Sustained pigmentation causes DNA damage and invokes translesion polymerase Polκ for repair in melanocytes.

Melanin protects skin cells from ultraviolet radiation-induced DNA damage. However, intermediates of eumelanin are highly reactive quinones that are potentially genotoxic. In this study, we systematically investigate the effect of sustained elevation of melanogenesis and map the consequent cellular repair response of melanocytes. Pigmentation increases γH2AX foci, DNA abasic sites, causes replication stress and invokes translesion polymerase Polκ in primary human melanocytes, as well as mouse melanoma cells. Confirming the causal link, CRISPR-based genetic ablation of tyrosinase results in depigmented cells with low Polκ levels. During pigmentation, Polκ activates replication stress response and keeps a check on uncontrolled proliferation of cells harboring melanin-damaged DNA. The mutational landscape observed in human melanoma could in part explain the error-prone bypass of DNA lesions by Polκ, whose absence would lead to genome instability. Thereby, translesion polymerase Polκ is a critical response of pigmenting melanocytes to combat melanin-induced DNA alterations. Our study illuminates the dark side of melanin and identifies (eu)melanogenesis as a key missing link between tanning response and mutagenesis, mediated via the necessary evil translesion polymerase, Polκ.

Induction of epithelial-mesenchymal transition in thyroid follicular cells is associated with cell adhesion alterations and low-dose hyper-radiosensitivity.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is associated with altered cellular adhesion. We previously demonstrated that cellular adhesion influences Low-dose Hyper-Radiosensitivity (HRS) in a variety of tumor cells. However, the relationship of low-dose HRS with the phenotypic plasticity incurred by EMT during the neoplastic transformation remains to be elucidated. OBJECTIVE: To investigate whether acquisition of EMT phenotype during progressive neoplastic transformation may affect low-dose radiation sensitivity. METHODS: Primary thyroid cells obtained from a human cystic thyroid nodule were first subjected to nutritional stress. This yielded immortalized INM-Thy1 cell strain, which was further treated with either multiple γ-radiation fractions (1.5 Gy each) or repetitive cycles of 3-methylcholanthrene and phorbol-12-myristate-13-acetate, yielding two progressive transformants, viz., INM-Thy1R and INM-Thy1C. Morphological alterations, chromosomal double-minutes, cell adhesion proteins, anchorage dependency, tumorigenicity in nude mice and cellular radiosensitivity were studied in these strains. RESULTS: Both transformants (INM-Thy1R, INM-Thy1C) displayed progressive tumorigenic features, viz., soft agar colony growth and solid tumor growth in nude mice, coupled with features of epithelial-mesenchymal transition and activated Wnt pathway. Incidentally, the chemical-induced transformant (INM-Thy1C) displayed a prominent HRS (αs/αr = 29.35) which remained unaffected at high cell density. However, the parental (INM-Thy1) cell line as well as radiation-induced transformant (INM-Thy1R) failed to show this hypersensitivity. CONCLUSION: The study shows that induction of EMT in thyroid follicular cells may accompany increased susceptibility to low-dose ionizing radiation, which was attenuated by adaptive resistance acquired during radiation-induced transformation.

A simplified protocol for gene expression-based biological dosimetry using peripheral whole blood.

PURPOSE: Assessing alterations in the expression of radiation-responsive genes in peripheral blood cells is considered a promising approach for high-throughput radiation biodosimetry. However, optimization of conditions for storage and transport of blood samples would be critical for obtaining reliable results. Recent studies involved the incubation of isolated peripheral blood mononuclear cells (in cell culture medium) and/or use of RNA stabilizing agents for sample storage, immediately after the ex vivo irradiation of whole blood. We used a simpler protocol by incubating undiluted peripheral whole blood without any RNA stabilizing agent, and studied the impact of storage temperature and incubation time on the expression levels of 19 known radiation responsive genes. MATERIALS & METHODS: Peripheral whole blood was γ-irradiated ex vivo at room temperature at low (0.5 Gy), moderate (1 Gy, 2 Gy) and high (4 Gy) doses and immediately incubated at two different temperatures at 4 °C or 37 °C for 2h, 4h and 24 h. Using qRT-PCR, mRNA expression levels of CDKN1A, DDB2, GADD45A, FDXR, BAX, BBC3, MYC, PCNA, XPC, ZMAT3, AEN, TRIAP1, CCNG1, RPS27L, CD70, EI24, C12orf5, TNFRSF10B, ASCC3 were analyzed at respective time-points and compared with the sham-irradiated controls. RESULTS: Transcriptional responses of all 19 genes did not alter significantly upon incubation of whole blood samples at 4 °C, as compared to untreated controls. However, incubation at 37 °C for 24 h resulted in significant radiation-induced overexpression in 14 out of the 19 genes analyzed (except CDKN1A, BBC3, MYC, CD 70 and EI24). Detailed patterns during incubation at 37 °C revealed time-dependent up-regulation of these genes, with DDB2 and FDXR showing significant up-regulation both at 4 and 24 h with the highest fold-change observed. CONCLUSION: Overall, the undiluted whole blood incubated at 37 °C for 24 h was found to elicit most optimal transcriptional response in the genes studied, with most profound overexpression of DDB2 and FDXR. We propose that sample storage/transport/post-transit incubation at the physiological temperature for up to 24 h may enhance the sensitivity of gene expression based biodosimetry and facilitate its usage for triage application.

Alterations in the expression pattern of RBC membrane associated proteins (RMAPs) in whole body γ-irradiated Sprague Dawley rats.

PURPOSE: Peripheral blood serum/plasma proteins are frequently studied for their potential use as radiation exposure biomarkers. Here we report RBC membrane associated proteins (RMAPs), which show alterations in expression level following whole-body γ-irradiation of rats at sub-lethal/lethal doses. MATERIALS AND METHODS: RBCs from peripheral blood of Sprague Dawley rats were segregated using the Ficoll-Hypaque method, and membrane fractions were hypotonically isolated at various time points (6 h, 24 h, 48 h) after γ-irradiation at 2 Gy, 5 Gy, and 7.5 Gy doses. Following purification of proteins from these fractions, two-dimensional electrophoresis (2-DE) was carried out. Treatment induced differentially expressed protein spots (≥2 fold increase/decrease) were picked up, trypsinized, and identified using LC-MS/MS analysis. Western immunoblots using protein specific antibodies were used to confirm the results. Gene ontology and interactions of these proteins were also studied. RESULTS: From a number of differentially expressed radiation-responsive 2-DE protein spots detected, eight were identified unequivocally using LC-MS/MS. Out of these, actin, cytoplasmic 1 (ACTB) showed detectable yet insignificant variation (<50%) in expression. In contrast, peroxiredoxin-2 (PRDX2) and 26S proteasome regulatory subunit RPN11 (PSMD14) were the two most prominently over-expressed proteins. Five more proteins, namely tropomyosin alpha-3 chain (TPM3), exosome component 6 (EXOSC6), isoform 4 of tropomyosin alpha-1 chain (TPM1), serum albumin (ALB), and the 55 kDa erythrocyte membrane protein (P55) showed distinct alteration in their expression at different time-points and doses. ALB, EXOSC6, and PSMD14 were the most responsive at 2 Gy, albeit at different time-points. While EXOSC6 and PSMD14 showed maximum over-expression (5-12 fold) at 6 h post-irradiation, ALB expression increased progressively (4 up to 7 fold) from 6 h to 48 h. TPM1 showed over-expression (2-3 fold) at all doses and time-points tested. TPM3 showed a dose-dependent response at all time-points studied; with no variation at 2 Gy, ∼2 fold increase at 5 Gy, and 3-6 fold at the highest dose used (7.5 Gy). The p55 protein was over-expressed (∼2.5 fold) only transiently at 24 h following the lethal (7.5 Gy) dose. CONCLUSION: This is the first study to report γ-radiation induced alterations in the RBC membrane associated proteins. We are further evaluating the potential of these proteins as radiation biomarkers. Due to the abundance and easy use of RBCs, this approach can prove very useful for detecting ionizing radiation exposure.

Design and Synthesis of Thiazole Scaffold-Based Small Molecules as Anticancer Agents Targeting the Human Lactate Dehydrogenase A Enzyme.

A new series of thiazole central scaffold-based small molecules of hLDHA inhibitors were designed using an in silico approach. Molecular docking analysis of designed molecules with hLDHA (PDB ID: 1I10) demonstrates that Ala 29, Val 30, Arg 98, Gln 99, Gly 96, and Thr 94 possessed strong interaction with the compounds. Compounds 8a, 8b, and 8d showed good binding affinity (-8.1 to -8.8 kcal/mol), whereas an additional interaction of NO2 at the ortho position in compounds 8c with Gln 99 through hydrogen bonding enhanced the affinity to -9.8 kcal/mol. Selected high-scored compounds were synthesized and screened for hLDHA inhibitory activities and in vitro anticancer activity in six cancer cell lines. Biochemical enzyme inhibition assays showed the highest hLDHA inhibitory activity observed with compounds 8b, 8c, and 8l. Compounds 8b, 8c, 8j, 8l, and 8m depicted significant anticancer activities, exhibiting IC50 values in the range of 1.65-8.60 µM in HeLa and SiHa cervical cancer cell lines. Compounds 8j and 8m exhibited notable anticancer activity with IC50 values of 7.90 and 5.15 µM, respectively, in liver cancer cells (HepG2). Interestingly, compounds 8j and 8m did not induce noticeable toxicity in the human embryonic kidney cells (HEK293). Insilico absorption, distribution, metabolism, and excretion profiling demonstrates that the compounds possess drug-likeness, and results may pave the way for the development of novel thiazole-based biologically active small molecules for therapeutics.

Identification of radiation responsive RBC membrane associated proteins (RMAPs) in whole-body γ-irradiated New Zealand white rabbits.

This study is aimed to identify radiation-responsive RBC Membrane Associated Proteins (RMAPs) in Rabbits in vivo. Male New Zealand White rabbits were exposed to a single acute total body γ-radiation dose of 2 Gy at a dose rate of 0.746 Gy/min. Following this, at early time points of 6 h till the 7 d, RMAPs were collected and analyzed by MALDI-TOF-MS. Bioinformatics analysis was conducted to explore the biological functions of these proteins. Based on fold change, radiation responsiveness, GO, pathway enrichment, and hub position in the PPI network, we identified seven RMAPs as potential biomarker candidates viz., PVALB, PRKCB, GPD1, CP2G1, CSNK2B, ATP1B1, TPI1. As per KEGG enrichment, most of the proteins were implicated in cellular radiation response, oxidative damage, DNA repair, apoptosis, immune response, and cell signaling. This study forms the foundation for RMAPs-based Proteomic strategies for high throughput radiation bio-dosimetry for triage in the case of a radiological/nuclear incident.

Isolated duodenal duplication cyst in a neonate.

Duodenal duplication cysts are a rare subtype of alimentary tract duplications cysts, consisting of 7% of all the duplications. We report a rare case of neonatal duodenal duplication cyst presenting as a palpable abdominal mass and features of gastric outlet obstruction. A 27-day-old male child presented with complaints of icterus, non-bilious vomiting after every feed and right-sided abdominal lump for the last 15 days. A computed tomography scan of the abdomen revealed well-defined peripherally enhancing cystic lesion noted in the subhepatic region extending up to the right lumbar region. On surgical exploration, a cystic mass was found attached to the pyloric part of the stomach along the mesenteric border of the first, second and third part of the duodenum, which was marsupialised, and no communication was found with the duodenum. On histopathological analysis, a duodenal duplication cyst was diagnosed without any heterotopic mucosa. The literature was reviewed and the approach to duodenal duplication cyst in neonates is discussed.

2-deoxy-D-glucose as an adjunct to standard of care in the medical management of COVID-19: a proof-of-concept and dose-ranging randomised phase II clinical trial.

BACKGROUND: At present, no single efficacious therapeutic exists for acute COVID-19 management and a multimodal approach may be necessary. 2-deoxy-D-glucose (2-DG) is a metabolic inhibitor that has been shown to limit multiplication of SARS-CoV-2 in-vitro. We evaluated the efficacy and safety of 2-DG as adjunct to standard care in the treatment of moderate to severe COVID-19 patients. METHODS: We conducted a randomized, open-label, phase II, clinical study to evaluate the efficacy, safety, and tolerability of 2-DG administered as adjunct to standard of care (SOC). A total of 110 patients between the ages of 18 and 65 years with moderate to severe COVID-19 were included. Patients were randomized to receive 63, 90, or 126 mg/kg/day 2-DG in addition to SOC or SOC only. Times to maintaining SpO2 ≥ 94% on room air, discharge, clinical recovery, vital signs normalisation, improvement by 1 and 2 points on WHO clinical progression scale, negative conversion on RT-PCR, requirement for intensive care, and mortality were analyzed to assess the efficacy. RESULTS: Patients treated with 90 mg/kg/day 2-DG plus SOC showed better outcomes. Time to maintaining SpO2 ≥ 94% was significantly shorter in the 2-DG 90 mg compared to SOC (median 2.5 days vs. 5 days, Hazard ratio [95% confidence interval] = 2.3 [1.14, 4.64], p = 0.0201). Times to discharge from isolation ward, to clinical recovery, and to vital signs normalization were significantly shorter for the 2-DG 90 mg group. All three doses of 2-DG were well tolerated. Thirty-three (30.3%) patients reported 65 adverse events and were mostly (86%) mild. CONCLUSIONS: 2-DG 90 mg/kg/day as adjunct to SOC showed clinical benefit over SOC alone in the treatment of moderate to severe COVID-19. The promising trends observed in current phase II study is encouraging for confirmatory evaluation of the efficacy and safety of 2-DG in a larger phase III trial. TRIAL REGISTRATION: CTRI, CTRI/2020/06/025664. Registered 5th June 2020, http://ctri.nic.in/Clinicaltrials/pdf_generate.php?trialid=44369&EncHid=&modid=&compid=%27,%2744369det%27 .

Glycolytic inhibitor 2-deoxy-d-glucose attenuates SARS-CoV-2 multiplication in host cells and weakens the infective potential of progeny virions.

AIMS: Virus-infected host cells switch their metabolism to a more glycolytic phenotype, required for new virion synthesis and packaging. Therefore, we investigated the effect and mechanistic action of glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) on virus multiplication in host cells following SARS-CoV-2 infection. MAIN METHODS: SARS-CoV-2 induced change in glycolysis was examined in Vero E6 cells. Effect of 2-DG on virus multiplication was evaluated by RT-PCR (N and RdRp genes) analysis, protein expression analysis of Nucleocapsid (N) and Spike (S) proteins and visual indication of cytopathy effect (CPE), The mass spectrometry analysis was performed to examine the 2-DG induced change in glycosylation status of receptor binding domain (RBD) in SARS-CoV-2 spike protein. KEY FINDINGS: We observed SARS-COV-2 infection induced increased glucose influx and glycolysis, resulting in selectively high accumulation of the fluorescent glucose analog, 2-NBDG in Vero E6 cells. 2-DG inhibited glycolysis, reduced virus multiplication and alleviated cells from virus-induced cytopathic effect (CPE) in SARS-CoV-2 infected cells. The progeny virions produced from 2-DG treated cells were found unglycosylated at crucial N-glycosites (N331 and N343) of the receptor-binding domain (RBD) in the spike protein, resulting in production of defective progeny virions with compromised infective potential. SIGNIFICANCE: The mechanistic study revealed that the inhibition of SARS-COV-2 multiplication is attributed to 2-DG induced glycolysis inhibition and possibly un-glycosylation of the spike protein, also. Therefore, based on its previous human trials in different types of Cancer and Herpes patients, it could be a potential molecule to study in COVID-19 patients.

Bilateral Single System Ectopic Ureters With Vaginal Insertion in a Female Child, A Rare Variant.

In most cases an ectopic ureter is associated with a duplicated renal collecting system while in only a few single systems is found. Bilateral single system ureteral ectopia is even rarer. A 9-year-old girl presented with urinary incontinence. Investigations pointed towards bilateral single system ectopic ureters with ectopic openings into vagina with a hypoplastic bladder. The left ureteric system was tortuous with malrotated and hypoplastic left kidney. A 4 × 2 cm hard calculus was found in the vagina. Right Ureteric reimplantation with left to right uretero-ureterostomy was done with satisfactory postoperative day time continence at 6 months without the need for bladder reconstruction or urinary diversion.

Establishing mechanisms affecting the individual response to ionizing radiation.

Purpose: Humans are increasingly exposed to ionizing radiation (IR). Both low (<100 mGy) and high doses can cause stochastic effects, including cancer; whereas doses above 100 mGy are needed to promote tissue or cell damage. 10-15% of radiotherapy (RT) patients suffer adverse reactions, described as displaying radiosensitivity (RS). Sensitivity to IR's stochastic effects is termed radiosusceptibility (RSu). To optimize radiation protection we need to understand the range of individual variability and underlying mechanisms. We review the potential mechanisms contributing to RS/RSu focusing on RS following RT, the most tractable RS group.Conclusions: The IR-induced DNA damage response (DDR) has been well characterized. Patients with mutations in the DDR have been identified and display marked RS but they represent only a small percentage of the RT patients with adverse reactions. We review the impacting mechanisms and additional factors influencing RS/RSu. We discuss whether RS/RSu might be genetically determined. As a recommendation, we propose that a prospective study be established to assess RS following RT. The study should detail tumor site and encompass a well-defined grading system. Predictive assays should be independently validated. Detailed analysis of the inflammatory, stress and immune responses, mitochondrial function and life style factors should be included. Existing cohorts should also be optimally exploited.

Delineating the Effects of Ionizing Radiation on Erythropoietic Lineage-Implications for Radiation Biodosimetry.

The overall lethality/morbidity of ionizing radiation exposure involves multiple forms of inhibitory or cytotoxic effects that may manifest in different tissues with a varying dose and time response. One of the major systemic effects leading to lethality of radiation includes its suppressive effect on hematopoiesis, which could be observed even at doses as low as 1-2 Gy, whereas effects on gastrointestinal and nervous systems appear at relatively higher doses in the same order. This article reviews the effects of radiation on the three distinct stages of erythropoiesis-formation of erythroid progenitor cells, differentiation of erythroid precursor cells, and terminal maturation. During these stepwise developmental processes, erythroid progenitor cells undergo rapid expansion to form terminally differentiated red blood cells that are continuously replenished from bone marrow into the circulating peripheral blood stream. Cellular radiation response depends upon many factors such as cell lineage, rate of proliferation, and differentiation status. Therefore, we discuss radiation-induced alterations during the progenitor, precursor, and terminal maturation stages and the implications thereof. Since biomarkers of ionizing radiation exposure in human populations are of great interest for assessing normal tissue injury as well as for biodosimetry in the event of accidental or incidental radiation exposures, we also highlight blood-based biomarkers that have potential utility for medical management.

Targeting miRNA for Therapeutics Using a Micronome Based Method for Identification of miRNA-mRNA Pairs and Validation of Key Regulator miRNA.

MicroRNAs are 18-22 bp long non-coding sequences and play a critical role in diverse biological processes, through modulation of gene expression at the post-transcriptional level by binding at the 3'-untranslated region of target mRNA. Consequent upon the discovery of structural and functional features of miRNA targeting, several molecular methods have been developed to identify miRNA targets. However, these methods suffer several drawbacks, including technical challenges, requirement of high cell volumes, inability to differentiate between direct and indirect targets, cell/tissue as well as experimental-specificity and imprecise binding site information. Alternatively in silico approach enables the exploration of the potential miRNA-mRNA pairs to investigate signature miRNA and proteins involved in the signaling of various diseases. Here, we describe micronome-based standard method for identification of miRNA-mRNA pairs as well as validation of key regulator miRNA.

Method for Detection of miRNAs in Non-Model Organisms with Unreported Database.

Non-model organisms are studied very frequently, as a simple accessible and convenient system to investigate the role of miRNAs in particular aspect of biology or disease. However, the unavailability of the annotated genome and hence miRNA database of these non-model organisms pose a major constraint for using them more efficiently. Here, we describe a new method to identify miRNAs in non-model organisms without complex sequencing strategies and using miRNAs from close relative organisms as proxy/reference sequences.

Multi-epitope DnaK peptide vaccine against S.Typhi: An in silico approach.

Salmonella is one of the key global causes of food and water borne enteric infections, responsible for significant morbidity and mortality worldwide especially in developing countries. Currently available vaccines against typhoid are moderately effective with several side effects and not efficacious against all Salmonella serovars. Due to limitations of these vaccines and emerging threats of multidrug resistance, developing an effective vaccine against these infections has increasingly become a priority. Heat shock proteins (Hsps), being evolutionarily conserved, represent dominant antigens in the host immune response. In continuation of our earlier studies on the development of S. Typhi DnaK and GroEL vaccine candidates, highly efficacious against Salmonella and multiple pathogens, in the present study, we have designed multi-epitope vaccine candidates common to multiple serovars of Salmonella using bioinformatics approach. Implementing various immunoinformatics tools such as IEDB, EpiJen, BCPRED, ElliPro and VaxiJen, led to the identification of many immunogenic B and T cell epitopes. The 3-D structure model of DnaK was generated to predict conformational B-cell epitopes using ElliPro server. Most promising T cell epitopes (29 CTLs, 18 T-helper cells) were selected based on their binding efficiency with commonly occurring MHC alleles. Finally we narrowed down to 5 protective antigenic peptides (PAPs), comprising highly conserved, antigenic and immunogenic B /T cell epitopes, least homologous with human host. These PAPs were predicted to be non-allergenic by allergenicity prediction tools (SORTALLER and AllerHunter). Hence, these immunogenic epitopes can be used for prophylactic or therapeutic usages specifically to defeat antibiotic-resistant Salmonella. These antigens have been reported for the first time and their conserved nature endow them as potential future vaccine candidates against other multiple pathogens as well.

A combination of resveratrol and 3,3'-diindolylmethane, a potent radioprotector.

PURPOSE: Exposure to ionizing radiation causes damage to the genomic integrity and stability of the cell. Though a large number of molecules have been studied for their radioprotective capability, no single agent is available today that meets all the requirements of a good radiprotector. In this study, we have investigated a combination of Resveratrol (RSV) and 3,3'-Diindolyl methane (DIM) for its efficacy for radioprotection. It is our hypothesis that this combination that possesses less toxicity than synthetic compounds, free radical scavenging potential, and the capacity to interfere with the several of the signaling cascades that trigger damage to cell by ionizing radiation may possess good radioprotective capability. MATERIALS AND METHODS: Mice were pre-treated with a combination of RSV and DIM and the 30-day mortality assay, endogenous antioxidant levels in intestinal mucosa, metaphase chromosomal aberrations, and micronuclei formation were assessed after exposed to ionizing radiation. RESULTS: The dose modifying factor (DRF) obtained for RSV, DIM, and the combination is 1.15, 1.17, and 1.3, respectively. Pre-treatment of mice with the combination results in significant (***p = .001) protection of the endogenous antioxidant levels, chromosomal aberrations, micronuclei formation, after exposure to ionizing radiation. CONCLUSIONS: Our findings suggest that pre-treatment with the combination of RSV and DIM protects effectively from the ionizing radiation-induced damage at the molecular, cellular, and tissue levels by counteracting both the direct and indirect effects.

Intrinsic attenuation of post-irradiation calcium and ER stress imparts significant radioprotection to lepidopteran insect cells.

Sf9 lepidopteran insect cells are 100-200 times more radioresistant than mammalian cells. This distinctive feature thus makes them suitable for studies exploring radioprotective molecular mechanisms. It has been established from previous studies of our group that downstream mitochondrial apoptotic signaling pathways in Sf9 cells are quite similar to mammalian cells, implicating the upstream signaling pathways in their extensive radioresistance. In the present study, intracellular and mitochondrial calcium levels remained unaltered in Sf9 cells in response to radiation, in sharp contrast to human (HEK293T) cells. The isolated mitochondria from Sf9 cells exhibited nearly 1.5 times greater calcium retention capacity than mammalian cells, highlighting their inherent stress resilience. Importantly, UPR/ER stress marker proteins (p-eIF2α, GRP4 and SERCA) remained unaltered by radiation and suggested highly attenuated ER and calcium stress. Lack of SERCA induction further corroborates the lack of radiation-induced calcium mobilization in these cells. The expression of CaMKII, an important effector molecule of calcium signaling, did not alter in response to radiation. Inhibiting CaMKII by KN-93 or suppressing CaM by siRNA failed to alter Sf9 cells response to radiation and suggests CaM-CaMKII independent radiation signaling. Therefore, this study suggests that attenuated calcium signaling/ER stress is an important determinant of lepidopteran cell radioresistance.

Nasal Glial Heterotopia with Cleft Palate.

Congenital midline nasal masses are rare anomalies of which nasal glial heterotopia represents an even rarer subset. We report a case of a 25-day-old male child with nasal glial heterotopia along with cleft palate suggesting embryonic fusion anomaly which was treated with excision and primary closure for nasal mass followed by palatal repair at later date.

Evidence for a radiation-responsive 'p53 gateway' contributing significantly to the radioresistance of lepidopteran insect cells.

Recently, we have demonstrated that microRNA-31 (miR-31) overexpression is inherent to radiation-induced cell death in the highly radioresistant Sf9 insect cells, and regulates pro-apoptotic Bax translocation to mitochondria. In the present study, we report that at sub-lethal radiation doses for Sf9 cells, miR-31 is significantly downregulated and is tightly regulated by an unusual mechanism involving p53. While ectopic overexpression of a well-conserved Sfp53 caused typical apoptosis, radiation-induced p53 accumulation observed selectively at sub-lethal doses failed to induce cell death. Further investigation of this paradoxical response revealed an intriguing phenomenon that sub-lethal radiation doses result in accumulation of a 'hyper-phosphorylated' Sfp53, which in turn binds to miR-31 genomic location and suppresses its expression to prevent cell death. Interestingly, priming cells with sub-lethal doses even prevented the apoptosis induced by lethal radiation or ectopic Sfp53 overexpression. On the other hand, silencing p53 increased radiation-induced cell death by inhibiting miR-31 downregulation. This study thus shows the existence of a unique radiation-responsive 'p53 gateway' preventing miR-31-mediated apoptosis in Sf9 cells. Since Sfp53 has a good functional homology with human p53, this study may have significant implications for effectively modulating the mammalian cell radioresistance.

Radioresistant Sf9 insect cells readily undergo an intrinsic mode of apoptosis in response to histone deacetylase (HDAC) inhibition.

Insect cell lines have been utilized as an important higher eukaryotic model system to decipher stress responses and cell death mechanisms. Lepidopteran Sf9 cells (derived from the ovaries of Spodoptera frugiperda) display nearly 100 times higher resistance to ionizing radiation in contrast to mammalian cells, which is partly contributed by an unusually high HDAC activity. However, their response to HDAC inhibition remains to be evaluated. In the present study, the effects of HDAC inhibitor (NaBt) on Sf9 cellular/nuclear morphology, cell cycle progression, DNA damage/repair, redox status, and mitochondrial perturbations were evaluated. NaBt-induced apoptosis was evident at 18 h in Sf9 cells at 2 mM concentration, primarily through mitochondrial induction of oxidative stress and subsequent DNA damage. Cell cycle analysis revealed appearance of sub-G1 DNA content at 12 h onwards and DNA fragmentation by 18 h. Initial few hours of treatment caused significant loss in MMP through oxidation of mitochondrial inner membrane protein, i.e., cardiolipin. HDAC inhibition-mediated apoptosis was associated with increased Bax/Bcl2 ratio, mitochondrial cytochrome-c release, and caspase-3 activation. The study thus infers that Sf9 cells, which can withstand very high radiation doses, are quite sensitive to the increase in the chromatin acetylation levels. In addition, HDAC inhibition also sensitized Sf9 cells to radiation-induced DNA damage, further corroborating our recent finding that chromatin compactness contributes significantly to their radioresistance. Therefore, the study demonstrates prominence of prevailing DNA/chromatin protective mechanisms in Lepidopteran insect cells.