Characteristics and outcomes of gallbladder cancer patients at the Tata Medical Center, Kolkata 2017-2019.

BACKGROUND: The north and north-eastern regions of India have among the highest incidence of gallbladder cancer (GBC) in the world. We report the clinicopathological charateristics and outcome of GBC patients in India. METHODS: Electronic medical records of patients diagnosed with GBC at Tata Medical Center, Kolkata between 2017 and 2019 were analyzed. RESULTS: There were 698 cases of confirmed GBC with a median age of 58 (IQR: 50-65) years and female:male ratio of 1.96. At presentation, 91% (496/544) had stage III/IV disease and 30% (189/640) had incidental GBC. The 2-year overall survival (OS) was 100% (95% CI: 100-100); 61% (95% CI: 45-83); 30% (95% CI: 21-43); and 9% (95% CI: 6-13) for stages I-IV, respectively (p = <0.0001).   For all patients, the 2-year OS in patients who had a radical cholecystectomy followed by adjuvant therapy (N = 36) was 50% (95% CI: 39-64), compared to 29% (95% CI: 22-38) for those who had a simple cholecystectomy and/or chemotherapy (N = 265) and 9% (95% CI: 6-14) in patients who were palliated (N = 107) (p = <0.0001). CONCLUSION: The combined surgical/chemotherapy approach for patients with stage II GBC showed the best outcomes. Early detection of GBC remains problematic with the majority of patients presenting with stage III-IV and who have a median survival of 9.1 months. Our data suggests that the tumor is chemoresponsive and multi-center collaborative clinical trials to identify alternative therapies are urgently required.

PLK-1 Interacting Checkpoint Helicase, PICH, Mediates Cellular Oxidative Stress Response.

Cells respond to oxidative stress by elevating the levels of antioxidants, signaling, and transcriptional regulation, often implemented by chromatin remodeling proteins. The study presented here shows that the expression of PICH, a Rad54-like helicase belonging to the ATP-dependent chromatin remodeling protein family, is upregulated during oxidative stress in HeLa cells. We also show that PICH regulates the expression of Nrf2, a transcription factor regulating antioxidant response in both the absence and presence of oxidative stress. The overexpression of PICH in PICH-depleted cells restored Nrf2 as well as antioxidant gene expression. In turn, Nrf2 regulated the expression of PICH in the presence of oxidative stress. ChIP experiments showed that PICH is present on the Nrf2 as well as antioxidant gene promoters, suggesting that the protein might be regulating the expression of these genes directly by binding to the DNA sequences. In addition, Nrf2 and histone acetylation (H3K27ac) also played a role in activating transcription in the presence of oxidative stress. Both Nrf2 and H3K27ac were found to be present on PICH and antioxidant promoters. Their occupancy was dependent on the PICH expression as fold enrichment was found to be decreased in PICH-depleted cells. PICH ablation led to the reduced expression of Nrf2 and impaired antioxidant response, leading to increased ROS content and thus showing PICH is essential for the cell to respond to oxidative stress.

Impact of prenatal maternal psychological distress on fetal biometric parameters in household air pollution-exposed Nigerian women.

RATIONALE: Studies identify prenatal household air pollution (HAP) exposure and maternal psychological distress (PMPD) as independent factors contributing to gestational ill-health and adverse birth outcomes. OBJECTIVE: We investigated the impact of PMPD on fetal biometric parameters (FBP) in HAP-exposed pregnant Nigerian women. METHODS: The randomized controlled trial (RCT; ClinicalTrials.gov NCT02394574) investigated effects of HAP exposure in pregnant Nigerian women (n = 324), who customarily cooked with polluting fuels (firewood or kerosene). Half of the women (intervention group) were given CleanCook ethanol stoves to use for 156 days during the study. Once a month, all women were administered an abridged version of the SF-12v2TM health-related quality of life questionnaire to assess psychological distress. Using mixed effects linear regression models, adjusted for relevant covariates, we analyzed associations between the women's exposure to PM2·5 (particulate matter with an aerodynamic diameter<2·5 microns) from HAP, their PMPD scores, and FBP (ultrasound estimated fetal weight [UEFW], head circumference [HC], abdominal circumference [AC], femur length [FL], biparietal diameter [BPD], estimated gestational age [GA] and intrauterine growth restriction [IUGR]), and birth anthropometric measures (birth weight [BW] and birth length [BL]). RESULTS: PMPD negatively impacted UEFW, HC, FL, BPD and BL (p<0·05). Controls (kerosene/firewood users) experienced significantly higher PMPD compared with ethanol-stove users (p<0·05). The mediation analysis revealed that the proportion of the outcome (fetal biometrics, birth anthropometrics, IUGR and GA), which can be explained via PMPD by groups (intervention vs. control) after adjusting for confounding variables was 6·2% (0·062). No significant correlation was observed between levels of PM2.5 exposure and PMPD scores. CONCLUSIONS: PMPD was an independent mediator of adverse fetal biometric parameters in pregnant women, who were exposed to HAP from burning of firewood/kerosene. Formulating preventative measures to alleviate maternal distress during pregnancy and reducing exposure to HAP is important from public health perspectives.

Fun30 and Rtt109 Mediate Epigenetic Regulation of the DNA Damage Response Pathway in C. albicans.

Fun30, an ATP-dependent chromatin remodeler from S. cerevisiae, is known to mediate both regulation of gene expression as well as DNA damage response/repair. The Fun30 from C. albicans has not yet been elucidated. We show that C. albicans Fun30 is functionally homologous to both S. cerevisiae Fun30 and human SMARCAD1. Further, C. albicans Fun30 can mediate double-strand break end resection as well as regulate gene expression. This protein regulates transcription of RTT109, TEL1, MEC1, and SNF2-genes that encode for proteins involved in DNA damage response and repair pathways. The regulation mediated by C. albicans Fun30 is dependent on its ATPase activity. The expression of FUN30, in turn, is regulated by histone H3K56 acetylation catalyzed by Rtt109 and encoded by RTT109. The RTT109Hz/FUN30Hz mutant strain shows sensitivity to oxidative stress and resistance to MMS as compared to the wild-type strain. Quantitative PCR showed that the sensitivity to oxidative stress results from downregulation of MEC1, RAD9, MRC1, and RAD5 expression; ChIP experiments showed that Fun30 but not H3K56ac regulates the expression of these genes in response to oxidative stress. In contrast, upon treatment with MMS, the expression of RAD9 is upregulated, which is modulated by both Fun30 and H3K56 acetylation. Thus, Fun30 and H3K56 acetylation mediate the response to genotoxic agents in C. albicans by regulating the expression of DNA damage response and repair pathway genes.

The interaction of oxidative stress with MAPK, PI3/AKT, NF-κB, and DNA damage kinases influences the fate of γ-radiation-induced bystander cells.

Oxidative stress is associated with the induction of a plethora of effects on cellular macromolecules and signaling cascades. The onset of oxidative imbalance characterizes irradiated cells. The present study investigates the effects of ionizing radiation on oxidative stress induction in bystander cells and their interactions with critical cell signaling mediators. The effect of irradiated cell-conditioned medium (ICCM) from γ-irradiated hepatocellular carcinoma (HepG2) cells were studied in bystander HepG2 and normal liver (BRL-3A) cells at early (1 h, 2 h) and later (24 h) time points post-irradiation. Although ROS generation and lipid peroxidation showed the highest effects in both bystander cell groups at the early time points, antioxidant enzymes superoxide dismutase and catalase showed the lowest activity. Oxidative stress was persistent up to 24 h, but the highest level was seen in 1 h ICCM treated 8By cells. Although the levels of all pro-survival signaling factors (p-PI3K, p-Akt, p-p38MAPK, p-JNK, and p-NFκB) increased in bystander HepG2 cells, they showed a significant decrease in bystander BRL-3A cells. JAK2-STAT3 activation, however, was reduced only in BRL-3A cells, with no effect in HepG2 cells. However, in both bystander cell groups, activation of DNA damage sensors ATM, ATR, and cell cycle inhibitor p21 increased. Elevated ROS levels down-regulated the activation of PI3K, Akt, JNK, and NF-κB in BRL-3A cells but enhanced the activation of ATM and p21. In contrast, in HepG2 cells, increased ROS level elevated the activation of PI3K, JNK, p38MAPK, NF-κB with no effect on p-ATM or p21. ROS differentially influenced the interactions between the signaling mediators in the bystander cells. p-ATR levels, although increased in both bystander cell groups, showed no association with other factors. ICCM from the same HepG2 cells differently affected signaling factors in two groups of cells, highlighting the critical significance of the study in the field of radiation biology.

CD Spectroscopy to Study DNA-Protein Interactions.

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The cross-talk between Bax, Bcl2, caspases, and DNA damage in bystander HepG2 cells is regulated by γ-radiation dose and time of conditioned media transfer.

Although radiation-induced bystander effects have been broadly explored in various biological systems, the molecular mechanisms and the consequences of different regulatory factors (dose, time, cell type) on bystander responses are not clearly understood. This study investigates the effects of irradiated cell-conditioned media (ICCM) collected at different times post-irradiation on bystander cancer cells regarding DNA damage and apoptosis induction. Human hepatocellular carcinoma HepG2 cells were exposed to γ-ray doses of 2 Gy, 5 Gy, and 8 Gy. In the early and late stages (1 h, 2 h, and 24 h) after irradiation, the ICCM was collected and transferred to unirradiated cells. Compared to control, bystander cells showed an increased level of H2AX phosphorylation, mitochondrial membrane depolarization, and elevation of intrinsic apoptotic pathway mediators such as p53, Bax, cas9, cas-3, and PARP cleavage. These results were confirmed by phosphatidylserine (PS) externalization and scanning electron microscopic observations, suggesting a rise in bystander HepG2 cell apoptosis. Anti-apoptotic Bcl2-level and viability were lower in bystander cells compared to control. The highest effects were observed in 8 Gy γ radiation-induced bystander cells. Even though the bystander effect was persistent at all time points of the study, ICCM at the early time points (1 or 2 h) had the most significant impact on the apoptosis markers in bystander cells. Nevertheless, 24 h ICCM induced the highest increase in H2AX and p53 phosphorylation and Bax levels. The effects of ICCM of irradiated HepG2 cells were additionally studied in normal liver cells BRL-3A to simulate actual radiotherapy conditions. The outcomes suggest that the expression of the signaling mediators in bystander cells is highly dynamic. A cross-talk between those signaling mediators regulates bystander responses depending on the radiation dose and time of incubation post-irradiation.

External modulators and redox homeostasis: Scenario in radiation-induced bystander cells.

Redox homeostasis is imperative to maintain normal physiologic and metabolic functions. Radiotherapy disturbs this balance and induces genomic instability in diseased cells. However, radiation-induced effects propagate beyond the targeted cells, affecting the adjacent non-targeted cells (bystander effects). The cellular impact of radiation, thus, encompasses both targeted and non-targeted effects. Use of external modulators along with radiation can increase radio-therapeutic efficiency. The modulators' classification as protectors or sensitizers depends on interactions with damaged DNA molecules. Thus, it is necessary to realize the functions of various radio-sensitizers or radio-protectors in both irradiated and bystander cells. This review focuses on some modulators of radiation-induced bystander effects (RIBE) and their action mechanisms. Knowledge about the underlying signaling cross-talk may promote selective sensitization of radiation-targeted cells and protection of bystander cells.

Household air pollution, ultrasound measurement, fetal biometric parameters and intrauterine growth restriction.

BACKGROUND: Low birthweight, intrauterine growth restriction (IUGR) and perinatal mortality have been associated with air pollution. However, intervention studies that use ultrasound measurements to assess the effects of household air pollution (HAP) on fetal biometric parameters (FBP) are rare. We investigated the effect of a cookstove intervention on FBP and IUGR in a randomized controlled trial (RCT) cohort of HAP-exposed pregnant Nigerian women. METHODS: We recruited 324 women early in the second trimester of pregnancy. Between 16 and 18 weeks, we randomized them to either continue cooking with firewood/kerosene (control group) or receive a CleanCook stove and ethanol fuel (intervention group). We measured fetal biparietal diameter (BPD), head circumference (HC), femur length (FL), abdominal circumference (AC) and ultrasound-estimated fetal weight (U-EFW) in the second and third trimesters. The women were clinically followed up at six regular time points during their pregnancies. Once during the women's second trimester and once during the third, we made 72-h continuous measurements of their personal exposures to particulate matter having aerodynamic diameter < 2.5 µm (PM2.5). We adopted a modified intent-to-treat approach for the analysis. Differences between the intervention and control groups on impact of HAP on fetal growth trajectories were analyzed using mixed effects regression models. RESULTS: There were no significant differences in fetal growth trajectories between the intervention and control groups. CONCLUSIONS: Larger studies in a setting of low ambient air pollution are required to further investigate the effect of transitioning to a cleaner fuel such as ethanol on intrauterine growth. TRIAL REGISTRATION: ClinicalTrials.gov NCT02394574 ; September 2012.

Impact of prenatal and postnatal household air pollution exposure on lung function of 2-year old Nigerian children by oscillometry.

RATIONALE: Lung function is adversely affected by exposure to household air pollution (HAP). Studies investigating the impact of prenatal and postnatal HAP exposure on early childhood lung development are limited, especially from Sub-Saharan Africa. OBJECTIVE: We used oscillometry to investigate the impact on lung function of prenatal and postnatal HAP exposure of children born to Nigerian women who participated in a randomized controlled cookstove intervention trial. METHODS: We performed oscillometric measurements (R: airway resistance; X: airway reactance; Fres: resonant frequency; AX: reactance area) in 223 children starting at age of 2 years (ethanol stove, n = 113; firewood/kerosene, n = 110). Personal exposure monitoring assessed mothers' prenatal exposure to particulate matter less than 2.5 µm in aerodynamic diameter (PM2.5). Postnatal HAP exposure was measured by determining household PM2.5 levels. We employed linear regression analysis to examine the association of prenatal and postnatal HAP exposures with children's lung function. Models were adjusted for age, gender, weight, height, group (intervention or control), birthweight and gestational age. RESULTS: Mean age of the children was 2.9 years (standard deviation = 0.3); 120 were boys (53.8%) and 103 were girls (46.2%). Higher postnatal PM2.5 exposures were significantly associated with higher airway reactance at 5 Hz (X5 Hz; p = 0.04) in adjusted models. There were no significant associations between prenatal or postnatal PM2.5 exposure levels and other oscillometry parameters in adjusted regression analysis. CONCLUSIONS: This is the first study to use oscillometry to explore the relationship between HAP exposure and lung function in children as young as 2 years. The findings provide some evidence that increased postnatal HAP exposure may result in poorer lung function in children, although larger studies are needed to confirm observed results. This study indicates that oscillometry is a low-cost and effective method to determine lung function in early childhood.

Optimal trapping stability of Escherichia coli in oscillating optical tweezers.

Single-beam oscillating optical tweezers can be used to trap rod-shaped bacterial cells and align them with their long axis lying within the focal plane. While such configuration is useful for imaging applications, the corresponding imaging resolution is limited by the fluctuations of the trapped cell. We study the fluctuations of four of the coordinates of the trapped cell, two for its center of mass position and two for its angular orientation, showing the way they depend on the trap length and the trapping beam power. We find that optimal trapping stability is obtained when the trap length is about the same as the cell length and that cell fluctuations in the focal plane decrease like the inverse of the trapping power.

Author Correction: Predicting the clinical impact of human mutation with deep neural networks.

In the version of this article originally published, the name of author Serafim Batzoglou was misspelled. The error has been corrected in the HTML and PDF versions of the article.

Druggability Simulations and X-Ray Crystallography Reveal a Ligand-Binding Site in the GluA3 AMPA Receptor N-Terminal Domain.

Ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission in the brain. Their dysfunction is implicated in many neurological disorders, rendering iGluRs potential drug targets. Here, we performed a systematic analysis of the druggability of two major iGluR subfamilies, using molecular dynamics simulations in the presence of drug-like molecules. We demonstrate the applicability of druggability simulations by faithfully identifying known agonist and modulator sites on AMPA receptors (AMPARs) and NMDA receptors. Simulations produced the expected allosteric changes of the AMPAR ligand-binding domain in response to agonist. We also identified a novel ligand-binding site specific to the GluA3 AMPAR N-terminal domain (NTD), resulting from its unique conformational flexibility that we explored further with crystal structures trapped in vastly different states. In addition to providing an in-depth analysis into iGluR NTD dynamics, our approach identifies druggable sites and permits the determination of pharmacophoric features toward novel iGluR modulators.

Household Air Pollution, Levels of Micronutrients and Heavy Metals in Cord and Maternal Blood, and Pregnancy Outcomes.

Cooking with kerosene emits toxic pollutants that may impact pregnancy outcomes. Sixty-eight women in their first trimester of pregnancy, kerosene users (n = 42) and liquefied natural gas (LNG) users (n = 26), were followed until birth. Maternal and cord blood were collected immediately after birth. Levels of micronutrients and heavy metals were quantified. Pregnancy outcomes (gestation age (GA), birth weight (BW), and chest and head circumference) were also measured. Mean (± standard deviation (SD)) age of mothers in kerosene and LNG groups were similar (p = 0.734). Mean (±SD) BW of newborns of LNG users was significantly higher compared to newborns of kerosene users (3.43 ± 0.32 vs. 3.02 ± 0.43, p < 0.001). Mean GA (in weeks) was similar between the two groups (p = 0.532). Women in the kerosene group had significantly higher cord blood levels of zinc, lead, mercury, iodine and vitamin B6 and lower levels of folic acid compared to LNG users (p < 0.05). Newborns of kerosene users had reduced levels of zinc, lead, mercury, iodine, vitamins B6 and B12, folic acid, and homocysteine compared with LNG users (p < 0.05). Also, cooking with kerosene was significantly associated with reduced birth weight after adjusting for potential confounders (ß ± standard error (SE) = -0.326 ± 0.155; p = 0.040). Smoke from kerosene stove was associated with reduced birth weight and micronutrients imbalance in mothers and newborns.

Predicting the clinical impact of human mutation with deep neural networks.

Millions of human genomes and exomes have been sequenced, but their clinical applications remain limited due to the difficulty of distinguishing disease-causing mutations from benign genetic variation. Here we demonstrate that common missense variants in other primate species are largely clinically benign in human, enabling pathogenic mutations to be systematically identified by the process of elimination. Using hundreds of thousands of common variants from population sequencing of six non-human primate species, we train a deep neural network that identifies pathogenic mutations in rare disease patients with 88% accuracy and enables the discovery of 14 new candidate genes in intellectual disability at genome-wide significance. Cataloging common variation from additional primate species would improve interpretation for millions of variants of uncertain significance, further advancing the clinical utility of human genome sequencing.

Cinnamon in Anticancer Armamentarium: A Molecular Approach.

In recent years, natural bioactive components draw a major attention for their potent anticarcinogenic activity. Cinnamon, one of the traditional spices, most frequently used in almost every household of tropical countries has got enormous efficacy to combat cancer. Cinnamon as a whole and/or its active components exhibited significant antineoplastic activity in different types of cancer. This review has been carried out to elicit the molecular mechanisms of action of cinnamon and its components on oncogenic regulators and related pathways. Web sites of Google Scholar, Medline, and PubMed were searched for articles written in English and published in peer-reviewed journals from 2003 to 2017. The anticarcinogenic potential of cinnamon varies with the type of cancer and also depends on the administered active compound individually or in combination with some chemicals or even extract of cinnamon as a whole. Some of its active components exert chemosensitization of well-known anticancer drugs. These outstanding properties of this spice necessitate its incorporation in both pharmaceuticals and nutraceuticals to explore possibilities of formulation of novel drug from this spice for treatment and prevention strategy of cancer.

Exosomal αvß6 integrin is required for monocyte M2 polarization in prostate cancer.

Therapeutic approaches aimed at curing prostate cancer are only partially successful given the occurrence of highly metastatic resistant phenotypes that frequently develop in response to therapies. Recently, we have described αvß6, a surface receptor of the integrin family as a novel therapeutic target for prostate cancer; this epithelial-specific molecule is an ideal target since, unlike other integrins, it is found in different types of cancer but not in normal tissues. We describe a novel αvß6-mediated signaling pathway that has profound effects on the microenvironment. We show that αvß6 is transferred from cancer cells to monocytes, including ß6-null monocytes, by exosomes and that monocytes from prostate cancer patients, but not from healthy volunteers, express αvß6. Cancer cell exosomes, purified via density gradients, promote M2 polarization, whereas αvß6 down-regulation in exosomes inhibits M2 polarization in recipient monocytes. Also, as evaluated by our proteomic analysis, αvß6 down-regulation causes a significant increase in donor cancer cells, and their exosomes, of two molecules that have a tumor suppressive role, STAT1 and MX1/2. Finally, using the Ptenpc-/- prostate cancer mouse model, which carries a prostate epithelial-specific Pten deletion, we demonstrate that αvß6 inhibition in vivo causes up-regulation of STAT1 in cancer cells. Our results provide evidence of a novel mechanism that regulates M2 polarization and prostate cancer progression through transfer of αvß6 from cancer cells to monocytes through exosomes.

Building a consumer market for ethanol-methanol cooking fuel in Lagos, Nigeria.

A recently completed randomized controlled study in Nigeria that transitioned pregnant women from traditional fuels to ethanol in their cook stoves demonstrated improved pregnancy outcomes in mothers and children. We subsequently conducted a pilot study of 30 households in Lagos, Nigeria, to determine the acceptability of blended ethanol/methanol as cooking fuel and willingness to pay for the Clean Cook stove. A third of the pilot participants expressed a willingness to purchase the stove for the minimum price of 42 USD or more. Fuel sales data suggest sustained, but non-exclusive, use of the CleanCook stove. These results will influence the final design and implementation of a planned 2500 stove commercial pilot that is scheduled to start in Nigeria in August 2018.

Pregnancy outcomes and ethanol cook stove intervention: A randomized-controlled trial in Ibadan, Nigeria.

BACKGROUND: Household air pollution (HAP) exposure has been linked to adverse pregnancy outcomes. OBJECTIVES: A randomized controlled trial was undertaken in Ibadan, Nigeria to determine the impact of cooking with ethanol on pregnancy outcomes. METHODS: Three-hundred-twenty-four pregnant women were randomized to either the control (continued cooking using kerosene/firewood stove, n=162) or intervention group (received ethanol stove, n=162). Primary outcome variables were birthweight, preterm delivery, intrauterine growth restriction (IUGR), and occurrence of miscarriage/stillbirth. RESULTS: Mean birthweights for ethanol and controls were 3076 and 2988g, respectively; the difference, 88g, (95% confidence interval: -18g to 194g), was not statistically significant (p=0.10). After adjusting for covariates, the difference reached significance (p=0.020). Rates of preterm delivery were 6.7% (ethanol) and 11.0% (control), (p=0.22). Number of miscarriages was 1(ethanol) vs. 4 (control) and stillbirths was 3 (ethanol) vs. 7 (control) (both non-significant). Average gestational age at delivery was significantly (p=0.015) higher in ethanol-users (39.2weeks) compared to controls (38.2weeks). Perinatal mortality (stillbirths and neonatal deaths) was twice as high in controls compared to ethanol-users (7.9% vs. 3.9%; p=0.045, after adjustment for covariates). We did not detect significant differences in exposure levels between the two treatment arms, perhaps due to large seasonal effects and high ambient air pollution levels. CONCLUSIONS: Transition from traditional biomass/kerosene fuel to ethanol reduced adverse pregnancy outcomes. However, the difference in birthweight was statistically significant only after covariate adjustment and the other significant differences were in tertiary endpoints. Our results are suggestive of a beneficial effect of ethanol use. Larger trials are required to validate these findings.