Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S ribosomal RNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant.These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors.

Vascular development begins when mesodermal cells differentiate into endothelial cells, which then form primitive vessels. It has been hypothesized that endothelial-specific gene expression may be regulated combinatorially, but the transcriptional mechanisms governing specificity in vascular gene expression remain incompletely understood. Here, we identify a 44 bp transcriptional enhancer that is sufficient to direct expression specifically and exclusively to the developing vascular endothelium. This enhancer is regulated by a composite cis-acting element, the FOX:ETS motif, which is bound and synergistically activated by Forkhead and Ets transcription factors. We demonstrate that coexpression of the Forkhead protein FoxC2 and the Ets protein Etv2 induces ectopic expression of vascular genes in Xenopus embryos, and that combinatorial knockdown of the orthologous genes in zebrafish embryos disrupts vascular development. Finally, we show that FOX:ETS motifs are present in many known endothelial-specific enhancers and that this motif is an efficient predictor of endothelial enhancers in the human genome.

Histopathological evaluation of cutaneous reactions to tattoos: Study at a tertiary care center.

BACKGROUND: Tattooing has been around for many years and is becoming an increasingly common fashion trend. As there are often few regulatory laws regarding the practice, an increase in the incidence of cutaneous reactions to tattoo inks is noted. These include allergic reactions, granulomatous dermatitis, infections, lichenoid dermatoses, and sometimes malignancy. The present study examines the histopathological changes seen in patients with cutaneous reactions to tattoo ink. METHOD: A prospective observational study was conducted over 18 months in the dermatology clinic of a tertiary care center in western India. The study population included 22 patients with cutaneous reactions over the tattoos. Punch biopsy specimens were sent to study the pattern of histopathological response. RESULTS: All 22 patients studied were between the ages of 17 and 35 years. The mean duration of development of reaction was 8.1 months. Most of the reactions were seen in black ink tattoos performed by amateurs. Perivascular and spongiotic dermatitis suggestive of allergic response was the most common feature on histopathology. Granulomatous response and lichenoid response were seen in five and three biopsies, respectively. CONCLUSION: Legalization is needed for this practice to prevent tattoo reactions. Histopathological evaluation is important as tattoo reactions may be associated with skin infections and malignancies.

Magnetic-Field-Dependent Equilibration of Fractional Quantum Hall Edge Modes.

Fractional conductance is measured by partitioning a ν=1 edge state using gate-tunable fractional quantum Hall (FQH) liquids of filling 1/3 or 2/3 for current injection and detection. We observe two sets of FQH plateaus 1/9, 2/9, 4/9 and 1/6, 1/3, 2/3 at low and high magnetic field ends of the ν=1 plateau, respectively. The findings are explained by magnetic field dependent equilibration of three FQH edge modes with conductance e^{2}/3h arising from edge reconstruction. The results reveal a remarkable enhancement of the equilibration lengths of the FQH edge modes with increasing magnetic field.

Acquired cutis laxa secondary to Sweet syndrome in a child (Marshall syndrome): A rare case report.

Sweet syndrome is rare in the pediatric population and usually responds well to treatment, resolving without sequelae. Marshall syndrome is a rare pediatric skin disease characterized by loss of elastic tissue (cutis laxa) secondary to acquired, localized neutrophilic dermatitis without any internal organ involvement. Only few cases of Marshall syndrome (acquired cutis laxa type II) have been reported. Systemic steroids and dapsone show excellent results in Sweet syndrome. Although there is no satisfactory treatment for cutis laxa, dapsone can be used in the acute phase for control of swelling.

Antidiabetic drug metformin affects the developmental competence of cleavage-stage embryos.

PURPOSE: Metformin is the most commonly prescribed drug in the management of metabolic disorders such as polycystic ovarian syndrome (PCOS) and gestational diabetes in women of reproductive age. Insulin-sensitizing effect of metformin helps in improving from PCOS features such as hyperandrogenism, anovulation, and infertility. However, its ability to cross placental barrier raises concern about safety of the drug on early embryonic development. In this study, we evaluated the effect of metformin on the ovarian function and embryo development. METHODS: Adult Swiss albino female mice were administered with metformin (0, 50, 100, and 200 mg/kg body weight) for 4 weeks and assessed for reproductive function and preimplantation embryo development. Further, effect of metformin (0, 10, 25, 50, 100, 250, and 500 µg/mL) exposure to 2-cell-stage embryos was tested under in vitro conditions. RESULTS: Metformin did not alter the body weight, blood glucose, ovarian weight, and follicular reserve. However, the early embryo development was significantly affected in mice treated with metformin in vivo at highest dose. Moreover, embryos which were exposed to metformin in vitro showed dose-dependent decline in blastocyst rate and hatching rate. Furthermore, at highest concentration of metformin (500 µg/mL), all the embryos were arrested at compaction stage. CONCLUSION: The study revealed that metformin affects the early embryonic development and raises concern about its use during conception.

Synthesis and Evaluation of Chirally Defined Side Chain Variants of 7-Chloro-4-Aminoquinoline To Overcome Drug Resistance in Malaria Chemotherapy.

A novel 4-aminoquinoline derivative [(S)-7-chloro-N-(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

In vitro susceptibility of Indian Plasmodium falciparum isolates to different antimalarial drugs & antibiotics.

Background & objectives: : The in vitro assays for susceptibility of Plasmodium falciparum to antimalarial drugs are important tools for monitoring drug resistance. During the present study, efforts were made to establish long-term continuous in vitro culture of Indian field isolates of P. falciparum and to determine their sensitivity to standard antimalarial drugs and antibiotics. Methods: Four (MZR-I, -II, -III and -IV) P. falciparum isolates were obtained from four patients who showed artemisinin-based combination therapy (ACT) from Mizoram, a north-eastern State of India, and characterized for their in vitro susceptibility to chloroquine diphosphate (CQ), quinine hydrochloride dehydrate, mefloquine, piperaquine, artemether, arteether, dihydro-artemisinin (DHA), lumefantrine and atovaquone and antibiotics, azithromycin and doxycycline. These patients showed ACT treatment failure. Two-fold serial dilutions of each drug were tested and the effect was evaluated using the malaria SYBR Green I fluorescence assay. K1 (chloroquine-resistant) and 3D7 (chloroquine-sensitive) reference strains were used as controls. Results: Growth profile of all field isolates was identical to that of reference parasites. The IC50 values of all the drugs were also similar against field isolates and reference parasite strains, except K1, exhibited high IC50 value (275±12.5 nM) of CQ for which it was resistant. All field isolates exhibited higher IC50 values of CQ, quinine hydrochloride dihydrate and DHA compared to reference strains. The resistance index of field isolates with respect to 3D7 ranged between 260.55 and 403.78 to CQ, 39.83 and 46.42 to quinine, and 2.98 and 4.16 to DHA, and with respect to K1 strain ranged between 6.51 and 10.08, 39.26 and 45.75, and 2.65 and 3.71. MZR-I isolate exhibited highest resistance index. Interpretation & conclusions: As the increase in IC50 and IC90 values of DHA against field isolates of P. falciparum was not significant, the tolerance to DHA-piperaquine (PPQ) combination might be because of PPQ only. Further study is required on more number of such isolates to generate data for a meaningful conclusion.

Benefits from retrieval practice are greater for students with lower working memory capacity.

We examined the effects of retrieval practice for students who varied in working memory capacity as a function of the lag between study of material and its initial test, whether or not feedback was given after the test, and the retention interval of the final test. We sought to determine whether a blend of these conditions exists that maximises benefits from retrieval practice for lower and higher working memory capacity students. College students learned general knowledge facts and then restudied the facts or were tested on them (with or without feedback) at lags of 0-9 intervening items. Final cued recall performance was better for tested items than for restudied items after both 10 minutes and 2 days, particularly for longer study-test lags. Furthermore, on the 2-day delayed test the benefits from retrieval practice with feedback were significantly greater for students with lower working memory capacity than for students with higher working memory capacity (r = -.42). Retrieval practice may be an especially effective learning strategy for lower ability students.

Correlation between in vitro and in vivo antimalarial activity of compounds using CQ-sensitive and CQ-resistant strains of Plasmodium falciparum and CQ-resistant strain of P. yoelii.

Present efforts have been made to establish a correlation between in vitro and in vivo antimalarial activity using MIC, IC50 and IC90 values against CQ-sensitive (3D7) and CQ-resistant (K1) strains of Plasmodium falciparum and in vivo activity against Plasmodium yoelii. The method of discriminant function analysis (DFA) was applied to analyze the data. It was observed that in vitro IC90 values against both 3D7 and K1 strains (p < 0.001) have strong correlation with in vivo curative activity. The respective IC50 and IC90 values of compounds, which cured mice (i.e., animals did not show recrudescence of parasitemia even after 60 days posttreatment), ranged between 3 and 14 nM and 14 and 186 nM against 3D7 and between 9 and 65 nM and 24 and 359 nM against the K1 strain of P. falciparum. Whereas the IC50 and IC90 values of compounds which exhibited in vivo suppressive activity in mice ranged between 10 and 307 nm and 61 and >965 nM, respectively, against 3D7 and 75 and >806 nm and 241 and >1232 nM against the K1 strain of P. falciparum. The findings suggest that IC90 values against both 3D7 and K1 strains (p < 0.02) are the main contributors for the prediction of in vivo curative activity of a new molecule. Apart from this, a reasonable correlation between MIC and IC50 values of compounds has also been established.

Mycobacterium tuberculosis can gain access to adipose depots of mice infected via the intra-nasal route and to lungs of mice with an infected subcutaneous fat implant.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis has the remarkable ability to persist as non-replicating forms in the host. These persisters are tolerant to drugs targeting actively replicating bacilli and hence are responsible for the need of an extended duration of anti-tubercular therapy. The anatomical locations and cell types housing Mtb persisters are being investigated in the recent times. Adipose tissue and the adipocytes are proposed niches of Mtb persisters. In the present study, we carried out experiments in the immunocompetent Swiss mice to see the dissemination of Mtb from lungs to adipose tissue and vice versa. Mice infected intra-nasally with ∼ 10(6), 10(4) or 10(2) bacilli harboured Mtb in various adipose depots distal to the lungs such as the visceral, subcutaneous and peri-renal depots. The dissemination was minimal at two weeks post-infection, as evident from culture negative adipose tissue samples. But at seven weeks post-infection, viable Mtb could be detected in 78%, 66% and 66% of the samples from high, moderate and low dose-infection groups respectively. In a separate experiment, Mtb-infected pre-adipocytes were implanted subcutaneously to un-infected mice. At five weeks post-implantation, the intact implants had a mean 7 ± 0.53 log10 CFUs/100 mg tissue, while the lungs had a mean 3.25 ± 0.32 log10 CFUs/100 mg tissue. In conclusion, the study shows that Mtb can disseminate from lungs to distant adipose depots and vice versa.

Comparison of Induced Sputum and Bronchoalveolar Lavage Fluid Examination in the Diagnosis of Sputum Negative Pulmonary Tuberculosis.

BACKGROUND: Tuberculosis (TB) is one of the common infections in the world, especially in developing countries like India. Therefore, early diagnosis is important. This study was undertaken to compare the yield of sputum, induction with bronchoalveolar lavage (BAL) in smear-negative suspected pulmonary TB patients in a tertiary care hospital in Agra. METHODS: Fifty patients were included in the study. In all patients, induced sputum, fibreoptic bronchoscopy and BAL fluid were subjected to diagnostic testing. RESULTS: On acid-fast smear examination, induced sputum and BAL fluid tested positive in 27/50 and 25/50 patients, respectively with a sensitivity of 83.3% and 90% respectively (p<0.0001). On comparing sputum induction versus BAL on culture, 30 patients were positive by sputum induction and 27 patients were positive on BAL fluid, with the sensitivity of 85.7% and 77.1%, respectively. The results showed that the sputum induction showed a significantly higher yield than that of BAL fluid (p=0.0013). CONCLUSION: Sputum induction offers an alternative approach in the diagnosis of smear-negative suspected pulmonary TB patients and would enhance sensitivity for the diagnosis of TB.

In Silico Exploration for New Antimalarials: Arylsulfonyloxy Acetimidamides as Prospective Agents.

A strategy is described to identify new antimalarial agents to overcome the drug resistance and/or failure issues through in silico screening of multiple biological targets. As a part of this, three enzymes namely CTPS, CK, and GST were selected, from among 56 drug targets of P. falciparum, and used them in virtual screening of ZINC database entries which led to the design and synthesis of arylsulfonyloxy acetimidamides as their consensus inhibitors. From these, two compounds showed good activity against sensitive (3D7; IC50, 1.10 and 1.45 µM) and resistant (K1; IC50, 2.10 and 2.13 µM) strains of the parasite, and they were further investigated through docking and molecular dynamics simulations. The findings of this study collectively paved the way for arylsulfonyloxy acetimidamides as a new class of antimalarial agents.

The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10.

Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. Here, we establish a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. We identify a transcriptional enhancer of Mef2c that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. We demonstrate that Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, we show that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes.

Antiplasmodial activity of new 4-aminoquinoline derivatives against chloroquine resistant strain.

Emergence and spread of multidrug resistant strains of Plasmodium falciparum has severely limited the antimalarial chemotherapeutic options. In order to overcome the obstacle, a set of new side-chain modified 4-aminoquinolines were synthesized and screened against chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of P. falciparum. The key feature of the designed molecules is the use of methylpiperazine linked α, ß(3)- and γ-amino acids to generate novel side chain modified 4-aminoquinoline analogues. Among the evaluated compounds, 20c and 30 were found more potent than CQ against K1 and displayed a four-fold and a three-fold higher activity respectively, with a good selectivity index (SI=5846 and 11,350). All synthesized compounds had resistance index between 1.06 and >14.13 as against 47.2 for chloroquine. Biophysical studies suggested that this series of compounds act on heme polymerization target.

Synthesis of chiral chloroquine and its analogues as antimalarial agents.

In this investigation, we describe a new approach to chiral synthesis of chloroquine and its analogues. All tested compounds displayed potent activity against chloroquine sensitive as well as chloroquine resistant strains of Plasmodium falciparum in vitro and Plasmodium yoelii in vivo. Compounds S-13 b, S-13c, S-13 d and S-13 i displayed excellent in vitro antimalarial activity with an IC50 value of 56.82, 60.41, 21.82 and 7.94 nM, respectively, in the case of resistant strain. Furthermore, compounds S-13a, S-13c and S-13 d showed in vivo suppression of 100% parasitaemia on day 4 in the mouse model against Plasmodium yoelii when administered orally. These results underscore the application of synthetic methodology and need for further lead optimization.

Collective memories of three wars in United States history in younger and older adults.

A collective memory is a representation of the past that is shared by members of a group. We investigated similarities and differences in the collective memories of younger and older adults for three major wars in U.S. history (the Civil War, World War II, and the Iraq War). Both groups were alive during the recent Iraq War, but only the older subjects were alive during World War II, and both groups learned about the Civil War from historical sources. Subjects recalled the 10 most important events that occurred during each war and then evaluated the emotional valence, the relative importance, and their level of knowledge for each event. They also estimated the percentage of people that would share their memory of each event within their age group and the other age group. Although most historical events were recalled by fewer than 25 % of subjects, younger and older adults commonly recalled a core set of events for each war that conform to a narrative structure that may be fundamental to collective remembering. Younger adults showed greater consensus in the events that they recalled for all three wars, relative to older adults, but there was less consensus in both groups for the Iraq War. Whereas younger adults recalled more specific events of short duration, older adults recalled more extended and summarized events of long duration. Our study shows that collective memories can be studied empirically and can differ depending on whether the events are experienced personally or learned from historical sources.

Standardized Quantification of Mast Cells in the Gastrointestinal Tract in Adults.

BACKGROUND: Current data on the normal quantity of mast cells throughout the adult gastrointestinal tract are limited in several domains. These include microanatomic localization of mast cells, standardization of staining and counting methods, and reporting of microscope field of view. OBJECTIVE: To address this lack of reliable reference ranges to facilitate the study of and diagnosis of emerging mast cell-mediated diseases. METHODS: We examined biopsies obtained from the esophagus, stomach, duodenum, and colon from an unselected cohort. Mean and peak mast cell density were determined on slides stained for tryptase and CD117, and were expressed per high power field (hpf) and surface area (mm2), thus deriving reference ranges (average ± 2 SDs). RESULTS: For the most common hpf surface area (0.238 mm2), upper limits of the derived reference ranges for average/peak mast cells were 0.15/3.67 (esophagus, tryptase), 0.70/5.98 (esophagus, CD117), 22.56/35.30 (stomach, tryptase), 31.32/53.10 (stomach, CD117), 30.28/49.77 (duodenal crypts, tryptase), 41.96/65.26 (duodenal crypts, CD117), 4.98/11.56 (duodenal villi, tryptase), 8.38/14.17 (duodenal villi, CD117), 26.58/41.08 (colon, tryptase), and 35.57/57.92 (colon, CD117). Interobserver variability was moderate to good. There was significant correlation between average and peak mast cell counts. CONCLUSIONS: These data help standardize mast cell reference ranges throughout the gastrointestinal tract in adults, which can be used to determine whether abnormal levels of mast cells are present in patients with suspected mast cell-mediated disease. Our data show that the commonly used cutoff of 20 mast cells per hpf irrespective of the gastrointestinal tract segment is an underestimate of an appropriate cutoff in stomach, duodenum (crypt area), and colon.

Assessment of knowledge, attitude and practice of fixed-dose combinations amongst attending physicians and residents: a cross-sectional evaluation.

Background: Fixed-dose combinations (FDCs) were brought into the market with the intent of providing benefits primarily to patients and physicians. Nevertheless, despite their multiple advantages, they have their own set of drawbacks, especially regarding irrational FDCs. If physicians continue to prescribe them, prohibiting their sale would become all the more challenging. This cross-sectional survey study was planned to comprehend the level of knowledge, attitude and practice of physicians regarding such FDCs at a tertiary care teaching institute of western Uttar Pradesh, India. Methodology: A pre-validated questionnaire was communicated electronically to all the attending physicians. For data analysis, descriptive statistics were applied and a χ2 test was performed for inter-group comparison. Results: Amongst the 108 respondents, participation was almost comparable from both medical and surgical branches, with most participants being junior residents (58%). Even with sound knowledge of FDCs, only 46.30% of them were aware of banned FDCs. Similarly, only 6.48% could correctly identify the disadvantages associated with the use of FDCs, and 33.18% could correctly recognize irrational FDCs. This finding was consistently reflected in their attitude and practice and only 15.74% of respondents cross-referenced FDCs with the available literature. Furthermore, despite 88.89% of respondents checking for rationality of FDCs before prescribing them, a compendium of irrational FDCs is routinely prescribed. Conclusion: To amend these shortcomings in prescribing of irrational FDCs, some recommendations are proposed by the authors herein.