Breast Cancer With HER2 Immunohistochemical Score 2 and Average HER2 Signals/Cell 6 or More and HER2/CEP17 Ratio Less Than 2 ('ISH Group 3'): A Multi-Institutional Cohort Analysis Emphasizing Outcome and Molecular Subtype.

Breast cancer (BC) with average human epidermal growth factor receptor 2 (HER2) signals/cell ≥6 and HER2/chromosome enumeration probe 17 (CEP17) ratio <2 (in situ hybridization [ISH] group 3) is very rare, accounting for 0.4% to 3.0% of cases sent for the dual-probe ISH assay. Although such patients are currently eligible for treatment with HER2-targeted therapy, their characteristics and outcomes remain poorly understood. Sixty-two BCs with equivocal HER2 immunohistochemical score (2+) and reflex ISH group 3 results were identified across 4 institutions. Available clinicopathologic characteristics, MammaPrint and BluePrint molecular results, and follow-up information were retrospectively analyzed. Most BCs with HER2 equivocal immunohistochemical and ISH group 3 results were histologic grade 2 or 3 (100%), estrogen receptor (ER) positive (90.3%), with an average HER2 signals/cell of 7.3. Molecular profiles revealed that 80% (16/20) of tumors were luminal subtypes, and HER2 molecular subtype was identified in 10% of tumors (2/20). Twelve (19.4%) out of 62 patients developed local recurrence and/or distant metastasis with a median follow-up of 50 months. One (10%) of 10 patients achieved pathologic complete response after neoadjuvant chemotherapy. Forty-nine (79%) out of 62 patients completed anti-HER2 agents, and exploratory analysis showed no statistically significant difference in disease outcomes between patients who completed anti-HER2 treatment and those who did not. Univariate analysis revealed advanced clinical stage, and ER/progesterone receptor negativity was associated with unfavorable disease outcomes, and exploratory multivariate analysis demonstrated that clinical stage was the most significant factor associated with disease outcomes in the studied population. These findings increase our understanding of this rare, but clinically important HER2 category. Large-scale prospective randomized studies are needed to further evaluate the role of perioperative HER2-targeted therapy in this patient population.

Identification of a new gregarine parasite associated with mass mortality events of freshwater pearl mussels (Margaritifera margaritifera) in Sweden.

Freshwater bivalves play key ecological roles in lakes and rivers, largely contributing to healthy ecosystems. The freshwater pearl mussel, Margaritifera margaritifera, is found in Europe and on the East coast of North America. Once common in oxygenated streams, M. margaritifera is rapidly declining and consequently assessed as a threatened species worldwide. Deterioration of water quality has been considered the main factor for the mass mortality events affecting this species. Yet, the role of parasitic infections has not been investigated. Here, we report the discovery of three novel protist lineages found in Swedish populations of M. margaritifera belonging to one of the terrestrial groups of gregarines (Eugregarinorida, Apicomplexa). These lineages are closely related-but clearly separated-from the tadpole parasite Nematopsis temporariae. In one lineage, which is specifically associated with mortality events of M. margaritifera, we found cysts containing single vermiform zoites in the gills and other organs of diseased individuals using microscopy and in situ hybridization. This represents the first report of a parasitic infection in M. margaritifera that may be linked to the decline of this mussel species. We propose a tentative life cycle with the distribution of different developmental stages and potential exit from the host into the environment.

Leukolectins support lifelong innate immunity in lower vertebrates, and reveal dichotomies of several leukocytic lineages.

Innate immunity is vital for animal homeostasis and survival. First-line immuno-defense for fish larvae involves mucus enriched with leukolectin (LL) secreted by dermal lectocytes. Later during the critical transition from yolk-nutrition to feeding, additional larval immuno-protection in zebrafish (zF) is provided by macrophages containing LL (lectophages). This work investigated new LL-expression in embryos and in blood, structures of fish leukocytic LL and LL-genes, and LL-presence in chicken leukocytes. In zF-embryos, lectophages appear ∼10 hpf, while later, cells co-expressing myeloperoxidase- and LL-mRNA were detected (∼19 hpf). Furthermore, protein-extracts of Atlantic salmon (Ssal) leukocytes contained LL-proteins, compartmentalized in the cytosol. Cloning and sequencing revealed 94 % nt-sequence identity between variants of Ssal-leukolectins. Highly conserved LLs allowed production of epitope-specific anti-LL IgGs. Immuno-fluorescence-analysis demonstrated that most Ssal-bloodcells were LL-negative, but both some large cells with protrusions and some small, rounded cells did express LL. Immunoperoxidase-staining method confirmed LL-expression in some Ssal-leukocytes, identified as macrophages, PMN-leukocytes, thrombocytes and dendritic cells. However, closer examination revealed a dichotomy of these cell-categories into either LL-positive, or LL-negative variants. In situ hybridization demonstrated profuse LL-expression in Ssal head kidney interstitial tissue, while LL-transcripts were absent in large kidney tubules. Both hematopoietic (non-pigmented) marrow cells and melano-macrophages expressed LL-mRNA, implying that leukolectins provide lifelong innate immuno-protection. PCR-amplification using Ssal-leukocytic DNA as template, and direct sequencing yielded a leukocytic ll-gene. Some cells in salmon, cod, halibut, oikopleura and zebrafish embryos express LL-proteins and/or LL-mRNA, and LL-mRNA is detected in salmon, cod and chicken leukocytes. However, current genomes for these species lack recognizable LL-loci except the Ssal_v3.1 Genome-assembly. The data demonstrate an unexpected dichotomy of some leukocyte lineages into LL-positive or LL-negative cell-variants. Such dichotomies suggest exploring differential impacts from the duplicated leukocyte-lineages in health and disease.

Role of iRhom2 in Olfaction: Implications for Odorant Receptor Regulation and Activity-Dependent Adaptation.

The cell surface metalloprotease ADAM17 (a disintegrin and metalloprotease 17) and its binding partners iRhom2 and iRhom1 (inactive Rhomboid-like proteins 1 and 2) modulate cell-cell interactions by mediating the release of membrane proteins such as TNFα (Tumor necrosis factor α) and EGFR (Epidermal growth factor receptor) ligands from the cell surface. Most cell types express both iRhoms, though myeloid cells exclusively express iRhom2, and iRhom1 is the main iRhom in the mouse brain. Here, we report that iRhom2 is uniquely expressed in olfactory sensory neurons (OSNs), highly specialized cells expressing one olfactory receptor (OR) from a repertoire of more than a thousand OR genes in mice. iRhom2-/- mice had no evident morphological defects in the olfactory epithelium (OE), yet RNAseq analysis revealed differential expression of a small subset of ORs. Notably, while the majority of ORs remain unaffected in iRhom2-/- OE, OSNs expressing ORs that are enriched in iRhom2-/- OE showed fewer gene expression changes upon odor environmental changes than the majority of OSNs. Moreover, we discovered an inverse correlation between the expression of iRhom2 compared to OSN activity genes and that odor exposure negatively regulates iRhom2 expression. Given that ORs are specialized G-protein coupled receptors (GPCRs) and many GPCRs activate iRhom2/ADAM17, we investigated if ORs could activate iRhom2/ADAM17. Activation of an olfactory receptor that is ectopically expressed in keratinocytes (OR2AT4) by its agonist Sandalore leads to ERK1/2 phosphorylation, likely via an iRhom2/ADAM17-dependent pathway. Taken together, these findings point to a mechanism by which odor stimulation of OSNs activates iRhom2/ADAM17 catalytic activity, resulting in downstream transcriptional changes to the OR repertoire and activity genes, and driving a negative feedback loop to downregulate iRhom2 expression.

Fission yeast Ish1 and Les1 interact with each other in the lumen of the nuclear envelope.

The nuclear envelope (NE) provides a permeable barrier that separates the eukaryotic genome from the cytoplasm. NE is a double membrane composed of inner and outer nuclear membranes. Ish1 is a stress-responsive NE protein in the fission yeast, Schizosaccharomyces pombe. Les1 is another NE protein that shares several similar domains with Ish1, but the relationship between them remains unknown. In this study, using fluorescence and electron microscopy, we found that most regions of these proteins were localized within the NE lumen. We also found that Ish1 interacted with Les1 via its C-terminal region in the NE lumen and that the NE localization of Ish1 depended on the C-terminal region of Les1. Ish1 and Les1 were co-localized at the NE in interphase cells, but when the nucleus divided at the end of mitosis (closed mitosis), they showed distinguishable localization at the midzone membrane domain. These results suggest the regulated interaction between Ish1 and Les1 in the NE lumen, although this interaction does not appear to be essential for cell survival.

Improved concordance of challenging human epidermal growth factor receptor 2 dual in-situ hybridisation cases with the use of a digital image analysis algorithm in breast cancer.

AIMS: Accurate assessment of human epidermal growth factor receptor 2 (HER2) expression by HER2 immunohistochemistry and in-situ hybridisation (ISH) is critical for the management of patients with breast cancer. The revised 2018 ASCO/CAP guidelines define 5 groups based on HER2 expression and copy number. Manual pathologist quantification by light microscopy of equivocal and less common HER2 ISH groups (groups 2-4) can be challenging, and there are no data on interobserver variability in reporting of these cases. We sought to determine whether a digital algorithm could improve interobserver variability in the assessment of difficult HER2 ISH cases. METHODS AND RESULTS: HER2 ISH was evaluated in a cohort enriched for less common HER2 patterns using standard light microscopy versus analysis of whole slide images using the Roche uPath HER2 dual ISH image analysis algorithm. Standard microscopy demonstrated significant interobserver variability with a Fleiss's kappa value of 0.471 (fair-moderate agreement) improving to 0.666 (moderate-good) with the use of the algorithm. For HER2 group designation (groups 1-5), there was poor-moderate reliability between pathologists by microscopy [intraclass correlation coefficient (ICC) = 0.526], improving to moderate-good agreement (ICC = 0.763) with the use of the algorithm. In subgroup analysis, the algorithm improved concordance particularly in groups 2, 4 and 5. Time to enumerate cases was also significantly reduced. CONCLUSION: This work demonstrates the potential of a digital image analysis algorithm to improve the concordance of pathologist HER2 amplification status reporting in less common HER2 groups. This has the potential to improve therapy selection and outcomes for patients with HER2-low and borderline HER2-amplified breast cancers.

Single mRNA detection of Wnt signaling pathway in the human limbus.

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the corneal stroma (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other limbal quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.

Pathogenesis of Bohle iridovirus infection in Krefft's freshwater turtle hatchlings (Emydura macquarii krefftii).

Ranaviruses have been detected in over 12 families of reptiles including many genera of turtles, tortoises, and terrapins, but the pathogenesis of these infections is still poorly understood. Krefft's river turtle hatchlings (N = 36; Emydura macquarii krefftii) were inoculated intramuscularly with Bohle iridovirus (BIV, Ranavirus, isolate) or saline, and euthanized at 9 timepoints (3 infected and 1 control per timepoint) over a 24-day period. Samples of lung, liver, kidney, and spleen were collected for quantitative polymerase chain reaction (PCR); internal organs, skin, and oral cavity samples were fixed for histopathological examination. The earliest lesions, at 8 days postinoculation (dpi), were lymphocytic inflammation of the skin and fibrinoid necrosis of regional vessels at the site of inoculation, and mild ulcerative necrosis with lymphocytic and heterophilic inflammation in the oral, nasal, and tongue mucosae. Fibrinonecrotic foci with heterophilic inflammation were detected in spleen and gonads at 16 dpi. Multifocal hepatic necrosis, heterophilic inflammation, and occasional basophilic intracytoplasmic inclusion bodies were observed at 20 dpi, along with ulcerative lymphocytic and heterophilic tracheitis and bronchitis. Tracheitis, bronchitis, and rare bone marrow necrosis were present at 24 dpi. Of the viscera tested for ranaviral DNA by PCR, the liver and spleen had the highest viral loads throughout infection, and thus appeared to be major targets of viral replication. Testing of whole blood by qPCR was the most-effective ante-mortem method for detecting ranaviral infection compared with oral swabs. This study represents the first time-dependent pathogenesis study of a ranaviral infection in turtles.

Autonomic regulation in different hypertensive phenotypes - the HELIUS study.

Background: Hypertension can be classified into different phenotypes based on systolic and diastolic blood pressure (BP) that carry a different prognosis and may therefore be differently associated with sympathetic activity. We assessed the association between cardiac autonomic function determined from continuous finger BP recordings and hypertensive phenotypes. Methods: We included 10,221 individuals aged between 18-70 years from the multi-ethnic HELIUS study. Finger BP was recorded continuously for 3-5 minutes from which cross-correlation baroreflex sensitivity (xBRS) and heart rate variability (HRV) were determined. Hypertension was classified into isolated systolic (ISH; ≥140/<90), diastolic (IDH; <140/≥90) and combined systolic and diastolic hypertension (SDH; ≥140/≥90). Differences were assessed after stratification by age (younger: ≤40, older: >40 years) and sex, using regression with correction for relevant covariates. For xBRS, values were log-transformed. Results: In younger adults with ISH, xBRS was comparable to normotensive individuals in men (ratio 0.92; 95%CI 0.84-1.01) and women (1.00; 95%CI 0.84-1.20), while xBRS was significantly lower in IDH and SDH (ratios between 0.67 and 0.80). In older adults, all hypertensive phenotypes had significantly lower xBRS compared to normotensives. We found a similar pattern for HRV in men, while in women HRV did not differ between phenotypes. Conclusions: In younger men and women ISH is not associated with a shift towards increased sympathetic control, while IDH and SDH in younger and all hypertensive phenotypes in older participants were associated with increased sympathetic control. This suggests that alterations in autonomic regulation could be a contributing factor to known prognostic disparities between hypertensive phenotypes.

Hypertension can be classified into different phenotypes based on systolic and diastolic blood pressure (BP) that carry a different prognosis. Impaired autonomic regulation is important in the pathogenesis of hypertension and independently associated with adverse cardiovascular outcomes.We analyzed 3-5 minutes continuous non-invasive finger blood pressure recordings performed in over 10.000 individuals participating in the HELIUS cohort study. From these measurements, short term heart rate variability (HRV) and cross correlation baroreflex sensitivity (xBRS) were determined using an automatic algorithm.In our analysis we observed pronounced differences in the relation between autonomic regulation and hypertensive phenotypes that depend on age and sex.Younger men and women (age 18-40 years) with isolated systolic hypertension had similar values for xBRS and HRV compared to normotensives, while isolated diastolic hypertension was associated with a shift towards increased sympathetic control. In contrast to our findings in younger individuals, all hypertensive phenotypes were associated with increased sympathetic control in older participants (age 40-70 years).This supports earlier studies showing prognostic differences and suggests that alterations in sympathovagal balance could be a contributing factor to the disparities between phenotypes.

Clinical correlation analysis of HPV infection in adult laryngeal papilloma and adverse events.

OBJECT: To explore the factors affecting dysplasia and carcinogenesis in adult patients with laryngeal papilloma, and the clinical differences between human papillomavirus (HPV)-positive and HPV-negative patients. METHODS: Clinical data of 80 adult patients with laryngeal papilloma and associated adverse events were collected retrospectively. They had undergone surgery in the Department of Otolaryngology Head and Neck, Beijing Tongren Hospital, Capital Medical University between January 2010 and December 2020. HPV infection was detected using RNA in situ hybridization. RESULTS: Regression analysis showed that multiple lesions and high Ki-67 expression were independent factors affecting the occurrence of adverse events. Differences between the HPV-positive and HPV-negative groups were compared. The age and Ki-67 expression in the HPV-negative group were significantly higher than those in the HPV-positive group. In the severe dysplasia to carcinogenesis subgroup, the proportion of HPV-negative patients was significantly higher than that in the mild to moderate dysplasia subgroup. There was a high correlation between positive p16 immunohistochemistry (IHC) and positive HPV. CONCLUSIONS: Multiple lesions and high Ki-67 expression are independent factors that are linked with adverse laryngeal papilloma progression. Elderly HPV-negative patients are at an increased risk of severe dysplasia and carcinogenesis. Positive p16 IHC was very accurate in detecting HPV infection.

Frequent expression of PD-L1 in lymphocyte-rich hepatocellular carcinoma: A report of 4 cases.

BACKGROUND: Programmed death ligand 1 (PD-L1) is an immune checkpoint inhibitor. PD-L1 binds to its receptor programmed death receptor (PD-1) expressed by immune cells and plays a key role in regulating immune responses. Engagement of PD-L1 on cancer cells and PD-1 on immune cells avoid destruction of tumour cells by immune cells. Immunostaining with PD-L1 has been suggested as a biomarker predictive of antiPD-L1 immunotherapy. Lymphocyte-rich hepatocellular carcinoma (LrHCC) is a rare histological HCC subtype which is characterised by neoplastic epithelial cells intermixed with numerous immune cells. METHODS: Here in we investigated immunohistochemical PD-L1 expression in 4 cases of LrHCC. Tumour proportion score (TPS) and immune cell score was recorded. Immunophenotypic characterization of the tumour and inflammatory cells was also done. Epstein-Barr encoding region (EBER) in situ hybridization (ISH) assay as performed in all four tumours. RESULTS: Expression of PD-L1 was demonstrated in tumour epithelial cells and immune cells in all four cases. Incomplete to membranous staining was demonstrated in the tumour cells. Tumour proportion score (TPS) was 1.2-20 %. Immune cells demonstrated membranous and cytoplasmic immunostaining. Immune cell score was ≥1 % to >10 %. CONCLUSION: PD-L1 expression in both tumour and immune cells suggests distinct immunogenic feature and potential role of antiPD-L1 therapies in cases with inoperable disease.

Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules.

The European grapevine (Vitis vinifera L.) is one of the world's most widely cultivated and economically important fruit crops. Seedless fruits are particularly desired for table grapes, with seedlessness resulting from stenospermocarpy being an important goal for cultivar improvement. The establishment of an RNA in situ hybridisation (ISH) system for grape berries and ovules is, therefore, important for understanding the molecular mechanisms of ovule abortion in stenospermocarpic seedless cultivars. We improved RNA in situ hybridisation procedures for developing berries and ovules by targeting two transcription factor genes, VvHB63 and VvTAU, using two seeded varieties, 'Red Globe' and 'Pinot Noir', and two seedless cultivars, 'Flame Seedless' and 'Thompson Seedless'. Optimisation focused on the time of proteinase K treatment, probe length, probe concentration, hybridisation temperature and post-hybridisation washing conditions. The objectives were to maximise hybridisation signals and minimise background interference, while still preserving tissue integrity. For the target genes and samples tested, the best results were obtained with a pre-hybridisation proteinase K treatment of 30 min, probe length of 150 bp and concentration of 100 ng/mL, hybridisation temperature of 50 °C, three washes with 0.2× saline sodium citrate (SSC) solution and blocking with 1% blocking reagent for 45 min during the subsequent hybridisation. The improved ISH system was used to study the spatiotemporal expression patterns of genes related to ovule development at a microscopic level.

Current and potential immunohistochemical biomarkers for prognosis and therapeutic stratification of breast carcinoma.

The identification of biomarkers on cancer tissue samples could be obtained through several technologies. In this setting, the immunohistochemistry and in situ hybridization are accessible in most pathology laboratories. Particularly, immunohistochemistry can be used not only for diagnostic issues, but also to define prognostic classes and to define response to specific therapies. Particularly the last applications have been firstly developed in the breast cancer pathology. In addition, the development of molecular classification proposed some prognostic/predictive classes that could be easily defined by immunohistochemistry. Thus, the role of the pathologists has become increasingly important in the definition of prognosis and in the choice therapy, because the immunohistochemical biomarkers are used to guide treatment, to classify breast cancer into biologically and prognostically distinct subtypes. In this review, we will provide information on the current application of the immunohistochemical biomarkers useful in the management of breast cancer patients. Moreover, we consider the application of immunohistochemistry in the definition of the most promising biomarkers derived from molecular studies of the breast cancer, that in the future could integrate the characterization of breast cancer into clinical practice.

Prevalence of Her2-neu status and its clinicopathological association in newly diagnosed gastric cancer patients.

BACKGROUND: This study aimed to report the prevalence of HER2-neu in newly diagnosed early or metastatic gastric cancer (GC) patients, to determine the percentage of patients achieving various IHC scores correlating with the ISH results and to establish a database for GC patients in Lebanon. METHODS: This was a national, multicenter, descriptive and cross-sectional study in patients with histologically confirmed early or metastatic GC newly diagnosed. All eligible patients underwent the IHC and ISH tests in a central laboratory. Demographics, medical history and histopathology data were collected. RESULTS: One hundred fifty-seven patients were included (mean age at diagnosis: 63 ± 14.1 years) during a 3.5 year period. The prevalence of HER2-neu over expression was 21% (95% CI: 15.3-27.4) using ICH and ISH. Agreement between IHC and ISH results was significantly substantial (kappa = 0.681; p-value < 0.001). Over expressed HER2-neu status was significantly associated with high ECOG performance status only. CONCLUSIONS: The prevalence of HER2-neu over expression in newly diagnosed early or metastatic GC patients seemed to be high in Lebanon. The database generated allows to monitor trends in the epidemiology and management of GC.

Marmoset Brain ISH Data Revealed Molecular Difference Between Cortical Folding Patterns.

Literature studies have demonstrated the structural, connectional, and functional differences between cortical folding patterns in mammalian brains, such as convex and concave patterns. However, the molecular underpinning of such convex/concave differences remains largely unknown. Thanks to public access to a recently released set of marmoset whole-brain in situ hybridization data by RIKEN, Japan; this data's accessibility empowers us to improve our understanding of the organization, regulation, and function of genes and their relation to macroscale metrics of brains. In this work, magnetic resonance imaging and diffusion tensor imaging macroscale neuroimaging data in this dataset were used to delineate convex/concave patterns in marmoset and to examine their structural features. Machine learning and visualization tools were employed to investigate the possible transcriptome difference between cortical convex and concave patterns. Experimental results demonstrated that a collection of genes is differentially expressed in convex and concave patterns, and their expression profiles can robustly characterize and differentiate the two folding patterns. More importantly, neuroscientific interpretations of these differentially expressed genes, as well as axonal guidance pathway analysis and gene enrichment analysis, offer novel understanding of structural and functional differences between cortical folding patterns in different regions from a molecular perspective.

Neurosecretory protein GL in GIFT tilapia (Oreochromis niloticus): cDNA cloning, tissue distribution and effects of feeding on its expression.

Neurosecretory protein GL (NPGL), a novel neuropeptide, has been identified in the hypothalamus of chicks and rodents. NPGL plays a crucial role in monitoring energetic status via the regulation of feeding and metabolism. However, no study on NPGL has been reported in fish thus far. In the present study, the full-length cDNA of NPGL was identified from the hypothalamus of GIFT tilapia (Oreochromis niloticus). The ORF of tilapia NPGL is 471 bp and encodes a precursor peptide with a size of 156 a.a, consisting of a 26 a.a signal peptide and an 82 a.a mature peptide. Tissue distribution profiles of npgl in tilapia were acquired using semiquantitative PCR and in situ hybridization (ISH). The results showed that the highest npgl mRNA is expressed in the telencephalic-preoptic complex, which comprises both the telencephalon and the anterior preoptic area (POA) of male tilapia, and in the ovary of female tilapia. In addition, in male tilapia, the ISH results showed that the cells containing npgl mRNA were distributed exclusively in the anterior periventricular pretectal nucleus (Ppa) of the POA. FISH results demonstrated that npgl mRNA is also expressed in the lateral tuberal nucleus of the hypothalamus (NLT). Real-time PCR showed that npgl mRNA significantly increased in the telencephalic-preoptic complex of male tilapia that were fasted for 24 h and then fed a full diet for 20 min compared with the unfed group. Results of the FISH study showed that parvocellular cells containing npgl mRNA in the Ppa of fed fish were apparently more abundant than those of the unfed group. Few npgl positive signals also appeared in the NLT after full feeding, where pomc mRNA is highly expressed. These results indicate that NPGL may be a short-term satiety factor in fish and that the coexpression of NPGL and POMC may be present in the hypothalamus of male tilapia.

RNAscope in situ hybridization and RT-PCR for detection of SARS-CoV-2 in chilblain-like lesions: A clinical, laboratory and histopathological study.

BACKGROUND: Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, an increasing number of chilblain-like lesions (ChLL) have been increasingly reported worldwide. To date, the causal link between ChLL and SARS-CoV-2 infection has not been unequivocally established. METHODS: In this case series, we present demographic, clinical, laboratory, and histopathological information regarding 27 young patients with a clinical diagnosis of ChLL who referred to the Dermatology Unit of Papa Giovanni XXIII Hospital, Bergamo, Italy, from 1 April 2020 to 1 June 2020. RESULTS: The mean age was 14.2 years, and 21 patients (78%) experienced mild systemic symptoms a median of 28 days before the onset of cutaneous lesions. ChLL mostly involved the feet (20 patients - 74%). Among acral lesions, we identified three different clinical patterns: (i) chilblains in 20 patients (74%); (ii) fixed erythematous macules in 4 children (15%); (iii) erythrocyanosis in 3 female patients (11%). Blood examinations and viral serologies, including parvovirus B19, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and coxsackievirus were normal in all. Three patients (11%) underwent nasopharyngeal swab for RT-PCR for SARS-CoV-2 showing only 1 positive. Histopathological examinations of 7 skin biopsies confirmed the clinical diagnosis of chilblains; vessel thrombi were observed only in 1 case. Our findings failed to demonstrate the direct presence of SARS-CoV-2 RNA in skin biopsies, both with real-time polymerase chain reaction (RT-PCR) and RNAscope in situ hybridization (ISH). LIMITATIONS: Limited number of cases, unavailability of laboratory confirmation of COVID-19 in all patients, potential methodological weakness, and latency of skin biopsies in comparison to cutaneous lesions onset. CONCLUSIONS: These observations may support the hypothesis of an inflammatory pathogenesis rather than the presence of peripheral viral particles. Although, we could not exclude an early phase of viral endothelial damage followed by an IFN-I or complement-mediated inflammatory phase. Further observations on a large number of patients are needed to confirm this hypothesis.

Novel DNA-based in situ hybridization method to detect Desmozoon lepeophtherii in Atlantic salmon tissues.

The microsporidian Desmozoon lepeophtherii Freeman and Sommerville, 2009 is considered significant in the pathogenesis of gill disease in Atlantic salmon (Salmo salar Linnaeus, 1758). Due to the difficulty in detecting D. lepeophtherii in tissue sections, infections are normally diagnosed by molecular methods, routine haematoxylin and eosin (H&E) stained gill tissue sections and the use of other histochemical stains and labels to confirm the presence of spores. An in situ hybridization (ISH) protocol specific for D. lepeophtherii was developed using DIG-labelled oligonucleotide probes. Diseased Atlantic salmon gills were analysed by ISH, calcofluor white (CW) and H&E. All methods showed high levels of specificity (100%) in their ability to detect D. lepeophtherii, but the sensitivity was higher with ISH (92%), compared with CW (64%) and the presence of microvesicles on H&E stained sections (52%). High levels of D. lepeophtherii spores were significantly associated (p < .05) with the development of D. lepeophtherii-associated pathology in the gills, with Ct values below 19 and over 100 microsporidia/10 mm2 of gill tissue (from the ISH counts) seemingly necessary for the development of microvesicles. The ISH method has the advantage over other histological techniques in that it allows all life stages of the microsporidian to be detected in infected salmon gill tissue sections.

[Adult T-cell leukemia/lymphoma diagnosed by RNA in situ hybridization for HTLV-1 bZIP factor].

A 62-year-old man visited the Department of Otorhinolaryngology at our hospital with a chief complaint of a pharyngeal mass. He was admitted to our department with a diagnosis of T-cell lymphoma based on a biopsy of a mesopharyngeal tumor. Although clonality analysis was not performed due to the lack of an appropriate sample, we considered the possibility of lymphoma-type (Lugano classification stage II) adult T-cell leukemia-lymphoma (ATL), as the anti-HTLV-1 antibody was positive. During the course of the disease, the peripheral blood smear revealed atypical lymphocytes with cleaved nuclei, and inverse PCR was performed with DNA extracted from those cells; however, the result showed that the pattern of HTLV-1 proviral DNA integration sites was polyclonal. Further, we performed RNA in situ hybridization targeting HTLV-1 bZIP factor (HBZ-ISH) using the formalin-fixed paraffin-embedded (FFPE) tissue samples of the mesopharyngeal tumor, and a high expression of HBZ was found in the tumor cells, leading to the diagnosis of ATL. These findings suggest the effectiveness of the novel diagnostic method using FFPE tissue samples for ATL.

Nurse-led cardiovascular diseases risk assessment and communication using WHO/ISH risk prediction chart in a tertiary care hospital of North India.

Cardiovascular diseases (CVDs) contribute to most of the potentially preventable burden through early risk assessment. Nurse-led CVD risk assessment is an effective strategy to address the human resource crisis for CVD prevention. An interventional study was conducted in medicine wards of a tertiary care hospital in North India to train nurses in CVD risk assessment and its communication. All bedside nurses (n = 30) of selected wards were enrolled and trained in CVD risk assessment and communication using WHO/ISH risk prediction charts. Once fully trained, each nurse enrolled patients (>40 years of age) from their respective wards to assess and communicate CVD risk. To calculate the reliability of risk assessment, investigator simultaneously assessed CVD risk with nurses. The mean age of nurses was 32.07 ± 6.31 years. The results revealed that training significantly increased the knowledge of nursing personnel (P < 0.001). There was perfect inter-rater reliability agreement (Cohen's k = 0.929) between nurses and investigators while assessing CVD risk. Nurses demonstrated good communication skills. The study concluded that nurses can be trained successfully in CVD risk assessment and communication. The study recommends the task shifting of CVD risk assessment to nurses after providing proper training.