The pine wood nematode Bursaphelenchus xylophilus is a highly invasive species responsible for the widespread pine wilt disease. Double-stranded RNA (dsRNA) biopesticides represent a novel strategy for controlling plant-parasitic nematodes. The B. xylophilus arginine kinase (BxAK) features a conserved ATP-binding domain and exhibits nematode-specific divergence in the phylogenetic tree. Notably, whole-mount in situ hybridization signals are evident in the nematode head and middle sections, particularly in the juvenile stage before sex differentiation. In this study, we developed a novel dsRNA-like small interfering RNA (siRNA) assembly that specifically targets BxAK and presents highly nematicidal effects. The RNA interference (RNAi) efficiency achieved a 95.9 % reduction in second-stage juveniles. In bioassays, the median lethal concentrations of this siRNA assembly against B. xylophilus were 168.5 ng/μl for juveniles and 603.8 ng/μl for adults within 48 h. Moreover, transcriptomic results revealed significantly downregulated expression levels of genes related to metabolism and development, suggesting that the mode of action of BxAK silencing is related to disruptions in energy homeostasis and juvenile development. In conclusion, BxAK is a molecular target for controlling B. xylophilus, and our siRNA assembly significantly enhances RNAi efficiency and lowers the lethal concentration required, making it a promising candidate for future biocontrol applications.

Gremlin 1 (GREM1) is an antagonist of bone morphogenetic protein (BMP). GREM1 is expressed in the stromal cells of various carcinomas and promotes tumor progression by suppressing BMP signaling. We designed this study to establish an evaluation strategy for GREM1 expression, focusing on the tumor stroma, and to examine its clinicopathological significance in gastric cancer (GC) progression. We employed RNA in situ hybridization (ISH) to evaluate the prognostic value of GREM1 expression in a cohort of 104 surgically resected GC cases and assessed ISH scores according to previous reports. GREM1 expression was observed in tumor stromal cells, including fibroblasts. We defined GREM1-positive cells as those expressing ISH score ≥ 3 and quantified the number of GREM1-positive cells using image analysis software. We examined the relationship between the number of GREM1-positive cells in the tumor stroma and clinicopathological features. The number of GREM1-positive cells per tumor stroma ranged from 0 to 714.7 cells/mm2 (median, 1.65 cells/mm2). We divided the 104 GC cases into GREM1-High and GREM1-Low expression groups based on the abovementioned median value. GREM1-High expression group was significantly associated with a more advanced pT grade, pN grade, lymphatic invasion, and venous invasion. Kaplan-Meier analysis showed significantly poorer survival in the GREM1-High expression group than in the GREM1-Low expression group. These results indicated that GREM1 expression in GC is localized in tumor stromal cells, and that high GREM1 expression in the tumor stroma could be a poor prognostic factor.

Polyploidy is thought to be an evolutionary and systematic mechanism for gene flow and phenotypic advancement in flowering plants. It is a natural phenomenon that promotes diversity by creating new permutations enhancing the prime potentials as compared to progenitors. Two different pathways have been recognized in studying polyploidy in nature; mitotic or somatic chromosome doubling and cytogenetics variation. Secondly, the vital influence of being polyploid is its heritable property (unreduced reproductive cells) formed during first and second-division restitution (FDR & SDR). Different approaches either chemical (Colchicine, Oryzalin, Caffeine, Trifuralin, or phosphoric amides) or gaseous i.e. Nitrous oxide have been deliberated as strong polyploidy causing agents. A wide range of cytogenetic practices like chromosomes study, ploidy, genome analysis, and plant morphology and anatomy have been studied in different plant species. Flow cytometry for ploidy and chromosome analysis through fluorescence and genomic in situ hybridization (FISH & GISH) are the basic methods to evaluate heredity substances sampled from leaves and roots. Many horticultural crops have been developed successfully and released commercially for consumption. Moreover, some deep detailed studies are needed to check the strong relationship between unique morphological features and genetic makeup concerning genes and hormonal expression in a strong approach.

The aim of the present study was to develop and validate a quantitative image analysis (IA) algorithm to aid pathologists in assessing bright-field HER2 in situ hybridization (ISH) tests in solid cancers. A cohort of 80 sequential cases (40 HER2-negative and 40 HER2-positive) were evaluated for HER2 gene amplification with bright-field ISH. We developed an IA algorithm using the ISH Module from HALO software to automatically quantify HER2 and CEP17 copy numbers per cell as well as the HER2/CEP17 ratio. We observed a high correlation of HER2/CEP17 ratio, an average of HER2 and CEP17 copy number per cell between visual and IA quantification (Pearson\'s correlation coefficient of 0.842, 0.916, and 0.765, respectively). IA was able to count from 124 cells to 47,044 cells (median of 5565 cells). The margin of error for the visual quantification of the HER2/CEP17 ratio and of the average of HER2 copy number per cell decreased from a median of 0.23 to 0.02 and from a median of 0.49 to 0.04, respectively, in IA. Curve estimation regression models showed that a minimum of 469 or 953 invasive cancer cells per case is needed to reach an average margin of error below 0.1 for the HER2/CEP17 ratio or for the average of HER2 copy number per cell, respectively. Lastly, on average, a case took 212.1 s to execute the IA, which means that it evaluates about 130 cells/s and requires 6.7 s/mm2. The concordance of the IA software with the visual scoring was 95%, with a sensitivity of 90% and a specificity of 100%. All four discordant cases were able to achieve concordant results after the region of interest adjustment. In conclusion, this validation study underscores the usefulness of IA in HER2 ISH testing, displaying excellent concordance with visual scoring and significantly reducing margins of error.

Multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore the cellular diversity and the architecture of tissues. We propose a sensitive, open-source, simple and flexible method for the generation of in situ expression maps of hundreds of genes. We use direct ligation of padlock probes on mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high-throughput and large multiplexing. We validate the method across a number of species and show its use in combination with orthogonal methods such as antibody staining, highlighting its potential value for developmental and tissue biology studies. Finally, we provide an end-to-end computational workflow that covers the steps of probe design, image processing, data extraction, cell segmentation, clustering and annotation of cell types. By enabling easier access to high-throughput spatially resolved transcriptomics, we hope to encourage a diversity of applications and the exploration of a wide range of biological questions.

Profiling gene expression while preserving cell locations aids in the comprehensive understanding of cell fates in multicellular organisms. However, simple and flexible isolation of microregions of interest (mROIs) for spatial transcriptomics is still challenging. We present a laser-induced forward transfer (LIFT)-based method combined with a full-length mRNA-sequencing protocol (LIFT-seq) for profiling region-specific tissues. LIFT-seq demonstrated that mROIs from two adjacent sections could reliably and sensitively detect and display gene expression. In addition, LIFT-seq can identify region-specific mROIs in the mouse cortex and hippocampus. Finally, LIFT-seq identified marker genes in different layers of the cortex with very similar expression patterns. These genes were then validated using in situ hybridization (ISH) results. Therefore, LIFT-seq will be a valuable and efficient technique for profiling the spatial transcriptome in various tissues.

Hepatitis B virus (HBV) developed highly intricates mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. With the aid of a series of classical analytical methods such as ultrafiltration, and Southern and Northern blots, a general framework of HBV life cycle has been established. However, this picture still lacks many key histological contexts which involves pathophysiological changes of hepatocytes, non-parenchymal cells, infiltrated leukocytes, and associated extracellular matrix. Here, we describe a CISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in liver tissue of chronic hepatitis B patients. By coupling it with immunohistochemistry and other histological stains, much richer information regarding the HBV-induced pathological changes can be harvested.

UNASSIGNED: Diagnosing brain tumors is critical due to their complex nature. This review explores the potential of in situ hybridization for diagnosing brain neoplasms, examining their attributes and applications in neurology and oncology.
UNASSIGNED: The review surveys literature and cross-references findings with the OMIM database, examining 513 records. It pinpoints mutations suitable for in situ hybridization and identifies common chromosomal and gene anomalies in brain tumors. Emphasis is placed on mutations\' clinical implications, including prognosis and drug sensitivity.
UNASSIGNED: Amplifications in EGFR, MDM2, and MDM4, along with Y chromosome loss, chromosome 7 polysomy, and deletions of PTEN, CDKN2/p16, TP53, and DMBT1, correlate with poor prognosis in glioma patients. Protective genetic changes in glioma include increased expression of ADGRB3/1, IL12B, DYRKA1, VEGFC, LRRC4, and BMP4. Elevated MMP24 expression worsens prognosis in glioma, oligodendroglioma, and meningioma patients. Meningioma exhibits common chromosomal anomalies like loss of chromosomes 1, 9, 17, and 22, with specific genes implicated in their development. Main occurrences in medulloblastoma include the formation of isochromosome 17q and SHH signaling pathway disruption. Increased expression of BARHL1 is associated with prolonged survival. Adenomas mutations were reviewed with a focus on adenoma-carcinoma transition and different subtypes, with MMP9 identified as the main metalloprotease implicated in tumor progression.
UNASSIGNED: Molecular-genetic diagnostics for common brain tumors involve diverse genetic anomalies. In situ hybridization shows promise for diagnosing and prognosticating tumors. Detecting tumor-specific alterations is vital for prognosis and treatment. However, many mutations require other methods, hindering in situ hybridization from becoming the primary diagnostic method.

BACKGROUND: Early diagnosis of neosporosis in dogs is challenging.
OBJECTIVE: To evaluate the feasibility of a compound multimodal testing approach for diagnosing in dogs neuromuscular and combined forms of neosporosis.
METHODS: A total of 16 dogs diagnosed with solely neuromuscular neosporosis or with a combination of neuromuscular and central nervous system neosporosis.
METHODS: Retrospective review of clinical signs, laboratory findings, treatment, and outcome with focus on the diagnostic utility of different tests. Development of a chromogenic in situ hybridization (ISH) assay for the identification of Neospora caninum in paraffin-embedded muscle samples.
RESULTS: 13/16 dogs had only neuromuscular signs of neosporosis, 3/16 had disease signs with concomitant central nervous system (CNS) involvement. Serology was performed in 15/16, with 10/15 showing titers >1 : 160 at admission. PCR on muscle samples detected N. caninum DNA in 11/16. Immunohistochemistry (IHC) detected N. caninum in 9/16 and ISH in 9/16. Histopathology revealed inflammatory myopathy in 10/16, necrotizing myopathy in 5/16, borderline changes in 1/16 and tachyzoites in 9/16. In 4 cases, N. caninum infection was confirmed with all 5 diagnostic methods, 3 cases with 4, 2 with 3, 6 with 2, and 1 animal with 1.
CONCLUSIONS: Diagnosis of N. caninum infection should rely on a multimodal diagnostic approach and negativity of 1 single test should not allow for exclusion. Serology in combination with direct parasite identification via histopathology, DNA via PCR, or both modalities, appears a reliable diagnostic approach.

A substantial percentage of the population remains at risk for cervical cancer due to pre-existing human papillomavirus (HPV) infections, despite prophylactic vaccines. Early diagnosis and treatment are crucial for better disease outcomes. The development of new treatments heavily relies on suitable preclinical model systems. Recently, we established a mouse papillomavirus (MmuPV1) model that is relevant to HPV genital pathogenesis. In the current study, we validated the use of Papanicolaou (Pap) smears, a valuable early diagnostic tool for detecting HPV cervical cancer, to monitor disease progression in the MmuPV1 mouse model. Biweekly cervicovaginal swabs were collected from the MmuPV1-infected mice for viral DNA quantitation and cytology assessment. The Pap smear slides were evaluated for signs of epithelial cell abnormalities using the 2014 Bethesda system criteria. Tissues from the infected mice were harvested at various times post-viral infection for additional histological and virological assays. Over time, increased viral replication was consistent with higher levels of viral DNA, and it coincided with an uptick in epithelial cell abnormalities with higher severity scores noted as early as 10 weeks after viral infection. The cytological results also correlated with the histological evaluation of tissues harvested simultaneously. Both immunocompromised and immunocompetent mice with squamous cell carcinoma (SCC) cytology also developed vaginal SCCs. Notably, samples from the MmuPV1-infected mice exhibited similar cellular abnormalities compared to the corresponding human samples at similar disease stages. Hence, Pap smear screening proves to be an effective tool for the longitudinal monitoring of disease progression in the MmuPV1 mouse model.
OBJECTIVE: Papanicolaou (Pap) smear has saved millions of women\'s lives as a valuable early screening tool for detecting human papillomavirus (HPV) cervical precancers and cancer. However, more than 200,000 women in the United States alone remain at risk for cervical cancer due to pre-existing HPV infection-induced precancers, as there are currently no effective treatments for HPV-associated precancers and cancers other than invasive procedures including a loop electrosurgical excision procedure (LEEP) to remove abnormal tissues. In the current study, we validated the use of Pap smears to monitor disease progression in our recently established mouse papillomavirus model. To the best of our knowledge, this is the first study that provides compelling evidence of applying Pap smears from cervicovaginal swabs to monitor disease progression in mice. This HPV-relevant cytology assay will enable us to develop and test novel antiviral and anti-tumor therapies using this model to eliminate HPV-associated diseases and cancers.