Probes for INS

Full size image [/article/10.1007/s42764-021-00038-x/figures/1] The findings described above support the statement that HPV infection is common, but, in comparison, cervical cancer is quite rare, leading to the conclusion that HPV infection alone is not sufficient to produce cancer, as tumor development and progression require the contribution of multiple factors. Among the risk factors for cancer development and progression in women infected with HR HPV are the determinants of persistent infection, as it is well established that only women in whom HR HPV infection persists are at risk for cervical lesions that may progress to cancer (Banister et al. 2015 and references therein). HPV persistence has also been linked to HPV-mediated disease in men (Bettampadi et al. 2020 [/article/10.1007/s42764-021-00038-x#ref-CR16]). This is an important area of study, because in principle, if we were able to determine at a single visit whether or not an incident HR HPV infection will persist, we could target HPV-mediated cancer surveillance resources to the people who present with persistent infection. Our own (unpublished) findings support the concept that women with persistent HPV infection fail to mount a strong immune response to HPV. In turn, immune responses to HPV are likely to be influenced by HLA and SNP profiles, both of which have been linked with cervical cancer susceptibility (Chen et al. 2014; Das Gosh et al. 2017). Among the many SNPs that have relevance for cervical cancer development, the Arg/Pro TP53 polymorphism at codon 72 has received considerable attention, as the homozygote Arg/Arg phenotype is associated with a higher risk of developing cervical cancer, at least in certain populations (Ojeda et al. 2003; Chuery et al. 2017). TP53 codon 72 polymorphism has been connected with higher HPV E6/E7 expression, which appears to correlate with the Arg/Arg genotype (Chuery et al. 2017). Despite the continuing controversies in this area, there is evidence that this particular polymorphism plays a role in cervical cancer development, albeit with additional intervening factors that may modulate its impact in different populations.

Introduction The control of energy balance involves communication of peripheral hormones with brain regions controlling food intake and energy expenditure such as the arcuate nucleus of the hypothalamus (ARC). Within the ARC, two primary neuronal subpopulations control energy balance: proopiomelanocortin (POMC) neurons, which reduce food intake and increase energy expenditure; and agouti-related protein (AgRP) neurons, which inhibit POMC neurons and conversely increase food intake and suppress energy expenditure. These circuits are typically disrupted by high fat diet (HFD) leading to a chronic state of energy imbalance and obesity. Accumulating evidence suggests that HFD-induced obesity is associated with deficiency of angiotensin (Ang)-(1-7), a protective renin-angiotensin system hormone. Our recent data show that systemically administered Ang-(1-7) induces adipose thermogenesis to enhance energy expenditure and promote weight loss. We propose that effects of Ang-(1-7) on energy balance involve activation of ARC neurocircuits, but this has not been tested. Additionally, the localization and neuronal subpopulations expressing Ang-(1-7) mas receptors (MasR) in the ARC is unknown. In this study, we hypothesized that: Ang-(1-7) activates ARC neurons; MasR are expressed in the ARC and are primarily colocalized with POMC neurons; and the ability of Ang-(1-7) to activate ARC neurons as well as co-localization of MasR with POMC neurons is disrupted following chronic HFD. Methods Male C57Bl/6J mice were fed a 60% HFD or matched control diet ad libitum for 12 weeks. Mice then received subcutaneous injection of Ang-(1-7) [2 mg/kg] to induce neuronal activation in the ARC, as measured by c-fos gene expression (n=4-6/group). In a second cohort of mice, RNAscope in situ hybridization was performed on coronal ARC sections to determine co-localization of MasR mRNA within POMC versus AgRP neurons (n=5/group). Results We found that Ang-(1-7) increases the number of c-fos positive cells in the ARC (39±6 vs. 19±3 saline; p=0.022) in control diet mice. Ang-(1-7)-mediated activation of ARC neurons was attenuated in HFD mice (34±3 vs. 23±4 saline; p=0.185). The rostral-medial-caudal distribution of ARC MasR was similar between control diet and HFD mice, with no difference in percentage of MasR positive neurons between groups (18±1 and 15±5%, respectively; p=0.733). MasR were more highly co-localized to POMC versus AgRP neurons, with HFD tending to reduce these co-localizations (MasR/POMC: 49±10 control vs. 33±5% HFD, p=0.199; MasR/AgRP: 36±11 control vs.16±7% HFD, p=0.209). Conclusions These findings suggest that chronic HFD reduces the ability of Ang-(1-7) to acutely activate neurons in the ARC. Further, HFD disrupts co-localization of MasR with POMC and AgRP neurons in the ARC indicating disconnect in the endogenous neurocircuitry controlling energy balance. Further studies are needed to explore the importance of MasR in these neuronal subpopulations for energy balance, to determine the potential for targeting of Ang-(1-7) as an innovative pharmacological strategy for obesity treatment.

Introduction: Acute kidney injury (AKI) is commonly associated with adverse outcomes among asphyxiated neonates with higher burden reported in middle and low income countries. With the many limitations accredited to the current assessment of renal function in asphyxiated neonates, other biomarkers amongst them being urinary b2-microglobulin can be evaluated as promising tests for AKI. We aimed to determine the accuracy of urinary b2-microglobulin for early detection of AKI in asphyxiated neonates at a tertiary hospital in northern Tanzania. Methods: A cross-sectional study was conducted from January to the end of June, 2019 at the hospital among term newborns aged between 0- 24 hours old with birth asphyxia. Urine and blood samples were collected for estimation of urinary b2-microglobulin and serum creatinine test respectively. Data was analyzed using Statistical Package for Social Sciences ( SPSS) version 23. Accuracy of urinary b2- microglobulin was determined by receiver operating characteristic curve. Results: Of the 66 participants enrolled, majority 37(56.1%) were male. The proportion of neonates with elevated urinary b2-microglobulin was 60 (90%) with greater part also being male 33(55%). The sensitivity of urinary b2-microglobulin was 100% while the specificity was 10.3% with Area Under Receiver Operating Characteristic Curve 61.5%. Conclusions: Kidney injury is an anticipated complication in asphyxiated neonates. Urinary b2-microglobulin is a sensitive early, noninvasive test for diagnosing Acute Kidney Injury in neonates who have suffered from birth asphyxia. However, for neonates who have suffered severe forms of birth asphyxia, other follow up confirmatory tests will be required to diagnose acute kidney injury in asphyxiated neonates. No conflict of interest

Collagen VI (COLVI) is a critical myomatrix protein for skeletal muscle health and maintenance. There are 6 COL6 genes (COL6A1-COL6A6). Pathogenic variants in COL6A1, COL6A2, or COL6A3 cause COLVI-related dystrophies (COL6-RDs) with early-onset muscle weakness and loss of ambulation. Identifying novel therapeutic targets is critical for developing COL6-RDs therapies. Here, using in situ hybridization, we aim to identify, quantify, and locate all cell types that express the wild-type (WT) COL6 genes and a common, recurrent pseudoexon (PE)-inserting COL6A1 mutation in limb skeletal muscles and diaphragms of WT and COL6-RDs male mice at 10-day-old, and 6- and 20-month-old. We first analyzed published mouse skeletal muscle RNA-seq datasets to identify potential sensitive and specific mRNA cell markers (3/cell type). Then, we conducted a pilot marker validation experiment with RNAscope. We quantified and assessed the expression of the selected 33 markers’ transcripts in 3 non-overlapping fields of a quadriceps section from 2-month-old and 6-month-old mice (n=1). The specificity was reported as % of cells that had a marker for one cell type and lacked the markers of all other cell types. Our preliminary data indicated markers that were specific (Pdgfra: Fibro-adipogenic progenitors (FAPs) (98-100% specific), Myod1: satellite cells (SCs) (88-96%), and Pecam1: endothelial cells (endo) (92-100%)), non-specific (Esam: endo (57-77%), and Asb5: SCs (55-81%)), or specific in only one age group (Dpt: FAPs (2-month: 73%; 6-month: 89%), and Cdh5: endo (2-month: 80%; 6-month: 100%). We are currently validating each marker's specificity and sensitivity. We will identify and locate the cells that express the Col6 genes and the PE and quantify their transcripts expression levels in the various models and age groups. The findings of this project will provide additional insights into the roles of COLVI-producing cells in the pathogenesis of COL6-RDs and help direct therapeutic approaches.

Background The increase in syphilis rates worldwide, particularly in people living with HIV (PLWH), as well as the challenging diagnosis that secondary syphilis represents, make essential the accurate recognition of its manifestations, particularly in easy-access sites like the oral mucosa. Objective To describe the clinicopathologic spectrum of oral secondary syphilis (OSS) in PLWH. Methods A cross-sectional and descriptive study that included PLWH with OSS from 3 HIV referral centers in Mexico City (2004-2020). Demographic and clinical data were obtained. A comprehensive oral examination was done. OSS was diagnosed following established criteria. Histopathologic/cytological procedures were performed to rule out specific oral lesions. In all patients, Venereal Disease Research Laboratory tests were assessed and, if possible, a confirmatory fluorescent treponemal antibody test or biopsy was performed. Statistical analysis was performed using SPSS v25. Results Forty-seven PLWH with OSS (97.8% male, median age: 32 years, 63.8% with acquired immunodeficiency syndrome) were included. Thirty-five were receiving combination antiretroviral therapy (74.5%; median of 1146 [Q1-Q3: 337.5-1971] days) with a median CD4+ count of 385 (Q1-Q3: 223-664) cells/mm3 and a Log10 HIV viral load of 4.1 (Q1-Q3: 3.7-5.3) copies/mL. Forty had a complete clinical-serological diagnosis (85.1%; 17 had histopathologic confirmation) and 7 had a clinical-histopathologic diagnosis. Twenty-nine individuals presented 1 lesion (61.7%), and mucous patch was the most common type mainly on oropharyngeal mucosa, followed by ulcers and macular lesions. Ten patients presented maculopapular dermatosis (21.3%). Conclusions In PLWH, oral lesions, particularly mucous patch and/or ulcers on the oral and oropharyngeal mucosa, must alert specialists to consider a diagnosis of syphilis and perform a comprehensive panel of confirmatory tests.

Vaping is suggested to be a risk factor for poor wound healing akin to smoking. However, the molecular and histologic mechanisms underlying this postulation remain unknown. Our study sought to compare molecular and histologic changes in cutaneous flap and non-flap tissue between vaping, smoking and control cohorts. Animal study of 15 male Sprague-Dawley rats was randomized to three cohorts: negative control (n = 5), e-cigarette (n = 5) and cigarette (n = 5) and exposed to their respective treatments with serum cotinine monitoring. After 30 days, random pattern flaps were raised and healed for 2 weeks after which skin punch biopsies of flap and non-flap tissues were collected for quantitative-reverse transcription-polymerase chain reaction of three selected wound healing genes (transforming growth factor β [TGF-β], vascular endothelial growth factor [VEGF], matrix metalloproteinase-1 [MMP-1]); then, immunohistochemistry for CD68 expression, α-smooth muscle actin looking at microvessel density (MVD) and in situ hybridization to localize VEGF production were undertaken. In flap tissue, vaping (mean[SEM]) (0.61[0.07]) and smoking (0.70[0.04]) were associated with decreased fold change of VEGF expression compared with controls (0.91[0.03]) (p < 0.05, p < 0.05, respectively). In non-flap tissue, only vaping was associated with decreased VEGF expression (mean[SEM]) (0.81[0.07]), compared with controls (1.17[0.10]) (p < 0.05) with expression primarily localized to basal keratinocytes and dermal capillaries. Immunohistochemistry showed decreased MVD in smoking (0.27[0.06]) and vaping (0.26[0.04]) flap tissue compared to matched controls (0.65[0.14]) (p < 0.05, p < 0.05, respectively) and decreased areas of fibrosis compared with controls on gross histology. Vaping and smoking were similarly associated with decreased VEGF expression, MVD and fibrotic changes in flap tissue. The results suggest attenuated angiogenesis via decreased VEGF expression as a mechanism for poor wound healing in vaping-exposed rats.

Small case series have evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-detection in formalin-fixed paraffin-embedded (FFPE) tissue using reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and/or RNA-in situ hybridization (RNAish). To compare droplet digital PCR (ddPCR), IHC, and RNAish to detect SARS-CoV-2 in FFPE tissue in a large series of lung specimens from coronavirus disease 2019 (COVID-19) patients. ddPCR and RNAish used commercially available probes; IHC utilized clone 1A9. Twenty-six autopsies of COVID-19 patients with FFPE tissue blocks of 62 lung specimens, 22 heart specimens, 2 brain specimens, and 1 liver, and 1 umbilical cord were included. Control cases included 9 autopsy lungs from patients with other infections/inflammation and virus-infected tissue or cell lines. ddPCR had the highest sensitivity for SARS-CoV-2 (96%) when compared to IHC (31%) and RNAish (36%). All 3 tests had a specificity of 100%. Agreement between ddPCR and IHC or RNAish was fair (κ=0.23, κ=0.35, respectively). Agreement between IHC and ISH was substantial (κ=0.75). Interobserver reliability was almost perfect for IHC (κ=0.91) and fair to moderate for RNAish (κ=0.38-0.59). Lung tissues from patients who died earlier after onset of symptoms revealed higher copy numbers by ddPCR (P=.03, pearson corr = -0.65) and were more likely to be positive by RNAish (P=.02) than lungs from patients who died later. SARS-CoV-2 was identified in hyaline membranes, pneumocytes, and rarely in respiratory epithelium. ddPCR showed low copy numbers in 7 autopsy hearts from ProteoGenex Inc. All other extrapulmonary tissues were negative. ddPCR was the most sensitive and highly specific test to identify SARS-CoV-2 in lung specimens from COVID-19 patients.

We studied the suitability of commercially available monoclonal antibodies (mAbs) for the immunohistochemical (IHC) detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) in standard archival specimens. Antibodies were screened on HEK293 cells transfected with viral nucleoprotein, S1 subunit and S2 subunit of spike protein and on untransfected cells, as well as a panel of normal tissue. Lung tissue with presence of SARS-CoV2 confirmed by in situ hybridization (ISH) was also used. A total of 7 mAbs were tested: (1) mAb 001 (Sino Biological, 40143-R001), (2) mAb 007 (Sino Biological, 40150-R007), (3) mAb 019 (Sino Biological, 40143-R019), (4) mAb 1A9 (GeneTex, GTX632604), (5) mAb ABM19C9 (Abeomics, 10-10007), (6) FIPV3-70 (Santa Cruz, SC-65653), and (7) mAb 6F10 (BioVision, A2060). Only 2 mAbs, clone 001 to the nucleoprotein and clone 1A9 to the S2 subunit spike protein displayed specific immunoreactivity. Both clones showed strong staining in the acute phase of COVID-19 pneumonia, mostly in areas of acute diffuse alveolar damage, but were not completely congruent. Viral protein was also found in kidney tubules, endothelia of multiple organs and a nasal swab of a patient with persistent SARS-CoV2 infection. The other tested reagents were either poorly reactive or demonstrated nonspecific staining in tissues and lesions not infected by SARS-CoV2. Our study demonstrates that rigid specificity testing is mandatory for the evaluation of mAbs to SARS-CoV2 and that clones 001 to nucleoprotein and 1A9 to S2 subunit spike protein are useful for the in situ detection of SARS-CoV2.

Most cells in the body are mononuclear whereas skeletal muscle fibres are uniquely multinuclear. The nuclei of muscle fibres (myonuclei) are usually situated peripherally which complicates the equitable distribution of gene products. Myonuclear abundance can also change under conditions such as hypertrophy and atrophy. Specialised zones in muscle fibres have different functions and thus distinct synthetic demands from myonuclei. The complex structure and regulatory requirements of multinuclear muscle cells understandably led to the hypothesis that myonuclei govern defined 'domains' to maintain homeostasis and facilitate adaptation. The purpose of this review is to provide historical context for the myonuclear domain and evaluate its veracity with respect to mRNA and protein distribution resulting from myonuclear transcription. We synthesise insights from past and current in vitro and in vivo genetically modified models for studying the myonuclear domain under dynamic conditions. We also cover the most contemporary knowledge on mRNA and protein transport in muscle cells. Insights from emerging technologies such as single myonuclear RNA-sequencing further inform our discussion of the myonuclear domain. We broadly conclude: (1) the myonuclear domain can be flexible during muscle fibre growth and atrophy, (2) the mechanisms and role of myonuclear loss and motility deserve further consideration, (3) mRNA in muscle is actively transported via microtubules and locally restricted, but proteins may travel far from a myonucleus of origin and (4) myonuclear transcriptional specialisation extends beyond the classic neuromuscular and myotendinous populations. A deeper understanding of the myonuclear domain in muscle may promote effective therapies for ageing and disease.

Background Prostate cancer (PCa) is one of the main causes of death in men all over the world. To date, the emerging issue is the search for new diagnostic and prognostic biomarkers to distinguish patients with different risk types. Recent studies of PCa biomarkers have drawn attention to the remarkable heterogeneity of this tumor. Tumor heterogeneity (TH) is the main limitation of the ability to use biomarkers’ gene panels in clinics because of different cell subclones within one tumor. Methods Here we describe the relationship between tumor cells in a model of heterogeneous prostate cancer. Our model was based on direct and indirect co-cultivation of 2 prostate cell lines with aggressive (PC3-GFP) and indolent (22Rv1) phenotypes. To evaluate the tumor aggressiveness, we described the surface phenotype of adhesion molecules, measured the expression of genes related to metastasis in prostate cancer (Ai et al., 2017; Fan et al., 2018) and performed the motility tests. Results In experiments with direct co-cultivation of PC3 (aggressive) and 22Rv1 (indolent) cancer cells, we found a decrease of CD29 (integrin beta 1) on PC3 cells within 3 days, while the expression of other surface adhesion molecules (CD54, CD38, CD24 and CD44) was not altered, the surface phenotype of 22Rv1 did not change. Next, we assessed the expression of genes related to metastasis and showed that after 3 days of direct co-cultivation the expression of FLNC, AMACR, SNCG, HPN genes increases at least 2.5 times in 22Rv1, while HPN and FASN are upregulated in PC3 cells (for all measurements p

Progressive respiratory muscle weakness and ineffective cough contributes to morbidity and mortality in children with neuromuscular diseases (NMD). Inspiratory muscle training (IMT) aims to preserve or improve respiratory muscle strength and reduce respiratory morbidity. This study aimed to determine the safety and efficacy of IMT in children with NMD. A randomised cross-over study compared three-month intervention (IMT) with control periods. During the intervention, children with NMD (5-18 years) from two provinces in South Africa performed 30 breaths (at 30% of inspiratory muscle strength (Pimax)) with an electronic threshold device, twice daily. During the control period participants did not perform any IMT. Twenty-three participants (median (IQR) age of 12.33 (10.03-14.17) years), mostly male (n=20) and non-ambulant (n=14) were included. No adverse events related to IMT were reported. There was no evidence of a difference in median patient hospitalisation and respiratory tract infection rates between control and intervention periods (p=0.60; p=0.21). During IMT, Pimax and peak cough flow improved with a mean (SD) of 14.57 (±15.67) cmH2O and 32.27 (±36.60) L/min, compared to a change of 3.04(±11.93)cmH2O (p=0.01) and -16.59 (±48.29) L/min (p=0.0005) during the control period. There was no evidence of change in spirometry, functional ability and total health-related quality of life scores following intervention. Patient satisfaction with IMT was high (median 8/10 (IQR 5-10)) and adherence was good. A three-month IMT programme in children with NMD is well tolerated, appears to be safe and is associated with a significant improvement in respiratory muscle strength and cough efficacy.

The field of precision medicine allows for tailor-made treatments specific to a patient and thereby improve the efficiency and accuracy of disease prevention, diagnosis, and treatment and at the same time would reduce the cost, redundant treatment, and side effects of current treatments. Here, the combination of organ-on-a-chip and bioprinting into engineering high-content in vitro tissue models is envisioned to address some precision medicine challenges. This strategy could be employed to tackle the current coronavirus disease 2019 (COVID-19), which has made a significant impact and paradigm shift in our society. Nevertheless, despite that vaccines against COVID-19 have been successfully developed and vaccination programs are already being deployed worldwide, it will likely require some time before it is available to everyone. Furthermore, there are still some uncertainties and lack of a full understanding of the virus as demonstrated in the high number new mutations arising worldwide and reinfections of already vaccinated individuals. To this end, efficient diagnostic tools and treatments are still urgently needed. In this context, the convergence of bioprinting and organ-on-a-chip technologies, either used alone or in combination, could possibly function as a prominent tool in addressing the current pandemic. This could enable facile advances of important tools, diagnostics, and better physiologically representative in vitro models specific to individuals allowing for faster and more accurate screening of therapeutics evaluating their efficacy and toxicity. This review will cover such technological advances and highlight what is needed for the field to mature for tackling the various needs for current and future pandemics as well as their relevancy towards precision medicine.