Gene expression analysis is pivotal in cancer research and clinical practice. Although traditional methods lack spatial context, RNA in situ hybridization (RNA-ISH) is a powerful technique that retains spatial tissue information. Here, RNAscope score, RT-droplet digital PCR, and automated QuantISH and QuPath were used for quantifying RNA-ISH expression values from formalin-fixed, paraffin-embedded samples. The methods were compared using high-grade serous ovarian carcinoma samples, focusing on CCNE1, WFDC2, and PPIB genes. The findings demonstrate good concordance between automated methods and RNAscope, with RT-droplet digital PCR showing less concordance. Additionally, QuantISH exhibits robust performance, even for low-expressed genes like CCNE1, showcasing its modular design and enhancing accessibility as a viable alternative for gene expression analysis.

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The article discusses the importance of accurately distinguishing HER2-low from HER2-negative breast cancer, as novel ADCs have demonstrated activity in a large population of patients with HER2-low-expressing BC. While current guidelines recommend a dichotomous classification of HER2 as either positive or negative, the emergence of the HER2-low concept calls for standardization of HER2 testing in breast cancer, using currently available assays to better discriminate HER2 levels. This review covers the evolution and latest updates of the ASCO/CAP guidelines relevant to this important biomarker in breast cancer, including still-evolving concepts such as HER2 low, HER2 heterogeneity, and HER2 evolution. Our group presents the latest Mexican recommendations for HER2 status evaluation in breast cancer, considering the ASCO/CAP guidelines and introducing the HER2-low concept. In the era of personalized medicine, accurate HER2 status assessment remains one of the most important biomarkers in breast cancer, and the commitment of Mexican pathologists to theragnostic biomarker quality is crucial for providing the most efficient care in oncology.

Background: Breast cancer (BC) is the most prevalent cancer among women worldwide. Although advances have been made in the identification of predictive biomarkers, current options for early diagnosis and prognostic analysis are still suboptimal. Recently, transfer-RNA-derived RNA fragments (tRFs) have emerged as a class of small noncoding RNAs that play a role in the cancer progression. The authors aimed to identify a specific class of tRFs as a molecular marker for BC diagnosis and prognosis in clinical management. Methods: The levels of 5\'-tRF-His-GTG were quantified in BC tissue (n = 101) and inflammatory normal breast tissue (n = 22) using in situ hybridization. Clinicopathological parameters were obtained, including age, tumor node metastasis stage, hormone receptor status, histopathological grade, lymphovascular invasion, and recurrence. The correlation between the expression of 5\'-tRF-His-GTG and these parameters in different BC subtypes was analyzed. Patient death and cancer progression were regarded as clinical endpoints in the survival analysis. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were also performed to predict the involvement in pivotal biological process. Results: The expression of 5\'-tRF-His-GTG was significantly downregulated in BC tissues and was in connection with T stage in human epidermal growth factor 2-positive and basal-like BC, as well as N stage and histopathological grade in luminal BC. Patients with low expression of 5\'-tRF-His-GTG had a poor overall survival rate. Statistics of GO and KEGG pathway revealed that cation channel activity, protein catabolic process, response to temperature stimulus, cell cycle, focal adhesion, and glycerophospholipid metabolism were significantly enriched. Conclusions: This study suggests that the assessment of 5\'-tRF-His-GTG expression could serve as a novel biomarker for individual diagnosis and prognosis in BC.

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.

Lymphoepithelioma-like carcinoma of the skin (LELCS) is a rare primary skin cancer, with an annual incidence of 1/100,000 and about 85 cases published in the literature. It is considered the cutaneous counterpart of undifferentiated nasopharyngeal carcinoma (UNC, Schmincke-Regaud tumor) but has no association with EBV. We present an interesting case with features of LELCS in a 93-year-old man, right frontal-orbital region, diagnosed histologically and with immunohistochemical features. We also emphasize contrasting morphologic features for correct nosographic classification and address current issues, suggesting potential insights. Finally, we briefly reviewed other cases described in the literature.

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.

The fast-growing demand for seedless table grapes has attracted the attention of scientists for the development of new seedless cultivars. Various genes and pathways have been identified which affect seedlessness. However, the detail of the mechanism(s) regulating seedless traits in grape is still unclear, and genes related to seedlessness in grape require further study. Transcriptomic and genomic analyses of Homeobox (HB) transcription factors have suggested the involvement of HB genes, especially of HB-KNOX members, in grape seed development. Here, we functionally characterize VvHB63 gene in grape and report its role in fruit and seed development. VvHB63 showed higher expressions levels in the chalaza and integument of ovules in seedless grapes, than in seeded ones. However, no differences were observed in the sequences of seedless and seeded grape cultivars. In situ hybridization (ISH) analysis showed that VvHB63 gene was expressed in the episperm cells and ovules of \'Thompson Seedless\'. Conserved domains KNOX1 and KNOX2 were important for the interaction of VvHB63 with VvHB06. Heterologous over-expression of VvHB63 (35 S::VvHB63-OE) in tomato induced smaller fruits and seeds than in wild type or SlTkn1-KO. The synergistic cooperation between VvHB63 and related proteins play an important role in ovule development.

Detection of phosphorylated proteins in tissue sections using immunohistochemistry (IHC) is a challenging task. The absence of tissue staining may be caused by either a lack of protein expression or a lack of protein activation via its phosphorylation. To address this problem, we employed Integrated Co-detection Workflow (ICW) protocol to analyze lung cancer tissue sections by combining in situ hybridization (ISH) with IHC. The target protein of interest was epidermal growth factor receptor (EGFR, also known as ErbB1 and HER1) which is the founding member of the ErbB family of receptor tyrosine kinases. Using phospho-specific antibodies specific for a phosphorylated site Y1173 of EGFR molecule allowed us to analyze IHC and ISH staining at a single cell level in lung cancer tissue. We have observed both a co-localization of IHC with ISH signals and ISH-positive cells lacking IHC labeling for phosphorylated EGFR. ICW appears to be a very powerful spatial biology technique for accurate localization of cancer cells with phosphorylated/activated and non-phosphorylated/nonactivated proteins.

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.