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C‐peptide therapy exerts several positive actions on nerves, vasculature, smooth muscle relaxation, kidney function and bone. To date, the role of C‐peptide in preventing type 1 diabetes‐related muscle atrophy has not been investigated. Our aim was to evaluate if C‐peptide infusion prevents muscle wasting in diabetic rats. Twenty‐three male Wistar rats were randomly divided into three groups: normal control group, diabetic group and diabetic group plus C‐peptide. Diabetes was induced by streptozotocin injection, and C‐peptide was administered subcutaneously for 6 weeks. The blood samples were obtained at baseline, before streptozotocin injection and at the end of the study to assess C‐peptide, ubiquitin and other laboratory parameters. We also tested the ability of C‐peptide to regulate the skeletal muscle mass, the ubiquitin–proteasome system, the autophagy pathway as well as to improve muscle quality. C‐peptide administration reversed hyperglycaemia (P= 0.02) and hypertriglyceridaemia (P= 0.01) in diabetic plus C‐peptide rats compared with diabetic control rats. The diabetic‐control animals displayed a lower weight of the muscles in the lower limb considered individually than the control rats and the diabetic plus C‐peptide rats (P= 0.03; P= 0.03; P= 0.04; P= 0.004, respectively). The diabetic‐control rats presented a significantly higher serum concentration of ubiquitin compared with the diabetic plus C‐peptide and the control animals (P= 0.02 and P= 0.01). In muscles of the lower limb, the pAmpk expression was higher in the diabetic plus C‐peptide than the diabetic‐control rats (in the gastrocnemius, P= 0.002; in the tibialis anterior P= 0.005). The protein expression of Atrogin‐1 in gastrocnemius and tibialis was lower in the diabetic plus C‐peptide than in diabetic‐control rats (P= 0.02, P= 0.03). After 42 days, the cross‐sectional area in the gastrocnemius of the diabetic plus C‐peptide group had been reduced by 6.6% while the diabetic‐control rats had a 39.5% reduction compared with the control animals (P= 0.02). The cross‐sectional area of the tibialis and the extensor digitorum longus muscles was reduced, in the diabetic plus C‐peptide rats, by 10% and 11%, respectively, while the diabetic‐control group had a reduction of 65% and 45% compared with the control animals (both P< 0.0001). Similar results were obtained for the minimum Feret's diameter and perimeter. C‐peptide administration in rats could protect skeletal muscle mass from atrophy induced by type 1 diabetes mellitus. Our findings could suggest that targeting the ubiquitin–proteasome system, Ampk and muscle‐specific E3 ubiquitin ligases such as Atrogin‐1 and Traf6 may be an effective strategy for molecular and clinical intervention in the muscle wasting pathological process in T1DM.

AbstractBackground: Oral nicotine pouches (NPs) are smokeless, tobacco-free products that have a potential role in tobacco harm reduction strategies.Methods: In a cross-sectional study in Sweden/Denmark, several recognised biomarkers of potential harm (BoPHs) linked to smoking-related diseases/their initiating biological processes, and biomarkers of exposure (BoEs) to tobacco/tobacco smoke toxicants were compared among exclusive adult users of Velo NPs and current/former/never smokers. Over 24 h, participants used their usual product (Velo NP or cigarette) as normal, and BoEs/BoPHs were assessed via blood/24-h urine/exhaled breath/physiological assessments.Results: Among the primary endpoints, total NNAL (16.9 ± 29.47 vs 187.4 ± 228.93 pg/24 h), white blood cell count (5.59 ± 1.223 vs 6.90 ± 1.758 × 109/L), and COHb (4.36 ± 0.525 vs 8.03 ± 2.173% saturation) were significantly lower among Velo users than among smokers (91%, 19% and 46% lower, respectively, all P < 0.0001), while fractional exhaled NO, previously shown to be lower in smokers, was significantly higher (23.18 ± 17.909 vs 11.20 ± 6.980 ppb) among Velo users (107% higher, P < 0.0001). Furthermore, sICAM-1 tended to be lower (185.9 ± 42.88 vs 204.5 ± 64.85 ng/mL) among Velo users than smokers (9% lower). Several secondary endpoints, including six BoEs (3-HPMA (246.7 ± 91.07 vs 1165.7 ± 718.35 μg/24 h), 3-OH-B[a]P (82.4 ± 217.58 vs 258.3 ± 190.20 pg/24 h), HMPMA (135.1 ± 77.85 vs 368.8 ± 183.15 μg/24 h), MHBMA (0.22 ± 0.166 vs 3.39 ± 2.943 μg/24 h), S-PMA (0.10 ± 0.059 vs 3.53 ± 2.736 µg/24 h) and total NNN (7.5 ± 24.84 vs 9.7 ± 5.93 ng/24 h)), were significantly lower among Velo users (78.8%, 68.1%, 63.4%, 93.5%, 97.2% and 22.7% lower, respectively, P < 0.0001–0.0011), while total nicotine equivalents was significantly higher among Velo users (22.6 ± 12.69 vs 12.1 ± 7.92 mg/24 h, P < 0.0001), although Velo user levels are comparable to those previously reported among oral tobacco users, and Velo user and smoker mean levels were similar in Denmark.Conclusion: As compared with smokers, exclusive users of Velo NPs have significantly less exposure to tobacco toxicants and more favourable BoPHs associated with initiating biological processes of smoking-related diseases.International Standard Registered Clinical Trial number: ISRCTN16988167

Tobacco heating products (THPs) have reduced toxicant emissions relative to cigarettes. THPs are continually evolving, but safety and efficacy studies on each new variant involve considerable resources. As employed by the pharmaceutical industry, a “bridging” process could be used to demonstrate product equivalence.

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.

Supplemental Digital Content is available in the text.Although diagnosis of high-grade uterine mesenchymal tumors (UMTs) exhibiting classic morphologic features is straightforward, diagnosis is more challenging in tumors in which prototypical features are poorly developed, focal, and/or coexist with features seen in other neoplasms. Here, we sought to define the repertoire of somatic genetic alterations in diagnostically challenging UMTs with myomelanocytic differentiation, including some reported as perivascular epithelioid cell tumors (PEComas). In 17 samples from 15 women, the tumors were histologically heterogenous. Immunohistochemical expression of at least 1 melanocytic marker (HMB45, Melan-A, or MiTF) was identified in all tumors, and of myogenic markers (desmin or smooth muscle actin) in most tumors. Targeted massively parallel sequencing revealed several genetic alterations, most commonly in TP53(41% mutation, 12% deletion), TSC2(29% mutation, 6% deletion), RB1(18% deletion), ATRX(24% mutation), MED12(12% mutation), BRCA2(12% deletion), CDKN2A(6% deletion) as well as FGFR3, NTRK1, and ERBB3amplification (each 6%). Gene rearrangements (JAZF1-SUZ12; DNAJB6-PLAG1; and SFPQ-TFE3) were identified in 3 tumors. Integrating histopathologic, immunohistochemical, and genetic findings, tumors from 4 patients were consistent with malignant PEComa (1 TFE3-rearranged); 6 were classified as leiomyosarcomas; 3 showed overlapping features of PEComa and other sarcoma types (leiomyosarcoma or low-grade endometrial stromal sarcoma); and 2 were classified as sarcoma, not otherwise specified. Our findings suggest that diagnostically challenging UMTs with myomelanocytic differentiation represent a heterogenous group of neoplasms which harbor a diverse repertoire of somatic genetic alterations; these genetic alterations can aid classification.

Tall cell carcinoma with reverse polarity is a rare subtype of breast carcinoma with solid and papillary growth and nuclear features reminiscent of those of the tall cell variant of papillary thyroid carcinoma. These tumors harbor recurrent IDH2R172 hotspot mutations or TET2mutations, co-occurring with mutations in PI3K pathway genes. Diagnosis of tall cell carcinomas with reverse polarity is challenging in view of their rarity and the range of differential diagnosis. We sought to determine the sensitivity and specificity of IDH2 R172 immunohistochemistry for the detection of IDH2R172 hotspot mutations in this entity. We evaluated 14 tall cell carcinomas with reverse polarity (ten excision and five core needle biopsy specimens), 13 intraductal papillomas, 16 solid papillary carcinomas, and 5 encapsulated papillary carcinomas by Sanger sequencing of the IDH2R172 hotspot locus and of exons 9 and 20 of PIK3CA, and by immunohistochemistry using monoclonal antibodies (11C8B1) to the IDH2R172S mutation. The 14 tall cell carcinomas with reverse polarity studied harbored IDH2R172 hotspot mutations, which co-occurred with PIK3CAhotspot mutations in 50% of cases. None of the other papillary neoplasms analyzed displayed IDH2R172 mutations, however PIK3CAhotspot mutations were detected in 54% of intraductal papillomas, 6% of solid papillary carcinomas, and 20% of encapsulated papillary carcinomas tested. Immunohistochemical analysis with anti-IDH2 R172S antibodies (11C8B1) detected IDH2 R172 mutated protein in 93% (14/15) of tall cell carcinomas with reverse polarity samples including excision (n= 9/10) and core needle biopsy specimens (n= 5), whereas the remaining papillary neoplasms (n= 34) were negative. Our findings demonstrate that immunohistochemical analysis of IDH2 R172 is highly sensitive and specific for the detection of IDH2R172 hotspot mutations, and likely suitable as a diagnostic tool in the evaluation of excision and core needle biopsy material of tall cell carcinomas with reverse polarity.

Intrahepatic cholangiocarcinoma (ICC) is a rare, heterogeneous, highly lethal tumor of the biliary tract. Due to the lack of effective treatments, an early identification of ICC is essential to achieve the best outcome in terms of therapy and prognosis aiming for a curative intent. ICC may arise on a normal liver or with an underlying liver disease, making the diagnosis more difficult and complex. Contrast-enhancement ultrasound (CEUS) is an accurate procedure able to detect ICC-specific contrast vascular pattern, and thus facilitating the correlation between radiological and histopathological findings with high specificity and sensitivity. CEUS has been shown to have a high diagnostic potential in the diagnosis of ICC thanks to the possibility of studying in real time the intralesional microcirculation and evaluating the precocity of the enhancement of the lesion during the arterial phase. All these features allow to differentiate the ICC from hepatocarcinoma (HCC) with high sensitivity and specificity. Furthermore, CEUS is a rapid, non-invasive, non-nephrotoxic or non-allergenic tool. The only limitations CEUS may have are related to the disease site and patient characteristics (obesity) and compliance, including the operator’s experience. A clinical evaluation of the patient, together with tumor markers and biochemical tests assessment, to differentiate ICC from HCC are highly suggested.

9p21 deletions involving MTAP/CDKN2Agenes are detected in diffuse pleural mesotheliomas (DPM) but are absent in benign mesothelial proliferations. Loss of MTAP expression by immunohistochemistry (IHC) is well accepted as a surrogate for 9p21 deletion to support a diagnosis of DPM. Accurate interpretation can be critical in the diagnosis of DPM, but variations in antibody performance may impact interpretation. The objectives of this study were to compare the performance of MTAP monoclonal antibodies (mAbs) EPR6893 and 1813 and to compare MTAP expression by IHC with 9p21 copy number status in DPM. Cytoplasmic expression of MTAP IHC with mAbs EPR6893 (ab126770; Abcam) and 1813 (NBP2-75730, Novus Biologicals) was evaluated in 56 DPM (47 epithelioid, 7 biphasic, and 2 sarcomatoid) profiled by targeted next-generation sequencing. 9p21 Copy number status was assessed by Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing (FACETS) analysis and also by CDKN2Afluorescence in situ hybridization in discrepant cases when material was available. MTAP mAb 1813 showed stronger immunoreactivity, more specific staining, and no equivocal interpretations compared to mAb EPR6893 which showed equivocal staining in 19 (34%) of cases due to weak or heterogenous immunoreactivity, lack of definitive internal positive control, and/or nonspecific background staining. MTAP expression with mAb 1813 showed near perfect agreement with 9p21 copy number by combined FACETS/fluorescence in situ hybridization calls (κ = 0.85; 95% CI, 0.71-0.99; P< .001). MTAP IHC with mAb 1813 was 96% sensitive, 86% specific, and 93% accurate for 9p21 homozygous deletion. The findings of this study suggest that interpretation of MTAP IHC is improved with mAb 1813 because mAb EPR6893 was often limited by equivocal interpretations. We show that MTAP IHC and molecular assays are complementary in detecting 9p21 homozygous deletion. MTAP IHC may be particularly useful for low tumor purity samples and in low-resource settings.