Double Recombinant Mycobacterium bovis BCG Strain for Screening of Primary and Rationale-Based Antimycobacterial Compounds

Conventional antimycobacterial screening involves CFU analysis, which poses a great challenge due to slow growth of mycobacteria. Recombinant strains carrying reporter genes under the influence of constitutive promoters allow rapid and wide screening of compounds but without revealing their modes of action. Reporter strains using pathway-specific promoters provide a better alternative but allow a limited screening of compounds interfering with only a particular metabolic pathway. This reduces these strains to merely a second-line screening system, as they fail to identify even the more potent compounds if they are not inhibiting the pathway of interest. In this study, we have generated a double recombinant Mycobacterium bovis BCG strain carrying firefly and Renilla luciferase genes as two reporters under the control of a constitutive and an inducible mycobacterial promoter. The presence of dual reporters allows simultaneous expression and analysis of two reporter enzymes within a single system. The expression profile of the firefly luciferase gene, rendered by a constitutive mycobacterial promoter, coincides with the decline in bacterial growth in response to a wide range of antimycobacterial drugs, while the enhanced expression of Renilla luciferase mirrors the selective induction of the reporter gene expression as a result of pathway-specific inhibition. Thus, the double recombinant strain allows the screening of both primary and rationally synthesized antimycobacterial compounds in a single assay. The inhibiting response of drugs was monitored with a dual-luciferase reporter assay which can be easily adapted in high-throughput mode.     

Visual Assessment of Brain Magnetic Resonance Imaging Detects Injury to Cognitive Regulatory Sites in Patients With Heart Failure

BackgroundHeart failure (HF) patients exhibit depression and executive function impairments that contribute to HF mortality. Using specialized magnetic resonance imaging (MRI) analysis procedures, brain changes appear in areas regulating these functions (mammillary bodies, hippocampi, and frontal cortex). However, specialized MRI procedures are not part of standard clinical assessment for HF (which is usually a visual evaluation), and it is unclear whether visual MRI examination can detect changes in these structures.Methods and ResultsUsing brain MRI, we visually examined the mammillary bodies and frontal cortex for global and hippocampi for global and regional tissue changes in 17 HF and 50 control subjects. Significantly global changes emerged in the right mammillary body (HF 1.18 ± 1.13 vs control 0.52 ± 0.74; P = .024), right hippocampus (HF 1.53 ± 0.94 vs control 0.80 ± 0.86; P = .005), and left frontal cortex (HF 1.76 ± 1.03 vs control 1.24 ± 0.77; P = .034). Comparison of the visual method with specialized MRI techniques corroborates right hippocampal and left frontal cortical, but not mammillary body, tissue changes.ConclusionsVisual examination of brain MRI can detect damage in HF in areas regulating depression and executive function, including the right hippocampus and left frontal cortex. Visual MRI assessment in HF may facilitate evaluation of injury to these structures and the assessment of the impact of potential treatments for this damage.     

Endoscopic ultrasound-guided fine-needle aspiration of enlarged adrenals in patients with pyrexia of unknown origin: A single-center experience of 52 cases

Background

Fine-needle aspiration (FNA) of adrenals is needed in patients with pyrexia of unknown origin (PUO) and adrenal enlargement in absence of other diagnostic clues. Adrenals are easily accessible by endoscopic ultrasound (EUS) due to proximity; however, there is no systemic study available on FNA of adrenals in patients with PUO. The aim of this study was to evaluate the diagnostic yield and safety of EUS-FNA of enlarged adrenal in patients with PUO.

Methods

Data was analyzed from October 2010 to September 2016 at a single tertiary care center in northern India. EUS-FNA of enlarged adrenals was done in 52 patients for the etiological diagnosis of PUO in whom a definitive diagnosis could not be made with other means.

Results

The mean age was 48±14 years; 36 were males and 16 were females. EUS-FNA was done from the left adrenal in 50 patients and from the right sample in 2 patients. A technical success was achieved in 100% cases. The 19-G needle was used in the majority (75%) to the presence of necrotic areas in adrenals; median numbers of passes were 2. The cytopathological diagnoses were tuberculosis (n?=?36), histoplasmosis (n?=?13), lymphoma (n?=?2), and metastasis from undiagnosed neuroendocrine tumor of lung (n?=?1). Thus, a diagnosis could be made in 52/52 (100%) patients. None of the patients had any procedure-related complications.

Conclusions

EUS-FNA is a safe and effective method for evaluating etiology of PUO in patients with adrenal enlargement.

     

Evaluations of HIV type 1 rev gene diversity and functional domains following perinatal transmission

The human immunodeficiency virus type 1 (HIV-1) rev exons 1 and 2 sequences were analyzed from six mother-infant pairs following perinatal transmission. The rev open reading frame was maintained with a frequency of 93.96% in six mother-infant pairs' sequences. There was a low degree of viral heterogeneity and estimates of genetic diversity in mother-infant pairs' rev sequences. However, the distances of rev sequences between epidemiologically unlinked individuals were greater than in epidemiologically linked mother-infant pairs. Furthermore, phylogenetic parameters revealed that the epidemiologically linked mother-infant pairs were closer evolutionarily to each other as compared with epidemiologically unlinked mother-infant pairs. Both mothers and infants were under positive selection pressure as determined by the ratios of nonsynonymous to synonymous substitutions. The functional domains required for Rev activity, including nuclear export of RNA, RNA binding domain, and nuclear import signals, were conserved in all mother-infant pairs' sequences. The conservation of functional domains of rev and a low degree of heterogeneity following vertical transmission are consistent with an indispensable role of rev in the HIV-1 life cycle.