Skin Resident Memory T Cell Dysfunction In The Tibia Fracture Model Of Complex Regional Pain Syndrome

The Journal of Pain

2023 Apr 01

Wickman, J;Shenoda, B;Van Duyne, R;Kline, Z;Ajit, S;
| DOI: 10.1016/j.jpain.2023.02.060

Complex regional pain syndrome (CRPS) is a debilitating chronic pain disorder that with no effective treatments. Several microRNA (miRNA) are commonly dysregulated in CRPS patient and tibia fracture model of CRPS (TFM) mice, including miR-25 which is associated with positive treatment outcomes in patients. Interestingly, these miRNAs are predicted to target several genes critical to resident memory T cell (Trm) function. We hypothesize that miRNA dysregulation contributes to the pathology of CRPS through regulation of skin Trm development and maintenance. Therapeutic strategies blocking Trm development or maintenance may be beneficial in treating this disease. Whole blood samples were obtained from CRPS patients or healthy controls. miRNA and gene expression changes in blood and T cells were assessed by qPCR. Animals were treated with therapeutic agents after development of TFM and monitored for behavioral outcomes and T cell populations of collected tissues were analyzed at different time points by flow cytometry. There was an inverse correlation of miR-25 and CD69 in blood samples from CRPS patients compared to controls. TFM hindlimb skin shows increased epidermal CD8+ and CD4+ Trm, dermal CD4+ Trm. Epidermal CD8+ Trm, dermal CD4+ Trm are marked by increases in CD103+CD49a+ populations, and along with splenic CD8+ Tem show increased CD122+ cells. Therapeutic studies are ongoing. miRNA signatures in CRPS patients and TFM mice show common alterations which are capable of regulating CD69, a core Trm marker. TFM hindlimb skin shows increased pathological Trm populations and treatments targeting Trm development and maintenance may be beneficial in treating CRPS. 1RF1NS130481-01.

Mfge8 is expressed by pericytes in human gastric antrum submucosa from obese patients

American journal of physiology. Cell physiology

2023 Mar 20

Perrino, BA;Malogan, J;Cobine, CA;Sasse, KC;
PMID: 36939201 | DOI: 10.1152/ajpcell.00043.2023

The main function of the stomach is to digest ingested food. Gastric antrum muscular contractions mix ingested food with digestive enzymes and stomach acid and propel the chyme through the pyloric sphincter at a rate in which the small intestine can process the chyme for optimal nutrient absorption. Mfge8 binding to α8β1 integrins helps regulate gastric emptying by reducing the force of antral smooth muscle contractions. The source of Mfge8 within gastric muscles is unclear. Since Mfge8 is a secreted protein, Mfge8 could be delivered via the circulation, or be locally secreted by cells within the muscle layers. In this study we identify a source of Mfge8 within human gastric antrum muscles using spatial transcriptomic analysis. We show that Mfge8 is expressed in subpopulations of Mef2c+ perivascular cells within the submucosa layer of the gastric antrum. Mef2c is expressed in subpopulations of NG2+ and PDGFRB+ pericytes. Mfge8 is expressed in NG2+/Mef2c+ pericytes, but not in NG2+/Mef2c-, PDGFRB+/Mef2c-, or PDGFRB+/Mef2c+ pericytes. Mfge8 is absent from CD34+ endothelial cells but is expressed in a small population of perivascular ACTA2+ cells. We also show that α8 integrin is not expressed by ICC, supporting the findings that Mfge8 attenuates gastric antrum smooth muscle contractions by binding to α8β1 integrins on enteric smooth muscle cells. These findings suggest a novel, supplementary mechanism of regulation of gastric antrum motility by cellular regulators of capillary blood flow, in addition to the regulation of gastric antrum motility by the enteric nervous system and the SIP syncytium.

The infertile individual analysis based on whole-exome sequencing in chinese multi-ethnic groups

Genes & genomics

2022 Sep 17

Zhao, Q;Li, Y;Liang, Q;Zhao, J;Kang, K;Hou, M;Zhang, X;Du, R;Kong, L;Liang, B;Huang, W;
PMID: 36115009 | DOI: 10.1007/s13258-022-01307-z

Infertility is a common and rapidly growing health issue around the world. The genetic analysis based on the infertile population is crucial for intervention and treatment.To find candidate gene locus led to azoospermia in Chinese multi-ethnic groups and provide theoretical guidance for the diagnosis of genetic diseases to progressively aggravated infertility patients and sterile offspring with ART.The study based on whole-exome sequencing (WES) was presented for genetic characteristic analysis of multi-ethnics and identification of variants related to infertility in Xinjiang area of China.The frequency of pathogenic variants showed significant ethnic differences among four main ethnics in Xinjiang. The population structure analysis confirmed that the Hui was close to the Han population, the Kazak was close to the Uygur population, and there are three ancestry components in the four ethnics. In addition, ten candidate variants potentially regulated azoospermia were detected, and KNTC1 (rs7968222: G > T) was chosen to validate the association. Through the analysis in the valid group, the frequency of rs7968222 (G > T) has a significant difference in the azoospermia population (11.76%, 8/68) and normospermia population (4.63%, 35/756) (P < 0.001). Interestingly, the proportion of people with abnormal follicle-stimulating hormone (FSH) level in the group carrying rs7968222 (G > T) was significantly higher than non-carriers (P < 0.05). Therefore, rs7968222 may regulate spermatogenesis through affecting hormone level.Our study establishes the genetics analysis of Northwest China and finds a candidate gene locus KNTC1 (rs7968222: G > T), which is one of the genetic susceptibility factors for male azoospermia.

Role of Ecogenomics in Conservation and Management

Plant Ecogenomics

2022 Aug 19

Islam, S;Mangral, Z;Tariq, L;Bhat, B;Mehraj, H;Islam, S;Dar, T;
| DOI: 10.1201/9781003282006-9

Ecogenomics is the scientific approach to understand the relationship between structural and functional aspects of genomes with biotic/abiotic environmental factors. The classification of ecology depends upon the overall complexity (behavioral and population ecology) of an organism (plant and animal ecology) and system under investigation (soil and forest ecology). The molecular techniques adopted by these various ecology branches result in a new field known as ecological genomics or ecogenomics that focuses on an organism’s overall development during the evolutionary period. It is an interdisciplinary research field covering ecological science, microbiology, 176environmental and molecular biology, toxicology, physiology, chemistry, etc. The study related to the ecology of plants has a direct relationship with the adaptation mechanism because plants have no alternative to cope with the environment in which they grow. The world is facing biodiversity loss at an alarming rate, with a loss of 90% crop varieties in a century from the field. It is essential to select the genes in biological pathways responsible for an organism’s stability in the ecological system. The challenge is to understand the basic phenomenon behind adaptation, migration, inbreeding mechanism of endangered or critically endangered species. in ecogenomics, we know that the genes are responsible for the effective management strategies from a conservation point of view. Therefore, in this chapter, we discuss various aspects of community shaping and visualization. The community’s function and structure need to be studied due to the availability of plenty of molecular data. Further, the role of ecogenomics and multiomic approaches in conservation and management will also be emphasized.

Quantitative Imaging Analysis of the Spatial Relationship between Antiretrovirals, Reverse Transcriptase Simian-Human Immunodeficiency Virus RNA, and Fibrosis in the Spleens of Nonhuman Primates

Antimicrobial agents and chemotherapy

2022 Jul 20

Devanathan, AS;White, NR;Desyaterik, Y;De la Cruz, G;Nekorchuk, M;Terry, M;Busman-Sahay, K;Adamson, L;Luciw, P;Fedoriw, Y;Estes, JD;Rosen, EP;Kashuba, ADM;
PMID: 35856680 | DOI: 10.1128/aac.00609-22

Although current antiretroviral therapy (ART) has increased life expectancy, a cure for human immunodeficiency virus (HIV) remains elusive due to the persistence of the virus in tissue reservoirs. In the present study, we sought to elucidate the relationship between antiretrovirals (ARVs) and viral expression in the spleen. We performed mass spectrometry imaging (MSI) of 6 different ARVs, RNAscope in situ hybridization of viral RNA, and immunohistochemistry of three different fibrosis markers in the spleens of 8 uninfected and 10 reverse transcriptase simian-human immunodeficiency virus (RT-SHIV)-infected rhesus macaques (infected for 6 weeks) that had been dosed for 10 days with combination ART. Using MATLAB, computational quantitative imaging analysis was performed to evaluate the spatial and pharmacological relationships between the 6 ARVs, viral RNA, and fibrotic deposition. In these spleens, >50% of the spleen tissue area was not covered by any detectable ARV response (any concentration above the limits of detection for individual ARVs). The median spatial ARV coverage across all tissues was driven by maraviroc followed by efavirenz. Yet >50% of RNA-positive cells were not exposed to any detectable ARV. Quantifiable maraviroc and efavirenz colocalization with RNA-positive cells was usually greater than the in vitro concentration inhibiting 50% replication (IC50). Fibrosis markers covered more than 50% of the spleen tissue area and had negative relationships with cumulative ARV coverages. Our findings suggest that a heterogeneous ARV spatial distribution must be considered when evaluating viral persistence in lymphoid tissue reservoirs.

Detection accuracy of the Cobas HPV assay for high-risk HPV in head and neck FNA biopsy specimens

Cancer cytopathology

2022 Feb 22

Guo, M;Khanna, A;Tinnirello, AA;Hwang, J;Zhang, P;Xu, L;Li, G;Dahlstrom, KR;Sturgis, EM;Stewart, J;
PMID: 35192231 | DOI: 10.1002/cncy.22563

This study evaluated the detection accuracy of the Cobas human papillomavirus (HPV) assay for high-risk human papillomavirus (hrHPV) and HPV-16 in head and neck fine-needle aspiration (FNA) specimens with squamous cell carcinoma.Head and neck FNA biopsy specimens from 2012 to 2020 were retrospectively collected. Cobas HPV testing was performed on 90 FNA specimens with valid Cervista HPV testing results. Results of Cobas HPV and Cervista HPV assays were compared. A Linear Array or SPF10-LiPA25 HPV genotyping assay resolved cases with discrepant results. The κ value and accuracy of Cobas HPV testing were calculated. The accuracy of the Cobas HPV assay was also determined in 42 FNA needle-rinse specimens.Cobas HPV was positive in 82% of the FNA specimens (74 of 90). The concordance between Cobas HPV and Cervista HPV test results was 88.9% (80 of 90) with substantial agreement (κ = 0.669; 95% CI, 0.481-0.856). With HPV genotyping confirmation in cases with discrepant results between the 2 HPV assays, Cobas HPV showed 100% sensitivity and specificity for hrHPV. HPV-16 was detected in 88% of HPV-positive cases (65 of 74). HPV genotyping confirmed 1 false-negative HPV-16 result and 1 false-positive HPV-16 result. Overall, the accuracy of Cobas HPV for HPV-16 was 97.8%. The accuracy of Cobas HPV in FNA needle-rinse specimens was 100%.The Cobas HPV assay is highly accurate for determining the HPV status in head and neck FNA specimens. FNA needle rinse is valid for Cobas HPV testing in patients with squamous cell carcinoma.

Spatial distribution of beta-klotho mRNA in the mouse hypothalamus, hippocampal region, subiculum, and amygdala

The Journal of comparative neurology

2022 Feb 10

Bono, BS;Koziel Ly, NK;Miller, PA;Williams-Ikhenoba, J;Dumiaty, Y;Chee, MJ;
PMID: 35143049 | DOI: 10.1002/cne.25306

Beta-klotho (KLB) is a coreceptor required for endocrine fibroblast growth factor (FGF) 15/19 and FGF21 signaling in the brain. Klb is prominent within the hypothalamus, which is consistent with its metabolic functions, but diverse roles for Klb are now emerging. Central Klb expression is low but discrete and may govern FGF-targeted sites. However, given its low expression, it is unclear if Klb mRNA is more widespread. We performed in situ hybridization to label Klb mRNA to generate spatial maps capturing the distribution and levels of Klb within the mouse hypothalamus, hippocampal region, subiculum, and amygdala. Semiquantitative analysis revealed that Klb-labeled cells may express low, medium, or high levels of Klb mRNA. Hypothalamic Klb hybridization was heterogeneous and varied rostrocaudally within the same region. Most Klb-labeled cells were found in the lateral hypothalamic zone, but the periventricular hypothalamic region, including the suprachiasmatic nucleus, contained the greatest proportion of cells expressing medium or high Klb levels. We also found heterogeneous Klb hybridization in the amygdala and subiculum, where Klb was especially distinct within the central amygdalar nucleus and ventral subiculum, respectively. By contrast, Klb-labeled cells in the hippocampal region only expressed low levels of Klb and were typically found in the pyramidal layer of Ammon's horn or dentate gyrus. The Klb-labeled regions identified in this study are consistent with reported roles of Klb in metabolism, taste preference, and neuroprotection. However, additional identified sites, including within the hypothalamus and amygdala, may suggest novel roles for FGF15/19 or FGF21 signaling.

Profiling senescent cells in human brains reveals neurons with CDKN2D/p19 and tau neuropathology

Nature Aging

2021 Dec 01

Dehkordi, S;Walker, J;Sah, E;Bennett, E;Atrian, F;Frost, B;Woost, B;Bennett, R;Orr, T;Zhou, Y;Andhey, P;Colonna, M;Sudmant, P;Xu, P;Wang, M;Zhang, B;Zare, H;Orr, M;
| DOI: 10.1038/s43587-021-00142-3

Senescent cells contribute to pathology and dysfunction in animal models1 [/articles/s43587-021-00142-3#ref-CR1]. Their sparse distribution and heterogenous phenotype have presented challenges to their detection in human tissues. We developed a senescence eigengene approach to identify these rare cells within large, diverse populations of postmortem human brain cells. Eigengenes are useful when no single gene reliably captures a phenotype, like senescence. They also help to reduce noise, which is important in large transcriptomic datasets where subtle signals from low-expressing genes can be lost. Each of our eigengenes detected ∼2% senescent cells from a population of ∼140,000 single nuclei derived from 76 postmortem human brains with various levels of Alzheimer’s disease (AD) pathology. More than 97% of the senescent cells were excitatory neurons and overlapped with neurons containing neurofibrillary tangle (NFT) tau pathology. Cyclin-dependent kinase inhibitor 2D (_CDKN2D/_p19) was predicted as the most significant contributor to the primary senescence eigengene. RNAscope and immunofluorescence confirmed its elevated expression in AD brain tissue. The p19-expressing neuron population had 1.8-fold larger nuclei and significantly more cells with lipofuscin than p19-negative neurons. These hallmark senescence phenotypes were further elevated in the presence of NFTs. Collectively, _CDKN2D/_p19-expressing neurons with NFTs represent a unique cellular population in human AD with a senescence-like phenotype. The eigengenes developed may be useful in future senescence profiling studies as they identified senescent cells accurately in snRNA-Seq datasets and predicted biomarkers for histological investigation.

IL-23 amplifies the epithelial-mesenchymal transition of mechanically conditioned alveolar epithelial cells in RA-ILD through mTOR/S6 signaling

American journal of physiology. Lung cellular and molecular physiology

2021 Sep 29

Zhang, C;Wang, S;Lau, J;Roden, AC;Matteson, EL;Sun, J;Luo, F;Tschumperlin, DJ;Vassallo, R;
PMID: 34585990 | DOI: 10.1152/ajplung.00292.2021

Epithelial-mesenchymal transition (EMT) creates an environment facilitating fibrosis following alveolar epithelial cell injury. IL-23 has important roles in chronic autoimmune conditions like rheumatoid arthritis (RA), but its role in the interstitial lung disease that affects RA patients is unclear. This study aimed to determine the pro-fibrogenic role of IL-23 on somatic alveolar type I (ATI) epithelial cells. Primary ATI cells were isolated from rats and cultured on plastic dishes for 1-3 weeks. After prolonged culture (≥14 days) on rigid culture dishes, primary ATI cells gradually acquired a mesenchymal phenotype, identified by decreased expression of caveolin-1, and reorganization of F-actin cytoskeleton, indicating the initiation of EMT by matrix stiffness. To determine how IL-23 promotes EMT in vitro, transitioning ATI cells, cultured on a stiff substrate for ≥14 days were stimulated with IL-23. The EMT phenotype was significantly enhanced by IL-23 which upregulated α-SMA, collagen I/III protein, and decreased caveolin-1. Furthermore, IL-23 significantly promoted cell invasion as well as apoptotic resistance on transitioning ATI cells. Mechanistically, IL-23 induced EMT was mTOR/S6 signaling dependent and reversible by rapamycin. Transcriptional sequencing analysis of human lung fibrosis biopsy tissue revealed key roles for IL-23 in RA-ILD. This result was further validated by significantly upregulated IL-23 expression at the mRNA level in RA-ILD lung sections. Notably, transitioning ATI epithelial cells were abundantly detected in RA-ILD tissue. Taken together, these data support a role for IL-23 in the pathogenesis of RA lung fibrosis by promoting EMT in alveolar epithelial cells through mTOR/S6 signaling.

Evidence that pubertal status impacts KNDy neurons in the gilt

Biology of reproduction

2021 Oct 13

Harlow, K;Renwick, AN;Shuping, SL;Sommer, JR;Lents, CA;Knauer, MT;Nestor, CC;
PMID: 34643223 | DOI: 10.1093/biolre/ioab189

Puberty onset is a complex physiological process which enables the capacity for reproduction through increased gonadotropin-releasing hormone (GnRH), and subsequently luteinizing hormone (LH), secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus (ARC) are believed to govern the timing of puberty, the degree to which KNDy neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the ARC. Fluorescent in situ hybridization revealed that the majority (> 74%) of ARC neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer ARC cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P < 0.05), but the number of ARC cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P < 0.05). Together, these data reveal novel information about KNDy neurons in gilts and supports the idea that NKB and dynorphin play a role in puberty onset in the female pig.

Reverse translation approach generates a signature of penetrating fibrosis in Crohn\'s disease that is associated with anti-TNF response

Gut

2021 Jul 14

Xiong, S;Whitehurst, CE;Li, L;Heo, GS;Lai, CW;Jain, U;Muegge, BD;Espenschied, ST;Musich, RJ;Chen, M;Liu, Y;Liu, TC;Stappenbeck, TS;
PMID: 34261752 | DOI: 10.1136/gutjnl-2020-323405

Fibrosis is a common feature of Crohn's disease (CD) which can involve the mesenteric fat. However, the molecular signature of this process remains unclear. Our goal was to define the transcriptional signature of mesenteric fibrosis in CD subjects and to model mesenteric fibrosis in mice to improve our understanding of CD pathogenesis.We performed histological and transcriptional analysis of fibrosis in CD samples. We modelled a CD-like fibrosis phenotype by performing repeated colonic biopsies in mice and analysed the model by histology, type I collagen-targeted positron emission tomography (PET) and global gene expression. We generated a gene set list of essential features of mesenteric fibrosis and compared it to mucosal biopsy datasets from inflammatory bowel disease patients to identify a refined gene set that correlated with clinical outcomes.Mesenteric fibrosis in CD was interconnected to areas of fibrosis in all layers of the intestine, defined as penetrating fibrosis. We found a transcriptional signature of differentially expressed genes enriched in areas of the mesenteric fat of CD subjects with high levels of fibrosis. Mice subjected to repeated colonic biopsies showed penetrating fibrosis as shown by histology, PET imaging and transcriptional analysis. Finally, we composed a composite 24-gene set list that was linked to inflammatory fibroblasts and correlated with treatment response.We linked histopathological and molecular features of CD penetrating fibrosis to a mouse model of repeated biopsy injuries. This experimental system provides an innovative approach for functional investigations of underlying profibrotic mechanisms and therapeutic concepts in CD.

Propofol differentially induces unconsciousness and respiratory depression through distinct interactions between GABAA receptor and GABAergic neuron in corresponding nuclei

Acta biochimica et biophysica Sinica

2021 Jun 17

Jiang, J;Jiao, Y;Gao, PO;Yin, W;Zhou, W;Zhang, Y;Liu, Y;Wen, D;Wang, Y;Zhou, L;Yu, T;Yu, W;
PMID: 34137445 | DOI: 10.1093/abbs/gmab084

Propofol is the most commonly used intravenous anesthetic worldwide. It can induce loss of consciousness prior to the occurrence of severe respiratory suppression, which is also a pharmacodynamic feature of all general anesthetics. However, the neural mechanisms underlying this natural phenomenon are controversial and highly related to patient safety. In the present study, we demonstrated that the pharmacodynamic effects of propofol (50 and 100 μM) on suppression of consciousness-related excitatory postsynaptic currents in the medial prefrontal cortex (mPFC) and centromedian nucleus of the thalamus (CMT) were lower than those in the kernel respiratory rhythmogenesis nucleus pre-Bötzinger complex (PrBo). Furthermore, we unexpectedly found that the GABAA receptor β3 subunit is the key target for propofol's action and that it is mutually and exclusively expressed in GABAergic neurons. It is also more abundant in the mPFC and CMT, but mainly co-localized with GABAergic neurons in the PrBo. As a result, the differentiated expression pattern should mediate more neuron suppression through the activation of GABAergic neurons in the mPFC and CMT at low doses of propofol (50 μM). However, PrBo GABAergic neurons were only activated by propofol at a high dose (100 μM). These results highlight the detailed pharmacodynamic effects of propofol on consciousness-related and respiration-related nuclei and provide the distinct interaction mechanism between the β3 subunit and GABAergic neurons in mediating the suppression of consciousness compared to the inhibition of respiration.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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