Chemical technology principles for selective bioconjugation of proteins and antibodies.

Proteins are multifunctional large organic compounds that constitute an essential component of a living system. Hence, control over their bioconjugation impacts science at the chemistry-biology-medicine interface. A chemical toolbox for their precision engineering can boost healthcare and open a gateway for directed or precision therapeutics. Such a chemical toolbox remained elusive for a long time due to the complexity presented by the large pool of functional groups. The precise single-site modification of a protein requires a method to address a combination of selectivity attributes. This review focuses on guiding principles that can segregate them to simplify the task for a chemical method. Such a disintegration systematically employs a multi-step chemical transformation to deconvolute the selectivity challenges. It constitutes a disintegrate (DIN) theory that offers additional control parameters for tuning precision in protein bioconjugation. This review outlines the selectivity hurdles faced by chemical methods. It elaborates on the developments in the perspective of DIN theory to demonstrate simultaneous regulation of reactivity, chemoselectivity, site-selectivity, modularity, residue specificity, and protein specificity. It discusses the progress of such methods to construct protein and antibody conjugates for biologics, including antibody-fluorophore and antibody-drug conjugates (AFCs and ADCs). It also briefs how this knowledge can assist in developing small molecule-based covalent inhibitors. In the process, it highlights an opportunity for hypothesis-driven routes to accelerate discoveries of selective methods and establish new targetome in the precision engineering of proteins and antibodies.

Utilization of modern menstrual methods and related unmet needs among college going women in Coimbatore district: a descriptive cross-sectional study.

OBJECTIVES: The primary objectives were to determine the proportion of modern menstrual method (MMM) users among college going women in Coimbatore district, Tamil Nadu; and to estimate the unmet needs associated with use of MMMs in comparison with other menstrual hygiene methods (MHMs). We also assessed the factors that determine MMM use among college going women. METHODS: This was a descriptive cross-sectional study conducted among college going women in Coimbatore district, Tamil Nadu, India between October 2022 and January 2023 using a purpose predesigned, pretested, semi-structured proforma that included validated Menstrual Practice Needs Scale (MPNS-36). RESULTS: Only 1.4% of the study participants used MMMs - menstrual cups (1.3%) and tampons (0.1%). Sanitary pads were the most common MHM of choice (96.3%); of which majority (98.6%) used disposable pads and more than half (50.4%) used non-biodegradable pads. Importantly, one in six (16.5%) were not aware of nature of sanitary pads (biodegradable or nonbiodegradable) used. The unmet needs associated with MMMs (menstrual cups and tampons) were significantly lower than that for other MHMs (including sanitary pads), in particular, the unmet material and home environment needs, unmet material reliability concerns, unmet reuse needs and unmet reuse insecurity. However, we found no significant difference between MMMs, sanitary pads and other MHMs in terms of unmet transport, college environment, change and disposal insecurity needs. The significant predictors of use of MMMs were age (more than 21 years of age), residence (urban), type of stay (off campus including home), socioeconomic status (upper), fathers' and mothers' education (high school and above), and presence of personal income. Discussions with friends (or peers) both before and after menarche regarding menstruation resulted in higher adoption of modern menstrual methods. CONCLUSION: MMMs provided comparative advantage with lesser unmet needs for material reliability and reuse insecurity concerns, particularly in home environment. However, none of the MHMs fulfilled the user expectations for transport and disposal insecurity concerns, particularly outdoors.

Depression among older adults in an urban slum of Raipur city - a community based cross-sectional study.

BACKGROUND: Older adults are at risk of chronic, silent depressive changes and the vulnerability of older adults in urban slums of India is rarely exposed. The objective of this study was to estimate the prevalence of depression among the older adults in the urban slums of India and to study the factors associated with it. METHODS: This was a community based analytical cross-sectional study conducted in Urban Field Practice Area of a tertiary care teaching hospital in Chhattisgarh, India among older adults more than or equal to 60 years of age selected using two stage, simple random sampling. The data was collected in a sample of 400 older adults by face-to-face interview using self-designed, semi-structured and pretested proforma that included validated Hindi version of Geriatric Depression Scale (GDS-15) and analyzed using SPSS v23. RESULTS: The prevalence of depression among older adults was 51.5% in the present study; with 27%, 12.8% and 11.8% having mild, moderate and severe depression respectively. Number of family members, living status of spouse, emotional attachment to family members, conflict in family, loneliness, social isolation, marginal friendship ties, functional status, physical exercise, active complains and diastolic BP were independent predictors of depression in older adults. CONCLUSION: Early identification of depression in older adults using GDS-15 and incorporation of social isolation and functionality assessment routinely by healthcare providers for all older adults attending the outpatient departments is the need of the hour. A holistic approach to care of older adults is vital as healthcare providers seek to understand the impact of multiple, complex, interconnected factors on overall health and well-being of older adults.

Functioning of a Nutritional Rehabilitation Center against acceptable levels of care.

Background: Establishment of Nutritional Rehabilitation Centers (NRCs) is an initiative under National Rural Health Mission to provide institutional care for children with severe acute malnutrition (SAM). The aim of the study was to assess the effectiveness of nutritional interventional measures in improving the nutritional status of children admitted to an NRC. Methods: A retrospective health facility-based descriptive study was conducted in the NRC, Rural Health Training Center, All India Institute of Medical Sciences, Raipur. Results: A total of 765 children were enrolled in the NRC between March 2015 and November 2019, and majority (87.97%) were admitted as per weight for height/length (<-3SD) criteria. The total number of SAM children with complications were 428 (55.94%). Of the 724 total discharges, 498 (68.78%) were cured, 197 (27.2%) were nonresponders and 28 (3.87%) were defaulters. Conclusion: The findings suggest factors affecting nutritional rehabilitation that are complex and require a more integrated management in the health system and community. Regular review, supportive supervision and identification of nutrient-dense food from locally available low-cost ingredients is the need of the hour.

Prevalence and determinants of social isolation among elderly in an urban slum of Raipur city-A community based cross-sectional study.

BACKGROUND: Social isolation is a predictor of mortality and morbidity, as well as an outcome of many health-related predictors. The aim of this study was to estimate the prevalence of social isolation among the elderly and to study factors associated with it. METHODS: This was an analytical cross-sectional study conducted in Urban Field Practice Area of a tertiary care teaching hospital in Chhattisgarh, India among elderly more than or equal to 60 years of age selected using random sampling. The data was collected in a sample of 400 elderly by face-to-face interview using predesigned, pretested, and semi-structured proforma that included Lubben Social Network Scale-6 (LSNS-6) and analyzed using Stata v16. RESULTS: The prevalence of social isolation was 34.3%; highest among those 65-74 years of age (44.5%) and females (56.9%). Increasing age, elderly with less than three family members, lower socio-economic status, no children and financial dependence among socio-demographic factors; insufficient time with children, lack of emotionally attachment and conflict in the family among family support factors; lack of social participation, poor perception of health and feeling lonely among psychosocial factors; lack of physical exercise, spiritual activity and sound sleep among lifestyle factors; and presence of active complaints, raised blood pressure and being anemic among medical factors were significantly associated with social isolation. Overall, the independent predictors of social isolation in elderly were age ≥75 years, lack of participation in social functions, feeling lonely and lack of sound sleep. CONCLUSION: The prevalence of social isolation among elderly from an urban slum in India was high and the existing evidence show a rising trend. Social isolation assessment and use of LSNS-6 by healthcare providers should be incorporated into elderly care at primary healthcare delivery points across the country.

Gestational exposure to titanium dioxide, diesel exhaust, and concentrated urban air particles affects levels of specialized pro-resolving mediators in response to allergen in asthma-susceptible neonate lungs.

Maternal gestational exposures to traffic and urban air pollutant particulates have been linked to increased risk and/or worsening asthma in children; however, mechanisms underlying this vertical transmission are not entirely understood. It was postulated that gestational particle exposure might affect the ability to elicit specialized proresolving mediator (SPM) responses upon allergen encounter in neonates. Lipidomic profiling of 50 SPMs was performed in lungs of neonates born to mice exposed to concentrated urban air particles (CAP), diesel exhaust particles (DEP), or less immunotoxic titanium dioxide particles (TiO2). While asthma-like phenotypes were induced with identical eosinophilia intensity across neonates of all particle-exposed mothers, levels of LXA4, HEPE and HETE isoforms, and HDoHe were only decreased by CAP and DEP only but not by TiO2. However, RvE2 and RvD1 were inhibited by all particles. In contrast, isomers of Maresin1 and Protectin D1 were variably elevated by CAP and DEP, whereas Protectin DX, PGE2, and TxB2 were increased in all groups. Only Protectin D1/DX, MaR1(n-3,DPA), 5(S),15(S)-DiHETE, PGE2, and RvE3 correlated with eosinophilia but the majority of other analytes, elevated or inhibited, showed no marked correlation with inflammation intensity. Evidence indicates that gestational particle exposure leads to both particle-specific and nonspecific effects on the SPM network.

Total knee arthroplasty with long tibial stem for tibial stress fractures with knee osteoarthritis: Two birds with one stone.

PURPOSE: The treatment and outcome of tibial stress fractures concomitant with knee osteoarthritis (OA) are complicated. The aim of this study was to evaluate the functional and radiological outcome of total knee arthroplasty with long tibial stem as a treatment for patients having knee OA and tibial stress fracture. METHODS: Patients who were diagnosed to have proximal tibia stress fracture along with knee OA at our institution between June 2013 and November 2018 were included in our study. All patients underwent total knee arthroplasty with long tibial stem. Preoperative and postoperative functional assessments were done according to range of movement of the knee joint, knee society score and knee injury and OA outcome score. Descriptive analysis was carried out by mean and standard deviation for quantitative variables, frequency and proportion for categorical variables. RESULTS: Twelve patients were included in the study. All patients were found to have stress fractures in the proximal half of tibia and extra-arthrosis. Four patients had non-union/delayed union, and 8 patients had acute fractures. The average preoperative range of movement was 88.1°, which improved to 116.3° at 3 months following surgery. It was found that the fracture has healed in all cases. Mean knee society score improved from 32.9 preoperatively to 89.3 at 1 year follow-up. Knee injury and OA outcome score improved from a mean score of 28.3 preoperatively to 81.1 at 1 year follow-up. CONCLUSION: Stress fractures can occur in the proximal tibia in patients with knee OA. Total knee arthroplasty with tibial stem provides a suitable solution for both conditions. Additional plating or bone graft is unlikely to be required.

Size at birth, lifecourse factors, and cognitive function in late life: findings from the MYsore study of Natal effects on Ageing and Health (MYNAH) cohort in South India.

OBJECTIVE: To examine if smaller size at birth, an indicator of growth restriction in utero, is associated with lower cognition in late life, and whether this may be mediated by impaired early life brain development and/or adverse cardiometabolic programming. DESIGN: Longitudinal follow-up of a birth cohort. SETTING: CSI Holdsworth Memorial Hospital (HMH), Mysore South India. PARTICIPANTS: 721 men and women (55-80 years) whose size at birth was recorded at HMH. Approximately 20 years earlier, a subset (n = 522) of them had assessments for cardiometabolic disorders in mid-life. MEASUREMENTS: Standardized measurement of cognitive function, depression, sociodemographic, and lifestyle factors; blood tests and assessments for cardiometabolic disorders. RESULTS: Participants who were heavier at birth had higher composite cognitive scores (0.12 SD per SD birth weight [95% CI 0.05, 0.19] p = 0.001) in late life. Other lifecourse factors independently positively related to cognition were maternal educational level and participants' own educational level, adult leg length, body mass index, and socioeconomic position, and negatively were diabetes in mid-life and current depression and stroke. The association of birth weight with cognition was independent cardiometabolic risk factors and was attenuated after adjustment for all lifecourse factors (0.08 SD per SD birth weight [95% CI -0.01, 0.18] p = 0.07). CONCLUSIONS: The findings are consistent with positive effects of early life environmental factors (better fetal growth, education, and childhood socioeconomic status) on brain development resulting in greater long-term cognitive function. The results do not support a pathway linking poorer fetal development with reduced late life cognitive function through cardiometabolic programming.

Is there an association between zinc and COVID-19-associated mucormycosis? Results of an experimental and clinical study.

BACKGROUND: The enormous increase in COVID-19-associated mucormycosis (CAM) in India lacks an explanation. Zinc supplementation during COVID-19 management is speculated as a contributor to mucormycosis. We conducted an experimental and clinical study to explore the association of zinc and mucormycosis. METHODS: We inoculated pure isolates of Rhizopus arrhizus obtained from subjects with CAM on dichloran rose Bengal chloramphenicol (DRBC) agar enriched with (three different concentrations) and without zinc. At 24 h, we counted the viable colonies and measured the dry weight of colonies at 24, 48 and 72 h. We also compared the clinical features and serum zinc levels in 29 CAM cases and 28 COVID-19 subjects without mucormycosis (controls). RESULTS: We tested eight isolates of R arrhizus and noted a visible increase in growth in zinc-enriched media. A viable count percentage showed a significantly increased growth in four of the eight isolates in zinc-augmented DRBC agar. A time- and concentration-dependent increase in the mean fungal biomass with zinc was observed in all three isolates tested. We enrolled 29 cases of CAM and 28 controls. The mean serum zinc concentration was below the reference range in all the subjects and was not significantly different between the cases and controls. CONCLUSIONS: Half of the R arrhizus isolates grew better with zinc enrichment in vitro. However, our study does not conclusively support the hypothesis that zinc supplementation contributed to the pathogenesis of mucormycosis. More data, both in vitro and in vivo, may resolve the role of zinc in the pathogenesis of CAM.

Severe Acute Respiratory Infection Surveillance during the Initial Phase of the COVID-19 Outbreak in North India: A Comparison of COVID-19 to Other SARI Causes.

INTRODUCTION: World Health Organization proposes severe acute respiratory infection (SARI) case definition for coronavirus disease 2019 (COVID-19) surveillance; however, early differentiation between SARI etiologies remains challenging. We aimed to investigate the spectrum and outcome of SARI and compare COVID-19 to non-COVID-19 causes. PATIENTS AND METHODS: A prospective cohort study was conducted between March 15, 2020, to August 15, 2020, at an adult medical emergency in North India. SARI was diagnosed using a "modified" case definition-febrile respiratory symptoms or radiographic evidence of pneumonia or acute respiratory distress syndrome of ≤14 days duration, along with a need for hospitalization and in the absence of an alternative etiology that fully explains the illness. COVID-19 was diagnosed with reverse transcription-polymerase chain reaction testing. RESULTS: In total, 95/212 (44.8%) cases had COVID-19. Community-acquired pneumonia (n = 57), exacerbation of chronic lung disease (n = 11), heart failure (n = 11), tropical febrile illnesses (n = 10), and influenza A (n = 5) were common non-COVID-19 causes. No between-group differences were apparent in age ≥60 years, comorbidities, oxygenation, leukocytosis, lymphopenia, acute physiology and chronic health evaluation (APACHE)-II score, CURB-65 score, and ventilator requirement at 24-hour. Bilateral lung distribution and middle-lower zones involvement in radiography predicted COVID-19. The median hospital stay was longer with COVID-19 (12 versus 5 days, p = 0.000); however, mortality was similar (31.6% versus 28.2%, p = 0.593). Independent mortality predictors were higher mean APACHE II in COVID-19 and early ventilator requirement in non-COVID-19 cases. CONCLUSIONS: COVID-19 has similar severity and mortality as non-COVID-19 SARI but requires an extended hospital stay. Including radiography in the SARI definition might improve COVID-19 surveillance. HOW TO CITE THIS ARTICLE: Pannu AK, Kumar M, Singh P, Shaji A, Ghosh A, Behera A, et al. Severe Acute Respiratory Infection Surveillance during the Initial Phase of the COVID-19 Outbreak in North India: A Comparison of COVID-19 to Other SARI Causes. Indian J Crit Care Med 2021;25(7):761-767.

Chemical methods for modification of proteins.

The library of chemical reactions for C-C and C-heteroatom bond formation is exceptional. The understanding of reactivity and diverse aspects of selectivity facilitates the functional group transformation of high complexity. However, the same is not valid for proteins as an organic substrate. Gratifyingly, we can translate some of the pre-existing reactions for developing methods for the modification of proteins. Also, there is enormous potential to create a new knowledge domain that will be unique to the densely functionalized architecture of proteins. At the outset, we outlined a few concepts that bridge the gap between chemical reactions with small molecules and proteins. Next, we introduced the key attributes and challenges associated with the selectivity that emerges due to the presence of multiple types and copies of functional groups. The examples with nucleophilic amino acids outline the chemoselectivity-associated features. Gradually, the discussion moves toward the concepts that led to the successful realization of site-selectivity and N-terminus residue-specificity. The attributes of organic chemistry that emerge due to the multifunctional organization of the substrate are marked. The last section overviews the analysis of protein bioconjugates by mass spectrometry. Also, the review outlines the unmet needs and opportunities.

Pd-Catalyzed Decarboxylation and Dual C(sp3)-H Functionalization Protocols for the Synthesis of 2,4-Diarylpyridines.

The Pd-catalyzed decarboxylation and dual C(sp3)-H bond functionalization approaches have been described for the preparation of symmetrical and unsymmetrical 2,4-diarylpyridines. The developed transformations were realized using nonactivated aromatic ketones and amino acids as C-N sources. The efficacy of the catalyst and reagent combination drives the transformation toward the formation of desired products with high yields and selectivity. The described reaction conditions have seduced the self-reaction of phenylalanine via [2 + 2 + 2] cycloaddition and minimized the formation of 3,5-phenylpyridine as a side product, whereas using glycine as a C-N source, the corresponding 2,6-diarylpyridines were formed as minor products.

A Case of Life-threatening Amlodipine and Atenolol Overdose.

Treating a patient of amlodipine-atenolol poisoning is nightmare for a physician. In high dose both the drugs individually cause severe bradycardia and hypotension. In combination they cause severe cardiovascular depression. Here we report a case of 66-year-old obese, hypertensive, depressed male, who presented to emergency 9 hours after consumption of 25 tablets of amlodipine-atenolol (5 mg+50 mg). On evaluation, he had refractory bradycardia, hypotension and acute kidney injury (AKI). Eventually he developed cardiac arrest. He was revived after 5 minutes of cardio-pulmonary resuscitation (CPR). He was successfully managed with gastric lavage, fluids, inotropes, atropine, isoprenaline and subsequently with calcium gluconate infusion, high-dose insulin euglycemia therapy (HIET) and lipid emulsion therapy. Glucagon infusion was also planned but it was not available. Patient hemodynamics improved and on 8th day he got the discharge. Our case exemplifies the importance of timely and aggressive management of lethal overdose of amlodipine-atenolol poisoning. How to cite this article: Tale S, Kumar M, Ghosh S, Bhalla A. A Case of Life-threatening Amlodipine and Atenolol Overdose. Indian J Crit Care Med 2019;23(6):281-283.

A topological transition from bimolecular quadruplex to G-triplex/tri-G-quadruplex exhibited by truncated double repeats of human telomere.

Human telomeric G-rich sequences can fold back into various conformations depending upon the salt (Na+ or K+) at physiological pH. On the basis of results obtained by native PAGE electrophoresis, circular dichroism, and UV-melting experiments, we report here that truncated sequences of human telomere (d-GGGTTAGGG; GM9, d-AGGGTTAGGG; GM10, d-TAGGGTTAGGG; GM11) adopt a varied range of quadruplex conformations as a function of the cation present. By correlating CD and gel electrophoresis experiments; it was concluded that the GM9 oligonucleotide can self-associate to form a tetramer quadruplex (antiparallel; AP) in Na+ solution and a mixture of G-triplex (AP) or tri-G-quadruplex (parallel; P) along with a tetramer G-quadruplex structure (AP) in K+. The GM10 oligonucleotide formed a bimolecular G-quadruplex in both Na+ and K+ solutions, while GM11 associated to form a bimolecular G-quadruplex (AP) structure in Na+ solution and a mixture of bimolecular G-quadruplex (AP) and bimolecular G-quadruplex (P) along with parallel G-triplex or antiparallel tri-G-quadruplex in K+. All the UV-melting profiles, thermal difference spectra, and CD melting curves suggested the formation of a variety of G-quadruplex conformations by the DNA sequences studied in Na+ and K+ ions. Hypothetical models for different conformations adopted by these DNA molecules have also been proposed, which may further enhance our knowledge about the divergent topologies of guanine quadruplexes.

Results of a randomized controlled trial of level IIb preserving neck dissection in clinically node-negative squamous carcinoma of the oral cavity.

BACKGROUND: The lymphatic spread from the cancers of the oral cavity follows an orderly progression and involvement of lower nodes without involvement of upper nodes and skip metastasis is rare. Selective neck dissections are increasingly being performed for node-positive patients; however, in node-negative patients the options of wait and watch, prophylactic radiotherapy, and prophylactic elective node dissections are debated. Quality of life and shoulder functions are important to choose the appropriate therapeutic modality. PATIENTS AND METHODS: Patients with oral squamous carcinoma with clinically and radiologically negative neck were randomized to IIb preserving superselective neck dissection or conventional supraomohyoid neck dissection. The primary end point of the study was recurrence of disease (clinical or radiological) and shoulder function as demonstrated by the clinical examination and electromyography. The secondary end point was quality of life as measured by the FACT-HN version 4 questionnaire at the end of 1 year follow-up. RESULTS: The mean number of lymph node harvested per patient was 25.6 (range 8-85). Of the 32 patients, 3 had histologically positive node in level Ib, one of these patients had single positive node while the remaining two had three positive nodes in level Ib. At median follow-up of 36 months disease-free survival in IIb, sparing group was 83% compared to 91% in control arm, the difference in survival between two groups was statistically not significant (p = 0.694). EMG of the shoulder showed denervation pattern in 45% patients undergoing IIb preserving surgery at 1 month follow-up compared to 95% in conventional surgery group, this recovered in all patients but one at 3 months and 100% recovery was seen at 6 months. CONCLUSIONS: The results of the present study indicate that superselective IIb preserving neck dissections are technically feasible and appear to be oncologically safe procedures when performed as elective prophylactic procedures in highly select group of patients. A significant number of occult metastasis seen in the present study suggests prophylactic dissection to be better than wait and watch policy. Results also show initial higher shoulder morbidity at 1 month in patients undergoing IIb preserving dissections; however, at the end of 1 year recovery is complete and both procedures are comparable. TRIAL REGISTRATION: The trial is registered at clinicaltrials.gov with registration no NCT00847717 ; registered on February 19, 2009.

Effect of neoadjuvant chemotherapy and its correlation with HPV status, EGFR, Her-2-neu, and GADD45 expression in oral squamous cell carcinoma.

BACKGROUND: Head and neck cancers are the commonest cancer in Southeast Asia. Despite being a surface cancer, it is associated with significant morbidity as despite early detection by the patients they often report for treatment late and hence are associated with poor prognosis. The role of neoadjuvant chemotherapy in head and neck cancer is still under evaluation; there is a large subgroup of population that does not respond to chemotherapy, and hence, most studies have failed to show any survival benefit. This study evaluated the role of neoadjuvant therapy with docetaxel and carboplatin in patients with oral cancer and correlated the response to human papilloma virus, EGFR1, EGFR2, and GADD45 expression. METHODS: A total of 24 locally advanced, non-metastatic oral cancer patients were included in the study. Tumor biopsies were taken prior to the start of neoadjuvant therapy for expression of EGFR, Her-2-Neu, and GADD45 by immunohistochemistry and for HPV by PCR. The response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) criteria after three cycles of chemotherapy. Statistical analysis was performed using correlation and Kaplan-Meier analysis; the difference in survival was calculated with log rank test. RESULTS: A total of 21 male and 3 female with a mean age of 53.12 years were enrolled. Sixty-five percent of these received three cycles of chemotherapy. Five patients were positive for HPV 16 and none for HPV 18. Twenty-two of 24 patients showed GADD45 expression, 3 showed expression of Her-2-Neu while all 24 showed expression for EGFR1 protein. Two-year overall survival was 81%; GADD45 expressions were found to significantly affect the overall and disease-free survival, while any of the other protein expression studied and HPV status was not significant. CONCLUSION: The result of the present study shows significant downgrading of the oral cancers with neoadjuvant chemotherapy suggesting its utility in borderline operable cases. However, the response of chemotherapy does not appear to be related to the expression of EGFR, Her-2-Neu, and GADD45 protein or presence of HPV. Bone involvement, perineural invasion, and GADD45 expression significantly predict OS and DFS. All patients who did not express Gadd45 died before 2 years. Study with more subjects and longer follow-up should be carried out to elucidate this relation further.

Hospitalization for ambulatory care sensitive conditions among urban Métis adults.

BACKGROUND: Hospitalizations for ambulatory care sensitive conditions (ACSCs) are potentially preventable, but may be required if these conditions are not managed well. National-level information about ACSC hospitalizations is available for Canada, but not for Aboriginal groups. This study describes ACSC hospitalizations among urban Métis adults relative to their non-Aboriginal counterparts. DATA AND METHODS: The 2006/2007-to-2008/2009 Discharge Abstract Database, which contains hospitalization records from all acute care facilities (excluding Quebec), was linked to the 2006 Census to obtain Aboriginal identity information. Age-standardized ACSC hospitalization rates (ASHRs) per 100,000 population and rate ratios were calculated for Métis aged 18 to 74 relative to non-Aboriginal people of the same ages. Odds of ACSC hospitalizations were estimated using logistic regression models, adjusting for demographic, geographic, and socioeconomic characteristics. RESULTS: The ASHR for ACSCs among urban Métis adults was twice that among non-Aboriginal adults (393 versus 184 per 100,000 population). Even when demographic, geographic, and socioeconomic characteristics were taken into account, Métis had higher odds of ACSC hospitalizations overall (OR 1.5). Most commonly, these hospitalizations were for diabetes (OR 1.8) or chronic obstructive pulmonary disease (OR 1.5). Modelled factors partly reduced differences between Métis and non-Aboriginal adults, but variations between the groups remained after all adjustments. INTERPRETATION: Rates of ACSC hospitalizations were higher among Métis than among non-Aboriginal adults who lived in urban areas. Further research using other data sources is warranted to assess the roles of factors not available for this analysis, such as primary care, co-morbidity, and health behaviours.

Mycobacterium tuberculosis UvrB Is a Robust DNA-Stimulated ATPase That Also Possesses Structure-Specific ATP-Dependent DNA Helicase Activity.

Much is known about the Escherichia coli nucleotide excision repair (NER) pathway; however, very little is understood about the proteins involved and the molecular mechanism of NER in mycobacteria. In this study, we show that Mycobacterium tuberculosis UvrB (MtUvrB), which exists in solution as a monomer, binds to DNA in a structure-dependent manner. A systematic examination of MtUvrB substrate specificity reveals that it associates preferentially with single-stranded DNA, duplexes with 3' or 5' overhangs, and linear duplex DNA with splayed arms. Whereas E. coli UvrB (EcUvrB) binds weakly to undamaged DNA and has no ATPase activity, MtUvrB possesses intrinsic ATPase activity that is greatly stimulated by both single- and double-stranded DNA. Strikingly, we found that MtUvrB, but not EcUvrB, possesses the DNA unwinding activity characteristic of an ATP-dependent DNA helicase. The helicase activity of MtUvrB proceeds in the 3' to 5' direction and is strongly modulated by a nontranslocating 5' single-stranded tail, indicating that in addition to the translocating strand it also interacts with the 5' end of the substrate. The fraction of DNA unwound by MtUvrB decreases significantly as the length of the duplex increases: it fails to unwind duplexes longer than 70 bp. These results, on one hand, reveal significant mechanistic differences between MtUvrB and EcUvrB and, on the other, support an alternative role for UvrB in the processing of key DNA replication intermediates. Altogether, our findings provide insights into the catalytic functions of UvrB and lay the foundation for further understanding of the NER pathway in M. tuberculosis.

The Protease Locus of Francisella tularensis LVS Is Required for Stress Tolerance and Infection in the Mammalian Host.

Francisella tularensis is the causative agent of tularemia and a category A potential agent of bioterrorism, but the pathogenic mechanisms of F. tularensis are largely unknown. Our previous transposon mutagenesis screen identified 95 lung infectivity-associated F. tularensis genes, including those encoding the Lon and ClpP proteases. The present study validates the importance of Lon and ClpP in intramacrophage growth and infection of the mammalian host by using unmarked deletion mutants of the F. tularensis live vaccine strain (LVS). Further experiments revealed that lon and clpP are also required for F. tularensis tolerance to stressful conditions. A quantitative proteomic comparison between heat-stressed LVS and the isogenic Lon-deficient mutant identified 29 putative Lon substrate proteins. The follow-up protein degradation experiments identified five substrates of the F. tularensis Lon protease (FTL578, FTL663, FTL1217, FTL1228, and FTL1957). FTL578 (ornithine cyclodeaminase), FTL663 (heat shock protein), and FTL1228 (iron-sulfur activator complex subunit SufD) have been previously described as virulence-associated factors in F. tularensis Identification of these Lon substrates has thus provided important clues for further understanding of the F. tularensis stress response and pathogenesis. The high-throughput approach developed in this study can be used for systematic identification of the Lon substrates in other prokaryotic and eukaryotic organisms.