggesting a unique regulatory mechanism for reestablishing normal expression of select σ54 target genes. Taken together, our findings reveal that C. trachomatis utilizes multiple novel survival strategies to cope with environmental stress and even to replicate. Future strategies that can specifically target and disrupt Chlamydia’s heat shock response will likely be of therapeutic value.

ABSTRACT Chlamydia trachomatis is an obligate intracellular bacterium whose unique developmental cycle consists of an infectious elementary body and a replicative reticulate body. Progression of this developmental cycle requires temporal control of the transcriptome. In addition to the three chlamydial sigma factors (σ66, σ28, and σ54) that recognize promoter sequences of genes, chlamydial transcription factors are expected to play crucial roles in transcriptional regulation. Here, we investigate the function of GrgA, a Chlamydia-specific transcription factor, in C. trachomatis transcriptomic expression. We show that 10 to 30 min of GrgA overexpression induces 13 genes, which likely comprise the direct regulon of GrgA. Significantly, σ66-dependent genes that code for two important transcription repressors are components of the direct regulon. One of these repressors is Euo, which prevents the expression of late genes during early phases. The other is HrcA, which regulates molecular chaperone expression and controls stress response. The direct regulon also includes a σ28-dependent gene that codes for the putative virulence factor PmpI. Furthermore, overexpression of GrgA leads to decreased expression of almost all tRNAs. Transcriptomic studies suggest that GrgA, Euo, and HrcA have distinct but overlapping indirect regulons. These findings, together with temporal expression patterns of grgA, euo, and hrcA, indicate that a transcriptional regulatory network of these three transcription factors plays critical roles in C. trachomatis growth and development. IMPORTANCE Chlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide and is a leading cause of preventable blindness in underdeveloped areas as well as some developed countries. Chlamydia carries genes that encode a limited number of known transcription factors. While Euo is thought to be critical for early chlamydial development, the functions of GrgA and HrcA in the developmental cycle are unclear. Activation of euo and hrcA immediately following GrgA overexpression indicates that GrgA functions as a master transcriptional regulator. In addition, by broadly inhibiting tRNA expression, GrgA serves as a key regulator of chlamydial protein synthesis. Furthermore, by upregulating pmpI, GrgA may act as an upstream virulence determinant. Finally, genes coregulated by GrgA, Euo, and HrcA likely play critical roles in chlamydial growth and developmental control.