Strong Expression Research Articles

The transcription factor combination MEF2 and KLF7 promotes axonal sprouting in the injured spinal cord with functional improvement and regeneration-associated gene expression

BackgroundAxon regeneration after injury to the central nervous system (CNS) is limited by an inhibitory environment but also because injured neurons fail to initiate expression of regeneration associated genes (RAGs). The potential of strong RAG expression to promote regeneration in the CNS is exemplified by the conditioning lesion model, whereby peripheral nerve injury promotes regeneration of centrally projecting branches of the injured neurons. RAG expression could potentially be induced by delivery of the right set of transcription factors (TFs). We here aim to identify TF combinations that activate this program.MethodsWe first analysed binding site motifs in promoters of the RAG program to identify nine candidate growth-promoting TFs. These were systematically screened in vitro to identify combinations that had potent neurite-growth promoting activity. Next, adeno-associated viral vectors were used to express these TF combinations in vivo in L4/L5 dorsal root ganglia to test whether they would promote regeneration in a spinal cord injury model (dorsal column lesion) in female rats. To determine whether they could activate the RAG program we carried out gene expression profiling on laser-dissected dorsal root ganglion neurons specifically expressing these TF combinations, and of DRG neurons that had been axotomized.ResultsPromoter analysis identified ATF3, Jun, CEBPD, KLF7, MEF2, SMAD1, SOX11, STAT3 and SRF as candidate RAG-activating TFs. In vitro screening identified two TF combinations, KLF7/MEF2 and ATF3/KLF7/MEF2, that had potent neurite-growth promoting activity, the latter being the more powerful. In vivo, KLF7/MEF2, but not ATF3/KLF7/MEF2 or KLF7 or MEF2 alone, promoted axonal sprouting into the dorsal column lesion site and led to improved functional recovery. Gene expression profiling revealed that unexpectedly, the MEF2-VP16 construct used had little transcriptional activity in vivo, suggesting additional steps may be required to achieve full MEF2 activity. All combinations except MEF2 alone induced RAG expression mirroring that induced by axotomy to significant extents, while ATF3/KLF7/MEF2, KLF7 and ATF3, but not KLF7/MEF2 also induced apoptosis-related genes which may hinder regeneration.ConclusionsThe TF combination KLF7/MEF2 partially mimics the conditioning lesion effect, inducing axonal sprouting into a dorsal column lesion and driving significant RAG expression, and also promotes functional improvement.

NTRK gene fusion and molecular pathological characteristics of mismatch repair deficient colorectal cancer

Objective: To investigate the expression pattern of pan-TRK protein in colorectal cancers with NTRK gene fusion and mismatch repair deficient (dMMR) and to analyze its molecular pathological characteristics. Methods: A total of 117 dMMR colorectal cancers diagnosed in the Department of Pathology of Henan Provincial People's Hospital, Zhengzhou, China from 2020 to 2023 were collected. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and DNA/RNA-based next-generation sequencing (NGS) were used to detect pan-TRK protein expression and fusion partner genes in tumors, and to further explore the correlation between pan-TRK staining patterns and partner genes. Results: IHC and FISH were performed successfully in formalin-fixed paraffin-embedded tissues from 117 dMMR colorectal cancer patients. There were 15 (15/117, 12.8%) cases with positive pan-TRK, including 6 cases with strong staining in tumor cell membrane and cytoplasm, 2 cases with weakly granular staining in tumor cytoplasm, 2 cases with moderate dot-like staining in near 5% tumor cell nuclei, 1 case with moderately to strongly granular staining in the cytoplasm and membrane of tumor cells, 1 case with moderately to weakly granular staining in about 60% of the tumor cells, 1 case with strongly staining in about 1% of the tumor cells, 1 case with moderately to strongly staining in about 3% of the tumor cells and 1 case with diffuse, moderate para-nuclear dot-like and weakly perinuclear granular staining. NTRK1 gene disruption was detected in 6 cases (6/117, 5.1%) and consistent with diffusely strong expression of pan-TRK. Based on DNA/RNA NGS, it was further confirmed that the 6 cases with NTRK1 gene disruption all carried TPM3-NTRK1 fusion gene, and all had high microsatellite instability and high tumor mutation burden. No KRAS, NRAS, BRAF V600E or TP53 gene mutations were detected. Four patients carried frame shift mutations in RNF43. Other molecular changes included 3 cases with ROS1 gene mutation, 2 cases with BRAC, ALK, and EGFR gene mutations, 2 cases with ATM gene mutation, and 2 cases with KIT gene mutation. These were missense/frame shift mutations that were associated with no clinical significance. The nine pan-TRK-positive cases without NTRK gene fusion detected with DNA-based NGS were further confirmed with RNA-based NGS, and no NTRK gene fusion was found. The sensitivity and specificity of NTRK gene fusion detected using IHC were 100.0% and 92.5%, respectively. The sensitivity and specificity of diffusely strong membranous/cytoplasmic staining were both 100.0%. Conclusions: Pan-TRK protein has various expression patterns in dMMR colorectal cancer. Its diffusely strong expression is highly suggestive of NTRK1 gene fusion. TPM3-NTRK1 gene fusion is a common form of NTRK gene fusion in dMMR colorectal cancer.

Hematopathological Patterns in Acute Myeloid Leukemia with Complications of Overt Disseminated Intravascular Coagulation

Background: Acute myeloid leukemia (AML) complicated by disseminated intravascular coagulation (DIC) poses major diagnostic and therapeutic challenges. While DIC is well documented in acute promyelocytic leukemia, its manifestations in non-APL AML remain underexplored, necessitating precise diagnostic strategies for effective management. Methods: AML patients with overt DIC were analyzed, including morphological, immunophenotypic, cytogenetic, and genetic evaluations. DIC was diagnosed using the ISTH scoring system, and AML subtypes were classified following WHO criteria. Results: Three diagnostic patterns were identified. (1) Acute promyelocytic leukemia: Leukemia characterized by PML::RARa rearrangements, FLT3 co-mutations, and frequent Auer rods and faggot bundles. Immunocytological analysis showed CD34 and HLA-DR negativity. (2) AML with FLT3 and/or NPM1 mutations: A high prevalence of cup-like blasts was found in 70% of cases. FLT3 mutations, often co-occurring with NPM1, dominated, while karyotypes were typically normal. Immunophenotyping revealed strong myeloid marker expression (MPO+, CD13+, and CD33+), with occasional CD34 negativity. (3) AML with monocytic differentiation: Leukemia defined by monoblastic/promonocytic morphology, DNMT3A mutations, and complex karyotypes or 11q23 rearrangements. Immunophenotyping demonstrated a dominance of monocytic markers (CD4+, CD14+, CD15+, and CD64+). Two patients presented unique profiles with no alignment to these patterns. Conclusions: This study highlights distinct hematopathological patterns of AML with overt DIC, providing a framework for early and precise diagnosis. Recognizing these patterns is critical for tailoring diagnostic and therapeutic approaches to improve outcomes in this high-risk population.

Adaptive Divergence and Functional Convergence: The Evolution of Pulmonary Gene Expression in Amphibians of the Qingzang Plateau.

The Qingzang Plateau, with its harsh environmental conditions-low oxygen, high ultraviolet radiation and significant temperature fluctuations-demands specialised adaptations for survival. While genetic adaptations have been extensively studied, gene expression's role in amphibian adaptation to high elevations remains understudied. This study analysed pulmonary gene expression in 119 amphibians across the plateau to explore how genetic and environmental factors shape expression evolution. Transcriptomic analyses revealed significant interspecies variation, driven by environmental factors like temperature, oxygen levels, UVB radiation and precipitation. Principal Component and Mantel analyses found no significant correlation between gene expression divergence and genetic distance. Instead, species-specific traits and environmental pressures were pivotal in shaping expression patterns. PERMANOVA analysis showed environmental factors had varying impacts on species. For instance, Bufo gargarizans exhibited a strong gene expression response to multiple environmental factors, while Scutiger boulengeri was less influenced, reflecting diverse adaptive strategies. Functional enrichment analysis highlighted convergence in key biological processes, such as energy metabolism, apoptosis and autophagy, despite species-specific gene expression differences. These processes are critical for surviving the plateau's extremes. The findings suggest that gene expression evolution in amphibians on the Qingzang Plateau is shaped by both genetic diversity and environmental pressures. Although gene expression profiles vary, they converge on essential functions, offering insights into adaptation mechanisms in extreme environments.

Evaluation of immunohistochemical TRPC3 and TRPC6 expression patterns in human endometriosis.

Although to date the pathogenesis of endometriosis remains largely unexplained, it is known that processes of migration, proliferation and revascularization and thus calcium as a messenger substance play an important role. Consecutively, the present study examines the immunohistochemical expression of the calcium transient receptor potential channels 3 and 6 (TRPC3 and TRPC6) in ectopically located (outside the uterine cavity) endometrial tissue. Laparoscopically collected and histomorphologically verified endometriosis tissues from several different intraabdominal locations were examined (n = 20) and immunohistochemical stainings were performed with anti-TRPC3 and anti-TRPC6 antibodies (Alomone Labs, Jerusalem). Hereby, eutopic endometrium served as a healthy control cohort (n = 6). Staining patterns were evaluated using a modified immunoreactive score (IRS) and exploratory statistical analysis was performed, aiming to determine relations of staining intensities with associated clinical parameters such as rASRM stage and location. We determined a strong cytoplasmatic TRPC3 and TRPC6 expression in all ectopic endometrial glandular formations, albeit with focally varying staining intensities. Within our cohort, we did not verify a statistically significant difference of TRPC3 and TRPC6 expression between endometriosis patients and a healthy control group or between different clinical affections (rASRM stages). Our study confirms - to our knowledge for the first time - the successful immunohistochemical assessment of TRPC3 and TRPC6 in endometriosis, setting the basis for future studies aiming at evaluating not only clinical aspects of TRPC3 and TRPC6 expression but also shedding light on its function in the pathophysiological context of endometriosis.

NADPH oxidase 4-SH3 domain-containing YSC84-like 1 complex participates liver inflammation and fibrosis.

There is growing evidence that NADPH oxidase 4 (Nox4) in hepatocytes contributes to liver inflammation and fibrosis during the development of metabolic dysfunction-associated steatohepatitis (MASH). However, how Nox4 is regulated and leads to liver pathogenesis is unclear. Our previous studies showed that the cytosolic protein SH3 domain-containing Ysc84-like 1 (SH3YL1) regulates Nox4 activity. Here, we asked whether SH3YL1 also participates in liver inflammation and fibrosis during MASH development. We generated that whole body SH3YL1 knockout (SH3YL1-/-), Nox4 knockout (Nox4-/-) mice, and the hepatocyte-specific SH3YL1 conditional knockout (Alb-Cre/SH3YL1fl/fl) mice were fed a methionine/choline-deficient (MCD) diet to induce liver inflammation and fibrosis in pathogenesis of MASH. Palmitate-stimulated primary SH3YL1- and Nox4-deficient hepatocytes and hepatic stellate cells (HSCs) did not generate H2O2. While the liver of MCD diet-fed wild type (WT) mice demonstrated elevated 3-nitrotyrosine as a protein oxidation and 4-hydroxynonenal adducts as a lipid oxidation and increased liver inflammation, hepatocyte apoptosis, and liver fibrosis, these events were markedly reduced in SH3YL1-/-, Nox4-/-, and Alb-Cre/SH3YL1fl/fl mice. The MCD diet-fed WT mice also showed elevated hepatocyte expression of SH3YL1 protein. Similarly, liver biopsies from MASH patients demonstrated strong hepatocyte SH3YL1 protein expression, whereas hepatocytes from patients with steatosis weakly expressed SH3YL1 and histologically normal patient hepatocytes exhibited very little SH3YL1 expression. The Nox4-SH3YL1 complex in murine hepatocytes elevates their H2O2 production, which promotes the liver inflammation, hepatocyte apoptosis, and liver fibrosis that characterize MASH. This axis may also participate in MASH in humans.

Expression of Trop2, Nectin-4, and FOLR1 cell surface markers in biliary tract cancers: Is it time to repurpose drugs?

637 Background: Biliary tract cancers (BTC), including gall bladder cancer, intrahepatic and extrahepatic cholangiocarcinoma are aggressive malignancies, with poor prognosis. Despite advances in frontline setting, and in biomarker selected population (IDH1, FGFR2, Her2, BRAF, HRD), there is a dire need to advance therapy in subsequent lines. Methods: We identified BTC patients (pts) at Roswell Park Cancer Center who underwent curative intent surgery between 01/2006-01/2020. After excluding duplicate samples and those with insufficient tissue, we found 78 pts with adequate follow up for analysis. Expression of trophoblast cell surface antigen 2 (Trop2), nectin4, folate receptor alpha (FOLR1) was assessed by immunohistochemistry and correlated with clinical outcomes. H score, a weighted index score that factor in, the % of cells across different intensity was calculated. H scores were classified as negative (0) and positive (>1). Positive H scores were grouped as weak (1-100), intermediate (101-200) and strong (201-300). Descriptive statistics were conducted, and multivariate analyses performed (R studio) to study any correlation between expression of markers and survival outcomes. Results: The median age of our cohort was 67.5 years (range 40-87) with 58% male; 24.4%, 30.8%, 20.5%, 7.7% had stages I, II, III, IV disease. Staging information was missing in 16.7% of pts. Of 73 analyzable samples for Trop2, all were positive for Trop2, with a mean H score of 86, SD of 68.9. 56.4% of patients had a weak H score, 30.8% had an intermediate H score and 6.4% had a strong H score for Trop2. Median overall survival (mOS) among low, intermediate and high expressing group of pts with Trop2 was 48.9 months (m), 23.9 m, and 41.9 m with median Progression Free Survival (mPFS) of 37.8m, 15.3 m, and 37.4 m, respectively. Of 75 analyzable samples for FOLR1 and Nectin4, FOLR1 was expressed in 51.3% with a mean H score of 6.97 (SD 21.9). Nectin4 was expressed in 78.2%, with a mean H score of 13.7 (SD 26.3). Pts with Trop2 expression had a mOS of 39.5 m and a mPFS of 28.7 m. There were no significant differences in mOS (48.9 m vs 33.3 m, p =0.14) and mPFS (37.8 m vs 26.2 m, p =0.35) among patients with positive vs negative FOLR1 respectively. There was no difference in mOS (41.6 m vs 38.3 m, p=0.92) and mPFS (32.8 vs 26.4 m, p-value=0.5) in patients with positive vs negative Nectin4. Multivariate analyses showed that pts with intermediate Trop2 (H score=101-200) had a high risk of death (HR 21.44, CI 2.81-163.4, p= 0.003). Conclusions: Though no difference was seen in clinical outcomes in pts with strong Trop2 expression, FOLR1, and Nectin4 expression, this could be explained by the small sample size. Trop2 is frequently expressed in BTCs and patients with Intermediate Trop2 H score had lower mOS and mPFS, with a higher risk of mortality. This warrants further investigation into potential use of Antibody Drug conjugates against Trop2 in BTC.

Microscopic and molecular analysis of the Urethral Plate: A Step Towards Improved Hypospadias Surgery Outcomes.

To investigate the microscopic and molecular structure of the urethral plate in hypospadias patients compared to normal urethral tissue, focusing on epithelial characteristics, cytokeratin expression, vascular patterns, and extracellular matrix components, including elastin fibers. We analyzed 8 full-thickness mid urethral plate samples from pediatric hypospadias patients using immunohistochemistry to evaluate epithelial structure, cytokeratin expression (PanCK, CK7, CK10, CK13, CK14), vascular patterns (vWF, CD31), and extracellular matrix components (elastin). Control tissues included fetal urethras and penile urethras from gender confirmation surgery. The hypospadias urethral plate exhibited a multi-layered epithelium, contrasting with the single-layered normal urethral epithelium. Cytokeratin expression differed significantly: CK7 was predominantly negative in hypospadias samples but strongly positive in controls; CK10 showed variable expression; CK13 and CK14 were consistently positive in hypospadias samples, with CK14 exhibiting strong expression in the basal layer. Vascular analysis revealed fewer subepithelial capillaries and smaller vascular spaces in hypospadias tissue. Elastin expression was positive in hypospadias samples, while fetal urethra showed lower expression and adult urethra displayed distinct elastin fiber bundles. Minimal smooth muscle tissue was observed in hypospadias and fetal samples compared to adult controls. The urethral plate in hypospadias patients demonstrates significant structural and molecular differences from normal urethral tissue, suggesting a developmental delay or arrest. These differences may contribute to surgical challenges and post-operative complications. Understanding these unique tissue characteristics could inform the development of novel surgical techniques or tissue engineering approaches to improve hypospadias repair outcomes.

Primary Visceral Ewing Sarcoma (Gastric and Bladder) with KIT Expression: A Report of Two Patients and a Differential Diagnostic Approach.

Extra-skeletal Ewing sarcoma is a rare tumor, most commonly occurring in the paravertebral region, extremities, retroperitoneum, and very rarely in visceral locations. We present two primary visceral Ewing sarcomas confirmed at the molecular level: one located in the stomach and the other in the bladder. Both tumors demonstrated strong and diffuse membranous CD99 expression, accompanied by variable KIT immunoreactivity. Additionally, we provide a review of the literature on primary gastric and bladder ES, including an analysis of differential diagnoses for gastric and bladder tumors exhibiting CD99 and KIT expression.

Stromal PDGFR-beta expression is a prognostic factor in high-grade serous ovarian cancer patients but is it also predictive for response to antiangiogenic treatment?

ObjectiveIn advanced ovarian cancer, the majority of patients receive anti-angiogenic treatment with bevacizumab. However, its use is often associated with severe side effects, and not all patients benefit from the therapy. Currently, there are no reliable biomarkers to predict response to treatment. Given their role as key regulators of angiogenesis, platelet-derived growth factor receptor-beta (PDGFR-beta) and vascular endothelial growth factor receptor-2 (VEGFR-2) are promising candidates for predictive biomarkers. This study evaluates their potential.MethodsPDGFR-beta and VEGFR-2 expression was evaluated using immunohistochemistry in a tissue microarray assay including 391 ovarian tissue samples. Correlation analyses with clinical and histopathological parameters were performed in a homogeneous cohort of 199 high grade serous ovarian cancer samples (HGSOC).ResultsIn HGSOC, strong stromal PDGFR-beta expression was associated with significantly shorter overall survival compared to weak/moderate expression. The impact of stromal PDGFR-beta expression on patient survival was however not restricted to the subgroup of patients receiving therapy with bevacizumab, and therefore cannot be considered as a predictive factor. For VEGFR-2, no or weak protein expression was found in the majority of the tumor samples. Survival analyses showed a more favorable prognosis with no or weak VEGFR-2 expression.ConclusionsHigh stromal expression levels of PDGFR-beta correlate with shorter overall survival in HGSOC. Thus, stromal PDGFR-beta might serve as a prognostic biomarker. No predictive effect in response to bevacizumab therapy could be attributed.

A case of de novo neuroendocrine prostate cancer presented with elevated level of serum CEA carrying BRCA2 mutation: case report and literature review

BackgroundDe novo neuroendocrine prostate cancer (NEPC) is a rare subtype of prostate cancer (PCa) and few markers are available for screening and monitoring. Potential circulating or fluid markers might facilitate early diagnosis thus improving prognosis of NEPC, especially for de novo NEPC.Case presentationA man of 71-year was presented with elevated level of serum carcinoembryonic antigen (CEA) (1296.5 ng/ml) and normal PSA (0.47ng/ml). Gastrointestinal endoscopy showed no signs of gastric or colorectal cancer. Fluorodeoxyglucose positron emission tomography-computed tomography (FDG PET-CT) and prostate-specific membrane antigen PET-CT (PSMA PET-CT) indicated prostate cancer with metastases including pelvic lymph nodes, bone as well as lung metastases. Biopsy of prostate revealed mixed carcinoma including small cell neuroendocrine carcinoma (SCNEC) and adenocarcinoma (Gleason score of 4 + 5). Immunohistochemistry (IHC) staining and next generation sequencing demonstrated a strong expression of chromogranin A (CgA), synaptophysin (SYN) and CEA, and a germline mutation in BRCA2, respectively. After a prostatic massage, an increased level of CEA (137 ng/ml vs 5 ng/ml) was detected in urine. Olaparib, a Poly ADP-ribose polymerase inhibitor (PARPi), combined with androgen deprivation therapy (ADT) were administrated. FDG PET-CT indicated tumor regression in both quantity and size three months later, and CEA levels of serum and urine decreased to 23 ng/ml and 2.4 ng/ml 4 months later, respectively.ConclusionThis is the first report of a de novo NEPC presented with an elevated level of CEA, in which CEA was also detected in urine specimen post a prostatic massage. After a combination treatment of ADT for 3 months, levels of CEA in both serum and urine decreased sharply when tumor regressed radiologically. CEA might be a marker of screening and monitoring of NEPC.

Multiplex immunohistochemistry to explore the tumor immune microenvironment in HCC patients with different GPC3 expression

ObjectivesGPC3 has been recognized as a promising target for immunotherapy in hepatocellular carcinoma (HCC). However, the GPC3-targeted immunotherapies have shown limited therapeutic efficacy. The use of anti-PD-1/PD-L1 monoclonal antibodies in HCC treatment is considerably constrained. Furthermore, there is still a notable lack of understanding concerning the immune landscape in HCC, especially regarding varied GPC3 expression levels. Therefore, thorough exploration of the intricate tumor immune microenvironment at different GPC3 expression levels is essential for guiding and improving HCC treatment strategies.MethodsSixty patients with HCC were enrolled in this study, receiving a first-line treatment that combined anti-angiogenesis targeted drugs and immunotherapy. Immunohistochemistry was used to assess the levels of GPC3 expression. Multiple immunohistochemical markers, such as CD8, PD-1, LAG3, TIGIT, TIM-3, CD103, Claudin18.2, PD-L1, CD4, Foxp3, CD68, CD163, GPC3, CD11C, CD14, CD66b, and HLA-DR, were used to characterize the immune microenvironment and spatial distribution of immune cells in HCC tumors with different levels of GPC3 expression. Cell expression levels and spatial distribution were determined by fluorescence staining and subsequent analysis of fluorescence intensity using the Panoramic Pathology Workstation (Pano ATLAS). This approach facilitated a detailed examination of cell characteristics and spatial information within the samples.ResultsBased on the result of GPC3 immunohistochemical analysis, patients with strong positive GPC3 expression were classified as high GPC3 expression, while the others were classified as low GPC3 expression. Patients in the low GPC3 expression group had longer overall survival (OS) than in the high group (P = 0.003, HR = 2.9240). Further exploration of the immune microenvironment based on different GPC3 expression levels revealed that in high GPC3 expression group, the proportions of CD8+ T cells(P = 0.0435), TIM-3+ T cells(P = 0.0447), CD103+CD8+ tissue-resident T cells(P = 0.0410), CD11C+CD14−DC cells(P = 0.0497), CD11C+HLA-DR−DC cells(P = 0.0309), CD11C+CD14−HLA-DR−DC cells(P = 0.0233), and CD11C+CD14−CD66b−DC cells(P = 0.0474) were all higher compared to low expression group. At spatial distances of 10 μm, 20 μm, and 30 μm, the levels of CD8+ T cells were higher in the high expression group compared to the low expression group (high vs. low: P = 0.0281, P = 0.0236, P = 0.0220).ConclusionsMultiple immunohistochemistry is a powerful technique for exploring the intricate immune microenvironment of hepatocellular carcinoma, enabling the precise identification of diverse cell subsets and their spatial distribution within the tumor microenvironment. This methodology provides valuable insights into the complex interactions and spatial organization of immune cells in the context of hepatocellular carcinoma progression. Low GPC3 expression in HCC patients indicates potential benefits from combined targeted and immunotherapy. Different levels of GPC3 expression levels can predict the effectiveness of targeted combination immunotherapy in HCC patients. Additionally, different GPC3 expression patterns in HCC patients correspond to unique tumor immune microenvironments, which have implications for guiding HCC treatment approaches.

IMMUNOHISTOCHEMICAL CHARACTERISTICS OF BASAL CELL CARCINOMA AT NATIONAL HOSPITAL OF DERMATOLOGY AND VENEREOLOGY, VIETNAM

Objective: To investigate the immunohistochemical characteristics of basal cell carcinoma at the National Hospital of Dermatology and Venereology. Methods: A descriptive, cross-sectional, retrospective, and prospective study was conducted on 312 patients diagnosed with basal cell carcinoma at the National Hospital of Dermatology and Venereology from January 2019 to September 2023. Histopathological and immunohistochemical analysis was performed using four immune markers: BerEP4, Bcl-2, p63, and EMA. Results: BerEP4 immunostaining was positive in all 312 cases of basal cell carcinoma, representing various histological subtypes. Bcl-2 immunostaining was also positive in all 312 cases of basal cell carcinoma, with notably stronger expression in peripheral tumor cells compared to the central region. P63 immunostaining yielded positive results in 310 cases of basal cell carcinoma, accounting for 99.4% of the cases studied. EMA immunostaining, on the other hand, was negative in 308 cases of basal cell carcinoma, equivalent to 87.2%. The four cases of EMA-positive basal cell carcinoma were all classified as the histopathological subtype of squamous basal cell carcinoma (within the invasive category). Conclusions: These results suggest that immunohistochemistry with the four immune markers, BerEP4, Bcl-2, p63, and EMA, can be used to support the definitive and differential diagnosis of basal cell carcinoma compared to other cutaneous carcinomas. Received 15 September 2023Revised 26 September 2023Accepted 03 November 2023

New Integrative Vectors Increase Agrobacterium rhizogenes Transformation and Help Characterise Roles for Soybean GmTML Gene Family Members.

Hairy-root transformation is widely used to generate transgenic plant roots for genetic functional characterisation studies. However, transformation efficiency can be limited, largely due to the use of binary vectors. Here, we report on the development of novel integrative vectors that significantly increase the transformation efficiency of hairy roots. This includes pHGUS7, for promoter::reporter visualisation studies, and pHOG13, for genetic insertion and overexpression studies. These vectors have been designed to simplify cloning workflows, enhance the selection of positively transformed Agrobacterium colonies, and increase the transformation efficiency and ease of selection of genetically modified hairy roots. To demonstrate the efficacy of the new vectors, Too Much Love (TML) encoding genes acting in the Autoregulation Of Nodulation (AON) pathway of soybeans were investigated. Both constructs provided significantly higher transformation rates than the binary vector control, often resulting in > 70% of the roots being transformed. This was achieved using either whole-plant seedlings or cotyledonary nodes in tissue culture. Overexpression of each individual TML encoding gene (GmTML1a, GmTML1b and GmTML2) using pHOG13 resulted in a significant reduction in nodule number, demonstrating the role of all three in inhibiting nodule organogenesis. Moreover, reporter-fusions with the promoter of each TML encoding gene using pHGUS7 revealed that each exhibits a unique pattern of expression in nodules, with GmTML1b displaying considerably stronger expression than GmTML1a or GmTML2. Taken together, these results demonstrate the utility and efficiency of the new pHOG13 and pHGUS7 integrative vectors in hairy-root transformation, and improve our understanding of the critical TML-encoding genes in soybean nodulation control.

Can PSMA PET detect intratumour heterogeneity in histological PSMA expression of primary prostate cancer? Analysis of [68Ga]Ga-PSMA-11 and [18F]PSMA-1007.

Prostate-specific membrane-antigen positron emission tomography (PSMA PET) is a promising candidate for non-invasive characterization of prostate cancer (PCa). This study evaluated whether PET with tracers [68Ga]Ga-PSMA-11 or [18F]PSMA-1007 is capable to depict intratumour heterogeneity of histological PSMA expression. Thirty-five patients with biopsy-proven primary PCa without evidence of metastatic disease nor prior interventions were prospectively enrolled. All patients underwent PSMA PET combined with computer tomography (CT) with either [68Ga]Ga-PSMA-11 (cohort I, 20 patients) or [18F]PSMA-1007 (cohort II, 15 patients) followed by radical prostatectomy. Specimens were scanned by ex-vivo CT and histologically prepared. On digitized whole-mount prostate sections, PCa areas with different morphologies were manually defined and H-Score of immunohistochemical PSMA expression was calculated with assistance by artificial intelligence (AI). PCa areas with similar H-Score were unified in segmentation on ex-vivo CT. After co-registration on PSMA PET-CT, Spearman's coefficients of PSMA expression to mean and maximum standardized uptake value (SUVmean and SUVmax) were calculated. Furthermore, the agreement of the co-registered tumour areas to gross tumour volume (GTV) in PSMA PET was analysed. Thirty-two patients were included in the final analysis. For histological PCa areas, immunohistochemical PSMA expression correlated significantly to SUVmean and SUVmax (p < 0.001, p = 0.001). An approximate linear correlation between H-Score and SUVmean / SUVmax was found for tumour areas larger than 400μm² in histology (p < 0.001). Tumour areas with strong PSMA expression showed a significantly larger overlap to GTV in PSMA PET after co-registration than tumour areas with very low PSMA expression (p < 0.01). No significant differences were found between the two tracer cohorts (p = 0.72). PSMA PET with both [68Ga]Ga-PSMA-11 or [18F]PSMA-1007 is able to detect changes in histological PSMA expression within PCa lesions allowing biologically targeted radiotherapy.

Methanol Leaf Extract of Voacanga Africana Protects Against Diethylnitrosamine and Carbon Tetrachloride-Induced Hepatotoxicity in Wistar Rats via Antioxidant and Anti-Inflammatory Activities

Background: Hepatotoxicity induced by chemical agents is a common and life-threatening disease that developed from acute or chronic exposure to environmental chemicals. Despite numerous approaches towards the treatment of liver toxicity, no safe and effective therapy exists. This study evaluated the antioxidative and anti-inflammatory effects of methanol leaf extract of Voacanga africana (VA) in diethylnitrosamine (DEN) and carbon tetrachloride (CCL4)-induced hepatotoxicity in rats. The animals were assigned into seven groups: Control, [DEN+CCL4], [DEN+CCL4] with VA (100, 200, 400 mg/kg), [DEN+CCL4] with Sorafenib (SFB), and SFB alone. Rats received DEN (200 mg/kg) once via i.p. and CCL4 (3 mL/kg) via subcutaneous route once in a week for six weeks. VA was administered orally from the second week until the 15th week. GC-FID analyses of VA revealed active components Voacinol, Voacangine, Amataine, and Ibogaine. [DEN+CCL4] caused a 26% increase in liver organo-somatic weight, which was reduced by 18%, 20%, and 9% with VA doses of 100, 200, and 400 mg/kg, respectively. [DEN+CCL4] also significantly increased serum gamma-glutamyl transferase, alanine, and aspartate aminotransferases by 55%, 25%, and 17%, and raised α-fetoprotein and total bilirubin levels by 2.0 and 2.5folds, respectively. Hepatic nitric oxide and myeloperoxidase activities were increased by 52% and 123%, respectively in the intoxicated rats. Additionally, malondialdehyde levels increased by 80% with decrease in glutathione peroxidase, glutathione-s-transferase, catalase and superoxide dismutase by 35%, 34%, 25% and 32%, respectively. Immunohistochemistry showed mild APC and strong Bcl-2 expression, while histology revealed severe hepatic necrosis. VA treatment mitigated oxidative stress, inflammation, and restored liver architecture.

An epiallele of a gene encoding a PfkB-type carbohydrate kinase affects plant architecture in maize.

Plant architecture greatly contributes to grain yield, but the epigenetic regulation of plant architecture remains elusive. Here, we identified the maize (Zea mays L.) mutant plant architecture 1 (par1), which shows reduced plant height, shorter and narrower leaves, and larger leaf angles than the wild type. Interestingly, par1 is an epiallele harboring a de novo CACTA insertion in the intron of the Par1 gene. High DNA methylation levels of the CACTA insertion are associated with strong Par1 expression and normal phenotypes. In contrast, low DNA methylation levels of this insertion are associated with weak Par1 expression and a mutant-like phenotype. The Par1 gene encodes a PfkB-type carbohydrate kinase that converts nucleosides to nucleoside monophosphates both in vitro and in vivo. Additional analyses showed that genes differentially expressed in the par1 mutant are enriched in jasmonic acid (JA) metabolism, and levels of JA metabolites were significantly higher in the mutant than in the wild type. Treatment with either nucleoside monophosphates or a synthetic inhibitor of JA biosynthesis reduced JA levels and partially rescued the mutant phenotype. In summary, we identified an epiallele of a gene encoding a PfkB-type carbohydrate kinase that might affect nucleoside monophosphate and JA levels, thus affecting maize growth.

Correlation Analysis Between Multi-Drug Resistance Phenotype and Virulence Factor Expression of Clinical Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA), as a common pathogen of nosocomial infections, has been experiencing an increasing rate of drug resistance with the widespread use and abuse of antimicrobial drugs. High-drug-resistance and high-virulence phenotypes are two distinctive features of the strong pathogenicity of multi-drug-resistant PA. Exploring the characterization of virulence factor expression and its relationship with the multi-drug resistance phenotype is essential to reduce the further development of resistance as well as a high standard of infection prevention and control. A total of 50 PA isolated from clinical practice were collected. The Kirby-Bauer test was used for drug-sensitive screening, and the results showed that 16 strains were resistant and 16 strains were sensitive. The drug resistance rate of multi-drug-resistant PA against cefepime, cefazolin, ampicillin, and imipenem was up to 100%. The multi-drug-resistant groups were superior in producing pyocyanin and forming biofilm to the sensitive groups. The distribution of isolates with different swarming motility capacities and elastase levels did not show pronounced differences among the multi-drug-resistant and sensitive groups. In addition, biofilm formation was moderately associated with imipenem resistance. Among the strains with strong virulence factor expression, the gene bands showed little difference, suggesting that the gene is highly homologous. The virulence factor matrix analysis showed that there were different degrees of correlation among the 4 virulence factors. The correlation between multidrug-resistant PA and virulence factor expression is complex. PA, which were good at producing pyocyain and forming biofilm, were highly resistant to cephalosporins, beta-lactams and carbepenems; hence, such drugs are not proper for anti-infective treatment in clinics.

Impact of HIF-1α, LOX and ITGA5 Synergistic Interaction in the Tumor Microenvironment on Colorectal Cancer Prognosis.

Background: As colorectal cancers are histopathologically and molecularly highly heterogeneous tumors, it is necessary to consider the tumor's microenvironment as well as its cellular characteristics in order to determine the biological behavior of the tumor. This study included 100 patients who underwent resection for colorectal cancer. We aimed to investigate the relationships between the expression status of the HIF-1α, LOX and ITGA5 proteins and clinicopathologic parameters. Methods: HIF-1α, LOX and ITGA5 antibodies were applied immunohistochemically to tissue microarrays prepared from tumor samples. Expression status in the tumor microenvironment were evaluated using a combined scoring system based on staining intensity and the percentage of positively stained cells. Nuclear HIF-1α expression in tumor cells was quantified, with >1% considered positive. The staining of HIF-1α, ITGA5 and LOX was analyzed in relation to prognostic and molecular features. Results: The staining of HIF-1α, ITGA5 and LOX in the tumor microenvironment demonstrated a positive correlation with one another and with HIF-1α and LOX expression in tumor cells. In patients with KRAS, NRAS or BRAF mutation and the moderate to strong expression of all three of these proteins in the tumor microenvironment, the number of metastatic lymph nodes was higher than in other patients. Stage IV patients with the moderate to strong expression of HIF-1α, ITGA5 or LOX in the microenvironment had lower progression-free survival than those with weak expression (p < 0.05). In addition, female gender; moderate to strong HIF-1α, LOX and ITGA5 stromal expression; and metastatic first line chemotherapy only were found to be independently associated with an increased risk of progression. Conclusions: These markers may be useful in predicting treatment responses and may also guide the development of alternative or combined treatments that specifically target molecules such as HIF and LOX. Our study should be supported by more comprehensive studies addressing the tumor stroma and its prognostic importance.

The aerial epidermis is a major site of quinolizidine alkaloid biosynthesis in narrow-leafed lupin.

Lupins are promising protein crops that accumulate toxic quinolizidine alkaloids (QAs) in the seeds, complicating their end-use. QAs are synthesized in green organs (leaves, stems, and pods) and a subset of them is transported to the seeds during fruit development. The exact sites of biosynthesis and accumulation remain unknown; however, mesophyll cells have been proposed as sources, and epidermal cells as sinks. We investigated the exact sites of QA biosynthesis and accumulation in biosynthetic organs of narrow-leafed lupin (Lupinus angustifolius) using mass spectrometry-based imaging (MSI), laser-capture microdissection coupled to RNA-Seq, and precursor feeding studies coupled to LC-MS and MSI. We found that the QAs that accumulate in seeds ('core' QAs) were evenly distributed across tissues; however, their esterified versions accumulated primarily in the epidermis. Surprisingly, RNA-Seq revealed strong biosynthetic gene expression in the epidermis, which was confirmed in leaves by quantitative real-time polymerase chain reaction. Finally, feeding studies using a stably labeled precursor showed that the lower leaf epidermis is highly biosynthetic. Our results indicate that the epidermis is a major site of QA biosynthesis in narrow-leafed lupin, challenging the current assumptions. Our work has direct implications for the elucidation of the QA biosynthesis pathway and the long-distance transport network from source to seed.