Pesti, A;Danics, K;Glasz, T;Várkonyi, T;Barbai, T;Reszegi, A;Kovalszky, I;Vályi-Nagy, I;Dobi, D;Lotz, G;Schaff, Z;Kiss, A;
PMID: 36527584 | DOI: 10.1007/s11357-022-00700-6
The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope ) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.
The Journal of clinical endocrinology and metabolism
Poma, AM;Proietti, A;Macerola, E;Bonuccelli, D;Conti, M;Salvetti, A;Dolo, V;Chillà, A;Basolo, A;Santini, F;Toniolo, A;Basolo, F;
PMID: 35567590 | DOI: 10.1210/clinem/dgac312
Involvement of the pituitary gland in SARS-CoV-2 infection has been clinically suggested by pituitary hormone deficiency in severe COVID-19 cases, by altered serum ACTH levels in hospitalized patients, and by cases of pituitary apoplexy. However, the direct viral infection of the gland has not been investigated.To evaluate whether the SARS-CoV-2 genome and antigens could be present in pituitary glands of lethal cases of COVID-19, and to assess possible changes in the expression of immune-related and pituitary-specific genes.SARS-CoV-2 genome and antigens were searched in the pituitary gland of 23 patients who died from COVID-19 and, as controls, in 12 subjects who died from trauma or sudden cardiac death. Real-time RT-PCR, in situ hybridization, immunohistochemistry and transmission electron microscopy were utilized. Levels of mRNA transcripts of immune-related and pituitary-specific genes were measured by the nCounter assay.The SARS-CoV-2 genome and antigens were detected in 14/23 (61%) pituitary glands of the COVID-19 group, not in controls. In SARS-CoV-2 positive pituitaries, the viral genome was consistently detected by PCR in the adeno- and the neurohypophysis. Immunohistochemistry, in situ hybridization and transmission electron microscopy confirmed the presence of SARS-CoV-2 in the pituitary. Activation of type I interferon signaling and enhanced levels of neutrophil and cytotoxic cell scores were found in virus-positive glands. mRNA transcripts of pituitary hormones and pituitary developmental/regulatory genes were suppressed in all COVID-19 cases irrespective of virus-positivity.Our study supports the tropism of SARS-CoV-2 for human pituitary and encourage to explore pituitary dysfunction post-COVID-19.
Appl Immunohistochem Mol Morphol. 2014 Sep;22(8):e32-40.
Kunju LP, Carskadon S, Siddiqui J, Tomlins SA, Chinnaiyan AM, Palanisamy N.
PMID: 25203299 | DOI: 10.1097/PAI.0000000000000095.
The genetic basis of 50% to 60% of prostate cancer (PCa) is attributable to rearrangements in E26 transformation-specific (ETS) (ERG, ETV1, ETV4, and ETV5), BRAF, and RAF1 genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of PCa for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERG rearrangement status by fluorescence in situ hybridization. However, specific antibodies for ETV1, ETV4, and ETV5 are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization-based assay for the in situ detection of ETV1, ETV4, and ETV5 in formalin-fixed paraffin-embedded tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of PCa with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright-field in situ detection of ETS gene aberrations in routine clinically available PCa specimens.
Covach A, Patel S, Hardin H, Lloyd RV.
PMID: 28660408 | DOI: 10.1007/s12022-017-9490-7
Oncocytic (Hürthle cell) and follicular neoplasms are related thyroid tumors with distinct molecular profiles. Diagnostic criteria separating adenomas and carcinomas for these two types of neoplasms are similar, but there may be some differences in the biological behavior of Hürthle cell and follicular carcinomas. Recent studies have shown that noncoding RNAs may have diagnostic and prognostic utility in separating benign and malignant Hürthle cell and follicular neoplasms. In this study, we examined expression of various noncoding RNAs including metastasis associated lung adenocarcinoma transcript 1 (MALAT1) and miR-RNA-885-5p (miR-885) in distinguishing between benign and malignant neoplasms. In addition, the expression of phosphorylated mechanistic receptor of rapamycin (p-mTOR) was also analyzed in these two groups of tumors. Tissue microarrays (TMAs) with archived tissue samples were analyzed using in situ hybridization (ISH) for MALAT1 and miR-885 and immunohistochemistry (IHC) for p-mTOR. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was also performed on a subset of the cases.MALAT1 and miR-885 were increased in all neoplastic groups compared to the normal thyroid tissues (p < 0.05). MALAT1 was more highly expressed in HCCs compared to FTCs, although the differences were not statistically significant (p = 0.06). MiR-885 was expressed at similar levels in FTCs and HCCs. P-mTOR protein was more highly expressed in FTCs than in HCCs (p<0.001). qRT-PCR analysis of noncoding RNAs supported the ISH findings. These results indicate that the noncoding RNAs MALAT1 and miR-885 show increased expression in neoplastic follicular and Hürthle cell thyroid neoplasms compared to normal thyroid tissues. P-mTOR was most highly expressed in FTC but was also increased in HCC, suggesting that drugs targeting this pathway may be useful for treatment of tumors unresponsive to conventional therapies.
Infection and transmission of SARS-CoV-2 and its alpha variant in pregnant white-tailed deer
bioRxiv : the preprint server for biology
Cool, K;Gaudreault, NN;Morozov, I;Trujillo, JD;Meekins, DA;McDowell, C;Carossino, M;Bold, D;Kwon, T;Balaraman, V;Madden, DW;Artiaga, BL;Pogranichniy, RM;Sosa, GR;Henningson, J;Wilson, WC;Balasuriya, UBR;García-Sastre, A;Richt, JA;
PMID: 34426811 | DOI: 10.1101/2021.08.15.456341
SARS-CoV-2, a novel Betacoronavirus, was first reported circulating in human populations in December 2019 and has since become a global pandemic. Recent history involving SARS-like coronavirus outbreaks (SARS-CoV and MERS-CoV) have demonstrated the significant role of intermediate and reservoir hosts in viral maintenance and transmission cycles. Evidence of SARS-CoV-2 natural infection and experimental infections of a wide variety of animal species has been demonstrated, and in silico and in vitro studies have indicated that deer are susceptible to SARS-CoV-2 infection. White-tailed deer (Odocoileus virginianus) are amongst the most abundant, densely populated, and geographically widespread wild ruminant species in the United States. Human interaction with white-tailed deer has resulted in the occurrence of disease in human populations in the past. Recently, white-tailed deer fawns were shown to be susceptible to SARS-CoV-2. In the present study, we investigated the susceptibility and transmission of SARS-CoV-2 in adult white-tailed deer. In addition, we examined the competition of two SARS-CoV-2 isolates, representatives of the ancestral lineage A (SARS-CoV-2/human/USA/WA1/2020) and the alpha variant of concern (VOC) B.1.1.7 (SARS-CoV-2/human/USA/CA_CDC_5574/2020), through co-infection of white-tailed deer. Next-generation sequencing was used to determine the presence and transmission of each strain in the co-infected and contact sentinel animals. Our results demonstrate that adult white-tailed deer are highly susceptible to SARS-CoV-2 infection and can transmit the virus through direct contact as well as vertically from doe to fetus. Additionally, we determined that the alpha VOC B.1.1.7 isolate of SARS-CoV-2 outcompetes the ancestral lineage A isolate in white-tailed deer, as demonstrated by the genome of the virus shed from nasal and oral cavities from principal infected and contact animals, and from virus present in tissues of principal infected deer, fetuses and contact animals.
Zhang R, Hardin H, Huang W, Chen J, Asioli S, Righi A, Maletta F, Sapino A, Lloyd RV.
PMID: 27696303 | DOI: 10.1007/s12022-016-9453-4
Long non-coding RNAs (lncRNAs) are important for transcription and for epigenetic or posttranscriptional regulation of gene expression and may contribute to carcinogenesis. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNA involved in the regulation of the cell cycle, cell proliferation, and cell migration, is known to be deregulated in multiple cancers. Here, we analyzed the expression of MALAT1 on 195 cases of benign and malignant thyroid neoplasms by using tissue microarrays for RNA in situ hybridization (ISH) and real-time PCR. MALAT1 is highly expressed in normal thyroid (NT) tissues and thyroid tumors, with increased expression during progression from NT to papillary thyroid carcinomas (PTCs) but is downregulated in poorly differentiated thyroid cancers (PDCs) and anaplastic thyroid carcinomas (ATCs) compared to NT. Induction of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta in a PTC cell line (TPC1) led to increased MALAT1 expression, supporting a role for MALAT1 in EMT in thyroid tumors. This is the first ISH study of MALAT1 expression in thyroid tissues. It also provides the first piece of evidence suggesting MALAT1 downregulation in certain thyroid malignancies. Our findings support the notion that ATCs may be molecularly distinct from low-grade thyroid malignancies and suggest that MALAT1 may function both as an oncogene and as a tumor suppressor in different types of thyroid tumors.
Zhang Z, Weaver DL, Olsen D, deKay J, Peng Z, Ashikaga T, Evans MF.
PMID: 26323944 | DOI: 10.1136/jclinpath-2015-203275
Abstract
AIM:
Long non-coding RNAs (lncRNAs) are potential biomarkers for breast cancer risk stratification. LncRNA expression has been investigated primarily by RNA sequencing, quantitative reverse transcription PCR or microarray techniques. In this study, six breast cancer-implicated lncRNAs were investigated by chromogenic in situ hybridisation (CISH).
METHODS:
Invasive breast carcinoma (IBC), ductal carcinoma in situ (DCIS) and normal adjacent (NA) breast tissues from 52 patients were screened by CISH. Staining was graded by modified Allred scoring.
RESULTS:
HOTAIR, H19 and KCNQ1OT1 had significantly higher expression levels in IBC and DCIS than NA (p<0.05), and HOTAIR and H19 were expressed more strongly in IBC than in DCIS tissues (p<0.05). HOTAIR and KCNQ101T were expressed in tumour cells; H19 and MEG3 were expressed in stromal microenvironment cells; MALAT1 was expressed in all cells strongly and ZFAS1 was negative or weakly expressed in all specimens.
CONCLUSION:
These data corroborate the involvement of three lncRNAs (HOTAIR, H19 and KCNQ1OT1) in breast tumourigenesis and support lncRNA CISH as a potential clinical assay. Importantly, CISH allows identification of the tissue compartment expressing lncRNA.
Aslan, GS;Jaé, N;Manavski, Y;Fouani, Y;Shumliakivska, M;Kettenhausen, L;Kirchhof, L;Günther, S;Fischer, A;Luxán, G;Dimmeler, S;
PMID: 36883566 | DOI: 10.1172/jci.insight.162124
The adult mammalian heart has limited regenerative capacity, while the neonatal heart fully regenerates during the first week of life. Postnatal regeneration is mainly driven by proliferation of preexisting cardiomyocytes and supported by proregenerative macrophages and angiogenesis. Although the process of regeneration has been well studied in the neonatal mouse, the molecular mechanisms that define the switch between regenerative and nonregenerative cardiomyocytes are not well understood. Here, using in vivo and in vitro approaches, we identified the lncRNA Malat1 as a key player in postnatal cardiac regeneration. Malat1 deletion prevented heart regeneration in mice after myocardial infarction on postnatal day 3 associated with a decline in cardiomyocyte proliferation and reparative angiogenesis. Interestingly, Malat1 deficiency increased cardiomyocyte binucleation even in the absence of cardiac injury. Cardiomyocyte-specific deletion of Malat1 was sufficient to block regeneration, supporting a critical role of Malat1 in regulating cardiomyocyte proliferation and binucleation, a landmark of mature nonregenerative cardiomyocytes. In vitro, Malat1 deficiency induced binucleation and the expression of a maturation gene program. Finally, the loss of hnRNP U, an interaction partner of Malat1, induced similar features in vitro, suggesting that Malat1 regulates cardiomyocyte proliferation and binucleation by hnRNP U to control the regenerative window in the heart.
Winkler, ES;Chen, RE;Alam, F;Yildiz, S;Case, JB;Uccellini, MB;Holtzman, MJ;Garcia-Sastre, A;Schotsaert, M;Diamond, MS;
PMID: 34668780 | DOI: 10.1128/JVI.01511-21
The development of mouse models for COVID-19 has enabled testing of vaccines and therapeutics and defining aspects of SARS-CoV-2 pathogenesis. SARS-CoV-2 disease is severe in K18 transgenic mice (K18-hACE2-Tg) expressing human ACE2 (hACE2), the SARS-CoV-2 receptor, under an ectopic cytokeratin promoter, with high levels of infection measured in the lung and brain. Here, we evaluated SARS-CoV-2 infection in hACE2 KI mice that express hACE2 under an endogenous promoter in place of murine ACE2 (mACE2). Intranasal inoculation of hACE2 KI mice with SARS-CoV-2 WA1/2020 resulted in substantial viral replication within the upper and lower respiratory tracts with limited spread to extra-pulmonary organs. However, SARS-CoV-2-infected hACE2 KI mice did not lose weight and developed limited pathology. Moreover, no significant differences in viral burden were observed in hACE2 KI mice infected with B.1.1.7 or B.1.351 variants compared to WA1/2020 strain. Because the entry mechanisms of SARS-CoV-2 in mice remains uncertain, we evaluated the impact of the naturally-occurring, mouse-adapting N501Y mutation by comparing infection of hACE2 KI, K18-hACE2-Tg, ACE2-deficient, and wild-type C57BL/6 mice. The N501Y mutation minimally affected SARS-CoV-2 infection in hACE2 KI mice but was required for viral replication in wild-type C57BL/6 mice in a mACE2-dependent manner and augmented pathogenesis in the K18-hACE2 Tg mice. Thus, the N501Y mutation likely enhances interactions with mACE2 or hACE2 in vivo. Overall, our study highlights the hACE2 KI mice as a model of mild SARS-CoV-2 infection and disease and clarifies the requirement of the N501Y mutation in mice. IMPORTANCE Mouse models of SARS-CoV-2 pathogenesis have facilitated the rapid evaluation of countermeasures. While the first generation of models developed pneumonia and severe disease after SARS-CoV-2 infection, they relied on ectopic expression of supraphysiological levels of human ACE2 (hACE2). This has raised issues with their relevance to humans as the hACE2 receptor shows a more restricted expression pattern in the respiratory tract. Here we evaluated SARS-CoV-2 infection and disease with viruses containing or lacking a key mouse-adapting mutation in the spike gene in hACE2 KI mice, which express hACE2 under an endogenous promoter in place of murine ACE2. While infection of hACE2 KI mice with multiple strains of SARS-CoV-2 including variants of concern resulted in viral replication within the upper and lower respiratory tracts, the animals did not sustain severe lung injury. Thus, hACE2 KI mice serve as a model of mild infection with both ancestral and emerging SARS-CoV-2 variant strains.
Computational intelligence and neuroscience
Zhao, Y;Yan, G;Mi, J;Wang, G;Yu, M;Jin, D;Tong, X;Wang, X;
PMID: 35528328 | DOI: 10.1155/2022/8400106
Long noncoding RNA (lncRNA) is involved in the occurrence and development of diabetic kidney disease (DKD). It is necessary to identify the expression of lncRNA from DKD patients through systematic reviews, and then carry out silico analyses to recognize the dysregulated lncRNA and their associated pathways.The study searched Pubmed, Embase, Cochrane Library, WanFang, VIP, CNKI, and CBM to find lncRNA studies on DKD published before March 1, 2021. Systematic review of the literature on this topic was conducted to determine the expression of lncRNA in DKD and non-DKD controls. For the dysregulated lncRNA in DKD patients, silico analysis was performed, and lncRNA2Target v2.0 and starBase were used to search for potential target genes of lncRNA. The Encyclopedia of Genomics (KEGG) pathway enrichment analysis was performed to better identify dysregulated lncRNAs in DKD and determine the associated signal pathways.According to the inclusion and exclusion criteria, 28 publications meeting the eligibility criteria were included in the systematic evaluation. A total of 3,394 patients were enrolled in this study, including 1,238 patients in DKD group, and 1,223 diabetic patients, and 933 healthy adults in control group. Compared with the control, there were eight lncRNA disorders in DKD patients (MALAT1, GAS5, MIAT, CASC2, NEAT1, NR_033515, ARAP1-AS2, and ARAP1-AS1). In addition, five lncRNAs (MALAT1, GAS5, MIAT, CASC2, and NEAT1) participated in disease-related signal pathways, indicating their role in DKD. Discussion. This study showed that there were eight lncRNAs in DKD that were persistently dysregulated, especially five lncRNAs which were closely related to the disease. Although systematic review included 28 studies that analyzed the expression of lncRNA in DKD-related tissues, the potential of these dysregulated lncRNAs as biomarkers or therapeutic targets for DKD remains to be further explored. Trial registration. PROSPERO (CRD42021248634).
Ritter, JM;Wilson, TM;Gary, JM;Seixas, JN;Martines, RB;Bhatnagar, J;Bollweg, BC;Lee, E;Estetter, L;Silva-Flannery, L;Bullock, HA;Towner, JS;Cossaboom, CM;Wendling, NM;Amman, BR;Harvey, RR;Taylor, D;Rettler, H;Barton Behravesh, C;Zaki, SR;
PMID: 35229669 | DOI: 10.1177/03009858221079665
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory disease in mink similar to human COVID-19. We characterized the pathological findings in 72 mink from US farms with SARS-CoV-2 outbreaks, localized SARS-CoV-2 and its host cellular receptor angiotensin-converting enzyme 2 (ACE2) in mink respiratory tissues, and evaluated the utility of various test methods and specimens for SARS-CoV-2 detection in necropsy tissues. Of SARS-CoV-2-positive animals found dead, 74% had bronchiolitis and diffuse alveolar damage (DAD). Of euthanized SARS-CoV-2-positive animals, 72% had only mild interstitial pneumonia or minimal nonspecific lung changes (congestion, edema, macrophages); similar findings were seen in SARS-CoV-2-negative animals. Suppurative rhinitis, lymphocytic perivascular inflammation in the lungs, and lymphocytic infiltrates in other tissues were common in both SARS-CoV-2-positive and SARS-CoV-2-negative animals. In formalin-fixed paraffin-embedded (FFPE) upper respiratory tract (URT) specimens, conventional reverse transcription-polymerase chain reaction (cRT-PCR) was more sensitive than in situ hybridization (ISH) or immunohistochemistry (IHC) for detection of SARS-CoV-2. FFPE lung specimens yielded less detection of virus than FFPE URT specimens by all test methods. By IHC and ISH, virus localized extensively to epithelial cells in the nasal turbinates, and prominently within intact epithelium; olfactory mucosa was mostly spared. The SARS-CoV-2 receptor ACE2 was extensively detected by IHC within turbinate epithelium, with decreased detection in lower respiratory tract epithelium and alveolar macrophages. This study expands on the knowledge of the pathology and pathogenesis of natural SARS-CoV-2 infection in mink and supports their further investigation as a potential animal model of SARS-CoV-2 infection in humans.
Experimental and Molecular Pathology
Chu YH, Hardin H, Schneider DF, Chen H, Lloyd RV.
PMID: 29107050 | DOI: 10.1016/j.yexmp.2017.10.002
Abstract
BACKGROUND:
Non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are well-recognized post-transcriptional regulators of gene expression. This study examines the expression of microRNA-21 (miR-21) and lncRNA MALAT1 in medullary thyroid carcinomas (MTCs) and their effects on tumor behavior.
METHODS:
Tissue microarrays (TMAs) were constructed using normal thyroid (n=39), primary tumors (N=39) and metastatic MTCs (N=18) from a total of 42 MTC cases diagnosed between 1987 and 2016. In situ hybridization with probes for miR-21 and MALAT1 was performed. PCR quantification of expression was performed in a subset of normal thyroid (N=10) and primary MTCs (N=32). An MTC-derived cell line (MZ-CRC-1) was transfected with small interfering RNAs (siRNAs) targeting miR-21 and MALAT1 to determine the effects on cell proliferation and invasion.
RESULTS:
In situ hybridization (ISH) showed strong (2+ to 3+) expression of miR-21 in 17 (44%) primary MTCs and strong MALAT1 expression in 37 (95%) primary MTCs. Real-time PCR expression of miR-21 (P<0.001) and MALAT1 (P=0.038) in primary MTCs were significantly higher than in normal thyroid, supporting the ISH findings. Experiments with siRNAs showed inhibition of miR-21 and MALAT1 expression in the MTC-derived cell line, leading to significant decreases in cell proliferation (P<0.05) and invasion (P<0.05).
CONCLUSION:
There is increased expression of miR-21 and MALAT1 in MTCs. This study also showed an in vitro pro-oncogenic effect of MALAT1 and miR-21 in MTCs. The results suggest that overexpression of miR-21 and MALAT1 may regulate MTC progression.
