The gene localization technique of
(pinewood nematode, PWN) is used for study gene expression in PWNs. Two
hybridization methods, namely, whole-mount
hybridization and the cut-off method are used ...widely. To compare the effects of these two
hybridization methods, the present study investigated the patterns of two functional genes expression in PWNs. The
gene (GenBank accession number: OR228482), related to pathogenicity, and the
gene (GenBank accession number: OR228481), related to sex determination, were selected to map related genes in the whole-mount and amputated PWNs at different ages using these
hybridization methods. Based on the overall statistical comparison, we found that compared to the cut-off method, the whole-mount method exhibited higher staining rates and correct staining rates for the
gene and
gene. However, considering the correct staining aspect, the cut-off method yielded better staining effects on pinewood nematode sections than the whole-mount method, with clearer hybridization signal locations and less non-specific staining. In other words, the cut-off method demonstrated more precise gene localization. Both methods are applicable for gene localization, but considering the overall staining pattern, analysis of experimental results, and comprehensive experimental operations, we believe that the whole-mount method is more suitable for gene localization and expression analysis of development-related genes in pinewood nematodes. This is because intact pinewood nematodes are better suited for showcasing the continuous developmental process of development-related genes. On the other hand, considering the experimental time, accuracy of staining site, and the amount of non-specific staining, the cut-off method is more suitable for disease-related genes. Additionally, to achieve better performance, the cut-off method can be selectively applied to samples during the experimental process.
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.
The present study reports outbreaks of tilapia lake virus (TiLV), an emerging pathogen causing syncytial hepatitis of tilapia (SHT), in farmed tilapia in Thailand. Occurrence of the virus was ...confirmed by RT-PCR and nucleotide sequence homology to the TiLV from Israel. Diseased fish exhibited typical histopathological features of syncytial giant cells in the liver examined through H&E and semi-thin sections. Presence of intracytoplasmic viral particles was revealed by TEM. In situ hybridization using a specific DIG-labeled probe derived from a partial genome segment 3 of TiLV genome revealed multiple tissues tropism of the virus including liver, kidney, brain, spleen, gills and connective tissue of muscle. An alternative semi-nested RT-PCR protocol has been developed in this study for disease diagnosis. Additionally, comparative genetic analysis revealed genetic variations of Thailand-originated virus to the Israel TiLV strains, sharing 96.28 to 97.52% nucleotide identity and 97.35 to 98.84% amino acid identity. Outbreaks of TiLV in different continents might signal a serious threat to tilapia aquaculture globally.
•Outbreaks of TiLV associated with massive mortalities in farmed tilapia in Thailand.•An alternative semi-nested RT-PCR has been developed for disease diagnosis.•In situ hybridization assay revealed multiple tissues tropism of the virus.•Transmission electron microscopy revealed intracytoplasmic viral particles.•Partial genome of TiLV from Thailand exhibited genetic variations.
•Viral segments RNA1 and RNA2 were localized in NNV-infected cells by ISH using labeled RNA probes.•RNA1 and RNA2 was expressed at an early stage, virions are then assembled through the initially ...expressed viral genes.•The RNA1 gene was expressed separately by double labeling RNA-ISH.•Although the packaging of infectious particles is efficient, the RNA1 gene was still over-produced.
Nervous necrosis virus (NNV) belongs to the genus Betanodavirus of family Nodaviridae. Its genome consists of two RNA segments, RNA1 and RNA2. Several studies have investigated NNV detection by in situ hybridization (ISH), but these have typically focused on the detection of the RNA2 gene. In this study, we localized both RNA1 and RNA2 NNV segments in viral-infected cells by ISH, using labeled RNA probes (RNA-ISH). Also, immunocytochemistry (ICC) assay was carried out for localization of viral particle by targeting the coat protein. Further, viral quantification assays were performed by quantitative RT-PCR and viral infectivity (TCID50) in SSN-1 cells. Viral segments were observed by RNA-ISH at 6 h post infection (hpi), while NNV particles were detected at 24 hpi by ICC. Use of double labeling RNA-ISH revealed the co-expression of the two viral segments in the same area of the cells, while RNA1 was also detected separately. Comparison of the level of viral genomic segments and viral infectivity revealed significantly more copies of RNA1 at each time points than copies of RNA2 and greater NNV titers. The results suggest that RNA1 might be expressed in the early stages of replication, with RNA2 expressed later. The virions then assemble through initially expressed viral genomic segments. Even though infectious particles displayed very efficient packaging, the RNA1 segment was still over-produced.
