Differential analysis of histopathological and genetic markers of cancer aggressiveness, and survival difference in EBV-positive and EBV-negative prostate carcinoma.

Several studies have shown an association between prostate carcinoma (PCa) and Epstein-Barr virus (EBV); however, none of the studies so far have identified the histopathological and genetic markers of cancer aggressiveness associated with EBV in PCa tissues. In this study, we used previously characterized EBV-PCR-positive (n = 39) and EBV-negative (n = 60) PCa tissues to perform an IHC-based assessment of key histopathological and molecular markers of PCa aggressiveness (EMT markers, AR expression, perineural invasion, and lymphocytic infiltration characterization). Additionally, we investigated the differential expression of key oncogenes, EMT-associated genes, and PCa-specific oncomiRs, in EBV-positive and -negative tissues, using the qPCR array. Finally, survival benefit analysis was also performed in EBV-positive and EBV-negative PCa patients. The EBV-positive PCa exhibited a higher percentage (80%) of perineural invasion (PNI) compared to EBV-negative PCa (67.3%) samples. Similarly, a higher lymphocytic infiltration was observed in EBV-LMP1-positive PCa samples. The subset characterization of T and B cell lymphocytic infiltration showed a trend of higher intratumoral and tumor stromal lymphocytic infiltration in EBV-negative tissues compared with EBV-positive tissues. The logistic regression analysis showed that EBV-positive status was associated with decreased odds (OR = 0.07; p-value < 0.019) of CD3 intratumoral lymphocytic infiltration in PCa tissues. The analysis of IHC-based expression patterns of EMT markers showed comparable expression of all EMT markers, except vimentin, which showed higher expression in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Furthermore, gene expression analysis showed a statistically significant difference (p < 0.05) in the expression of CDH1, AR, CHEK-2, CDKN-1B, and CDC-20 and oncomiRs miR-126, miR-152-3p, miR-452, miR-145-3p, miR-196a, miR-183-3p, and miR-146b in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Overall, the survival proportion was comparable in both groups. The presence of EBV in the PCa tissues results in an increased expression of certain oncogenes, oncomiRs, and EMT marker (vimentin) and a decrease in CD3 ITL, which may be associated with the aggressive forms of PCa.

Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma.

High-grade gliomas are extremely fatal tumors, marked by severe hypoxia and therapeutic resistance. Autophagy is a cellular degradative process that can be activated by hypoxia, ultimately resulting in tumor advancement and chemo-resistance. Our study aimed to examine the link between autophagy markers' expression in low-grade gliomas (LGGs) and high-grade gliomas (HGGs). In 39 glioma cases, we assessed the protein expression of autophagy markers LC3B, SQSTM1/p62, and DRAM by immunohistochemistry (IHC) and the mRNA expression of the autophagy genes PTEN, PI3K, AKT, mTOR, ULK1, ULK2, UVRAG, Beclin 1, and VPS34 using RT-qPCR. LC3B, SQSTM1/p62, and DRAM expression were positive in 64.1%, 51.3%, and 28.2% of glioma cases, respectively. The expression of LC3B and SQSTM1/p62 was notably higher in HGGs compared to LGGs. VPS34 exhibited a significant differential expression, displaying increased fold change in HGGs compared to LGGs. Additionally, it exhibited robust positive associations with Beclin1 (rs = 0.768), UVRAG (rs = 0.802), and ULK2 (rs = 0.786) in HGGs. This underscores a potential association between autophagy and the progression of gliomas. We provide preliminary data for the functional analysis of autophagy using a cell culture model and to identify potential targets for therapeutic interventions.

Association of Kaiso and partner proteins in oral squamous cell carcinoma.

Objectives: 1. Identification of protein expression and subcellular localization of E-cadherin (E-cad), p120 catenin (P120ctn), and Kaiso in oral cancer (OC). 2. To study the protein expression of cyclin D1 and c-Myc (Kaiso targets) and determine their relationship with the expression and localization of Kaiso. Methods: Histological grading was performed in accordance with Broder's criteria. Expression and localization data for E-cad, p120ctn, Kaiso, cyclin D1, and c-Myc were acquired using immunohistochemistry. Data were analyzed using SPSS version 21. The chi-square test was used to measure the statistical significance of associations, with p < 0.05 as statistically significant. Results: Of 47 OC cases, 36% showed low E-cad expression and 34% showed low p120ctn. Low Kaiso expression was recognized in 78% of tumor specimens. Aberrant cytoplasmic localization of p120ctn was seen in 80.8% cases. Cytoplasmic Kaiso localization was appreciated in 87% of tumor tissues, whereas 29.7% lacked any nuclear Kaiso. Kaiso expression was significantly associated with the expression of cyclin D1 but not with c-Myc. Conclusion: The present study identified a change in the localization of Kaiso in OC. The significance of this in relation to OC and tumor prognosis needs to be investigated with further studies using larger sample sizes and more sensitive molecular tools.

Analysis of differential gene expression of pro-inflammatory cytokines in the nasopharyngeal milieu of mild & severe COVID-19 cases.

INTRODUCTION: A subset of individuals with COVID-19 can suffer from a severe form of the disease requiring breathing support for respiratory failure and even death due to disease complications. COVID-19 disease severity can be attributed to numerous factors, where several studies have associated changes in the expression of serum pro-inflammatory cytokines with disease severity. However, very few studies have associated the changes in expression of pro-inflammatory changes in the nasopharyngeal milieu with disease severity. Therefore, in the current study, we performed differential gene expression analysis of various pro-inflammatory cytokines in the nasopharyngeal milieu of mild & severe COVID-19 cases. MATERIAL AND METHOD: For this retrospective, cross-sectional study, a total of 118 nasopharyngeal swab samples, previously collected from mild and severe (based on the WHO criteria) COVID-19 patients were used. A real-time qPCR was performed to determine the viral loads and also evaluate the mRNA expression of eight cytokines (IL-1, IL-2, IL-4, IL-6, IL-10, IFN-γ, TGF-ß1, and TNF-α). Subsequently, an unpaired T-test was applied to compare the statistical difference in mean expression of viral loads and each cytokine between the mild and severe groups, while the Pearson correlation test was applied to establish a correlation between disease severity, viral load, and cytokines expression. Similarly, a multivariable logistic regression analysis was performed to assess the relationship between different variables from the data and disease severity. RESULTS: Out of 118 samples, 71 were mild, while 47 were severe. The mean viral load between the mild and severe groups was comparable (mild group: 27.07± 5.22; severe group: 26.37 ±7.89). The mRNA expression of cytokines IL-2, IL-6, IFN- γ, and TNF-α was significantly different in the two groups (p<0.05), where the Log2 normalized expression of IL-2, IL-6, IFN- γ, and TNF-α was found to be 2.2-, 16-, 2.3-, and 1.73-fold less in the severe group as compared to the mild group. Furthermore, we also observed a significant positive correlation between all the cytokines in the severe group. The multivariate analysis showed a significant relationship between age, IL-6, and disease severity. CONCLUSION: This decreased expression of certain cytokines (IL-2, IL-6, TNF-α, and IFN-γ) in the nasopharyngeal milieu may be considered early biomarkers for disease severity in COVID-19 patients.

Association between the baseline gene expression profile in periapical granuloma and periapical wound healing after surgical endodontic treatment.

In this study, we have investigated the association between the baseline gene expression profile in periapical granuloma and periapical wound healing after surgical endodontic treatment. Twenty-seven patients aged between 15 and 57 years underwent periapical surgery. The retrieved periapical tissue sample was used for mRNA expression analysis of COL1A1, VTN, ITGA5, IL-4, TNF, ANGPT, VEGFA, and CTGF. All patients were recalled after 6 and 12 months for periapical healing evaluation. Healing was then correlated with baseline gene expression. Healing was observed in 15 patients at the end of 6 months, which increased to 21 patients after 12 months. Six patients showed no healing even after 12 months. Analysis of baseline expression levels of the tested genes with healing status showed the mean relative expression of VTN, VEGFA, ANGPT, TNF, and CTGF to be significantly different (p < 0.05) between the healing group (6 and 12 months) (72.99%) and the non-healing (94.42%) group. Periapical Index scores 3-5 exhibited a positive correlation with ITGA-5 expression. Overexpression of ANGPT and a strong positive correlation between ITGA5 and PAI scores in the non-healing group of patients may suggest these genes to be a potential prognostic biomarker for periapical wound non-healing after surgical endodontic treatment.

Detection and characterization of latency stage of EBV and histopathological analysis of prostatic adenocarcinoma tissues.

The pathophysiology of prostate cancer involves both genetic and acquired factors, including pathogens, such as viruses. A limited number of studies have shown the presence of Epstein-Barr virus (EBV) in prostate cancer tissues. However, there is a dearth of data exploring EBV latency profile in prostate cancer, and the relationship of EBV with histopathological features of prostate cancer. In this study, prostate cancer and benign prostatic hyperplasia (BPH) samples were screened for the presence of EBV, followed by the characterization of the EBV latency profile and analysis of histopathological parameters in EBV-positive and EBV-negative groups. A conventional PCR strategy was employed using virus-specific primers to screen EBV in 99 formalin-fixed paraffin-embedded (FFPE) prostate cancer and 33 BPH samples received for histopathological analysis during the years 2019-2020. Subsequently, cDNA samples were used in a qPCR array to analyze the expression of EBV latency-associated genes to map the latency profile EBV maintains in the samples. Finally, statistical analyses were performed to determine the correlation between EBV and several histopathological features of the samples. EBV was detected in 39% of prostate cancer and 24% of BPH samples. The histopathological analysis of prostate cancer samples identified all samples as prostatic adenocarcinoma of acinar type, while statistical analyses revealed EBV-positive samples to exhibit significantly higher (p < 0.05) Gleason major and total Gleason scores as compared to EBV-negative samples. In the EBV-positive samples, variable expression patterns of latency-associated genes were observed, where most of the samples exhibited EBV latency II/III-like profiles in prostate cancer, while latency-II-like profiles in BPH samples. This study suggests a high prevalence of EBV in prostate samples, where EBV exhibited latency II/III-like profiles. Furthermore, EBV-positive samples exhibited a higher Gleason score suggesting a possible link between EBV and the onset/progression of prostate cancers. However, future functional studies are required to understand the role of the EBV gene expression profile in the onset/progression of prostate cancer.

Baseline MMP expression in periapical granuloma and its relationship with periapical wound healing after surgical endodontic treatment.

BACKGROUND: Matrix metalloproteinases (MMPs) catalyzes the degradation of the extracellular matrix components and have a major role in many physiological processes including wound healing. In the current study, we examined the correlation of baseline MMPs 1, 2, 7, and 9 expressions with periapical wound healing after surgical endodontic treatment. METHODS: 27 patients aged between 15 and 57 years presenting with chronic apical periodontitis or chronic apical abscess of an anterior tooth with previously attempted or failed root canal treatment were included in this study. During surgical endodontic treatment, tissue from the periapical lesion sample was collected and used for gross histopathological analysis as well as mRNA expression analysis of MMPs 1, 2, 7, and 9. Patients were recalled for follow-up after 6  months to evaluate the healing status both clinically and radiographically and healing was correlated with baseline MMP expression. RESULTS: Out of 27 patients, healing was observed in 15 patients at the end of 6 months, and in 21 patients after 12 months.. Six patients showed no healing even after 12 months. Analysis of baseline MMP 1, 2, 7, and 9 expression levels with healing status showed the mean relative expression of MMP2 and MMP9 to be considerably increased in the non-healing group as compared to the healing group. CONCLUSION: Overexpression of MMP2 and MMP9 may be considered as a potential prognostic biomarker for periapical wound healing after surgical endodontic treatment. However, further studies are desirable to establish its precise relationship with periapical wound healing.

Risk factors of Oral cancer- A hospital based case control study.

BACKGROUND: Oral cancer is a highly prevalent malignancy in Pakistan. Among various risk factors associated with this neoplasm, habits such as smoked and smokeless tobacco usage, betel quid, and betel nut consumption are the major culprits in our society. In the present study, we aimed to ascertain prevalent risk factors for OC in our population and to compare our findings with healthy controls to establish their significance. MATERIAL AND METHODS: A hospital-based case control study was conducted at Dow University of Health Sciences, Pakistan from January 2015 - September 2016. Information pertaining to unhealthy oral habits was obtained from 62 oral cancer patients (cases) and 62 healthy controls on specifically designed proforma by the principal investigator. RESULTS: Smokeless tobacco is strong, independent risk factor for oral cancer development in our study population. Buccal mucosa is the predominantly affected site (71%) which corresponds with high smokeless tobacco use. All studied habits increase risk of oral cancer as demonstrated by high odds ratio. CONCLUSIONS: Despite advancement in our knowledge and understanding of carcinogenic potential of these hazardous substances not enough efforts have been put forth to effectively control their widespread sale and consumption, particularly by the youth in our society. Key words:Betel Quid, Gutka, Oral Cancer Risk, Smokeless Tobacco.

The Epstein-Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins.

Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate-dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation.

The Gly-Ala repeat modulates the interaction of Epstein-Barr virus nuclear antigen-1 with cellular chromatin.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) plays a pivotal role in EBV infection by anchoring the viral episome to cellular DNA, which regulates replication and partitioning in dividing cells. Here, we have used fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) techniques to study the interaction of EBNA1 with cellular chromatin in interphase and mitosis. This analysis revealed that while EBNA1 is highly mobile in both conditions, mobility is significantly reduced in mitosis when an immobile fraction is also detected. The N-terminal chromatin-targeting module of EBNA1 includes two Gly-Arg rich domains (GR1 and GR2) separated by a Gly-Ala repeat (GAr) of variable length. Using a set of deletion mutants and GFP-fusion reporters, we found that the GR domains cooperatively determine the mobility of EBNA1, whereas mobility is increased by the interposed GAr in a length-dependent manner. These findings highlight a previously unrecognized property of the interaction of EBNA1 with cellular chromatin that may fine-tune its function in the maintenance of viral latency.

High avidity binding to DNA protects ubiquitylated substrates from proteasomal degradation.

Protein domains that act as degradation and stabilization signals regulate the rate of turnover of proteasomal substrates. Here we report that the bipartite Gly-Arg repeat of the Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 acts as a stabilization signal that inhibits proteasomal degradation in the nucleus by promoting binding to cellular DNA. Protection can be transferred by grafting the domain to unrelated proteasomal substrates and does not involve changes of ubiquitylation. Protection is also afforded by other protein domains that, similar to the Gly-Arg repeat, mediate high avidity binding to DNA, as exemplified by resistance to detergent extraction. Our findings identify high avidity binding to DNA as a portable inhibitory signal that counteracts proteasomal degradation.