Reverse Transcriptase-polymerase Chain Reaction Analysis Research Articles

Deletion of the c2515 and c2516 Genes Affects Iron Uptake and Virulence of APEC O1 Strain E516.

Avian pathogenic Escherichia coli (APEC), widely spread among poultry, is well-known to cause colibacillosis in chickens, which results in significant losses in poultry industry. The ability to uptake iron in the extra-intestinal environment is prerequisite for APEC survival. For adaptation to the low-iron environments, the bacteria have evolved multiple iron acquisition systems to ensure optimal iron uptake. However, many components of these iron acquisition pathways are still not clearly known. An in silico analysis of the genome of a septicemic APEC O1 strain E516 identified two putative iron transport genes homologous to the c2515 and c2516 genes from uropathogenic E. coli CFT073. In this study, we constructed the single and double gene deletion mutants, and studied their biological characteristic and pathogenic traits through in vitro and in vivo assays. Reverse transcriptase PCR (RT-PCR) analyses demonstrated that the mutations destroying the reading frame of the target genes abolished their transcription. Deletion of the single or double genes of c2515 and c2516 in APEC E516 weakened its ability to produce siderophore. Consistently, the mutants exhibited growth defect under iron-depleted conditions and the intracellular iron levels in the mutants were decreased in comparison with that of the wild-type (WT). Cell infection assays showed that the iron uptake defective mutants were more easily eliminated by the macrophage. Inactivation of the c2515 and c2516 genes affected bacterial colonization of chicken tissues, as well as the 50% lethal dose levels compared with the WT strain. Moreover, the expression levels of several iron uptake-related genes were significantly decreased in the double-deletion mutant. In total, the c2515 and c2516 may involve in siderophore-mediated iron uptake and participate in the pathogenesis of APEC O1 strain E516.

Clinical presentation and course of the novel coronavirus disease 2019 in patients with various types of cancer: A retrospective case-control analysis of an experienced cancer center in Turkey.

Cancers have been reported to worsen the clinical course of coronavirus disease 2019 (COVID-19) infection. We aimed to demonstrate the real-life data on health outcomes in COVID-19-infected cancer patients. We analyzed the data of 43 COVID-19-infected cancer patients in our COVID-19 clinics between March 25, 2020, and May 9, 2020, retrospectively. We determined that 1051 patients were followed up with COVID-19 infection and 43 (4%) of them were cancer patients. The mean age of the patients was 64.3 ± 12.3 years. Lung cancer is the most common cancer type among the patients (23.2%). Dyspnea (51.2%) was the most common symptom in the first admission. Typical ground-glass consolidation or patchy appearance with peribronchial thickening resembling bronchopneumonia on high-resolution computed tomography (HRCT) was present in 29 (67.4%) patients. COVID-19 was diagnosed in 14 (32.5%) patients based on reverse transcriptase-polymerase chain reaction analysis of nose-throat swab samples without any sign of lung involvement on HRCT. Total mortality of the COVID-19 infection was 46.5% (n = 20). Presence of heart disease (hazard ratio [HR]: 3.5; 95% confidence interval [CI]: 1.29-9.4), previous surgeries to the respiratory system (HR: 6.95; 95% CI: 1.29-27.7), and presence of dyspnea at admission (HR: 4; 95% CI: 1.31-12.3) were statistically significantly associated with death (P = 0.01, 0.02, and 0.01, respectively). Our practices supported that cancer patients were more affected by COVID-19 disease than the normal population. However, our findings can not be generalized due to being retrospective and single centered study, Also, we did not compare the findings with noncancer patients with COVID19 disease.

Evidence of the pathogenic HERV-W envelope expression in T lymphocytes in association with the respiratory outcome of COVID-19 patients

BackgroundDespite an impressive effort in clinical research, no standard therapeutic approach for coronavirus disease 2019 (COVID-19) patients has been established, highlighting the need to identify early biomarkers for predicting disease progression and new therapeutic interventions for patient management. The present study aimed to evaluate the involvement of the human endogenous retrovirus -W envelope (HERV-W ENV) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection considering recent findings that HERVs are activated in response to infectious agents and lead to various immunopathological effects. We analysed HERV-W ENV expression in blood cells of COVID-19 patients in correlation with clinical characteristics and have discussed its potential role in the outcome of the disease. MethodsWe analysed HERV-W ENV expression in blood samples of COVID-19 patients and healthy donors by flow cytometry and quantitative reverse transcriptase PCR analysis, and evaluated its correlation with clinical signs, inflammatory markers, cytokine expression, and disease progression. FindingsHERV-W ENV was highly expressed in the leukocytes of COVID-19 patients but not in those of healthy donors. Its expression correlated with the markers of T-cell differentiation and exhaustion and blood cytokine levels. The percentage of HERV-W ENV-positive lymphocytes correlated with inflammatory markers and pneumonia severity in COVID-19 patients. Notably, HERV-W ENV expression reflects the respiratory outcome of patients during hospitalization. InterpretationGiven the known immuno- and neuro-pathogenicity of HERV-W ENV protein, it could promote certain pathogenic features of COVID-19 and therefore serve as a biomarker to predict clinical progression of disease and open to further studies for therapeutic intervention. FundingInformation available at the end of the manuscript.

Proinflammatory Cytokine Modulates Intracellular Calcium Handling and Enhances Ventricular Arrhythmia Susceptibility.

Background: The mechanism of Interleukin-17 (IL-17) induced ventricular arrhythmia (VA) remains unclear. This study aimed to investigate the effect of intracellular calcium (Cai) handling and VA susceptibility by IL-17.Methods: The electrophysiological properties of isolated perfused rabbit hearts under IL-17 (20 ng/ml, N = 6) and the IL-17 with neutralizer (0.4 μg/ml, N = 6) were evaluated using an optical mapping system. The action potential duration (APD) and Cai transient duration (CaiTD) were examined, and semiquantitative reverse transcriptase-polymerase chain reaction analysis of ion channels was performed.Results: There were longer APD80, CaiTD80 and increased thresholds of APD and CaiTD alternans, the maximum slope of APD restitution and induction of VA threshold in IL-17 group compared with those in IL-17 neutralizer and baseline groups. During ventricular fibrillation, the number of phase singularities and dominant frequency were both significantly greater in IL-17 group than in baseline group. The mRNA expressions of the Na+/Ca2+ exchanger, phospholamban, and ryanodine receptor Ca2+ release channel were upregulated, and the subunit of L-type Ca2+ current and sarcoplasmic reticulum Ca2+-ATPase 2a were significantly reduced in IL-17 group compared to baseline and IL-17 neutralizer group.Conclusions: IL-17 enhanced CaiTD and APD alternans through disturbances in calcium handling, which may increase VA susceptibility.

Characterization of DtrJ as an IncC plasmid conjugative DNA transfer component

Incompatibility group C (IncC) plasmids are large (50-400kb), broad host range plasmids that drive the spread of genes conferring resistance to all classes of antibiotics, most notably the blaNDM gene that confers resistance to last-line carbapenems and the mcr-3 gene that confers resistance to colistin. Several recent studies have improved our understanding of the basic biological mechanisms driving the success of IncC, in particular the identification of multiple novel IncC conjugation genes by transposon directed insertion-site sequencing. Here, one of these genes, dtrJ, was examined in further detail. The dtrJ gene is located in the DNA transfer locus on the IncC backbone, and quantitative reverse-transcriptase PCR analysis revealed it is transcribed in the same operon as the DNA transfer genes traI and traD (encoding the relaxase and coupling protein, respectively) and activated by the AcaDC regulatory complex. We confirmed that DtrJ is not required for pilus biogenesis or mate pair formation. Instead, DtrJ localizes to the membrane, where it interacts with the coupling protein TraD and functions as an IncC DNA transfer protein. Overall, this work has defined the role of DtrJ in DNA transfer of IncC plasmids during conjugation.

M2 macrophage-derived exosomal microRNA-155-5p promotes the immune escape of colon cancer by downregulating ZC3H12B

Previous evidence has highlighted M2 macrophage regulation of cancer cells via exosome shuttling of microRNAs (miRNAs or miRs). The current study set out to explore the possible role of M2 macrophage-derived exosomal miR-155-5p in regard to immune escape of colon cancer cells. Experimental data from quantitative reverse-transcriptase PCR (qRT-PCR) and western blot analysis revealed highly expressed miR-155-5p and interleukin (IL)-6 and poorly expressed ZC3H12B in M2 macrophage-derived exosomes. Additionally, miR-155-5p could be transferred by M2 macrophage-isolated exosomes to colon cancer cells, which targeted ZC3H12B by binding to the 3¢ UTR, as identified by dual luciferase reporter gene. Meanwhile, gain- and loss-of function experimentation on miR-155-5p and ZC3H12B in SW48 and HT29 cells cocultured with M2 macrophage-secreted exosomes demonstrated that miR-155-5p overexpression or ZC3H12B silencing promoted the proliferation and antiapoptosis ability of SW48 and HT29 cells, as well as augmenting the CD3+ T cell proliferation and the proportion of interferon (IFN)-γ+ T cells. Xenograft models confirmed that M2 macrophage-derived exosomal miR-155-5p reduced the ZC3H12B expression to upregulate IL-6, which consequently induced immune escape and tumor formation. Collectively, our findings indicated that M2 macrophage-derived exosomal miR-155-5p can potentially promote the immune escape of colon cancer by impairing ZC3H12B-mediated IL-6 stability reduction, thereby promoting the occurrence and development of colon cancer.

Overexpression of chromodomain helicase DNA binding protein 5 (CHD5) inhibits cell proliferation and induces cell cycle arrest and apoptosis in chronic myeloid leukemia.

BackgroundChromodomain helicase DNA binding protein 5 (CHD5) was reported to be a tumor suppressor and our previous work showed CHD5 was epigenetically inactivated in human chronic myeloid leukemia (CML). This study aimed to investigate the effect of its overexpression on CML tumorigenesis.MethodsQuantitative reverse-transcriptase PCR and Western blotting analysis were used to detect the expression of CHD5 in human CML cell lines. The endogenous CHD5 expression was activated in two CML cell lines by CRISPR/dCas9-SAM system. In vitro cell function experiments were performed including proliferation, colony formation, apoptosis, autophagy, senescence and differentiation assays. Furthermore, tumorigenicity was evaluated in vivo in nude mice xenograft model.ResultsCHD5 was down-regulated in CML cell lines compare to normal bone marrow mononuclear cells (MCs). Cell proliferation after activating CHD5 was significantly inhibited. Moreover, overexpression of CHD5 induced G2/M phase arrest and apoptosis in CML cells. In a tumor xenograft mouse model, CHD5 restoration was found to sharply repress tumor growth. Compared with the control group, overexpression of CHD5 enhanced the expression of p21 and cdc2 phosphorylation, whereas decreased the protein level of Cyclin B1. Furthermore, experiments revealed that up-regulation of CHD5 activated caspase-3, while anti-apoptosis protein Bcl-2 expression was reduced in CML cells.ConclusionsCHD5 plays a role of anti-tumorigenic effects involved in CML cell proliferation, cell cycle arrest and apoptosis.

No secret hiding place? Absence of SARS-CoV-2 on the ocular surface of 1145 hospitalized patients in a pandemic area

ObjectivesThe aims of this study were to evaluate the isolated prevalence of real-time reverse transcriptase-polymerase chain reaction (RT-PCR)–confirmed SARS-CoV-2 on the ocular surface without systemic infection in hospitalized asymptomatic patients and to determine the risk for ophthalmologists and medical staff to be infected by prescreened asymptomatic patients in a tertiary eye care center.MethodsIn this prospective, observational study, bilateral swaps of the conjunctiva in the lower fornices as well as nasopharyngeal swaps were collected in 1145 hospitalized asymptomatic patients of a tertiary eye care center. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed for each swap to evaluate the prevalence of SARS-CoV-2. Demographic data and potential risk factors for an isolated infection of the ocular surface were noted.ResultsTwo thousand two hundred eighty-eight (99.9%) of all 2290 tested eyes had negative results in the RT-PCR analysis of the conjunctival swabs. One patient had bilateral false-positive results in the conjunctival swabs. None of the 1145 patients had any positive RT-PCR-confirmed result in the nasopharyngeal swabs.ConclusionsThe risk for an isolated conjunctival viral activity in patients with a negative nasopharyngeal swab-based RT-PCR seems to be absent or extremely low, suggesting no need to perform additional conjunctival swabs in patients with negative nasopharyngeal swabs. Furthermore, the risk of a work-related SARS-CoV-2 infection due to direct contact with preselected asymptomatic patients in an eye care center is very low, especially when additional hygiene standards and safe distances are respected carefully. This might reassure medical staff and reduce the fear of SARS-CoV-2 infection.

First Evidence of Pathogenic HERV-W Envelope Expression in T Lymphocytes in Association with the Respiratory Outcome of COVID-19 Patients

Background: Despite an impressive effort in clinical research, no standard therapeutic approach for coronavirus disease 2019 (COVID-19) patients has been established, highlighting the need to identify early biomarkers for predicting disease progression and new therapeutic intervention for patient management. The present study aimed to evaluate the involvement of the human endogenous retrovirus -W envelope (HERV-W ENV) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection considering recent findings that HERVs are activated in response to infectious agents and lead to various immunopathological effects. We analysed HERV-W ENV expression in blood cells of COVID-19 patients in correlation with clinical characteristics and have discussed its potential role in the outcome of the disease.Methods: We analysed HERV-W ENV expression in blood samples of Covid-19 patients and healthy donors by flow cytometry and quantitative reverse transcriptase PCR analysis, and evaluated its correlation with clinical signs, inflammatory markers, cytokine expression, and disease progression.Findings: HERV-W ENV was highly expressed in the leukocytes of COVID-19 patients but not in those of healthy donors. Its expression correlated with the markers of T-cell differentiation and exhaustion and blood cytokine levels. The percentage of HERV-W ENV-positive lymphocytes correlated with inflammatory markers and pneumonia severity in COVID-19 patients. Notably, HERV-W ENV expression reflects the respiratory outcome of patients during hospitalization.Interpretation: Given the known immuno- and neuro-pathogenicity of HERV-W ENV protein, it could promote certain pathogenic features of COVID-19 and therefore serve as a biomarker to predict clinical progression of disease and as a potential therapeutic target.Funding: Genero InnovationDeclaration of Interests: C.M. reports grant from Gilead, outside the submitted work; L.S. reports personal fees and other from Merck, Gilead, and Abbvie, grants from Gilead, outside the submitted work; HP and BC receive compensation for their work by Geneuro-Innovation. The other authors declare no competing interests.Ethics Approval Statement: Ethical approval for the collection and use of human samples was obtained from the ethical board of the Hospital, COronaVIrus Disease: Safety and Efficacy of Experimental Treatment (COVID_SEET prot.7562/2020, 9th April 2020, experimental register 46.20).

A case report of a pregnant woman with coronavirus disease 2019 (COVID-19) and her live-born infant

In December 2019, an outbreak of coronavirus disease 2019 (COVID-19) occurred in Wuhan, China. The disease, which is now a global pandemic, is reportedly associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we report a case of COVID-19 in a 31-year-old pregnant woman who showed symptoms that included fever, a four-day history of dry cough, and myalgia. Real-time reverse transcriptase-polymerase chain reaction analysis of naso- and oropharyngeal samples was positive for the SARS-CoV-2. A cesarean section was performed during the acute phase of COVID-19; the full-term infant was isolated from his mother and underwent formula feeding. He was healthy and negative for the SARS-CoV-2. This report describes the clinical features, diagnosis, and treatment of the mother’s illness and its effects on her live-born infant.

Molecular differentiation between velogenic isolates and lentogenic LaSota strain of Newcastle disease virus

Objective: Isolation, serological and molecular identification and differentiation between velogenic and lentogenic of Newcastle disease virus (NDV) strains. Design: Descriptive study. Procedures: In this study, A total of 24 Pooled samples were collected from Dakahlia, Egypt, from 2017 to 2018 .The virus was isolated in 10 days old (ECEs) and serologically identified in third egg passage allantoic fluid by Haemagglutination (HA), Haemagglutination inhibition test (HIT) and agar gel precipitation test (AGPT). Molecular confirmation was done by reverse transcriptase-polymerase chain reaction (RT-PCR). Genetic diversity between velogenic and lentogenic NDV strains was done by RT-PCR amplification of 254bp F gene fragment from velogenic NDVs and sequence analysis of 362bp NDV F gene fragment. Results: Out of 24 tested samples, 20 samples were collected from allontoic fluid of 3rd passage positive by HA test, 18 samples by HIT, 16 samples by AGPT and 18 isolates were positive with RT-PCR amplification of 362bp F gene fragment. LaSota strain gave positive results in HA, HIT, AGPT and RT-PCR amplification of 362bp fragment. Interestingly, 254bp fragment failed to be amplified from LaSota strain. The phylogenic analysis revealed that our isolates were related to Egyptian velogenic genotype VII NDVs. The deduced amino acid sequence of the F protein cleavage site of our isolates was characteristic of velogenic NDVs (112R-R-Q-K-R↓F117) while LaSota strain had characteristic lentogenic NDVs cleavage site (112G-R-Q-G-R↓L117). Conclusion and clinical relevance: RT-PCR and sequence analysis in this study can be used for differentiation between velogenic and lentogenic NDV strains.

BMP Ligand Trap ALK3-Fc Attenuates Osteogenesis and Heterotopic Ossification in Blast-Related Lower Extremity Trauma.

Traumatic heterotopic ossification (tHO) commonly develops in wounded service members who sustain high-energy and blast-related traumatic amputations. Currently, no safe and effective preventive measures have been identified for this patient population. Bone morphogenetic protein (BMP) signaling blockade has previously been shown to reduce ectopic bone formation in genetic models of HO. In this study, we demonstrate the efficacy of small-molecule inhibition with LDN193189 (ALK2/ALK3 inhibition), LDN212854 (ALK2-biased inhibition), and BMP ligand trap ALK3-Fc at inhibiting early and late osteogenic differentiation of tissue-resident mesenchymal progenitor cells (MPCs) harvested from mice subjected to burn/tenotomy, a well-characterized trauma-induced model of HO. Using an established rat tHO model of blast-related extremity trauma and methicillin-resistant Staphylococcus aureus infection, a significant decrease in ectopic bone volume was observed by micro-computed tomography imaging following treatment with LDN193189, LDN212854, and ALK3-Fc. The efficacy of LDN193189 and LDN212854 in this model was associated with weight loss (17%-19%) within the first two postoperative weeks, and in the case of LDN193189, delayed wound healing and metastatic infection was observed, while ALK3-Fc was well tolerated. At day 14 following injury, RNA-Seq and quantitative reverse transcriptase-polymerase chain reaction analysis revealed that ALK3-Fc enhanced the expression of skeletal muscle structural genes and myogenic transcriptional factors while inhibiting the expression of inflammatory genes. Tissue-resident MPCs harvested from rats treated with ALK3-Fc exhibited reduced osteogenic differentiation, proliferation, and self-renewal capacity and diminished expression of genes associated with endochondral ossification and SMAD-dependent signaling pathways. Together, these results confirm the contribution of BMP signaling in osteogenic differentiation and ectopic bone formation and that a selective ligand-trap approach such as ALK3-Fc may be an effective and tolerable prophylactic strategy for tHO.

Changes in Mucin Production in Human Airway Epithelial Cells After Exposure to Electronic Cigarette Vapor With or Without Nicotine

ObjectivesThe emergence of electronic cigarettes (e-cigarettes) has created new perceptions of the tobacco market. Unlike traditional tobacco, the greatest advantage of e-cigarettes is that they have less smell and are convenient and inexpensive. Most e-cigarette smokers believe that e-cigarette smoking is less harmful than traditional smoking. Information on the effects of e-cigarettes on human health is limited, and the issue remains controversial.MethodsWe studied the effects of e-cigarette vapor on mucin (MUC5AC and MUC5B) and the change of MUC5AC and MUC5B from e-cigarette liquid with or without nicotine in respiratory epithelial cells. The effects of e-cigarette vapor with or without nicotine on mucin, along with the involved signaling pathways, were investigated using reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, enzyme immunoassays, and immunoblot analysis with several specific inhibitors and small interfering RNA.ResultsE-cigarette vapor with or without nicotine stimulated MUC5AC, but not MUC5B, expression in respiratory epithelial cells. In addition, we showed that e-cigarette vapor with and without nicotine induced MUC5AC expression via activation of the mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase [ERK] 1/2 and p38) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways in human airway epithelial cells.Conclusion E-cigarette vapor with and with nicotine significantly increased MUC5AC expression in human airway epithelial cells.

Consistent Detection of TLS/FUS-ERG Chimeric Transcripts in Acute Myeloid Leukemia With t(16; 21)(p11; q22) and Identification of a Novel Transcript

Abstract16; 21 translocation is a recurrent primary abnormality in acute myeloid leukemia (AML). The genes involved in this translocation are ERG on chromosome 21 and TLS/FUS on chromosome 16. The rearrangement of the two chromosomes forms the TLS/FUS-ERG fusion gene and produces a consistent chimeric transcript on the der (21) chromosome. In this study, we analyzed the clinical characteristics of 19 patients with t(16; 21)-AML, including 2 patients who evolved from myelodysplastic syndrome, and detected the chimeric transcripts of the TLS/FUS-ERG fusion gene in the patients during various clinical stages by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. We found that the patients with t(16; 21) are characterized by a relatively younger age (median age, 22 years old), involvement of various subtypes of French-American-British classification and a poor prognosis: 18 of the 19 patients died of the disease (median survival was 16 months). Four types of TLS/FUS-ERG chimeric transcripts including a novel type were noted in the RT-PCR analysis. The novel transcript contained an additional 138 nucleotides consisting of TLS/FUS exon 8 and ERG exons 7 and 8 and had an in-frame fusion. These chimeric transcripts were consistently detectable in the samples obtained not only at diagnosis and relapse but also in short and long complete remission, suggesting that t(16; 21)-AML is resistant to conventional chemotherapy. Thus, we recommend that t(16; 21) should be monitored by RT-PCR even in clinical remission and the patients should be treated by other more powerful modality like stem-cell transplantation in the first remission.

Shiga-Like Toxin-1 Receptor on Human Breast Cancer, Lymphoma, and Myeloma and Absence From CD34+ Hematopoietic Stem Cells: Implications for Ex Vivo Tumor Purging and Autologous Stem Cell Transplantation

The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1), targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow cytometry and immunocytochemistry on lymphoma and breast cancer cells recovered from biopsies of primary human cancers as well as on B cells or plasma cells present in blood/bone marrow samples of multiple myeloma patients. Breast cancer cell lines also expressed receptors for the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma, B-cell chronic lymphocytic leukemia, and myeloma B or plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold, as measured by flow cytometry. Depletion of myeloma plasma cells was confirmed using a cellular limiting dilution assay followed by reverse transcriptase-polymerase chain reaction analysis of clonotypic IgH transcripts, which showed a greater than 3 log reduction in clonotypic myeloma cells after SLT-1 treatment. Receptors for the toxin were not detected on human CD34(+) hematopoietic progenitor cells (HPC). HPC were pretreated with a concentration of SLT-1 known to purge primary malignant B cells and cultured for 6 days. The number of HPC was comparable in toxin-treated and untreated cultures. HPC were functionally intact as well. Colony-forming units (CFU) were present at an identical frequency in untreated and SLT-1 pretreated cultures, confirming that CFU escape SLT-1 toxicity. The results suggest the ex vivo use of SLT-1 in purging SLT-1 receptor-expressing malignant cells from autologous stem cell grafts of breast cancer, lymphoma, and myeloma patients.

Plasma cell–directed therapies in monoclonal gammopathy–associated scleromyxedema

AbstractScleromyxedema is a rare skin and systemic mucinosis that is usually associated with monoclonal gammopathy (MG). In this French multicenter retrospective study of 33 patients, we investigated the clinical and therapeutic features of MG-associated scleromyxedema. Skin molecular signatures were analyzed using a transcriptomic approach. Skin symptoms included papular eruptions (100%), sclerodermoid features (91%), and leonine facies (39%). MG involved an immunoglobulin G isotype in all patients, with a predominant λ light chain (73%). Associated hematologic malignancies were diagnosed in 4 of 33 patients (12%) (smoldering myeloma, n = 2; chronic lymphoid leukemia, n = 1; and refractory cytopenia with multilineage dysplasia, n = 1). Carpal tunnel syndrome (33%), arthralgia (25%), and dermato-neuro syndrome (DNS) (18%) were the most common systemic complications. One patient with mucinous cardiopathy died of acute heart failure. High-dose IV immunoglobulin (HDIVig), alone or in combination with steroids, appeared to be quite effective in nonsevere cases (clinical complete response achieved in 13/31 patients). Plasma cell–directed therapies using lenalidomide and/or bortezomib with dexamethasone and HDIVig led to a significant improvement in severe cases (HDIVig refractory or cases with central nervous system or cardiac involvement). The emergency treatment of DNS with combined plasmapheresis, HDIVig, and high-dose corticosteroids induced the complete remission of neurological symptoms in 4 of 5 patients. Quantitative reverse-transcriptase polymerase chain reaction analysis of 6 scleromyxedema skin samples showed significantly higher profibrotic pathway levels (transforming growth factor β and collagen-1) than in healthy skin. Prospective studies targeting plasma cell clones and/or fibrotic pathways are warranted for long-term scleromyxedema management.

A Circular RNA Derived from Golgi Glycoprotein 1 mRNA Regulates KRAS Expression and Promotes Colorectal Cancer Progression by Targeting microRNA-622.

BackgroundCircular RNAs (circRNAs) represent a distinct class of non-coding RNAs that have attracted substantial research attention in recent years. We identified a novel circRNA derived from golgi glycoprotein 1 mRNA (circ_GLG1), the role of which is unknown in colorectal cancer (CRC). The purpose of this study was to explore the potential roles and mechanisms of circ_GLG1 in CRC.Materials and MethodsQuantitative reverse transcriptase-polymerase chain reaction analysis was performed to quantify circ_GLG1 expression in 40 pairs of CRC tissues and adjacent normal tissues as well as CRC cell lines. DLD1 CRC cells were transfected with a small-interfering RNA against circ_GLG1, after which cell proliferation, viability, invasion, and migration were measured through cell counting kit-8 colony-formation, transwell, and wound-healing assays, respectively. Dual-luciferase reporter assays were performed to explore the binding sites among circ_GLG1, miR-622, and Kirsten rat sarcoma (KRAS) transcripts. KRAS protein expression was detected using Western blot analysis.ResultsCirc_GLG1 expression was significantly higher in CRC tissues than in adjacent normal tissues. Knocking down circ_GLG1 in DLD1 cells inhibited tumor cell viability, proliferation, invasion, and migration, and these effects were reversed by co-transfecting an miR-622 inhibitor. Circ_GLG1 promoted KRAS expression at both the mRNA and protein levels by acting as an miR-622 sponge. Dual-luciferase reporter assays demonstrated that miR-622 interacted with circ_GLG1 and KRAS mRNA.ConclusionOur study revealed the role of the circ_GLG1–miR-622–KRAS axis in CRC. Moreover, our findings provide insight into the molecular mechanism of circ_GLG1 in CRC and suggest potential new biomarkers for diagnosing this disease.

Familial Pseudohypoparathyroidism Type IB Associated with an SVA Retrotransposon Insertion in the GNAS Locus.

Loss of methylation (LOM) at GNAS-A/B:TSS-differentially methylated regions (DMRs) in the GNAS locus is observed in pseudohypoparathyroidism type 1B (PHP1B). Many PHP1B cases are sporadic, but autosomal dominant-PHP1B has a deletion involving NESP55 expressed from the maternal allele or STX16 located upstream of the GNAS locus on the maternal allele. We report the possible first familial PHP1B cases with retrotransposon insertion in the GNAS locus on the maternal allele. To our knowledge, they are the possible first cases with imprinting disorders caused by retrotransposon insertion. The two sibling cases experienced tetany and/or cramps from school age and had hypocalcemia and an increased serum intact parathyroid hormone (PTH) level together with overweight, round face, and normal intellectual levels. Methylation analysis for DMRs in the GNAS locus showed only LOM of the GNAS-A/B:TSS-DMR. Copy number abnormalities at STX16 and the GNAS locus were not detected by array comparative genomic hybridization. Whole-genome sequencing and Sanger sequencing revealed an approximately 1000-bp SVA retrotransposon insertion upstream of the first exon of A/B on the GNAS locus in these siblings. Whole-genome methylome analysis by Enzymatic Methyl-Seq in the siblings showed normal methylation status in the region surrounding the insertion site and mild LOM of the GNAS-A/B:TSS-DMR. We conducted transcriptome analysis using mRNA from skin fibroblasts and induced pluripotent stem cells (iPSCs) derived from the siblings and detected no aberrant NESP55 transcripts. Quantitative reverse-transcriptase PCR (qRT-PCR) analysis in skin fibroblasts showed increased A/B expression in the patients and no NESP55 expression, even in a control. qRT-PCR analysis in iPSCs showed decreased NESP55 expression with normal methylation status of the GNAS-NESP:TSS-DMR in the patients. The retrotransposon insertion in the siblings likely caused decreased NESP55 expression that could lead to increased A/B expression via LOM of the GNAS-A/B:TSS-DMR, subsequent reduced Gsα expression, and finally, PHP1B development. © 2022 American Society for Bone and Mineral Research (ASBMR).

SIRT1 alleviates hepatic ischemia-reperfusion injury via the miR-182-mediated XBP1/NLRP3 pathway

The hepatoprotection of histone deacetylase sirtuin 1 (SIRT1) has been identified to attenuate ischemia-reperfusion (IR)-triggered inflammation and liver damage. This study was performed to characterize the function of SIRT1 in hepatic IR injury. In in vivo assays on liver-specific knockout mice of SIRT1, we first validated the effect of SIRT1 knockout on liver damage and XBP1/NLRP3 inflammasome activation. Next, we examined whether knockdown of XBP1/NLRP3 or miR-182 agomir could reverse the effect of SIRT1 knockout. In in vitro assays, NCTC1469 cells subjected to hypoxia/reoxygenation (H/R) were transduced with small interfering RNA (siRNA)/activator of SIRT1 or miR-182 agomir to confirm the effect of SIRT1 on NCTC1469 cell behaviors as well as the regulation of miR-182 and the XBP1/NLRP3 signaling pathway. Hepatic IR injury was appreciably aggravated in SIRT1 knockout mice, and SIRT1 knockdown abolished the inhibition of XBP1/NLRP3 inflammasome activation, which was reversed by NLRP3 knockdown, XBP1 knockdown, or miR-182 agomir. Mechanistically, miR-182 expression was positively regulated by SIRT1 in hepatic IR injury in mice, and miR-182 inhibited the expression of XBP1 by binding to the 3′ untranslated region (UTR) of XBP1. The histone deacetylase SIRT1 inhibits the downstream XBP1/NLRP3 inflammatory pathway by activating miR-182, thus alleviating hepatic IR injury in mice.

Identification and characterization of circular RNAs in mammary gland tissue from sheep at peak lactation and during the nonlactating period

Circular RNAs are a class of noncoding RNA with a widespread occurrence in eukaryote tissues, and with some having been demonstrated to have clear biological function. In sheep, little is known about the role of circular RNAs in mammary gland tissue, and therefore an RNA sequencing approach was used to compare mammary gland tissue expression of circular RNAs in 9 Small Tail Han sheep at peak lactation, and subsequently when they were not lactating. These 9 sheep had their RNA pooled for analysis into 3 libraries from peak lactation and 3 from the nonlactating period. A total of 3,278 and 1,756 circular RNAs were identified in the peak lactation and nonlactating mammary gland tissues, respectively, and the expression and identity of 9 of them was confirmed using reverse transcriptase-polymerase chain reaction analysis and DNA sequencing. The type, chromosomal location and length of the circular RNAs identified were ascertained. Forty upregulated and one downregulated circular RNAs were characterized in the mammary gland tissue at peak lactation compared with the nonlactating mammary gland tissue. Gene ontology enrichment analysis revealed that the parental genes of these differentially expressed circular RNAs were related to molecular function, binding, protein binding, ATP binding, and ion binding. Five differentially expression circular RNAs were selected for further analysis to predict their target microRNAs, and some microRNAs reportedly associated with the development of the mammary gland were found in the constructed circular RNA-microRNA network. This study reveals the expression profiles and characterization of circular RNAs at 2 key stages of mammary gland activity, thereby providing an improved understanding of the roles of circular RNAs in the mammary gland of sheep.