Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms

Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes

Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP

A Versatile ΦC31 Based Reporter System for Measuring AP-1 and Nrf2 Signaling in Drosophila and in Tissue Culture

This paper describes the construction and characterization of a system of transcriptional reporter genes for monitoring the activity of signaling pathways and gene regulation mechanisms in intact Drosophila, dissected tissues or cultured cells. Transgenic integration of the reporters into the Drosophila germline was performed in a site-directed manner, using ΦC31 integrase. This strategy avoids variable position effects and assures low base level activity and high signal responsiveness. Defined integration sites furthermore enable the experimenter to compare the activity of different reporters in one organism. The reporter constructs have a modular design to facilitate the combination of promoter elements (synthetic transcription factor binding sites or natural regulatory sequences), reporter genes (eGFP, or DsRed.T4), and genomic integration sites. The system was used to analyze and compare the activity and signal response profiles of two stress inducible transcription factors, AP-1 and Nrf2. To complement the transgenic reporter fly lines, tissue culture assays were developed in which the same synthetic ARE and TRE elements control the expression of firefly luciferase

Prevalence and determinants of delay in diagnosis of pulmonary tuberculosis in Darjeeling district of West Bengal

Background: Delayed diagnosis of tuberculosis (TB) is a significant problem both in individual as well as community level. Different studies around globe revealed that these diagnostic delays are attributed to both patient delay and health system-related delay. Aims: This study aims to assess the magnitude of delay in diagnosis and the association with sociodemographic profile among new sputum-positive pulmonary TB patients in Darjeeling district. Materials and Methods: A cross-sectional study was conducted among 374 TB patients from October 2011 to March 2012 using a predesigned pretested schedule by face-to-face interview. Statistical Analysis: Logistic regression analysis, odds ratios (OR), adjusted ORs. Results: Patient delay, health system delay and total diagnostic delay were 27 days, 20.1 days, and 20.6 days; mean delays were 23.64, 5.71, and 29.46 days, and median delays were 25, 5, and 32 days, respectively. Risk factors associated with patient delay were female gender, rural residence, illiteracy, smoking, alcohol consumption, taking two, or more alternate treatments; for health system delay were female sex, rural residence, time to reach health facility, time spent per visit; and for total diagnostic delay were female sex, alcoholism, and seeking more than two alternate treatment. Conclusions: The risk factors for delay identified may be the subject of future interventions

Risk factors associated with default among tuberculosis patients in Darjeeling district of West Bengal, India

Background: The treatment outcome "default" under Revised National Tuberculosis Control Program (RNTCP) is a patient who after treatment initiation has interrupted treatment consecutively for more than 2 months. Aims: To assess the timing, characteristics and distribution of the reasons for default with relation to some sociodemographic variables among new sputum-positive (NSP) tuberculosis (TB) patients in Darjeeling District, West Bengal. Settings and Design: A case-control study was conducted in three tuberculosis units (TUs) of Darjeeling from August′2011 to December′2011 among NSP TB patients enrolled for treatment in the TB register from 1 st Qtr′09 to 2 nd Qtr′10. Patients defaulted from treatment were considered as "cases" and those completed treatment as "controls" (79 cases and 79 controls). Materials and Methods: The enrolled cases and controls were interviewed by the health workers using a predesigned structured pro-forma. Statistical Analysis Used: Logistic regression analysis, odds ratios (OR), adjusted odds ratios (AOR). Results: 75% of the default occurred in the intensive phase (IP); 54.24% retrieval action was done within 1 day during IP and 75% within 1 week during continuation phase (CP); cent percent of the documented retrieval actions were undertaken by the contractual TB program staffs. Most commonly cited reasons for default were alcohol consumption (29.11%), adverse effects of drugs (25.32%), and long distance of DOT center (21.52%). In the logistic regression analysis, the factors independently associated were consumption of alcohol, inadequate knowledge about TB, inadequate patient provider interaction, instances of missed doses, adverse reactions of anti-TB drugs, Government Directly Observed Treatment (DOT) provider and smoking. Conclusions: Most defaults occurred in the intensive phase; pre-treatment counseling and initial home visit play very important role in this regard. Proper counseling by health care workers in patient provider meeting is needed

Cytoplasmic chromatin fragments—from mechanisms to therapeutic potential

Senescent cells, damaged cells that permanently exit the cell cycle, play important roles in development, tissue homeostasis, and tumorigenesis. Although many of these roles are beneficial in acute responses to stress and damage, the persistent accumulation of senescent cells is associated with many chronic diseases through their proinflammatory senescence-associated secretory phenotype (SASP). SASP expression is linked to DNA damage; however, the mechanisms that control the SASP are incompletely understood. More recently, it has been shown that senescent cells shed fragments of nuclear chromatin into the cytoplasm, so called cytoplasmic chromatin fragments (CCF). Here, we provide an overview of the current evidence linking DNA damage to the SASP through the formation of CCF. We describe mechanisms of CCF generation and their functional role in senescent cells, with emphasis on therapeutic potential

Mass Measles Vaccination Campaign in Aila Cyclone-Affected Areas of West Bengal, India: An In-depth Analysis and Experiences

Disaster-affected populations are highly vulnerable to outbreaks of measles. Therefore, a mass vaccination against measles was conducted in Aila cyclone-affected blocks of West Bengal, India in July 2009. The objectives of the present report were to conduct an in depth analysis of the campaign, and to discuss the major challenges. A block level micro-plan, which included mapping of the villages, health facilities, temporary settlements of disaster-affected population, communications available, formation of vaccination team, information education communication, vaccine storage, waste disposal, surveillance for adverse events following immunization, supervision and monitoring was developed. The rate of six months to five years old children, who were vaccinated by measles vaccine, was 70.7% and that of those who received one dose of vitamin A was 71.3%. Wastage factor for vaccine doses and auto-disable syringes were 1.09 and 1.07, respectively. Only 13 cases of adverse events following immunization were reported. An average of 0.91 puncture-proof containers per vaccination session was used. Despite the major challenges faced due to difficult to reach areas, inadequate infrastructure, manpower and communication, problems of vaccine storage and transport, the campaign achieved a remarkable success regarding measles vaccine coverage, improvements of cold chain infrastructure, formulating an efficient surveillance and reporting system for adverse events following immunization, building self-confidence of the stakeholder