Zika virus causes acute infection and inflammation in the ovary of mice without apparent defects in fertility.

J Infect Dis.

2019 May 07

Caine EA, Scheaffer SM, Broughton DE, Salazar V, Govero J, Poddar S, Osula A, Halabi J, Skaznik-Wikiel ME, Diamond MS, Moley KH.
PMID: 31063544 | DOI: 10.1093/infdis/jiz239

Zika virus (ZIKV) has become a global concern because infection of pregnant mothers was linked to congenital birth defects. ZIKV is unique from other flaviviruses, as it is transmitted vertically and sexually in addition to by mosquito vectors. Prior studies in mice, non-human primates, and humans have shown that ZIKV targets the testis in males, resulting in persistent infection and oligospermia. However, its effects on the corresponding female gonads have not been evaluated. Here, we assessed the effects of ZIKV on the ovary in non-pregnant mice. During the acute phase, ZIKV productively infected the ovary causing accumulation of CD4+ and virus-specific CD8+ T cells. T cells protected against ZIKV infection in the ovary, as higher viral burden was measured in CD8-/- and TCRβδ-/- mice. Increased cell death and tissue inflammation in the ovary was observed during the acute phase of infection, but this normalized over time. In contrast to that observed with males, minimal persistence and no long-term consequences of ZIKV infection on ovarian follicular reserve or fertility were demonstrated in this model. Thus, although ZIKV replicates in cells of the ovary and causes acute oophoritis, there is rapid resolution and no long-term effects on fertility, at least in mice.

Integrative Analysis of Programmed Death-Ligand 1 DNA, mRNA, and Protein Status and their Clinicopathological Correlation in Diffuse Large B-cell Lymphoma.

Histopathology. 2018 Oct 4.

2018 Oct 04

Sun C, Jia Y, Wang W, Bi R, Wu L, Bai Q, Zhou X.
PMID: 30286249 | DOI: 10.1111/his.13765

Abstract AIMS: The Protein expression of Programmed Death-Ligand 1 (PD-L1) has been recognized a poor prognostic biomarker in diffuse large B-cell lymphoma (DLBCL). We aim to detect PD-L1 DNA and mRNA status, and explore whether they contribute to protein expression and their clinicopathological correlation in DLBCL. METHODS AND RESULTS: In the study, we detected PD-L1 status in three different levels by Fluorescence in situ hybridization, RNA in situ hybridization and immunohistochemistry in 287 DLBCL samples with follow-ups, respectively. Their correlation and clinical pathological relevance was further analyzed. Our results showed that 1.7% (3/175) patients had PD-L1 amplification, 19.9% (57/287) PD-L1 mRNA high expression and 11.8% (34/287) high protein expression. Both mRNA and protein high expression of PD-L1 was significantly elevated in non-GCB than that in GCB DLBCL (P<0.05). In addition, the patients with PD-L1 mRNA or protein high expression but not DNA amplification have significantly poorer overall survival (OS) than that with PD-L1 low expression (P<0.05). Furthermore, we found that PD-L1 mRNA and protein expression are highly correlated (P=0.012), which was observed in all three samples with PD-L1 DNA amplification. CONCLUSIONS: PD-L1 DNA amplification is a rare event, PD-L1 mRNA mainly contribute to the protein high expression, and the latter two will serve as important biomarkers for predicting prognosis and selecting patients for immunotherapy in DLBCL.

Divergent functions of histone acetyltransferases KAT2A and KAT2B in keratinocyte self-renewal and differentiation

Journal of cell science

2023 Jun 15

Walters, BW;Tan, TJ;Tan, CT;Dube, CT;Lee, KT;Koh, J;Ong, YHB;Tan, VXH;Jahan, FRS;Lim, XN;Wan, Y;Lim, CY;
PMID: 37259855 | DOI: 10.1242/jcs.260723

The mammalian epidermis undergoes constant renewal, replenished by a pool of stem cells and terminal differentiation of their progeny. This is accompanied by changes in gene expression and morphology that are orchestrated, in part, by epigenetic modifiers. Here, we define the role of the histone acetyltransferase KAT2A in epidermal homeostasis and provide a comparative analysis that reveals key functional divergence with its paralog KAT2B. In contrast to the reported function of KAT2B in epidermal differentiation, KAT2A supports the undifferentiated state in keratinocytes. RNA-seq analysis of KAT2A- and KAT2B- depleted keratinocytes revealed dysregulated epidermal differentiation. Depletion of KAT2A led to premature expression of epidermal differentiation genes in the absence of inductive signals, whereas loss of KAT2B delayed differentiation. KAT2A acetyltransferase activity was indispensable in regulating epidermal differentiation gene expression. The metazoan-specific N terminus of KAT2A was also required to support its function in keratinocytes. We further showed that the interplay between KAT2A- and KAT2B-mediated regulation was important for normal cutaneous wound healing in vivo. Overall, these findings reveal a distinct mechanism in which keratinocytes use a pair of highly homologous histone acetyltransferases to support divergent functions in self-renewal and differentiation processes.

Distribution of bile acid receptor TGR5 in the gastrointestinal tract of dogs.

Histol Histopathol. 2018 Jul 12:18025.

2018 Jul 12

Giaretta PR, Suchodolski JS, Blick AK, Steiner JM, Lidbury JA, Rech RR.
PMID: 29999170 | DOI: 10.14670/HH-18-025

Takeda-G-protein-receptor-5 (TGR5) is a receptor for bile acids and its expression has been described in a variety of tissues and species. Characterization of TGR5 distribution and function has been investigated in drug discovery for the treatment of metabolic diseases in humans. Because dogs are one of the species used in biomedical research and share some similarities with human gastrointestinal diseases, the objective of this study was to characterize the distribution of TGR5 receptor in the canine species. This study characterizes the distribution of TGR5 receptor in the gastrointestinal tract, liver, gallbladder, and pancreas of 8 dogs. The distribution of TGR5 antigen and mRNA expression was investigated using immunohistochemistry and RNA in situ hybridization, respectively. TGR5 immunolabeling was located in the cell membrane or in the cell membrane and cytoplasm. TGR5 immunolabeling was broadly distributed in macrophages, endothelial cells, ganglion cells, and leiomyocytes throughout all the examined tissues. Epithelial cells from tongue, stomach to rectum, as well as from gallbladder, biliary and pancreatic ducts demonstrated TGR5 immunolabeling. In endocrine cells, TGR5 immunolabeling was observed in intestinal enteroendocrine cells and islets of Langerhans in the pancreas. The hepatocytes had a unique pattern of immunolabeling located on the canalicular surface of the cell membrane. TGR5 mRNA expression was located mainly in the nucleus and the only negative cells throughout all examined tissues were striated muscle from tongue and esophagus, muscularis mucosae, esophageal glands, and hepatic sinusoids. These findings indicate that the bile acid receptor TGR5 is ubiquitously distributed in the canine gastrointestinal tract.

Effects and sites of action of a M1 receptor positive allosteric modulator on colonic motility in rats and dogs compared with 5-HT4 agonism and cholinesterase inhibition

Neurogastroenterol Motil.

2020 Apr 26

Tsukimi Y, Pustovit RV, Harrington AM, Garcia-Caraballo S, Brierley SM, Di Natale M, Molero JC, Furness JB2
PMID: 32337809 | DOI: 10.1111/nmo.13866

BACKGROUND: Muscarinic receptor 1 positive allosteric modulators (M1PAMs) enhance colonic propulsive contractions and defecation through the facilitation of M1 receptor (M1R)-mediated signaling. We examined M1R expression in the colons of 5 species and compared colonic propulsion and defecation caused by the M1PAM, T440, the 5-HT4 agonist, prucalopride, and the cholinesterase inhibitor, neostigmine, in rats and dogs. METHODS: M1R expression was profiled by immunostaining and in situ hybridization. In vivo studies utilized male SD rats and beagle dogs. Colonic propulsive contractions were recorded by manometry in anesthetized rats. Gut contractions in dogs were assessed using implanted force transducers in the ileum, proximal, mid, and distal colons. KEY RESULTS: M1R was localized to neurons of myenteric and submucosal plexuses and the epithelium of the human colon. A similar receptor localization was observed in rat, dog, mouse, and pig. T440 enhanced normal defecation in rats in a dose-dependent manner. Prucalopride also enhanced defecation in rats, but the maximum effect was half that of T440. Neostigmine and T440 were similarly effective in enhancing defecation, but the effective dose of neostigmine was close to its lethal dose. In rats, all 3 compounds induced colonic contractions, but the associated propulsion was strongest with T440. In dogs, intestinal contractions elicited by T440 propagated from ileum to distal colon. Prucalopride and neostigmine also induced intestinal contractions, but these were less well coordinated. No loss of effectiveness of T440 on defecation occurred after 5 days of repeated dosing. CONCLUSION AND INFERENCES: These results suggest that M1PAMs produce highly coordinated propagating contraction by actions on the enteric nervous system of the colon. The localization of M1R to enteric neurons in both animals and humans suggests that the M1PAM effects would be translatable to human. M1PAMs provide a potential novel therapeutic option for constipation disorders

Advanced Safety and Genetic Stability in Mice of a Novel DNA-Launched Venezuelan Equine Encephalitis Virus Vaccine with Rearranged Structural Genes

Vaccines

2020 Mar 02

Johnson DM, Sokoloski KJ, Jokinen JD, Pfeffer TL, Chu YK, Adcock RS, Chung D, Tretyakova I, Pushko P, Lukashevich IS
PMID: 32121666 | DOI: 10.3390/vaccines8010114

The safety and genetic stability of V4020, a novel Venezuelan Equine Encephalitis Virus (VEEV) vaccine based on the investigational VEEV TC-83 strain, was evaluated in mice. V4020 was generated from infectious DNA, contains a stabilizing mutation in the E2-120 glycoprotein, and includes rearrangement of structural genes. After intracranial inoculation (IC), replication of V4020 was more attenuated than TC-83, as documented by low clinical scores, inflammation, viral load in brain, and earlier viral clearance. During the first 9 days post-inoculation (DPI), genes involved in inflammation, cytokine signaling, adaptive immune responses, and apoptosis were upregulated in both groups. However, the magnitude of upregulation was greater in TC-83 than V4020 mice, and this pattern persisted till 13 DPI, while V4020 gene expression profiles declined to mock-infected levels. In addition, genetic markers of macrophages, DCs, and microglia were strongly upregulated in TC-83 mice. During five serial passages in the brain, less severe clinical manifestations and a lower viral load were observed in V4020 mice and all animals survived. In contrast, 13.3% of mice met euthanasia criteria during the passages in TC-83 group. At 2 DPI, RNA-Seq analysis of brain tissues revealed that V4020 mice had lower rates of mutations throughout five passages. A higher synonymous mutation ratio was observed in the nsP4 (RdRP) gene of TC-83 compared to V4020 mice. At 2 DPI, both viruses induced different expression profiles of host genes involved in neuro-regeneration. Taken together, these results provide evidence for the improved safety and genetic stability of the experimental V4020 VEEV vaccine in a murine model

Hippocampal Expression of Cytochrome P450 1B1 in Penetrating Traumatic Brain Injury

International journal of molecular sciences

2022 Jan 10

Lidin, E;Sköld, MK;Angéria, M;Davidsson, J;Risling, M;
PMID: 35054909 | DOI: 10.3390/ijms23020722

Hippocampal dysfunction contributes to multiple traumatic brain injury sequala. Female rodents' outcome is superior to male which has been ascribed the neuroprotective sex hormones 17β-estradiol and progesterone. Cytochrome P450 1B1 (CYP1B1) is an oxidative enzyme influencing the neuroinflammatory response by creating inflammatory mediators and metabolizing neuroprotective 17β-estradiol and progesterone. In this study, we aimed to describe hippocampal CYP1B1 mRNA expression, protein presence of CYP1B1 and its key redox partner Cytochrome P450 reductase (CPR) in both sexes, as well as the effect of penetrating traumatic brain injury (pTBI). A total 64 adult Sprague Dawley rats divided by sex received pTBI or sham-surgery and were assigned survival times of 1-, 3-, 5- or 7 days. CYP1B1 mRNA was quantified using in-situ hybridization and immunohistochemistry performed to verify protein colocalization. CYP1B1 mRNA expression was present in all subregions but greatest in CA2 irrespective of sex, survival time or intervention. At 3-, 5- and 7 days post-injury, expression in CA2 was reduced in male rats subjected to pTBI compared to sham-surgery. Females subjected to pTBI instead exhibited increased expression in all CA subregions 3 days post-injury, the only time point expression in CA2 was greater in females than in males. Immunohistochemical analysis confirmed neuronal CYP1B1 protein in all hippocampal subregions, while CPR was limited to CA1 and CA2. CYP1B1 mRNA is constitutively expressed in both sexes. In response to pTBI, females displayed a more urgent but brief regulatory response than males. This indicates there may be sex-dependent differences in CYP1B1 activity, possibly influencing inflammation and neuroprotection in pTBI.

Protein arginine methyltransferase 1 regulates cell proliferation and differentiation in adult mouse adult intestine

Cell & bioscience

2021 Jun 22

Xue, L;Bao, L;Roediger, J;Su, Y;Shi, B;Shi, YB;
PMID: 34158114 | DOI: 10.1186/s13578-021-00627-z

Adult stem cells play an essential role in adult organ physiology and tissue repair and regeneration. While much has been learnt about the property and function of various adult stem cells, the mechanisms of their development remain poorly understood in mammals. Earlier studies suggest that the formation of adult mouse intestinal stem cells takes place during the first few weeks after birth, the postembryonic period when plasma thyroid hormone (T3) levels are high. Furthermore, deficiency in T3 signaling leads to defects in adult mouse intestine, including reduced cell proliferation in the intestinal crypts, where stem cells reside. Our earlier studies have shown that protein arginine methyltransferase 1 (PRMT1), a T3 receptor coactivator, is highly expressed during intestinal maturation in mouse.We have analyzed the expression of PRMT1 by immunohistochemistry and studied the effect of tissue-specific knockout of PRMT1 in the intestinal epithelium.We show that PRMT1 is expressed highly in the proliferating transit amplifying cells and crypt base stem cells. By using a conditional knockout mouse line, we have demonstrated that the expression of PRMT1 in the intestinal epithelium is critical for the development of the adult mouse intestine. Specific removal of PRMT1 in the intestinal epithelium results in, surprisingly, more elongated adult intestinal crypts with increased cell proliferation. In addition, epithelial cell migration along the crypt-villus axis and cell death on the villus are also increased. Furthermore, there are increased Goblet cells and reduced Paneth cells in the crypt while the number of crypt base stem cells remains unchanged.Our finding that PRMT1 knockout increases cell proliferation is surprising considering the role of PRMT1 in T3-signaling and the importance of T3 for intestinal development, and suggests that PRMT1 likely regulates pathways in addition to T3-signaling to affect intestinal development and/or homeostasis, thus affecting cell proliferating and epithelial turn over in the adult.

CK2β-regulated signaling controls B cell differentiation and function

Frontiers in immunology

2023 Jan 11

Quotti Tubi, L;Mandato, E;Canovas Nunes, S;Arjomand, A;Zaffino, F;Manni, S;Casellato, A;Macaccaro, P;Vitulo, N;Zumerle, S;Filhol, O;Boldyreff, B;Siebel, CW;Viola, A;Valle, G;Mainoldi, F;Casola, S;Cancila, V;Gulino, A;Tripodo, C;Pizzi, M;Dei Tos, AP;Trentin, L;Semenzato, G;Piazza, F;
PMID: 36713383 | DOI: 10.3389/fimmu.2022.959138

Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) mouse model of the β regulatory subunit of CK2. CK2βKO mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2βKO animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2βKO mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins. In vitro assays highlight that B cells lacking CK2β have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2βKO mice suggesting the importance of the β subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation.

LINC01133 promotes hepatocellular carcinoma progression by sponging miR-199a-5p and activating annexin A2

Clinical and translational medicine

2021 May 01

Yin, D;Hu, ZQ;Luo, CB;Wang, XY;Xin, HY;Sun, RQ;Wang, PC;Li, J;Fan, J;Zhou, ZJ;Zhou, J;Zhou, SL;
PMID: 34047479 | DOI: 10.1002/ctm2.409

Long noncoding RNAs (lncRNAs) are functionally associated with cancer development and progression. Although gene copy number variation (CNV) is common in hepatocellular carcinoma (HCC), it is not known how CNV in lncRNAs affects HCC progression and recurrence. We aimed to identify a CNV-related lncRNA involved in HCC progression and recurrence and illustrate its underlying mechanisms and prognostic value. We analyzed the whole genome sequencing (WGS) data of matched cancerous and noncancerous liver samples from 49 patients with HCC to identify lncRNAs with CNV. The results were validated in another cohort of 238 paired HCC and nontumor samples by TaqMan copy number assay. We preformed Kaplan-Meier analysis and log-rank test to identify lncRNA CNV with prognostic value. We conducted loss- and gain-of-function studies to explore the biological functions of LINC01133 in vitro and in vivo. The competing endogenous RNAs (ceRNAs) mechanism was clarified by microRNA sequencing (miR-seq), quantitative real-time PCR (qRT-PCR), western blot, and dual-luciferase reporter assays. We confirmed the binding mechanism between lncRNA and protein by RNA pull-down, RNA immunoprecipitation, qRT-PCR, and western blot analyses. Genomic copy numbers of LINC01133 were increased in HCC, which were positively related with the elevated expression of LINC01133. Increased copy number of LINC01133 predicted the poor prognosis in HCC patients. LINC01133 overexpression in HCC cells promoted proliferation and aggressive phenotypes in vitro, and facilitated tumor growth and lung metastasis in vivo, whereas LINC01133 knockdown had the opposite effects. LINC01133 sponged miR-199a-5p, resulting in enhanced expression of SNAI1, which induced epithelial-to-mesenchymal transition (EMT) in HCC cells. In addition, LINC01133 interacted with Annexin A2 (ANXA2) to activate the ANXA2/STAT3 signaling pathway. LINC01133 promotes HCC progression by sponging miR-199a-5p and interacting with ANXA2. LINC01133 CNV gain is predictive of poor prognosis in patients with HCC.

Impairment in renal medulla development underlies salt wasting in Clc-k2 channel deficiency

JCI insight

2021 Sep 09

Lin, MH;Chen, JC;Tian, X;Lee, CM;Yu, IS;Lo, YF;Uchida, S;Huang, CL;Chen, BC;Cheng, CJ;
PMID: 34499620 | DOI: 10.1172/jci.insight.151039

The prevailing view is that ClC-Ka chloride channel (mouse Clc-k1) functions in thin ascending limb for urine concentration, whereas ClC-Kb (mouse Clc-k2) in thick ascending limb (TAL) for salt reabsorption, respectively. Mutations of ClC-Kb cause classic Bartter syndrome with renal salt wasting with onset from perinatal to adolescent. We study the roles of Clc-k channels in perinatal mouse kidneys using constitutive or inducible kidney-specific gene ablation and 2-D and advanced 3-D imaging of optically cleared kidneys. We show that Clc-k1 and -k2 are broadly expressed and colocalized in perinatal kidneys. Deletion of Clc-k1 and -k2 reveals that both participate in NKCC2- and NCC-mediated NaCl reabsorption in neonatal kidneys. Embryonic deletion of Clc-k2 causes tubular injury and impairs renal medulla and TAL development. Inducible deletion of Clc-k2 begins after medulla maturation produces mild salt wasting resulting from reduced NCC activity. Thus, both Clc-k1 and -k2 contribute to salt reabsorption in TAL and DCT in neonates, potentially explaining less severe phenotypes in classic Bartter. As opposed to the current understanding that salt wasting in adult Bartter patients is due to Clc-k2 deficiency in adult TAL, our results suggest that it is mainly originated from medulla and TAL defects during development.

Characterization of LGR5 expression in poorly differentiated colorectal carcinoma with mismatch repair protein deficiency

BMC Cancer

2020 Apr 15

Nakajima T, Uehara T, Iwaya M, Kobayashi Y, Maruyama Y, Ota H
PMID: 32293346 | DOI: 10.1186/s12885-020-06791-8

BACKGROUND: Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a promising intestinal stem cell and carcinoma stem cell marker. We examined the relationship between mismatch repair (MMR) protein deficiency and LGR5 expression in poorly differentiated (PD) colorectal carcinoma (CRC). METHODS: In 29 cases of PD-CRC, deficiencies in MMR proteins (MLH1, PMS2, MSH2, MSH6) and ?-catenin expression were identified by immunohistochemistry (IHC). LGR5 expression was examined by the RNAscope assay in tissue microarrays. RESULTS: LGR5 H-scores in MMR-deficient (MMR-D) cases were significantly lower than those in MMR-proficient (MMR-P) cases (P?=?0.0033). Nuclear ?-catenin IHC scores in MMR-D cases were significantly lower than those in MMR-P cases (P?=?0.0024). In all cases, there was a positive correlation between LGR5 H-score and nuclear ?-catenin IHC score (r?=?0.6796, P?

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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
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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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