Membrane insertion mechanism of the caveola coat protein Cavin1.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Topological Persistence Guided Knowledge Distillation for Wearable Sensor Data.

Deep learning methods have achieved a lot of success in various applications involving converting wearable sensor data to actionable health insights. A common application areas is activity recognition, where deep-learning methods still suffer from limitations such as sensitivity to signal quality, sensor characteristic variations, and variability between subjects. To mitigate these issues, robust features obtained by topological data analysis (TDA) have been suggested as a potential solution. However, there are two significant obstacles to using topological features in deep learning: (1) large computational load to extract topological features using TDA, and (2) different signal representations obtained from deep learning and TDA which makes fusion difficult. In this paper, to enable integration of the strengths of topological methods in deep-learning for time-series data, we propose to use two teacher networks - one trained on the raw time-series data, and another trained on persistence images generated by TDA methods. These two teachers are jointly used to distill a single student model, which utilizes only the raw time-series data at test-time. This approach addresses both issues. The use of KD with multiple teachers utilizes complementary information, and results in a compact model with strong supervisory features and an integrated richer representation. To assimilate desirable information from different modalities, we design new constraints, including orthogonality imposed on feature correlation maps for improving feature expressiveness and allowing the student to easily learn from the teacher. Also, we apply an annealing strategy in KD for fast saturation and better accommodation from different features, while the knowledge gap between the teachers and student is reduced. Finally, a robust student model is distilled, which can at test-time uses only the time-series data as an input, while implicitly preserving topological features. The experimental results demonstrate the effectiveness of the proposed method on wearable sensor data. The proposed method shows 71.74% in classification accuracy on GENEActiv with WRN16-1 (1D CNNs) student, which outperforms baselines and takes much less processing time (less than 17 sec) than teachers on 6k testing samples.

Gender roles perceptions and ideal number of children: case study of Emirati youth.

BACKGROUND: The United Arab Emirates (UAE) is a traditional society with patriarchal values. The country has been experiencing a decline in fertility rates, bringing the total fertility rate for the national population to 3.3 children per woman, the lowest since 1970s. Existing literature indicates that having gender-egalitarian attitudes is associated with lower fertility rates. Therefore, this study aims to investigate the perceptions of gender roles among the highly educated Emirati youth and examine whether these attitudes influence their desire to have children. By doing so, we aim to gain insights into the factors contributing to the country's declining fertility rates. METHODS: This study utilized data from a cross-sectional quantitative survey. The survey was developed and administered in February-March 2019 to a purposive sample of 300 young Emirati males and females aged 18-30 years, studying at the UAE University. Both bivariate and multivariate analysis were performed to examine the levels of youths' perception towards gender roles and desired fertility. RESULTS: The data collected from Emirati youths revealed that 50% of them supported the traditional perspective on marriage, 30% considered motherhood is the most important thing for women, and a small percentage supported economic independence of women and husband participation in household chores/child-rearing. On average Emirati youth ideally wished to have 5.4 children in their future families, with a preference for sons over daughters. Youths who favoured women's economic independence and equal participation in household work contribution by spouses desired a lower number of children which is in line with the modernization theory and cultural evolution. CONCLUSION: The UAE has been facing continuous decline in fertility rates. The study indicates that ideal number of children is much higher than the current fertility rates in the country. To bridge this gap, the government could implement family policies that create an environment conducive to fulfilling the ideal desires of young Emiratis regarding family size. Moreover, present findings indicate that perceptions of equal gender roles could be a contributing factor to the declining fertility rates among the young generation. Promoting gender equality attitudes and strengthening institutional support for childbearing could become key strategy to address these issues.

Constrained Adaptive Distillation Based on Topological Persistence for Wearable Sensor Data.

Wearable sensor data analysis with persistence features generated by topological data analysis (TDA) has achieved great successes in various applications, however, it suffers from large computational and time resources for extracting topological features. In this paper, our approach utilizes knowledge distillation (KD) that involves the use of multiple teacher networks trained with the raw time-series and persistence images generated by TDA, respectively. However, direct transfer of knowledge from the teacher models utilizing different characteristics as inputs to the student model results in a knowledge gap and limited performance. To address this problem, we introduce a robust framework that integrates multimodal features from two different teachers and enables a student to learn desirable knowledge effectively. To account for statistical differences in multimodalities, entropy based constrained adaptive weighting mechanism is leveraged to automatically balance the effects of teachers and encourage the student model to adequately adopt the knowledge from two teachers. To assimilate dissimilar structural information generated by different style models for distillation, batch and channel similarities within a mini-batch are used. We demonstrate the effectiveness of the proposed method on wearable sensor data.

M. tuberculosis class II apurinic/ apyrimidinic-endonuclease/3'-5' exonuclease (XthA) engages with NAD+-dependent DNA ligase A (LigA) to counter futile cleavage and ligation cycles in base excision repair.

Class-II AP-endonuclease (XthA) and NAD+-dependent DNA ligase (LigA) are involved in initial and terminal stages of bacterial DNA base excision repair (BER), respectively. XthA acts on abasic sites of damaged DNA to create nicks with 3'OH and 5'-deoxyribose phosphate (5'-dRP) moieties. Co-immunoprecipitation using mycobacterial cell-lysate, identified MtbLigA-MtbXthA complex formation. Pull-down experiments using purified wild-type, and domain-deleted MtbLigA mutants show that LigA-XthA interactions are mediated by the BRCT-domain of LigA. Small-Angle-X-ray scattering, 15N/1H-HSQC chemical shift perturbation experiments and mutational analysis identified the BRCT-domain region that interacts with a novel 104DGQPSWSGKP113 motif on XthA for complex-formation. Isothermal-titration calorimetry experiments show that a synthetic peptide with this sequence interacts with MtbLigA and disrupts XthA-LigA interactions. In vitro assays involving DNA substrate and product analogs show that LigA can efficiently reseal 3'OH and 5'dRP DNA termini created by XthA at abasic sites. Assays and SAXS experiments performed in the presence and absence of DNA, show that XthA inhibits LigA by specifically engaging with the latter's BRCT-domain to prevent it from encircling substrate DNA. Overall, the study suggests a coordinating function for XthA whereby it engages initially with LigA to prevent the undesirable consequences of futile cleavage and ligation cycles that might derail bacterial BER.

Quality of care in abortion in the era of technological and medical advancements and self-care.

BACKGROUND: Discussions around quality of abortion care have been focused mainly on service-delivery aspects inside healthcare facilities. More recently, with availability of medical abortion (MA), increase in its self-use, and emergence of other delivery platforms such as telemedicine, the responsibility of quality care has broadened to actors outside of facilities. BODY OF TEXT: This commentary discusses the meaning of quality of abortion care with the paradigm shift brought by medical and technological advancement in abortions, and raises questions on the role of the state in ensuring quality in abortion management-especially in settings where abortion is decriminalized, but also in countries where abortion is permitted under certain circumstances. It consolidates the experience gained thus far in the provision of safe abortion services and also serves as a forward-thinking tool to keep pace with the uptake of newer health technologies (e.g., availability of medical abortion drugs), service delivery platforms (e.g., telemedicine, online pharmacies), and abortion care providers (e.g., community based pharmacists). CONCLUSIONS: This commentary provides context and rationale, and identifies areas for action that different stakeholders, including health advocates, policymakers, program managers, and women themselves, can adopt to fit into an alternative regime of abortion care.

Machine-Learning Approach for Automatic Detection of Wild Beluga Whales from Hand-Held Camera Pictures.

A key aspect of ocean protection consists in estimating the abundance of marine mammal population density within their habitat, which is usually accomplished using visual inspection and cameras from line-transect ships, small boats, and aircraft. However, marine mammal observation through vessel surveys requires significant workforce resources, including for the post-processing of pictures, and is further challenged due to animal bodies being partially hidden underwater, small-scale object size, occlusion among objects, and distracter objects (e.g., waves, sun glare, etc.). To relieve the human expert's workload while improving the observation accuracy, we propose a novel system for automating the detection of beluga whales (Delphinapterus leucas) in the wild from pictures. Our system relies on a dataset named Beluga-5k, containing more than 5.5 thousand pictures of belugas. First, to improve the dataset's annotation, we have designed a semi-manual strategy for annotating candidates in images with single (i.e., one beluga) and multiple (i.e., two or more belugas) candidate subjects efficiently. Second, we have studied the performance of three off-the-shelf object-detection algorithms, namely, Mask-RCNN, SSD, and YOLO v3-Tiny, on the Beluga-5k dataset. Afterward, we have set YOLO v3-Tiny as the detector, integrating single- and multiple-individual images into the model training. Our fine-tuned CNN-backbone detector trained with semi-manual annotations is able to detect belugas despite the presence of distracter objects with high accuracy (i.e., 97.05 mAP@0.5). Finally, our proposed method is able to detect overlapped/occluded multiple individuals in images (beluga whales that swim in groups). For instance, it is able to detect 688 out of 706 belugas encountered in 200 multiple images, achieving 98.29% precision and 99.14% recall.

Structure based identification of first-in-class fragment inhibitors that target the NMN pocket of M. tuberculosis NAD+-dependent DNA ligase A.

NAD+-dependent DNA ligase (LigA) is the essential replicative ligase in bacteria and differs from ATP-dependent counterparts like the human DNA ligase I (HligI) in several aspects. LigA uses NAD+ as the co-factor while the latter uses ATP. Further, the LigA carries out enzymatic activity with a single divalent metal ion in the active site while ATP-dependent ligases use two metal ions. Instead of the second metal ion, LigA have a unique NMN binding subdomain that facilitates the orientation of the ß-phosphate and NMN leaving group. LigA are therefore attractive targets for new anti-bacterial therapeutic development. Others and our group have earlier identified several LigA inhibitors that mainly bind to AMP binding site of LigA. However, no inhibitor is known to bind to the unique NMN binding subdomain. We initiated a fragment inhibitor discovery campaign against the M. tuberculosis LigA based on our co-crystal structure of adenylation domain with AMP and NMN. The study identified two fragments, 4-(4-fluorophenyl)-4,5,6,7-tetrahydro-3H imidazo[4,5-c] pyridine and N-(4-methylbenzyl)-1H-pyrrole-2-carboxamide, that bind to the NMN site. The fragments inhibit LigA with IC50 of 16.9 and 28.7 µM respectively and exhibit MIC of ~20 and 60 µg/ml against a temperature sensitive E. coli GR501 ligAts strain, rescued by MtbLigA. Co-crystal structures of the fragments with the adenylation domain of LigA show that they mimic the interactions of NMN. Overall, our results suggest that the NMN binding-site is a druggable target site for developing anti-LigA therapeutic strategies.

Structural-functional diversity of malaria parasite's PfHSP70-1 and PfHSP40 chaperone pair gives an edge over human orthologs in chaperone-assisted protein folding.

Plasmodium falciparum, the human malaria parasite harbors a metastable proteome which is vulnerable to proteotoxic stress conditions encountered during its lifecycle. How parasite's chaperone machinery is able to maintain its aggregation-prone proteome in functional state, is poorly understood. As HSP70-40 system forms the central hub in cellular proteostasis, we investigated the protein folding capacity of PfHSP70-1 and PfHSP40 chaperone pair and compared it with human orthologs (HSPA1A and DNAJA1). Despite the structural similarity, we observed that parasite chaperones and their human orthologs exhibit striking differences in conformational dynamics. Comprehensive biochemical investigations revealed that PfHSP70-1 and PfHSP40 chaperone pair has better protein folding, aggregation inhibition, oligomer remodeling and disaggregase activities than their human orthologs. Chaperone-swapping experiments suggest that PfHSP40 can also efficiently cooperate with human HSP70 to facilitate the folding of client-substrate. SPR-derived kinetic parameters reveal that PfHSP40 has higher binding affinity towards unfolded substrate than DNAJA1. Interestingly, the observed slow dissociation rate of PfHSP40-substrate interaction allows PfHSP40 to maintain the substrate in folding-competent state to minimize its misfolding. Structural investigation through small angle x-ray scattering gave insights into the conformational architecture of PfHSP70-1 (monomer), PfHSP40 (dimer) and their complex. Overall, our data suggest that the parasite has evolved functionally diverged and efficient chaperone machinery which allows the human malaria parasite to survive in hostile conditions. The distinct allosteric landscapes and interaction kinetics of plasmodial chaperones open avenues for the exploration of small-molecule based antimalarial interventions.

Contraception for married adolescents (15-19 years) in India: insights from the National Family Health Survey-4 (NFHS-4).

PURPOSE: Despite the fact that marriage below the age of 18 years is illegal in India, a considerable number of females get married and start childbearing during their adolescent years. There is low prevalence of contraceptive methods and high unmet need for family planning (FP). Realizing this, new government programs have been launched to increase the uptake of sexual and reproductive health services among adolescents. However, evidence specific to this age group remains scarce. AIM AND OBJECTIVES: The present study was conducted to assess the prevalence of modern contraceptives among married adolescents, and to determine its association with sociodemographic variables, health worker outreach, and media exposure to FP messages in India. METHODS: Data for this analysis was drawn from the fourth round of the National Family Health Survey (NFHS-4) conducted in India during 2015-16. The sample size is restricted to 13,232 currently married adolescent girls aged 15-19 years, who were not pregnant at the time of the survey. Bivariate and multivariate analysis were conducted to assess the levels of contraceptive use and its predictors among married adolescents. RESULTS: The use of modern contraceptives among married adolescents increased from 4 to 10% between 1992-93 and 2015-16. The uptake of modern contraceptives was found to be low among the uneducated, those residing in rural areas, among backward classes, those practising Hindu religion, women in the poorest wealth quintile, women without children, and those with no exposure to FP messages via media or health care workers. Among those who met health care workers and discussed FP issues with them, 34.11% were using modern contraceptives as compared to 11.53% of those who did not have discussions with health care workers. CONCLUSIONS: The evidence suggests that contact with health care workers significantly influences the use of modern contraceptives. Further focus on increasing contact between married adolescents' and health care workers, and improving the quality of counselling will protect adolescents from early marriage and pregnancy.

Complications related to early pregnancies and childbirth are a leading cause of death among adolescent girls. Still, a considerable number of young girls in India get married and start childbearing in adolescence. Data shows that 9% of the girls aged 15­19 years in rural areas and 5% in urban areas have already begun childbearing. The World Health Organisation (WHO) recommends access to high-quality sexual and reproductive health information and services as a fundamental right of adolescents. In recent years, the Indian government has also taken several initiatives to cater to the needs of adolescents.The present study focuses on the levels of contraceptive use and the influence of health care workers and media outreach on the uptake of modern contraceptives among married adolescents in India.Findings from the present study suggest that though there has been an increase in the use of modern contraceptives among married adolescents over time, overall the level of contraceptive use is very low. There is visible variation in the use of modern contraceptives depending on the educational status, residence, religion, caste, and household wealth of adolescent girls. Contact of married adolescents with health care workers positively influences the uptake of modern contraceptives. However, the proportion of married adolescents who had contact with health care workers is very low.

Share of current unmet need for modern contraceptive methods attributed to past users of these methods in India.

Despite persistent efforts, unmet need for contraceptives in India has declined only slightly from 14% to 13% between 2005-06 and 2015-16. Many women using a family planning method discontinue it without switching to another method and continue to have unmet need. This study quantified the share of current unmet need for modern contraceptive methods attributed to past users of these methods in India. Data were drawn from two rounds of the National Family Health Survey conducted in 2005-06 and 2015-16. Using information on women with current unmet need, and whether they used any modern method in the past, the share of past users with current unmet need for modern methods was calculated. Bivariate and multivariate analyses were performed. Among 46 million women with unmet need, 11 million were past users of modern methods in 2015-16. The share of current unmet need attributed to past users of modern contraceptive methods declined from 27% in 2005-06 to 24% in 2015-16. Share of current unmet need attributed to past users was associated with reversible method use. This share rose with increased use of modern reversible methods. With the Indian family planning programme's focus on increasing modern reversible method use, the share of unmet need attributed to past users of modern methods is likely to increase in the future. The programme's emphasis on continuation of contraceptive use, along with bringing in new users, could be one of the key strategies for India to achieve the FP2020 goals.

Services for women's sexual and reproductive health in India: an analysis of treatment-seeking for symptoms of reproductive tract infections in a nationally representative survey.

BACKGROUND: Women's health policy in India has had a longstanding focus on maternal health and family planning. Recent policy highlights the importance of expanding women's access to a broader range of sexual and reproductive health services. However, there has been very limited analysis of national survey data to examine the current status of treatment utilisation, variation across states and progress over time. METHODS: This paper examines women's treatment patterns for reproductive tract infections in India, based on data collected in the National Family Health Survey, a cross-sectional, nationally representative household survey conducted between 2015-16. The survey covered 699,686 women between the ages 15 and 49, of which 91,818 ever sexually active women responded to questions related to symptoms of reproductive tract infections. We estimate prevalence of reported symptoms and treatment-seeking, describe regional variation and utilise multivariable logistic regression to identify factors associated with women's treatment-seeking patterns. RESULTS: Thirty-nine percent of women who reported symptoms of reproductive tract infections sought any advice or treatment. Women's reported treatment-seeking in India has not changed since the last national survey a decade earlier. Reported symptoms and treatment-seeking varied widely across India, ranging from 64% in Punjab to 8% in Nagaland, with no clear regional pattern that emerged. Seventeen percent of symptomatic women sought services in the public sector, an improvement from 11% in 2005-06. Twenty-two percent utilised the private sector, with wide variation by states. National-level multivariable logistic regression indicated that treatment-seeking was associated with age, higher education, higher household wealth and having been employed in the past year. Women in the 25-35 age group had higher odds (aOR1.27; 95% CI: 1.10,1.50) of seeking treatment compared to both younger (15-19 years) and older (35 years and above) women, along with women with more than eight years of schooling (aOR: 1.23; 95% CI: 1.05,1.44) and from richer wealth quintiles (aOR: 1.53; 95% CI: 1.35,1.83). CONCLUSION: Women's use of services for reproductive tract infections remains a challenge in most parts of India. Our findings highlight the need to address barriers to seeking care and to improve measurement of gynaecological ailments in national surveys.

Health workers' outreach and intention to use contraceptives among married women in India.

BACKGROUND: The influence of health workers on uptake of maternal healthcare services is well documented; however, their outreach for family planning (FP) services and influence on the intention to use contraceptives is less explored in the Indian context. This study examined the extent of health worker outreach for FP service and its effects on intention to use contraceptives among currently married women aged 15-49 years. METHODS: This study used data from two rounds of the National Family Health Survey (NFHS) of India, conducted during 2005-06 and 2015-16 respectively. Bivariate analysis and multivariate logistic regression were used to understand the level of and change in health worker outreach for FP services over time, and its association with intention to use contraceptives among currently married women. RESULTS: In the past 10 years, health workers' outreach for FP service has significantly increased by about 10 percentage points, although the level is not optimal and only 28% of non-users were reached by health workers in 2015-16. Increase in the outreach to younger and low parity women was higher than their respective counterparts. Intention to use contraceptive among women who were not using any method was 41% when health workers contacted and discussed FP, compared to only 20% when there was no such contact with health workers. Multivariable analysis suggests that contact with health workers has significant positive effects on intention to use contraceptive (AOR = 3.05; p < 0.001; 95% CI 2.85-3.27). CONCLUSION: Increased scope of outreach of frontline health workers to provide FP communication and services will not only help in building knowledge of contraceptive methods but will also increase women's intention to use a method. For India, this may be the most promising way to achieve the Sustainable Development Goals 3.7, which calls for universal access to reproductive health services.

Is economic inequality in family planning in India associated with the private sector?

This study examined the pattern of economic disparity in the modern contraceptive prevalence rate (mCPR) among women receiving contraceptives from the public and private health sectors in India, using data from all four rounds of the National Family Health Survey conducted between 1992-93 and 2015-16. The mCPR was measured for currently married women aged 15-49 years. A concentration index was calculated and a pooled binary logistic regression analysis conducted to assess economic disparity (by household wealth quintiles) in modern contraceptive use between the public and private health sectors. The analyses were stratified by rural-urban place of residence. The results indicated that mCPR had increased in India over time. However, in 2015-16 only half of women - 48% (33% from the public sector, 12% from the private sector, 3% from other sources) - were using any modern contraceptive in India. Over time, the economic disparity in modern contraceptive use reduced across both public and private health sectors. However, the extent of the disparity was greater when women obtained the services from the private sector: the value of the concentration index for mCPR was 0.429 when obtained from the private sector and 0.133 when from the public sector in 2015-16. Multivariate analysis confirmed a similar pattern of the economic disparity across public and private sectors. Economic disparity in the mCPR has reduced considerably in India. While the economic disparity in 2015-16 was minimal among those accessing contraceptives from the public sector, it continued to exist among those receiving services from the private sector. While taking appropriate steps to plan and monitor private sector services for family planning, continued and increased engagement of public providers in the family planning programme in India is required to further reduce the economic disparity among those accessing contraceptive services from the private sector.

CD11a expression distinguishes infiltrating myeloid cells from plaque-associated microglia in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of age-related neurodegeneration and is characterized neuropathologically by the accumulation of insoluble beta-amyloid (Aß) peptides. In AD brains, plaque-associated myeloid (PAM) cells cluster around Aß plaques but fail to effectively clear Aß by phagocytosis. PAM cells were originally thought to be brain-resident microglia. However, several studies have also suggested that Aß-induced inflammation causes peripheral monocytes to enter the otherwise immune-privileged brain. The relationship between AD progression and inflammation in the brain remains ambiguous because microglia and monocyte-derived macrophages are extremely difficult to distinguish from one another in an inflamed brain. Whether PAM cells are microglia, peripheral macrophages, or a mixture of both remains unclear. CD11a is a component of the ß2 integrin LFA1. We have determined that CD11a is highly expressed on peripheral immune cells, including macrophages, but is not expressed by mouse microglia. These expression patterns remain consistent in LPS-treated inflamed mice, as well as in two mouse models of AD. Thus, CD11a can be used as a marker to distinguish murine microglia from infiltrating peripheral immune cells. Using CD11a, we show that PAM cells in AD transgenic brains are comprised entirely of microglia. We also demonstrate a novel fluorescence-assisted quantification technique (FAQT), which reveals a significant increase in T lymphocytes, especially in the brains of female AD mice. Our findings support the notion that microglia are the lead myeloid players in AD and that rejuvenating their phagocytic potential may be an important therapeutic strategy.

Critical determinants for substrate recognition and catalysis in the M. tuberculosis class II AP-endonuclease/3'-5' exonuclease III.

The Mycobacterium tuberculosis AP-endonuclease/3'-5' exodeoxyribonuclease (MtbXthA) is an important player in DNA base excision repair (BER). We demonstrate that the enzyme has robust apurinic/apyrimidinic (AP) endonuclease activity, 3'-5' exonuclease, phosphatase, and phosphodiesterase activities. The enzyme functions as an AP-endonuclease at high ionic environments, while the 3'-5'-exonuclease activity is predominant at low ionic environments. Our molecular modelling and mutational experiments show that E57 and D251 are critical for catalysis. Although nicked DNA and gapped DNA are fair substrates of MtbXthA, the gap-size did not affect the excision activity and furthermore, a substrate with a recessed 3'-end is preferred. To understand the determinants of abasic-site recognition, we examined the possible roles of (i) the base opposite the abasic site, (ii) the abasic ribose ring itself, (iii) local distortions in the AP-site, and (iv) conserved residues located near the active site. Our experiments demonstrate that the first three determinants do not play a role in MtbXthA, and in fact the enzyme exhibits robust endonucleolytic activity against single-stranded AP DNA also. Regarding the fourth determinant, it is known that the catalytic-site of AP endonucleases is surrounded by conserved aromatic residues and intriguingly, the exact residues that are directly involved in abasic site recognition vary with the individual proteins. We therefore, used a combination of mutational analysis, kinetic assays, and structure-based modelling, to identify that Y237, supported by Y137, mediates the formation of the MtbXthA-AP-DNA complex and AP-site incision.

Tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can specifically target bacterial DNA ligases and can distinguish them from human DNA ligase I.

DNA ligases are critical components for DNA metabolism in all organisms. NAD(+)-dependent DNA ligases (LigA) found exclusively in bacteria and certain entomopoxviruses are drawing increasing attention as therapeutic targets as they differ in their cofactor requirement from ATP-dependent eukaryotic homologs. Due to the similarities in the cofactor binding sites of the two classes of DNA ligases, it is necessary to find determinants that can distinguish between them for the exploitation of LigA as an anti-bacterial target. In the present endeavour, we have synthesized and evaluated a series of tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives for their ability to distinguish between bacterial and human DNA ligases. The in vivo inhibition assays that employed LigA deficient E. coli GR501 and S. typhimurium LT2 bacterial strains, rescued by ATP-dependent T4 DNA ligase or Mycobacterium tuberculosis NAD(+)-dependent DNA ligase (Mtb LigA), respectively, showed that the compounds can specifically inhibit bacterial LigA. The in vitro enzyme inhibition assays using purified MtbLigA, human DNA ligase I & T4 DNA ligase showed specific inhibition of MtbLigA at low micromolar range. Our results demonstrate that tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can distinguish between bacterial and human DNA ligases by ∼5-folds. In silico docking and enzyme inhibition assays identified that the compounds bind to the cofactor binding site and compete with the cofactor. Ethidium bromide displacement and gel-shift assays showed that the inhibitors do not exhibit any unwanted general interactions with the substrate DNA. These results set the stage for the detailed exploration of this compound class for development as antibacterials.