Background: With the advent of many new advanced techniques, sequences of a number of proteins have been made available. But the relative paucity of the experimentally determined three-dimensional structures of these proteins has paved way for the development of computational structure prediction methods. Protein secondary structure prediction is an essential step in modeling the tertiary structure. Among the various secondary structure prediction methods available, three different methods with unique working principles, namely, GOR, HNN, and SOPMA were evaluated for their efficiency to predict secondary structures. Methods: A set of 90 different proteins with known secondary structures from three major classes namely, mainly alpha, mainly beta, and mainly alpha beta was used as reference. Secondary structure data of these proteins obtained through experimental methods were compared with that of predictions made by GOR, HNN, and SOPMA respectively by employing various statistical analyses, namely paired sample test, correlation coefficient, standard deviation, standard error mean and scatter plots. Results: The secondary structure prediction tools namely, GOR and HNN were found to predict helical structures more accurately than the sheets. SOPMA was observed to predict sheets more accurately than helices. Conclusion: Based on the observed results, it could be concluded that there is no single tool that consistently predicts all the secondary structures accurately. It could also be anticipated that a combined use of these secondary prediction tools could further enhance the efficacy of in silico protein secondary structure prediction methods.

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Background: GeneXpert MTB/RIF (Xpert), the fully automated cartridge-based nucleic acid amplification test for simultaneous identification of Mycobacterium tuberculosis complex and rifampicin resistance (RR), directly from samples is considered as a game changer for tuberculosis (TB) control programs worldwide. Methods: We are reporting serious issues with repeatability among a subgroup of Xpert (Version 4) identified RR results from South Indian state recently switched to Xpert by the National TB control program. Results: We have demonstrated that poor repeatability is frequently associated with those Xpert derived RR results, identified by detection of delayed amplification of any probe in the presence of positive analyte results for all probes. Another significant contributing factor was found to be lower bacterial loads in samples. The repeat tests were done by Xpert and/or by line probe assay depending on smear positivity. The finding is worrying as Xpert is recommended over other tests due to its reportedly better performance among low bacterial load samples such as pediatric, extra-pulmonary, HIV-TB co-infected, and smear negative pulmonary TB and the same samples, it seems are more likely to cause error prone RR results. Conclusions: We recommend for additional genotypic tests with specific mutant probes for detecting mutations at rpoB hot sites and growth based tests for all Xpert derived RR-TB cases identified by the above algorithm for confirmation of the presence of mutation, based on our available data.

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Background: Pregnancy is associated with significant but reversible changes in thyroid function. Abnormal thyroid hormone levels could give rise to increased malplacentation increase to malplacentation that underlies the association between maternal thyroid dysfunction and adverse obstetric outcomes such as fetal brain damage, preterm births, and fetal death. Aim: The present study was planned to predict the risk of abortion in pregnancy by studying the relation between thyroid profile and rate of abortion. Methods: This prospective observational study was carried out in 100 pregnant women with a singleton pregnancy of 6–12 weeks of gestation in the Department of Biochemistry in collaboration with the Department of Obstetrics and Gynaecology, Pt. B.D. Sharma, PGIMS, Rohtak. The patients were divided into three groups, namely, Group 1 (n = 30, control group) included normal pregnant women with no history of abortion; Group 2 (n = 35) included pregnant females with previous history of abortion; and Group 3 (n = 35) comprised pregnant women coming with chief complaint of bleeding per vaginum. A volume of 5 mL blood sample was taken and serum was separated using centrifugation. Triiodothyronine (T₃), thyroxine (T₄), and thyroid stimulating hormone (TSH) levels were estimated by chemiluminiscence. Results: T₃, T₄ and levels were comparable in all the three groups and the difference was not statistically significant. TSH levels were found to be negatively correlated with T₃ and T₄ values in the three groups. Only, 13% of the patients had spontaneous abortion. Nearly 6.66% of the patients in Group 1, 11.42% in Group 2 and 19.99% of patients in Group 3 ended up in abortion. TSH was found to be strongly associated with abortion in all the three groups (P ≤ 0.001). TSH levels were significantly higher in the first trimester in women who had an abortion than pregnant women who had a successful continuation of pregnancy. Conclusions: TSH was strongly associated with abortion in the first trimester in all the three groups.

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Background: The purpose of this study was to investigate two-body wear resistance and hardness of different three modern composite materials. Methods: In the conditions recommended by the manufacturer five pieces were produced from each material. All samples were kept in distill water for 1 week and determined Vickers hardness (HV) before two-body wear test. Then, two-body wear tests were performed using a computer controlled chewing simulator. In this study, the chewing simulator was programmed to provide a 2 mm vertical movement and a 0.7 mm horizontal movement. For each wear test, Al₂O₃ with a diameter of 6 mm was used as antagonist material. The mean volume loss of all samples after the wear tests was determined with three dimensional profilometry. In addition to a random specimen was selected from each test group and SEM images were taken for analysis of wear tracks. Mean and standard deviation values were calculated and evaluated with the one-way ANOVA. Results: The hardness values of the materials tested in this study were measured between about 49 HV and 78 HV. Significantly, lowest mean volume loss was detected for the Heliomolar at about 3.1 μm3 whereas the highest mean volume loss was detected for the Silorane at about 6.4 μm3 after 360.000 chewing cycles. Conclusions: However, among the materials tested in this study suggested correlations between HV and two-body wear resistance are not significant.

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Chronic obstructive pulmonary disease and asthma affect millions of people worldwide. Conventional treatments are not sufficient at preventing the progression of these diseases. The treatments are unspecific, and when administered systematically, and in high doses, they have adverse health effects. Further, the current diagnostic methods are poor and they are not capable of identifying the accurate state of the disease. Nanoparticle-based theranostics (NBTs) are well-established systems that simultaneously provide treatment and diagnostics. Various nanocarriers can be used in NBT and are designed according to the need. In this review, liposome-based theranostic systems are evaluated and possible modifications that could provide better drug delivery and accumulation and diagnosis are exemplified. These systems can be applied to conventional therapeutics to avoid side effects and perhaps overcome the challenges that interfere with their cellular accumulation.

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Nontuberculous mycobacteria (NTM) infections are ever more important in recent years for leading causes of morbidity and mortality worldwide. Clinical appearance of Mycobacterium tuberculosis (TB) complex and NTM is same, but the treatment regimen is always different. NTM is challenging for both diagnostic and therapeutic with reason that it mimic pathological, microbiological, immunological, and radiological findings of TB. Newer molecular diagnostic methods allow for a better identification of NTM infections in patients not responding to antitubercular treatment and falsely categorized as drug-resistant TB. This article will explore the recent methods for the diagnosis and identification of NTM infections in clinical practice. In the future, the molecular-based diagnosis will significantly reduce the turnaround time of the diagnosis and thereby improving patient outcome.

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Background: Cholesterol degradation pathway is one of the important pathways in survival of Mycobacterium tuberculosis (Mtb) bacilli, and steroid C26-monooxygenase (CYP125) enzyme of Mtb associated with this pathway is reported to be novel drug target. This study aims to find out novel, safe, and effective inhibitors against CYP125 from natural phytochemicals with reported anti-tubercular activity. Methods: Bioinformatics approach such as homology modeling, virtual screening, and molecular dynamics (MD) simulation was applied to identify best hits among all the shortlisted 148 compounds. The Mtb H37Ra bacilli growth was measured at optical density at 600 nm in minimal media supplemented with cholesterol and monitored for 10 days. Two promising compounds, namely, sesamin and β-sitosterol, were studied to determine their effective minimum inhibitory concentrations (MICs) in Mtb H37Ra bacilli culture. Results: In virtual screening, 15 compounds showed comparatively better binding affinity than natural substrate (choletst-4-en-3-one). In MD simulation study, the protein structure was observed to be stable in alls the interaction complexes, i.e., with choletst-4-en-3-one, sesamin, and β-sitosterol. The MICs of sesamin and β-sitosterol were observed to be 2 μg/ml, inhibiting the growth of the Mtb bacilli by 51% and 53%, respectively. Conclusions: From the above experimental findings, sesamin and β-sitosterol may be proposed as safe and potential inhibitors of CYP125 resulting in diminished growth of Mtb bacilli.

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Background: Fluoroquinolones (FQs) are among the most important second-line anti-tuberculosis (TB) drugs, of which 8-methyl FQs; moxifloxacin (MFX) and gatifloxacin are considered to have the most mycobactericidal and “sterilizing action.” Because of the wide and often illogical usage, FQ resistance has evolved and challenged the multidrug-resistant (MDR)/extensively drug-resistant TB (XDRTB) control activities worldwide. We have compared the baseline ofloxacin (OFX) resistance among MDR/rifampicin-resistant (RR) TB cases identified from South Tamil Nadu and Kerala states in India and then assessed the cross-resistance with different concentrations of MFX using MGIT 960. Methods: Bactec MGIT 960 method and the standard protocol as per the manufacturer modified for the multiple concentrations of the drugs were used for the susceptibility testing. Results: We found that samples from newly identified MDR/RR cases of both states have baseline OFX resistance at 16–17%. MFX cross-resistance was 33%–35% (2 mg/L), 59%–70% (1 mg/L), and 87%–93% (0.5 mg/L). Conclusions: As the cross-resistances to MFX 1 mg/L (minimum inhibitory concentration between 1 and 2 mg/L) and 2 mg/L are very high, 59%–70% and 33%–35%, respectively, among newly identified OFX-resistant MDRTB cases, we assume the sterilizing activity of MFX-containing regimens may be seriously compromised leading to higher relapse rates despite having decent cure rates. This may pose considerable technical as well as cost-wise challenges for TB control program in the future.

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Backgrand: For patients with long segment of tracheal stenosis, resection and replacement is necessary. Tracheal reconstruction associated with complications such as stenosis, insufficiency of blood supply and surgical complication. Methods: In this study, we prepared collagen-chitosan scaffold and sheep's decellularized trachea plus culture medium with nanocup contain growth factors for chondrocyte and fibroblast-epithelial cell culture in rotary bioreactor. After attachment of cells, engineered trachea put into the omentum. Peritoneal stem cell interact with the epithelial and chondrocytes with attendance of growth factors released from nanocup. The bioengineered trachea with omental pedicle transposed from behind the sternum and transplanted in the position of resected trachea. In third experiment, we introduce the in situ tracheal repair technique with injectable matrix for reconstruction of long-segmental stenosis of trachea in a 29-year-old woman. Results: Severe tortuosity in the first experiment and mild stenosis was seen in the second experiment. In third experiment, normal shape in tracheal diameter was seen at injection sites. The interior portion of the trachea at virtual computed tomography (CT) scan and bronchoscopy were similarity to normal after four month fallowing up of injection. Conclusions: Severe tortuosity, mild stenosis in whole length of trachea in this study and disadvantage of trachea transplantation include open surgery, fatal consequences of anastomosis leakage, and rupture besides large mediastinal vessels; hence, we decided to introduce the novel in situ tracheal repair technique. Injectable bioresorbable scaffolds may be used as a temporary scaffolding for transplanted cells and thereby allow the cells to secrete extracellular matrix of their own to enable, in the long term, a complete and natural tissue replacement.

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Background: To diagnose tuberculosis (TB), it is necessary to demonstrate the presence of Mycobacterium tuberculosis in a clinical specimen such as sputum. A simple, low-cost rapid test for blood or urine is urgently needed but remains elusive. Tests for host response-derived biomarkers or secreted bacterial compounds have so far failed to provide sufficient sensitivity or specificity and alternative approaches are needed. Carboxymycobactins are amphiphilic siderophores unique to the mycobacteria that have the ability to transverse mammalian cell walls. They have insufficient mass to induce an antibody response and there are currently no simple sensitive tests for their detection. Methods: We report the development of an enzyme-linked immunosorbent assay (ELISA) for carboxymycobactin. Polyclonal antibodies were raised in rabbits following conjugation to a carrier protein, bovine serum albumin allowing development of a sensitive indirect double antibody ELISA. A second keyhole limpet hemocyanin conjugate was manufactured to adhere the hapten to the wells of the microplate. We established a limit of detection for the assay of 1 pg/ml. Carboxymycobactin was detected in culture supernatant from mycobacteria including clinical isolates of M. tuberculosis, but not from cultures of Rhodococcus, Nocardia, Streptomyces, Bacillus cereus, Klebsiella oxytoca, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Sample concentration was achieved following extraction with chloroform. Results: We have demonstrated for the first time the direct detection of carboxymycobactin in culture supernatant by immunoassay. Conclusions: We recommend testing samples from humans and animals to establish the potential utility of carboxymycobactin as a diagnostic marker of active mycobacterial disease.

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Background: Leprosy was eliminated from India in December 2005 imprinting a prevalence rate of <1/10,000 population. Still some endemic pockets exist in India where the new cases of leprosy continue to occur at a constant rate. It means the active transmission of leprosy is still continuing. In the present study, we aimed to elucidate the transmission pattern of Mycobacterium leprae using three microsatellite loci. Methods: Slit-skin samples from 36 newly diagnosed multi-bacillary leprosy cases from Ghatampur, Kanpur Nagar, Uttar Pradesh, India, were collected in a sterile centrifuge tube containing Tris-EDTA (TE) buffer. DNA was isolated, and the microsatellite loci, namely, (GT)₆, (TA)₁₈, and (TA)₈CA₃ were amplified using the in-house designed primers. The amplified products were recovered through polymerase chain reaction cleanup kit and sequenced by Sangers method in a 16 capillary genetic analyzer. Results: Sequences were searched by Basic Local Alignment Search Tool, and phylogenetic analysis was done to trace out the transmission pattern of M. leprae. Out of the three microsatellite loci, (TA)₁₈ was unable to define the transmission pattern while (GT)₆ and (TA)₈CA₃ were endowed with distinct signature for transmission of M. leprae in the study segment of the population. Conclusions: We found that nine types of M. leprae strains were circulating in the Ghatampur area and the same type of strains were found in the same villages or in neighboring villages. To conclude the study, we accentuated that (TA)₈CA₃ is a better microsatellite locus for strain typing of M. leprae.

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Background: We present our experience with optimization and diagnostic use of quantitative real-time polymerase chain reaction (PCR) targeting the lytA gene of Streptococcus pneumoniae for the detection of S. pneumoniae in whole blood of children <5 years of age. The assay was optimized to detect ≥5 CFU/10 μl or 1 copy of DNA/2 μl of blood. Methods: This assay was applied on 1912 whole blood specimens collected from children <5 years of age with pneumonia, of which 35 specimens were lytA positive. The bacterial loads were determined through categorization of load into five different categories, i.e., very high load, high load, moderate load, low load, and very low load. Results: Of the 35 lytA-positive samples, 9 (25.71%), 4 (11.42%), 1 (2.85%), 13 (37.14%), and 8 (22.85%) were categorized as very high load, high load, moderate load, low load, and very low load, respectively. Extracted samples were also subjected to serotyping by the Centers for Disease Control and Prevention PCR scheme. Positive samples with very high load and high load category were serotyped successfully in all instances. A high proportion of samples with low and very low load (61.53% and 75%, respectively) remained untypeable by the currently proposed schemes. Conclusions: LytA PCR assay in whole blood provides rapid and sensitive results for the diagnosis of invasive S. pneumoniae disease in a resource-limited setting, while also being amenable to quantitation and serotyping.

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Background: Bacterial contamination is common to all wounds. The bacterial burden of wounds has been found to have an inverse relationship with chronic wound healing. In seeking to develop a better understanding of the evolution of Buruli ulcer (BU) wounds, we performed a longitudinal study to quantify the bacterial burden of BU wounds during the course of streptomycin/rifampicin (SR) treatment. Methods: Twenty-one IS2404 polymerase chain reaction confirmed patients were longitudinally followed during the course of their treatment. Swab or tissue samples obtained from the lesions were quantitatively analyzed to determine the bacterial burden pre-, during, and post-SR treatment. Furthermore, the species of bacterial isolates obtained at these time points were also identified. Results: Based on the determination of the bacterial burden, 18/22 (81.8%) pretreatment, 15/25 (57.7%) during treatment, and 36/48 (75.0%) posttreatment samples were classified as superinfected, respectively. Thirty bacterial species including two species of anaerobic Clostridia (Clostridium perfringens and Clostridium sporogenes) were identified among 114 isolates. While Enterococcus faecalis, Pseudomonas aeruginosa, and Chryseomonas luteola dominated pretreatment, P. aeruginosa dominated during and posttreatment. Conclusions: Most BU patients presented with lesions with a high bacterial load which increased significantly posttreatment. Therefore, good wound care is necessary to control the microbial burden of BU wounds, especially posttreatment to minimize complications.

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The children just as adults are exposed to contract and develop the multi-resistant forms of tuberculosis (TB), constituting a major issue for the disease control. The children <5 years of age are the most exposed to present the most serious and more often deadly forms of the illness. Further, in many developing countries, the lack of pediatric forms of the TB drugs makes it difficult to control the problem. The TB diagnosis among the children is based on a set of arguments: the presence of a tuberculous person excreting bacillus, exposition and receptivity conditions of the child (the level of his immunity, the level of undernutrition, and associated pathologies). The diagnosis is also based on the research of the symptoms and other signs suggestive of TB: tuberculin skin test, thoracic radiography, and interferon-gamma test. The aim of this study is to describe and analyze the features and difficulties of the biological diagnosis of TB among the children and to find a strategy for the improvement of the results.

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Background: By invention of the atomic force microscope (AFM) in 1986, the imaging of surfaces objects at nanometer-scale resolutions becomes possible. Although, in the beginning, AFM was applied almost exclusively to characterize the surfaces of nonbiological materials, at present the application of the AFM to biological and biomedical research has increased exponentially. Methods: In this study, we tried to investigate and visualize Mycobacterium tuberculosis under AFM. The transformation of bacterial shape and the formation of “hard” shell in resistant or dormant conditions were characterized and identified. Results: Application of AFM for the study of antibiotic-resistant forms of M. tuberculosis revealed the presence of round-shaped bacteria along with conventional rod-shaped ones. There has also been concluded changing of the surface charge of the cell membrane for mutated forms since round-shaped bacteria fixed on the charged surface less strongly and can be moved along the surface by a microscope tip with easily. Conclusions: In brief, this article highlights the optimal operation modes and base principal to study dangerous bacilli under AFM.

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