Determinants of substance use among young people attending primary health centers in India.

Background: Substance use is a complex condition with multidimensional determinants. The present study aims to find the prevalence and determinants of substance use among young people attending primary healthcare centers in India. Methods: A multicentric cross-sectional study was conducted across 15 states in India on 1,630 young people (10-24 years) attending primary health centers. The Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST) was used to capture data on substance use. The degree of substance involvement was assessed and multivariate regression analysis was conducted to determine the risk factors of substance use. Results: The prevalence of substance use was 32.8%, with a median substance initiation age of 18 years. Among the substance users, 75.5% began before completing adolescence. Tobacco (26.4%), alcohol (26.1%) and cannabis (9.5%) were commonly consumed. Sociodemographic determinants included higher age, male gender, urban residence, positive family history, northeastern state residence and lower socioeconomic class. Over 80% of users had moderate or high involvement. Conclusions: High substance use prevalence among young people in Indian healthcare centers underscores the urgency of targeted intervention. Insights on determinants guide effective prevention strategies for this complex public health issue.

Anesthetic Efficacy of Endo-Ice and Intrapulpal Ice Sticks After Inferior Alveolar Nerve Block in Symptomatic Irreversible Pulpitis: A Randomized Controlled Study.

BACKGROUND: There is a dearth of studies to assess the effectiveness of cryotherapy as a possible adjuvant in anesthesia. Therefore, this study aimed to compare the impact of pretreatment intraoral ice packs, application of Endo-ice, and a combination of Endo-ice and intrapulpal ice sticks use on the effectiveness of pulpal anesthesia as an adjunct in the management of pain and dental anxiety in mandibular second molars with symptomatic irreversible pulpitis (SIP) against traditional inferior alveolar nerve block (IANB) technique. MATERIALS AND METHODS: The randomized controlled double-blind clinical study comprised of 200 subjects within the age group of 20-45 years with SIP involving mandibular second molars and divided into four groups of 50 each. Conventional IANB was administered in group 1. Intraoral compact ice packs, Endo-ice, and combined use of Endo-ice and intrapulpal ice sticks were employed following conventional IANB in groups 2, 3, and 4, respectively. The intensity of pain and anxiety before and after the intervention was documented using Heft-Parker visual analog scale (HP-VAS) and Corah's Dental Anxiety Scale-Revised (DAS-R). RESULTS: The average age of the overall study sample was 30.8±2.08 years, and the differences in age and gender distribution were found to be statistically insignificant. The mean HP-VAS scores on access opening and pulpectomy, using ANOVA paired with Tukey's post hoc multiple comparison tests were evaluated to be statistically highly significant (p<0.001). The greatest reduction in HP-VAS score was observed in group 4 receiving combined use of both Endo-ice and intrapulpal ice sticks. While the pretreatment DAS-R score was determined to be statistically insignificant between groups, group 1 subjects had the highest DAS-R score postoperatively (p<0.001). The effectiveness of pulpal anesthesia was found to be 84%, 96%, 92%, and 98% for groups 1 through 4, respectively, and demonstrated a statistically significant difference. CONCLUSION: The combined use of Endo-ice and intrapulpal ice sticks used as an adjuvant to local anesthesia (LA) was found to be significantly effective in lowering pain compared to the control groups in molars with SIP. Cold administration before and during the procedure was more effective than conventional LA in lowering intraoperative anxiety. Furthermore, the use of Endo-ice and intrapulpal cold after IANB significantly improves the effectiveness of pulpal anesthesia in mandibular second molars with SIP.

Prevention of radiation-induced bystander effects by agents that inactivate cell-free chromatin released from irradiated dying cells.

Radiation-induced bystander effect (RIBE) is a poorly understood phenomenon wherein non-targeted cells exhibit effects of radiation. We have reported that cell-free chromatin (cfCh) particles that are released from dying cells can integrate into genomes of surrounding healthy cells to induce DNA damage and inflammation. This raised the possibility that RIBE might be induced by cfCh released from irradiated dying cells. When conditioned media from BrdU-labeled irradiated cells were passed through filters of pore size 0.22 µm and incubated with unexposed cells, BrdU-labeled cfCh particles could be seen to readily enter their nuclei to activate H2AX, active Caspase-3, NFκB, and IL-6. A direct relationship was observed with respect to activation of RIBE biomarkers and radiation dose in the range of 0.1-0 Gy. We confirmed by FISH and cytogenetic analysis that cfCh had stably integrated into chromosomes of bystander cells and had led to extensive chromosomal instability. The above RIBE effects could be abrogated when conditioned media were pre-treated with agents that inactivate cfCh, namely, anti-histone antibody complexed nanoparticles (CNPs), DNase I and a novel DNA degrading agent Resveratrol-copper (R-Cu). Lower hemi-body irradiation with γ-rays (0.1-50 Gy) led to activation of H2AX, active Caspase-3, NFκB, and IL-6 in brain cells in a dose-dependent manner. Activation of these RIBE biomarkers could be abrogated by concurrent treatment with CNPs, DNase I and R-Cu indicating that activation of RIBE was not due to radiation scatter to the brain. RIBE activation was seen even when mini-beam radiation was delivered to the umbilical region of mice wherein radiation scatter to brain was negligible and could be abrogated by cfCh inactivating agents. These results indicate that cfCh released from radiation-induced dying cells are activators of RIBE and that it can be prevented by treatment with appropriate cfCh inactivating agents.

Physical shearing imparts biological activity to DNA and ability to transmit itself horizontally across species and kingdom boundaries.

BACKGROUND: We have recently reported that cell-free DNA (cfDNA) fragments derived from dying cells that circulate in blood are biologically active molecules and can readily enter into healthy cells to activate DNA damage and apoptotic responses in the recipients. However, DNA is not conventionally known to spontaneously enter into cells or to have any intrinsic biological activity. We hypothesized that cellular entry and acquisition of biological properties are functions of the size of DNA. RESULTS: To test this hypothesis, we generated small DNA fragments by sonicating high molecular weight DNA (HMW DNA) to mimic circulating cfDNA. Sonication of HMW DNA isolated from cancerous and non-cancerous human cells, bacteria and plant generated fragments 300-3000 bp in size which are similar to that reported for circulating cfDNA. We show here that while HMW DNAs were incapable of entering into cells, sonicated DNA (sDNA) from different sources could do so indiscriminately without heed to species or kingdom boundaries. Thus, sDNA from human cells and those from bacteria and plant could enter into nuclei of mouse cells and sDNA from human, bacterial and plant sources could spontaneously enter into bacteria. The intracellular sDNA associated themselves with host cell chromosomes and integrated into their genomes. Furthermore, sDNA, but not HMW DNA, from all four sources could phosphorylate H2AX and activate the pro-inflammatory transcription factor NFκB in mouse cells, indicating that sDNAs had acquired biological activities. CONCLUSIONS: Our results show that small fragments of DNA from different sources can indiscriminately enter into other cells across species and kingdom boundaries to integrate into their genomes and activate biological processes. This raises the possibility that fragmented DNA that are generated following organismal cell-death may have evolutionary implications by acting as mobile genetic elements that are involved in horizontal gene transfer.

Cell-free chromatin from dying cancer cells integrate into genomes of bystander healthy cells to induce DNA damage and inflammation.

Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell-cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3 mouse fibroblast cells, numerous cfCh emerged from them and rapidly entered into nuclei of bystander NIH3T3 cells to integrate into their genomes. This led to activation of H2AX and inflammatory cytokines NFκB, IL-6, TNFα and IFNγ. Genomic integration of cfCh triggered global deregulation of transcription and upregulation of pathways related to phagocytosis, DNA damage and inflammation. None of these activities were observed when living cancer cells were co-cultivated with NIH3T3 cells. However, upon intravenous injection into mice, both dead and live cells were found to be active. Living cancer cells are known to undergo extensive cell death when injected intravenously, and we observed that cfCh emerging from both types of cells integrated into genomes of cells of distant organs and induced DNA damage and inflammation. γH2AX and NFκB were frequently co-expressed in the same cells suggesting that DNA damage and inflammation are closely linked pathologies. As concurrent DNA damage and inflammation is a potent stimulus for oncogenic transformation, our results suggest that cfCh from dying cancer cells can transform cells of the microenvironment both locally and in distant organs providing a novel mechanism of tumor invasion and metastasis. The afore-described pro-oncogenic pathologies could be abrogated by concurrent treatment with chromatin neutralizing/degrading agents suggesting therapeutic possibilities.

Evidence for cell-free nucleic acids as continuously arising endogenous DNA mutagens.

There is extensive literature to show that nucleic acids can be taken up by cells under experimental conditions and that foetal DNA can be detected in maternal tissues. The uptaken DNA can integrate into host cell genomes and can be transcribed and translated into proteins. They can also cause chromosomal damage and karyotype alterations. Cell-free nucleic acids (cfNAs)-based non-invasive DNA diagnostic techniques are being extensively researched in the field of cancer with the potential to advance new prognostic parameters and direct treatment decisions. However, whether extracellular cfNAs that are released into circulation from dying cells as a consequence of normal physiology have any functional significance has not been explored. A recent study has demonstrated that circulating cfNAs have the ability to cause DNA damage and mutagenesis by illegitimately integrating into healthy cells of the body, thereby acting as mobile genetic elements. Fluorescently-labeled cfNAs isolated from sera of cancer patients and healthy volunteers were shown to be readily taken up by host cells followed by activation of a DNA-damage-repair-response which led their large scale integration into the host cell genomes. The latter caused dsDNA breaks and apoptosis in cells in vitro and in those of vital organs when injected intravenously into mice. Cell-free chromatin was consistently more active than cell-free DNA, while cfNAs derived from cancer patients were significantly more active than those from healthy volunteers. This study suggests that circulating extracellular cfNAs act as physiological continuously arising DNA mutagens with implications for ageing, cancer and a host of other degenerative human pathologies.

Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes.

Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

A paradoxical relationship between Resveratrol and copper (II) with respect to degradation of DNA and RNA.

Resveratrol (R), a plant polyphenol, is known to reduce Cu (II) to Cu (I) generating reactive oxygen species that can cleave plasmid DNA. Here we report a surprising observation of a paradoxical relationship between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu increased progressively as the ratio of R to Cu was increased i.e., the concentration of Cu was successively reduced with respect to a fixed concentration R. Whereas cleavage of plasmid DNA occurred at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA was achieved. By further increasing the ratio, whereby the concentration of Cu was reduced to very low levels, the DNA degrading activity of R-Cu was lost. This paradoxical relationship is also seen with respect to eukaryotic genomic DNA and RNA. Since R-Cu may have anti-cancer and anti-viral activities, our findings may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.

Analysis of scattering from complex dielectric objects using the generalized method of moments.

Integral equation-based analysis of scattering from dielectric objects has been a topic of research for many decades. Different integral equation formulations, discretization methods, and comparative data of their relative advantages have been well studied. Traditional discretization methods typically rely on a tight coupling between the underlying geometry discretization and the approximation function space that is defined on this discretization. As a result, it is difficult to stitch together different approximation spaces or nonconformal domains or match basis sets to local physics. Furthermore, the basis sets most commonly used in discretizing dielectric boundary integral operators impose limits on the variety of integral equation formulations that can be employed. We recently published a methodology [J. Opt. Soc. Am. A28, 328 (2011)10.1364/JOSAA.28.000328JOAOD61084-7529] that overcomes several of these bottlenecks. In the present paper, we introduce several extensions to these concepts for dielectric scattering problems. Specifically, we present a method that (i) uses mixed higher order local geometric descriptions and (ii) mixes multiple basis sets defined on this geometry, including higher order polynomials and classical Rao-Wilton-Glisson functions. Furthermore, we provide a unified description of different integral equation formulations that can be used for the analysis of scattering from dielectric objects, and show that the present approach admits a larger range of formulations than existing methods. A number of results demonstrating the efficiency of the method (in terms of accuracy and capability) together with applicability to different formulations are presented.

Role of muscle pedicle bone graft as an adjunct to open reduction and internal fixation in the management of neglected and ununited femoral neck fracture in young adults: a prospective study of 17 cases.

AIM: To evaluate the role of quadratus femoris muscle pedicle bone grafting along with open reduction and internal fixation in the treatment of neglected and ununited femoral neck fractures in young adults. MATERIALS AND METHODS: Young adults within the age group of 15-45 years with ununited, old neglected fractures of femoral neck were included in the study. All patients underwent quadratus femoris muscle pedicle bone grafting along with open reduction and internal fixation. The post-operative complications and functional recovery was assessed using Charnley's modification of Merle D'Aubigne and Postel hip scoring. RESULTS: A total of 17 patients were included in the study. The average time of union following the procedure was found to be approximately 8 months. There were two cases of delayed union and no cases of avascular necrosis. Ten patients (58.8%) had excellent or very good post-operative functional results and 6 patients (35.3%) had good or satisfactory results. CONCLUSION: Muscle pedicle bone grafting is a useful adjunct to open reduction and internal fixation for ununited femoral neck fractures. It achieves good final functional results comparable with other methods, but requires greater expertise.

Nucleic acids in circulation: are they harmful to the host?

It has been estimated that 10(11) -10(12) cells, primarily of haematogenous origin, die in the adult human body daily, and a similar number is regenerated to maintain homeostasis. Despite the presence of an efficient scavenging system for dead cells, considerable amounts of fragmented genetic material enter the circulation in healthy individuals. Elevated blood levels of extracellular nucleic acids have been reported in various disease conditions; such as ageing and age-related degenerative disorders, cancer; acute and chronic inflammatory conditions, severe trauma and autoimmune disorders. In addition to genomic DNA and nucleosomes, mitochondrial DNA is also found in circulation, as are RNA and microRNA. There is extensive literature that suggests that extraneously added nucleic acids have biological actions. They can enter into cells in vitro and in vivo and induce genetic transformation and cellular and chromosomal damage; and experimentally added nucleic acids are capable of activating both innate and adaptive immune systems and inducing a sterile inflammatory response. The possibility as to whether circulating nucleic acids may, likewise, have biological activities has not been explored. In this review we raise the question as to whether circulating nucleic acids may have damaging effects on the host and be implicated in ageing and diverse acute and chronic human pathologies.

Generalized method of moments: a framework for analyzing scattering from homogeneous dielectric bodies.

In this paper, we present a novel framework for discretizing integral equations--specifically, those used for analyzing scattering from dielectric bodies. The candidate integral equations chosen for the analysis are the well-known Poggio-Miller-Chang-Harrington-Wu-Tsai (PMCHWT) and the Müller equations. Discrete solutions to these equations are typically obtained by representing the spatial variation of the currents using the Rao-Wilton-Glisson (RWG) basis functions or their higher order equivalents. In this paper, we propose a framework for defining basis functions that departs significantly from those of RWG functions in that approximation functions can be chosen independent of continuity constraints. We will show that using this framework together with a quasi-Helmholtz type representation has a number of benefits. Namely, (i) it shows excellent convergence, (ii) it permits inclusion of different orders of polynomials or different functions as basis functions without imposition of additional constraints, (iii) the method can easily handle nonconformal meshes, and (iv) the method is well conditioned at all frequencies. These features will be demonstrated via a number of numerical experiments.

Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants.

The genetic aspects of oriC replication initiation in Mycobacterium tuberculosis are largely unknown. A two-step genetic screen was utilized for isolating M. tuberculosis dnaA cold-sensitive (cos) mutants. First, a resident plasmid expressing functional dnaA integrated at the attB locus in dnaA null background was exchanged with an incoming plasmid bearing a mutagenized dnaA gene. Next, the mutants that were defective for growth at 30 degrees C, a non-permissive temperature, but resumed growth and DNA synthesis when shifted to 37 degrees C, a permissive temperature, were subsequently selected. Nucleotide sequencing analysis located mutations to different regions of the dnaA gene. Modulation of the growth temperatures led to synchronized DNA synthesis. The dnaA expression under synchronized DNA replication conditions continued to increase during the replication period, but decreased thereafter reflecting autoregulation. The dnaAcos mutants at 30 degrees C were elongated suggesting that they may possibly be blocked during the cell division. The DnaA115 protein is defective in its ability to interact with ATP at 30 degrees C, but not at 37 degrees C. Our results suggest that the optimal cell cycle progression and replication initiation in M. tuberculosis requires that the dnaA promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.

Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two-component response regulator.

Paired two-component regulatory systems consisting of a sensor kinase and a response regulator are the major means by which bacteria sense and respond to different stimuli. The role of essential response regulator, MtrA, in Mycobacterium tuberculosis proliferation is unknown. We showed that elevating the intracellular levels of MtrA prevented M. tuberculosis from multiplying in macrophages, mice lungs and spleens, but did not affect its growth in broth. Intracellular trafficking analysis revealed that a vast majority of MtrA overproducing merodiploids were associated with lysosomal associated membrane protein (LAMP-1) positive vacuoles, indicating that intracellular growth attenuation is, in part, due to an impaired ability to block phagosome-lysosome fusion. A merodiploid strain producing elevated levels of phosphorylation-defective MtrA (MtrA(D53N)) was partially replicative in macrophages, but was attenuated in mice. Quantitative real-time PCR analyses revealed that expression of dnaA, an essential replication gene, was sharply upregulated during intramacrophage growth in the MtrA overproducer in a phosphorylation-dependent manner. Chromatin immunoprecipitation using anti-MtrA antibodies provided direct evidence that MtrA regulator binds to dnaA promoter in vivo indicating that dnaA promoter is a MtrA target. Simultaneous overexpression of mtrA regulator and its cognate mtrB kinase neither inhibited growth nor sharply increased the expression levels of dnaA in macrophages. We propose that proliferation of M. tuberculosis in vivo depends, in part, on the optimal ratio of phosphorylated to non-phosphorylated MtrA response regulator.