Screening of single nucleotide polymorphism in matrix metalloproteinase-2 (MMP2) and tetraspanin CD63 genes in Chlamydia trachomatis-infected tubal ectopic pregnancy patients.

OBJECTIVE: Tubal ectopic pregnancy (EP) is a leading cause of maternal morbidity and mortality. Studies have suggested that infection-induced inflammatory responses are major risk factors for EP. The aim of the present study was to find an association between MMP2 and CD63 gene variants and risk of EP during Chlamydia trachomatis infection in an Indian population. METHODS: Fallopian tube samples of 120 EP and 120 tubal ligation women were collected. C. trachomatis was detected by PCR. The genotyping of MMP2 (rs17859882 G/T, rs7201A/C) and CD63(rs2231464 C/T, rs376086542 A/G) gene variants was done by qualitative real-time PCR using allelic discrimination method (VIC- and FAM-labeled). RESULTS: The frequency of GG or GT genotype of MMP2 G/T polymorphism (rs17859882) was 66.6% in infected EP and 36.7% in uninfected EP and 22% in tubal ligation controls (P < 0.0001), while the frequency of AC or CC genotype of MMP2 A/C polymorphism (rs7201) was 66.6% in infected EP and 20.6% in uninfected EP and 13.5% in tubal ligation controls (P < 0.0001). The frequency of CT or TT genotype of CD63 C/T polymorphism (rs2231464) was 74% in infected EP and 21.8% in uninfected EP and 11.8% tubal ligation controls (P < 0.0001), while the frequency of AG or GG genotype of CD63 A/G polymorphism (rs376086542) was 48.1% in infected EP and 41.3% in uninfected EP and 18.6% tubal ligation controls (P < 0.0001). CONCLUSIONS: The present study revealed a strong association between the presence of gene variants MMP2 (rs17859882 G/T, rs7201A/C) and CD63 (rs2231464 C/T, rs376086542 A/G) and risk of tubal EP during C. trachomatis infection.

Clinical and histopathological spectrum of cranial small round cell tumors: An experience from a tertiary care center.

BACKGROUND: Small round cell tumors (SRCTs) are a group of malignant neoplasms with minimal or no differentiation, characterized by the presence of round cells with high nuclear-cytoplasmic ratio. Although SRCTs can occur in any part of the body, involvement of central nervous system (CNS) is uncommon. AIM: We aimed to study the clinicopathological spectrum of cranial SRCT diagnosed in our institute over a period of four years (2016-2019). MATERIAL AND METHODS: A retrospective review of medical records (2016-2019) with a morphological diagnosis of cranial SRCT was made. Both intra-axial and extra-axial tumors were included. A total of 60 cases were retrieved, and the clinical and histopathological features were studied. Special cytochemical staining and immunohistochemistry were performed, where needed. RESULTS: The mean age at presentation was 18.4 years (range, 1-60 years), with a male-to-female ratio of 2.5:1. The most common site was posterior fossa of brain (n = 28, 47%), followed by dorso-lumbar spine (n = 9, 15%). The most common type of tumor was medulloblastoma (n = 29, 48.3%), followed by Ewing sarcoma (ES)/peripheral primitive neuroectodermal tumor (pPNET) (n = 11, 18.3%), non-Hodgkin lymphoma (NHL) (n = 9, 15%), neuroblastoma (n = 3, 5%), and CNS embryonal tumor, NOS (n = 2, 3.3%). One case each of atypical teratoid rhabdoid tumor (ATRT), rhabdomyosarcoma, pineoblastoma, melanoma, rhabdomyosarcoma, and undifferentiated pleomorphic sarcoma was also documented. CONCLUSIONS: SRCTs have a variable age of presentation. Their incidence in CNS is low as compared to other organ systems. On light microscopy, the histopathology of these lesions is overlapping, posing a great diagnostic dilemma for the pathologist. The use of ancillary techniques like immunohistochemistry helps in arriving at the correct diagnosis. Treatment strategy and tumor prognosis also vary along the entire spectrum of SRCT, thus making exact characterization essential for proper management.

MicroRNA mediated regulation of oxidative stress and cytokines in Chlamydia trachomatis-infected recurrent spontaneous abortion: A case-control study.

PROBLEM: Increased oxidative stress (OS) and inflammatory responses are major underlying factors behind Chlamydia trachomatis-associated recurrent spontaneous abortion (RSA). miRNAs are known to regulate inflammation and OS and their dysregulation has been associated with compromised pregnancies. Therefore, aim of this study was to investigate the expression/correlation of OS biomarkers, cytokines and miRNAs in C. trachomatis-associated RSA. METHOD OF STUDY: Urine and non-heparinized blood samples were collected from RSA patients with history of >3 consecutive abortions (cases) and non-pregnant women with history of >2 successful deliveries (controls) attending Department of Obstetrics and Gynaecology, Safdarjung hospital, New Delhi. C. trachomatis detection was done in urine by PCR. miRNA expression was studied by microarray analysis and validated by real time-PCR. Evaluation of cytokines and antioxidant genes expression were done by real-time PCR. Level of OS biomarkers 8-hydroxy guanosine (8-OHdG) and 8-isporostane (8-IP) were measured by ELISA. RESULTS: Fifty circulating miRNAs were differentially expressed in infected patients compared with controls. Of these, four were overexpressed and 46 downregulated. Thirteen differentially expressed circulating miRNAs were selected to validate microarray results. miRs-8069, -3663-3p showed maximum upregulation/downregulation in infected versus control group. Expression of cytokines (IL-8, TNF-α, IFN-γ), antioxidant genes SOD2 and OS biomarkers (8-OHdG,8-IP) were increased while SOD1 was decreased in infected patients. miR-8069 showed significant positive correlation with cytokines, SOD2, 8-OHdG and 8-IP. miR-3663-3p showed significant positive correlation with SOD1. CONCLUSIONS: Overall results indicate circulating miRNAs are involved in pathogenesis of C. trachomatis-associated RSA and are potential modulators of cytokine signalling and OS in infected RSA.

Recent advances and future directions in etiopathogenesis and mechanisms of reactive oxygen species in cancer treatment.

A class of exceptionally bioactive molecules known as reactive oxygen species (ROS) have been widely studied in the context of cancer. They play a significant role in the etiopathogenesis for cancer. Implication of ROS in cancer biology is an evolving area, considering the recent advances; insights into their generation, role of genomic and epigenetic regulators for ROS, earlier thought to be a chemical process, with interrelations with cell death pathways- Apoptosis, ferroptosis, necroptosis and autophagy has been explored for newer targets that shift the balance of ROS towards cancer cell death. ROS are signal transducers that induce angiogenesis, invasion, cell migration, and proliferation at low to moderate concentrations and are considered normal by-products of a range of biological activities. Although ROS is known to exist in the oncology domain since time immemorial, its excessive quantities are known to damage organelles, membranes, lipids, proteins, and nucleic acids, resulting in cell death. In the last two decades, numerous studies have demonstrated immunotherapies and other anticancer treatments that modulate ROS levels have promising in vitro and in vivo effects. This review also explores recent targets for therapeutic interventions in cancer that are based on ROS generation or inhibition to disrupt the cell oxidative stress balance. Examples include-metabolic targets, targeted therapy with biomarkers, natural extracts and nutraceuticals and targets developed in the area of nano medicine. In this review, we present the molecular pathways which can be used to create therapy plans that target cancer by regulating ROS levels, particularly current developments and potential prospects for the effective implementation of ROS-mediated therapies in clinical settings. The recent advances in complex interaction with apoptosis especially ferroptosis and its role in epigenomics and modifications are a new paradigm, to just mechanical action of ROS, as highlighted in this review. Their inhibition by nutraceuticals and natural extracts has been a scientific challenging avenue that is explored. Also, the inhibition of generation of ROS by inhibitors, immune modulators and inhibitors of apoptosis and ferroptosis is explored in this review.

Primary cutaneous large B cell lymphoma masquerading as lupus vulgaris.

Primary cutaneous large B cell lymphoma, leg type is a rare and aggressive variant of cutaneous B cell lymphoma. It predominantly affects elderly women, with the lower limb being the most common site of presentation. The overall prognosis is poor, compared to other cutaneous B cell lymphomas. A 47-year-old man presented with a progressively enlarging nodule over the medial aspect of the left foot since 2 months. Clinical examination revealed a nodular plaque-like lesion with central ulceration that measured 7 × 7 cm, firm in consistency, and with ill-defined margins. The initial clinical diagnosis was lupus vulgaris. An incision biopsy was done, which on histopathology and immunohistochemistry revealed a rare diagnosis of primary cutaneous B cell lymphoma, leg type. The patient was started on chemotherapy; however, he succumbed to his illness about 1 year after the initial presentation. It is a rare type of cutaneous lymphoma, which may masquerade infectious disorders such as lupus vulgaris. A detailed histopathological and immunohistochemical analysis is essential for its correct diagnosis and management. Only a handful of cases of this rare condition are reported to date. This case has been reported in view of its rarity and unusual clinical presentation.

Immune regulation by activation markers at feto-maternal interface in infection-associated spontaneous preterm birth.

BACKGROUND: Spontaneous preterm birth (sPTB) is a global health concern. Studies reveal infections are majorly responsible for sPTB and immune activation markers play a role in regulation of maternal immune responses against pathogens during sPTB. AIM: To study the mRNA expression and correlation of activation markers (CD66a, ICAM1, ITGB1, TIM3, CD25, CD95) and associated cytokines (IL-1ß and IL-17)/prostaglandin receptors (EP2 and IP) in the placenta of Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum-infected sPTB women. METHODS: Placental samples were collected from 160 sPTB and 160 term birth women. PCR was used for the detection of C. trachomatis, M. hominis, U. urealyticum. The mRNA expression of activation markers, cytokines and prostaglandin receptors was evaluated by real-time qPCR. RESULTS: The fold-change expression of CD66a, ICAM1, TIM3, CD25 and CD95 was 2.89, 5.5, 4.95, 6.44 and 6.95-fold (p < 0.001), respectively; while for cytokines- IL-1ß and IL-17 was 5.41 and 4.71-fold (p < 0.001), respectively and for prostaglandin receptors- EP2 and IP was 5.5 and 5-fold (p < 0.001) upregulated, respectively in infected sPTB women. Significant positive correlation was obtained among ICAM-1 and IL-1ß/EP2/IL-17, TIM3 and IP/IL-17. Significant negative correlation was obtained between CD66a and EP2/IL-17, CD25 and IL-1ß/EP2, CD95 and IL-1ß/EP2 in infected sPTB women. CONCLUSIONS: CD66a, ICAM1 and TIM3 may play role in inflammation and have potential for the clinical beginning of preterm labour during infection while CD25 and CD95 are possibly involved in immunotolerance at feto-maternal interface during C. trachomatis, M. hominis and U. urealyticum infection.

Intronic variants of LGALS13 gene encoding placental protein (PP13) are linked with increased risk of infection-associated spontaneous preterm birth.

PROBLEM: Spontaneous preterm birth (sPTB) is a global health issue. Studies suggest infection and infection-based inflammatory responses are major risk factors for sPTB. Considering the important role of anti-inflammatory proteins in pregnancy, the study aimed to find the association between anti-inflammatory LGALS13 gene variants IVS2-22 A/G (rs2233706) and IVS3+72 T/A (rs2233708) and the risk of sPTB during Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum infection in Indian population. METHOD OF STUDY: Placental samples of 160 sPTB and 160 term women were collected. Pathogens were detected by PCR. The genotyping of LGALS13 gene variants IVS2-22 A/G (rs2233706) and IVS3+72 T/A (rs2233708) was done by qualitative real-time PCR using allelic discrimination method (VIC- and FAM-labeled). RESULTS: The frequency of AG or GG genotype of LGALS13 IVS2-22A/G polymorphism (rs2233706) was 75.5% in infected sPTB cases and 14.4% in uninfected sPTB cases and 7.3% in term birth controls (p < .0001), while the frequency of TA or AA genotype of LGALS13 IVS3+72T/A polymorphism (rs2233708) was 83.6% in infected sPTB cases and 18% in uninfected sPTB cases and 12.7% in term birth controls (p < .0001). The genotypic frequencies for both the variants of LGALS13 were statistically significant (p < .0001) in the infected sPTB versus uninfected sPTB and term birth controls. CONCLUSIONS: Study reveals strong association between the presence of immunological gene variants LGALS13 IVS2-22 A/G (rs2233706) and LGALS13 IVS3+72 T/A (rs2233708) and risk of sPTB during C. trachomatis, M. hominis and U. urealyticum infection.

Determination of chlamydial load in recurrent miscarriage in relation to some female sex hormones: a case-control study.

INTRODUCTION: Chlamydia trachomatis is a frequent cause of adverse pregnancy outcomes including recurrent spontaneous abortion (RSA). However, regulation of infectious load by host immune response is unknown. Female sex hormones are known to affect C. trachomatis infection. The aim of this study was to determine correlation of chlamydial infectious load and gestational age with concentration of progesterone/estrogen in RSA. METHODOLOGY: Urine and non-heparinized blood were collected from patients with history of > 3 spontaneous abortions (n = 150, cases) and those with history of > 2 successful deliveries (n = 150, controls) from Department of Obstetrics and Gynecology, Safdarjung hospital, New Delhi, India. C. trachomatis positivity was determined by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) and chlamydial load by real-time PCR. Estrogen and progesterone concentrations were estimated by ELISA and correlated with chlamydial load. RESULTS: 22/150 case patients were positive for C. trachomatis. 2,000-10,000 copies/mL of chlamydial load were detected in infected RSA patients. Progesterone concentration showed significant decrease while estrogen concentration was significantly increased in C. trachomatis-positive RSA patients versus controls. Chlamydial load and estrogen concentration were positively correlated while progesterone concentration was negatively correlated with chlamydial load. Gestational age was positively correlated with concentration of estrogen and negatively correlated with concentration of progesterone in infected-RSA women. CONCLUSIONS: Overall findings suggest that interplay between chlamydial copy load, hormonal changes such as increased expression of estrogen and decreased expression of progesterone, and advanced gestational age may be contributing as deciding factors for ensuing RSA during C. trachomatis-infection.

Microarray profiling of serum micro-RNAs in women with Chlamydia trachomatis-associated recurrent spontaneous abortion: A case control study.

Chlamydia trachomatis infection is a major cause of sexually transmitted diseases and adverse pregnancy outcomes such as recurrent spontaneous abortion (RSA). Micro-RNAs (miRNAs) have been known to be upregulated/downregulated in various reproductive-associated diseases such as ectopic pregnancy, preterm birth and pre-eclampsia. However, there is paucity of literature on miRNA profile in C. trachomatis-infected RSA. The present study aimed to determine the profile of serum miRNAs followed by their validation in C. trachomatis-infected RSA and to find target genes involved in biological pathways. Non-heparinized blood and first void urine were collected from 30 non-pregnant women with RSA and 30 non-pregnant women with ≥2 successful deliveries (controls) attending Department of Obstetrics and Gynaecology, Safdarjung hospital, New Delhi, India. C. trachomatis detection was done in urine by PCR and chlamydial load was determined by quantitative real-time PCR (qRT-PCR). miRNA expression was studied by microarray analysis followed by in vitro validation by qRT-PCR. Analysis of target genes/pathways was characterized in silico. 06 RSA patients were infected with C. trachomatis, while chlamydial load was found to be 6000-12,000 copies/ml. 110 circulating miRNAs were expressed differentially in infected RSA patients compared with controls. Of these, 16 were overexpressed and 94 downregulated. 06 differentially expressed circulating miRNAs were selected to validate the microarray results. qRT-PCR data confirmed the reliability of the microarray results: miR-4443, -5100, -7975 showed statistically significant upregulation, while miR-6808-5p, -3148, -6727-5p were significantly downregulated in infected RSA patients versus controls. Chlamydial load was positively correlated with these upregulated miRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that target genes of miRNAs in RSA are involved in AMPK, Akt and mTOR signaling pathways. Overall results indicate that differentially expressed circulating miRNAs are involved in pathogenesis of C. trachomatis-associated RSA and have the potential to be used as biomarkers for predicting RSA.

Chlamydia trachomatis infection regulates the expression of tetraspanins, activin-A, and inhibin-A in tubal ectopic pregnancy.

Mechanism of Chlamydia trachomatis causing tubal ectopic pregnancy (EP) is not well understood. Tetraspanins (tspans), activin-A, and inhibin-A might play a role in the development of pathological conditions leading to EP. The study aimed to elucidate the expression of tspans, activin-A, and inhibin-A with a role of associated cytokines in C. trachomatis-associated EP and analyze interacting partners of DEGs, with an expression of a few important interacting genes. Fallopian tissue and serum were collected from 100 EP (Group I) and 100 controls (Group II) from SJH, New Delhi, India. Detection of C. trachomatis was done by polymerase chain reaction (PCR) and IgG antibodies were detected by enzyme-linked immunosorbent assay. Expression of tspans, activin-A, inhibin-A, and cytokines was analyzed by real time (RT)-PCR and their interacting genes were assessed by STRING. Expression of few disease-associated interacting genes was studied by RT-PCR. A total of 29% (Group I) were C. trachomatis positive. Tspans and activin-A were significantly upregulated, while inhibin-A was significantly downregulated in Group Ia. ITGA1, TLR-2, ITGB2, and Smad-3 were a few interacting genes. Expression of ITGA1, TLR-2, and Smad-3 was significantly upregulated in C. trachomatis-positive EP. Results suggested dysregulated tspans, activin-A, and inhibin-A might play a role in C. trachomatis-infected tubal EP.

Diagnostic algorithm for pathological evaluation of gliomas in a resource-constrained setting.

Introduction: Gliomas are the most common primary intracranial tumors. The current World Health Organization (WHO) classification of central nervous system tumors recommends integrated histo-molecular diagnosis of gliomas. However, molecular testing is not available in even most of the advanced centers of our country, and histopathology aided with immunohistochemistry (IHC) is still widely used for diagnosis. Immunohistochemical markers such as iso-citrate dehydrogenase1 (IDH1) and Alpha Thalassemia/Mental Retardation Syndrome X-linked (ATRX) can be reliably used for the correct diagnosis, prognosis, and treatment of gliomas. Aim: We aimed to develop a diagnostic algorithm by integrating morphology, IDH1, and ATRX status of gliomas seen in our institute for 1 year. Settings and Design: Analytical cross-sectional study. Materials and Methods: This study included 60 histopathologically confirmed cases of astrocytic (n = 51) and oligodendroglial tumors (n = 9). Clinical, radiological, and histopathological features were noted and tumor grades assigned according to the WHO recommendations. IDH1 and ATRX mutation status was evaluated using IHC. The tumors were divided into three molecular groups on the basis of their IDH1 and ATRX mutation status: (1) Group 1: IDH1 negative and ATRX positive, (2) Group 2: IDH1 positive and ATRX positive, (3) Group 3: IDH1 positive and ATRX negative. Results: The mean age of presentation was 45.0 ± 15.8 years with a male-to-female ratio of 2:1. Seizures, headache, and hemiparesis were the most common modes of presentation. The tumor subtypes studied were glioblastoma (n = 32), anaplastic astrocytoma (n = 7), diffuse astrocytoma (n = 6), oligodendroglioma (n = 6), pilocytic astrocytoma (n = 6), and anaplastic oligodendroglioma (n = 3). IDH1 mutation was present in 26 cases including anaplastic astrocytoma (n = 7), diffuse astrocytoma (n = 6), oligodendroglioma (n = 5), secondary glioblastoma (n = 5), and anaplastic oligodendroglioma (n = 3). ATRX mutation, i. e., loss of ATRX was observed in 17 cases including diffuse astrocytoma (n = 5), anaplastic astocytoma (n = 5), anaplastic oligodendroglioma (n = 3), oligodendroglioma (n = 3), and secondary glioblastoma (n = 1). All six cases of pilocytic astrocytoma were negative for IDH1 and ATRX mutation. There were 34 patients in Group 1 (IDH1- and ATRX +), nine cases in Group 2 (IDH1 + and ATRX +), and 17 patients in Group 3 (IDH1 + and ATRX-). Conclusion: Diagnosis of gliomas should be based on a detailed clinicoradiological and histopathological assessment, followed by genotypic characterization. Evaluation for IDH1and ATRX status has both diagnostic and prognostic value as it helps in differentiating gliomas from reactive gliosis, primary glioblastoma from secondary glioblastoma, and pilocytic astrocytoma (WHO grade I) from diffuse astrocytoma (WHO grade II). Tumors with IDH1 mutations have a better outcome than those with wild-type IDH. IHC can serve as a useful surrogate to conventional molecular tests in resource-constrained settings. By devising an algorithm based on morphological and IHC features, we were able to stratify gliomas into three prognostic subgroups.

Immunomodulation of cytokine signalling at feto-maternal interface by microRNA-223 and -150-5p in infection-associated spontaneous preterm birth.

Spontaneous preterm birth (sPTB) is a global health concern and it is the most prevalent cause of infant mortality and morbidity with occurrence rate of 5 - 18% worldwide. Studies suggest infection and infection-driven activation of inflammatory responses are the potential risk factors for sPTB. MicroRNAs (miRNAs) are thought to control the expression of several immune genes, making them crucial components of the intricate immune regulatory network and the dysregulation of miRNAs in placenta has been associated to several pregnancy-related complications. However, studies on possible role of miRNAs in immunomodulation of cytokine signalling in infection-associated sPTB are scarce. Present study aimed to investigate expression/ correlation of a few circulating miRNAs (miR-223, -150-5p, -185-5p, -191-5p), miRNA target genes and associated cytokines in sPTB women found infected with Chlamydia trachomatis/ Mycoplasma hominis/ Ureaplasma urealyticum. Non-heparinized blood and placental sample were collected from 140 sPTB and 140 term women visiting Safdarjung hospital, New Delhi (India) for conducting PCR and RT-PCR for pathogen detection and miRNA/ target gene/ cytokine expression, respectively. Common target genes of differentially expressed miRNAs were obtained from databases. The correlation between select target genes/ cytokines and serum miRNAs was determined by Spearman's rank correlation. 43 sPTB were infected with either pathogen and a significant upregulation of serum miRNAs was observed. However, miR-223 and 150-5p showed maximum fold-change (4.78 and 5.58, respectively) in PTB versus control group. IL-6ST, TGF-ß R3 and MMP-14 were important target genes among 454 common targets, whereas, IL-6 and TGF-ß were associated cytokines. miR-223 and 150-5p showed significant negative correlation with IL-6ST/ IL-6/ MMP-14 and positive correlation with TGF-ß R3/ TGF-ß. A significant positive correlation was found between IL-6ST and IL-6, TGF-ß R3 and TGF-ß. However, miR-185-5p and 191-5p were not significantly correlated. Although post-transcriptional validation is required, yet on the basis of mRNA findings, the study concludes that miR-223 and 150-5p are apparently of clinical importance in regulation of inflammatory processes during infection-associated sPTB.

RNA Sequencing of Intraoperative Peritumoral Tissues Reveals Potential Pathways Involved in Glioma-Related Seizures.

Tumor-induced changes in the peritumoral neocortex play a crucial role in generation of seizures. This study aimed to investigate the molecular mechanisms potentially involved in peritumoral epilepsy in low-grade gliomas (LGGs). Intraoperative peritumoral brain tissues resected from LGG patients with seizures (pGRS) or without seizures (pGNS) were used for RNA sequencing (RNA-seq). Comparative transcriptomics was performed to identify differentially expressed genes (DEGs) in pGRS compared to pGNS using deseq2 and edgeR packages (R). Gene set enrichment analysis (GSEA) using Gene Ontology terms and Kyoto Encyclopedia of Genes & Genomes (KEGG) pathways was performed using the clusterProfiler package (R). The expression of key genes was validated at the transcript and protein levels in the peritumoral region using real-time PCR and immunohistochemistry, respectively. A total of 1073 DEGs were identified in pGRS compared to pGNS, of which 559 genes were upregulated and 514 genes were downregulated (log2 fold-change ≥ 2, padj < 0.001). The DEGs in pGRS were highly enriched in the "Glutamatergic Synapse" and "Spliceosome" pathways, with increased expression of GRIN2A (NR2A), GRIN2B (NR2B), GRIA1 (GLUR1), GRIA3 (GLUR3), GRM5, CACNA1C, CACNA1A, and ITPR2. Moreover, increased immunoreactivity was observed for NR2A, NR2B, and GLUR1 proteins in the peritumoral tissues of GRS. These findings suggest that altered glutamatergic signaling and perturbed Ca2+ homeostasis may be potential causes of peritumoral epilepsy in gliomas. This explorative study identifies important genes/pathways that merit further characterization for their potential involvement in glioma-related seizures.

Non-POU Domain-Containing Octomer-Binding (NONO) protein expression and stability promotes the tumorigenicity and activation of Akt/MAPK/ß-catenin pathways in human breast cancer cells.

Breast cancer is one of the most common cancers with a high mortality rate, underscoring the need to identify new therapeutic targets. Here we report that non-POU domain-containing octamer-binding (NONO) protein is overexpressed in breast cancer and validated the interaction of the WW domain of PIN1 with c-terminal threonine-proline (thr-pro) motifs of NONO. The interaction of NONO with PIN1 increases the stability of NONO by inhibiting its proteasomal degradation, and this identifies PIN1 as a positive regulator of NONO in promoting breast tumor development. Functionally, silencing of NONO inhibits the growth, survival, migration, invasion, epithelial to mesenchymal transition (EMT), and stemness of breast cancer cells in vitro. A human metastatic breast cancer cell xenograft was established in transparent zebrafish (Danio rerio) embryos to study the metastatic inability of NONO-silenced breast cancer cells in vivo. Mechanistically, NONO depletion promotes the expression of the PDL1 cell-surface protein in breast cancer cells. The identification of novel interactions of NONO with c-Jun and ß-catenin proteins and activation of the Akt/MAPK/ß-catenin signaling suggests that NONO is a novel regulator of Akt/MAPK/ß-catenin signaling pathways. Taken together, our results indicated an essential role of NONO in the tumorigenicity of breast cancer and could be a potential target for anti-cancerous drugs. Video Abstract.

Galectins are critical regulators of cytokine signalling at feto-maternal interface in infection-associated spontaneous preterm birth.

INTRODUCTION: Spontaneous preterm birth (sPTB) is a global health issue. Studies suggest infections are chiefly associated with sPTB and galectins (gals) play a role in regulation of innate and adaptive maternal immune response against pathogens during sPTB. The aim of this study was to describe the gene expression of gal -1, -3, -8, -9, -13 in relation to gene expression of cyclooxygenase-2 (COX-2) and the cytokines IL-8, IL-10, TNF-α, IFN-ϒ in the setting of sPTB and confirmed infection with Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum. METHODS: Placental samples were collected from 120 term control and 120 sPTB pregnancies. PCR was used to detect specific pathogens. Gene expression of galectins, cytokines, and COX-2 was performed using real time qPCR. RESULTS: Fold-change expression of gal -1, -3, -8, -9, -13 was 5.13, 6.11, 1.14, 5.23 and 7.16 (p<0.001), respectively; while IL-10, IL-8, TNF-α, IFN-ϒ and COX-2 was 6.29, 6.55, 6.35, 6.36 and 2.73-fold upregulated (p<0.05), respectively in infected sPTB. Gal-1 was positively correlated with IL-10 (r=0.49, p=0.003) while gal-3 showed significant correlation with IL-8 (r=0.42, p=0.0113), TNF-α (r=0.65, p=< 0.001) and COX-2 (r=0.72, p=0.001). However, gal-8 was not significantly correlated with any cytokine. Gal-9, -13 were negatively correlated with IFN-ϒ (r=-0.45, p=0.006) and IL-8 (r=-0.39, p=0.018). DISCUSSION: Gal-1, -9, -13 are anti-inflammatory and might play role in immune-tolerance while gal-3 is pro-inflammatory and possibly responsible for immunogenic response, having potential to anticipate the clinical beginning of preterm labour during infection.

Tissue proteome analysis for profiling proteins associated with lymph node metastasis in gallbladder cancer.

Lymph node (LN) metastasis is the earliest sign of metastatic spread and an established predictor of poor outcome in gallbladder cancer (GBC). Patients with LN positive GBC have a significantly worse survival (median survival- 7 months) than patients with LN negative disease (median survival- ~ 23 months) in spite of standard treatment which includes extended surgery followed by chemotherapy, radiotherapy and targeted therapy. This study aims at understanding the underlying molecular processes associated with LN metastasis in GBC. Here, we used iTRAQ-based quantitative proteomic analysis using tissue cohort comprising of primary tumor of LN negative GBC (n = 3), LN positive GBC (n = 4) and non-tumor controls (Gallstone disease, n = 4), to identify proteins associated with LN metastasis. A total of 58 differentially expressed proteins (DEPs) were found to be specifically associated with LN positive GBC based on the criteria of p value ≤ 0.05, fold change ≥ 2 and unique peptides ≥ 2. These include the cytoskeleton and associated proteins such as keratin, type II cytoskeletal 7 (KRT7), keratin type I cytoskeletal 19 (KRT19), vimentin (VIM), sorcin (SRI) and nuclear proteins such as nucleophosmin Isoform 1 (NPM1), heterogeneous nuclear ribonucleoproteins A2/B1 isoform X1 (HNRNPA2B1). Some of them are reported to be involved in promoting cell invasion and metastasis. Bioinformatic analysis of the deregulated proteins in LN positive GBC using STRING database identified 'neutrophil degranulation' and 'HIF1 activation' to be among the top deregulated pathways. Western blot and IHC analysis showed a significant overexpression of KRT7 and SRI in LN positive GBC in comparison to LN negative GBC. KRT7, SRI and other proteins may be further explored for their diagnostics and therapeutic applications in LN positive GBC.

Immunohistochemical Expression of Chemokine Receptor CXCR3 and its Ligand CXCL 11 in Basal Cell Carcinoma.

Introduction: Basal cell carcinoma (BCC) is the most common type of skin cancer worldwide. The pathogenesis of BCC involves interplay between various environmental and genetic factors. It is believed that chemokines play a significant role in the modulation of cancer growth by generating autocrine and paracrine signaling effects. The present study was conducted to elucidate the expression of chemokine, CXCL11, and its receptor CXCR3, and their interaction with tumor cells and peri-tumoral stroma in various subtypes of BCC. Aim and Objectives: The aim of this study was to evaluate the immunohistochemical expression of chemokine CXCL11 and its receptor CXCR3 in various subtypes of BCC. Materials and Methods: The study included 40 cases of histopathologically confirmed BCC. Clinical and histopathological features of various tumor subtypes were noted. Immunohistochemistry was performed using antibodies against CXCL11 and CXCR3, and these were assigned scores 0, 1, and 2 on the basis of immunohistochemical expression. Results: The median age of study participants was 65.0 ± 12.2 years with a male-to-female ratio of 1.5:1. The most common site was face, followed by neck, scalp, and back. The tumor subtypes included in the study were nodular (n = 20), pigmented (n = 8), infiltrating (n = 5), superficial (n = 4), and adenoid (n = 3). On immunohistochemistry, CXCR3 expression was seen in 34 (85%) cases with stromal inflammatory cells immunopositivity in 29 (72.5%) cases and tumor cells immunopositivity in 5 (12.5%) cases. CXCL11 expression was seen in 36 (90%) cases with weak expression in stroma and tumor in 18 cases and strong expression in the rest 18 cases. In individual subtypes, higher immunopositivity for CXCR3 and CXCL11 in tumor cells and peri-tumoral stroma was seen for nodular, infiltrating, and pigmented subtypes, compared to adenoid and superficial subtypes. Conclusion: Our study shows the enhanced expression of chemokine CXCL11 and its receptor CXCR3 in tumor cells and peri-tumoral stroma of BCC. This expression is greater in tumor cells of aggressive subtypes, i.e. nodular, infiltrating, and pigmented types. This suggests that receptor ligand pathway involving CXCR3 and CXCL11 plays a key role in pathogenesis of BCC, and blocking this pathway may result in inhibition of tumor growth. Thus, these chemokines may serve as future potential targets in developing novel therapeutic regimens against BCC.

Community-Minimal Invasive Tissue Sampling (cMITS) using a modified ambulance for ascertaining the cause of death: A novel approach piloted in a remote inaccessible rural area in India.

BACKGROUND: Melghat in India is a hilly, forested, difficult to access, impoverished rural area in northeast part of Maharashtra (Central India) with difficult healthcare access. Melghat has very high Mortality rates, because of grossly inadequate medical facilities. (1) Home deaths contribute to 67% of deaths,(2) which are difficult to track and where cause of death is mostly unknown. METHODS: A feasibility study was carried out in 93 rural villages and 5 hospitals to assess feasibility of tracking real-time community mortality and to ascertain cause of death in 0-60 months and 16-60 years age group using Minimal Invasive Tissue Sampling (MITS) in purpose-modified ambulance. We used the network of village health workers (VHW)s, to establish real-time community mortality tracking. Upon receipt of reports of home death, we performed MITS within 4 h of death in the vicinity of the village. RESULTS: We conducted 16 MITS. Nine, in MITS ambulance in community and seven at MAHAN hospital. The acceptance rate of MITS was 59.26%. Standard operating procedure (SOP) of conducting community MITS in an ambulance, is established. Major challenges were, Covid19 lockdown, reluctance of tribal parents for consent for MITS due to illiteracy, superstitions and fear of organ removal. Ambulance was an easy to reach transport means in remote area, provided a well-designed and discrete facility to perform MITS in community, winning the confidence of bereaved family. This has reduced time interval between time of death and performing MITS. CONCLUSIONS: MITS in purpose-modified Ambulance can be used worldwide for community MITS especially in areas which are remote and lack healthcare access. This solution needs to be assessed in different cultural settings to document culture specific issues.

Mechanistic insights into immunopathogenesis of murine cerebral malaria: Cues from "young" C57BL/6J and BALB/c mice.

Cerebral malaria (CM), a major cause of mortality in children <5 years, presents disparity in pathophysiological features and poor prognosis compared to adults. Adult C57BL/6J mice infected with Plasmodium berghei ANKA (PbA) are widely used to understand CM pathogenesis compared to relatively less prone BALB/c mice; however, age and immune status of the host also influence disease sequelae and cerebral manifestations. Murine models of CM known so far do not project complete disease spectrum of pediatric CM. The present study was designed to dissect and differentiate CM immunopathogenesis in "young" BALB/c and C57BL/6J mice infected with PbA, in search of a competent mouse model mimicking pediatric CM. Multipronged approach including the analysis of blood-brain barrier (BBB) permeability and parasite infiltration, histopathology, nitric oxide levels, and pro/anti-inflammatory (TNF-α, IFN-γ, IL-4, and IL-10) cytokine expression were compared in the cortices of both young BALB/c and C57BL/6J mice. The results illustrate severe course of infection and typical CM like histopathological alterations including monocytic plugging in PbA-infected "young" BALB/c compared to C57BL/6J mice. The decreased expression of tight junction proteins (ZO-1 and Claudin-3) and Evan's blue extravasation was also more evident in BALB/c mice indicating a more permeable BBB. The increased cortical expression of TNF-α, IFN-γ, IL-4, IL-10, iNOS, eNOS, nNOS, and associated activation of brain resident cells in cortices of BALB/c with progressive parasitaemia depicts the cumulative involvement of host immune responses and parasite accumulation in progression of CM. Thus, the incongruity of cytokine balance resulted in worsening of disease manifestation in "young" BALB/c similar to pediatric CM.