Cell state transition analysis identifies interventions that improve control of Mycobacterium tuberculosis infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from the tuberculosis (TB) susceptible and resistant mice. We then apply the cSTAR (cell state transition assessment and regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the transcriptional state of tumor necrosis factor (TNF)-activated TB-susceptible macrophages toward that of TB-resistant cells, i.e., prevents their aberrant activation without suppressing beneficial TNF responses. Last, we demonstrate that the compounds identified with this approach enhance the resistance of the TB-susceptible mouse macrophages to virulent Mycobacterium tuberculosis.

Cell state transition analysis identifies interventions that improve control of M. tuberculosis infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from TB-susceptible and TB-resistant mice. We then apply the cSTAR (cell State Transition Assessment and Regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the. transcriptional state of the TB-susceptible macrophages towards that of TB-resistant cells. Finally, we demonstrate that the compounds identified with this approach enhance resistance of the TB-susceptible mouse macrophages to virulent M. tuberculosis .

Medium throughput protocol for genome-based quantification of intracellular mycobacterial loads and macrophage survival during in vitro infection.

Here, we present a streamlined protocol for assessing intracellular Mycobacterium tuberculosis (Mtb) loads in macrophages. This protocol describes the simultaneous assessment of macrophage viability using automated microscopy. Further, we detail the quantification of mycobacterial loads using a rapid, inexpensive, and accurate approach for mycobacterial DNA isolation from paraformaldehyde-fixed macrophages. Simultaneous assessment of the bacterial loads using internal standard and macrophage viability allows for precise quantification of the effects of perturbations on Mtb and host cells while accounting for technical artifacts. For complete details on the use and execution of this protocol, please refer to Chatterjee et al. (2021).

A TOPSIS-Inspired Ranking Method Using Constrained Crowd Opinions for Urban Planning.

Crowdsourcing has become an important tool for gathering knowledge for urban planning problems. The questions posted to the crowd for urban planning problems are quite different from the traditional crowdsourcing models. Unlike the traditional crowdsourcing models, due to the constraints among the multiple components (e.g., multiple locations of facilities) in a single question and non-availability of the defined option sets, aggregating of multiple diverse opinions that satisfy the constraints as well as finding the ranking of the crowd workers becomes challenging. Moreover, owing to the presence of the conflicting nature of features, the traditional ranking methods such as the Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS) cannot always be feasible as the optimal solutions in terms of multiple objectives cannot occur simultaneously for the conflicting cases (e.g., benefit and cost criteria) for urban planning problems. Therefore, in this work, a multi-objective approach is proposed to produce better compromised solutions in terms of conflicting features from the general crowd. In addition, the solutions are employed to obtain a proper ideal solution for ranking the crowd. The experimental results are validated using two constrained crowd opinion datasets for real-world urban planning problems and compared with the state-of-the-art TOPSIS models.

Fuzzy association analysis for identifying climatic and socio-demographic factors impacting the spread of COVID-19.

Recently, the whole world witnessed the fatal outbreak of COVID-19 epidemic originating at Wuhan, Hubei province, China, during a mass gathering in a film festival. World Health Organization (WHO) has declared this COVID-19 as a pandemic due to its rapid spread across different countries within a few days. Several research works are being performed to understand the various influential factors responsible for spreading COVID. However, limited studies have been performed on how climatic and socio-demographic conditions may impact the spread of the virus. In this work, we aim to find the relationship of socio-demographic conditions, such as temperature, humidity, and population density of the regions, with the spread of COVID-19. The COVID data for different countries along with the social data are collected. For the experimental purpose, Fuzzy association rule mining is employed to infer the various relationships from the data. Moreover, to examine the seasonal effect, a streaming setting is also considered. The experimental results demonstrate various interesting insights to understand the impact of different factors on spreading COVID-19.

Channeling macrophage polarization by rocaglates increases macrophage resistance to Mycobacterium tuberculosis.

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatment of primary macrophages with rocaglates enhanced phagosome-lysosome fusion and control of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization toward the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g., chronic bacterial infections, allergies, and, possibly, certain tumors.

Mitral valve repair in children with rheumatic heart disease.

PURPOSE: Rheumatic heart disease is the most common acquired heart disease in children in developing countries. The heart valve lesions produce severe hemodynamic changes due to scarring of the valves over time. Around 15.6 million people are affected by rheumatic heart disease (RHD), and 230,000 die around the globe annually. Valve repair should be the primary goal, although it is technically challenging because of the fact that rheumatic process evolves making repair outcomes variable. METHODS: We reviewed the literature for the various techniques done for mitral valve repair in children with rheumatic heart disease. Early and late results of repair were compared with the results found for mitral valve repair done for such children. RESULTS: Prosthetic heart valve implantation in children has major negative impact on their immediate- and long-term survival as well as on quality of their life. Valve repair is associated with improved ventricular function because the normal valve tissue and subvalvular apparatus are preserved, reduced complications related to prosthetic valve, and lower in-hospital and late mortality. CONCLUSION: In children, the results of mitral valve replacement were found to be inferior to those of mitral valve repair. The reoperation rates are similar in patients undergoing initial repair or replacement, which favors repair as an option. In developing world, rheumatic mitral valve disease is more prevalent where adequate facilities for monitoring of prosthetic valve function and management of anticoagulation therapy are not easily available. Valve repair therefore should be the primary goal.

Neonatal ascending aorta thrombosis: A rare and lethal entity.

Ascending aorta thrombosis (AAT) in a neonate is a rare and lethal event. To date, AAT has been reported in around 25 patients, out of whom only eight patients had concomitant arch involvement. We report a case of one such patient with ascending aorta and arch thrombosis and present a brief review of the available literature.

The integrated stress response mediates necrosis in murine Mycobacterium tuberculosis granulomas.

The mechanism by which only some individuals infected with Mycobacterium tuberculosis develop necrotic granulomas with progressive disease while others form controlled granulomas that contain the infection remains poorly defined. Mice carrying the sst1-suscepible (sst1S) genotype develop necrotic inflammatory lung lesions, similar to human tuberculosis (TB) granulomas, which are linked to macrophage dysfunction, while their congenic counterpart (B6) mice do not. In this study we report that (a) sst1S macrophages developed aberrant, biphasic responses to TNF characterized by superinduction of stress and type I interferon pathways after prolonged TNF stimulation; (b) the late-stage TNF response was driven via a JNK/IFN-ß/protein kinase R (PKR) circuit; and (c) induced the integrated stress response (ISR) via PKR-mediated eIF2α phosphorylation and the subsequent hyperinduction of ATF3 and ISR-target genes Chac1, Trib3, and Ddit4. The administration of ISRIB, a small-molecule inhibitor of the ISR, blocked the development of necrosis in lung granulomas of M. tuberculosis-infected sst1S mice and concomitantly reduced the bacterial burden. Hence, induction of the ISR and the locked-in state of escalating stress driven by the type I IFN pathway in sst1S macrophages play a causal role in the development of necrosis in TB granulomas. Interruption of the aberrant stress response with inhibitors such as ISRIB may offer novel host-directed therapy strategies.

Left isomerism with bilateral superior vena cava, interrupted inferior vena cava and tetralogy of Fallot.

The association of left isomerism with tetralogy of Fallot (TOF) is rare and reported as scattered case reports. Complex congenital cardiac and noncardiac malformations are known to be associated with left isomerism, but right-sided obstructive lesions are rare. We present our experience with a case of left isomerism with bilateral superior vena cava, interrupted inferior vena cava, and TOF managed by atrial septation and intracardiac repair.

Outcome of 40 consecutive cases of modified Ross procedure using novel Dacron valved conduit.

OBJECTIVE: The Ross procedure is an established option for aortic valve disease in children. Due to limited availability of pulmonary homograft, we devised a novel technique for right ventricular outflow tract (RVOT) reconstruction by preparing indigenous Dacron valved conduit. METHODS: Forty consecutive cases of modified Ross procedure done at our center (2013-2018) were analyzed. Thirty-seven patients (95%) were followed up with median duration of 2.5 (0.08-5.5) years. Median age was 12 (5-39) years. Nineteen (47.5%) patients had rheumatic aortic valve disease, while 19 (47.5%) had congenital aortic valve disease. Aortic root replacement with pulmonary autograft was performed in all patients. Dacron conduit for RVOT reconstruction was used with on table sewn bileaflet valve using Dacron patch (n = 22), expanded polytetrafluoroethylene (ePTFE) membrane (n = 10), bioprosthetic valve (n = 4), and pericardium (n = 4). Additional surgical procedures included mitral valve repair (n = 10), septal myectomy (n = 2), ascending aorta replacement (n = 1), ruptured sinus of valsalva (RSOV) repair (n = 1), and ventricular septal defect (VSD) closure (n = 1). RESULTS: There was one in-hospital mortality while one late death occurred at 3.5 years postoperatively. The neo-aortic valve regurgitation on echocardiographic evaluation at last follow-up was trivial (n = 28), mild (n = 7), and moderate (n = 2). Mild RVOT obstruction was present in 8 patients while 18 patients had mild pulmonary regurgitation. No patient required reintervention during follow-up. CONCLUSION: Our early results of modified Ross procedure are encouraging, however, long-term follow-up is required.

Pulmonary root translocation and Senning procedure for congenitally corrected transposition of great arteries with pulmonary stenosis: a case report.

Congenitally corrected transposition of the great arteries (CCTGAs) is a condition, which includes atrioventricular and ventriculoarterial discordant connections along with ventricular septal defect (VSD), pulmonary stenosis (PS), or pulmonary atresia (PA). Without treatment, progressive systemic ventricular failure begins, which is followed by sudden cardiac death by the fourth or fifth decade. We report a case of a 4-year-old with CCTGA, VSD, and PS operated by Senning procedure and pulmonary root translocation (PRT) with uneventful postoperative recovery. PRT overcomes problems with the right ventricle to the pulmonary artery conduit and maintains pulmonary valve function and growth capacity. Our initial experience with PRT in CCTGA indicates that it is a feasible surgical alternative for such patients.

Comparison of del Nido and histidine-tryptophan-ketoglutarate cardioplegia solutions in pediatric patients undergoing open heart surgery: A prospective randomized clinical trial.

OBJECTIVES: We conducted a prospective randomized controlled trial to compare del Nido (DN) and histidine-tryptophan-ketoglutarate (HTK) cardioplegia solution in pediatric patients undergoing intracardiac tetralogy of Fallot repair. METHODS: One hundred consecutive patients 12 years of age or younger, undergoing intracardiac repair of tetralogy of Fallot were randomized into DN (n = 50) and HTK (n = 50) groups. Cardioplegia strategy consisted of a single dose of DN (20 mL/kg) or HTK (6 mL/kg/min for 6 minutes). Primary outcome was cardiac index (CI). Secondary outcomes were ventricular arrhythmias post cross-clamp release, time to peripheral rewarming, duration of mechanical ventilation, inotropic score, intensive care unit and hospital stay, and serum levels of troponin-I, interleukin-6, and tumor necrosis factor-α. Ultrastructural changes in the myocardium were assessed. RESULTS: CI was significantly higher in the DN group compared with the HTK group at 6 (P = .005) and 24 hours (P < .001) after surgery. It was on an average 0.44 L/min/m2 higher in the DN group at any time point (P = .004). Time for complete cessation of electrical activity was longer in the HTK group (P = .01) and more patients in the HTK group had ventricular arrhythmias post cross-clamp release (P = .03). Duration of mechanical ventilation (P = .006), intensive care unit stay (P = .05), and hospital stay (P < .001) were lower in the DN group. Patients in the DN group had lower troponin I levels 24 hours after cardiopulmonary bypass (P < .001). Electron microscopic studies showed more myocardial edema (P = .02) and myofibrillar disarray (P = .04) in the HTK group along with lower glycogen stores (P = .04). DN cardioplegia was more cost-effective than HTK cardioplegia (P < .001). CONCLUSIONS: DN cardioplegia was associated with better preservation of CI, less duration of mechanical ventilation, shorter intensive care unit and hospital stays, lower inotropic scores, and less release of troponin-I. Electron microscopy showed less myocardial edema and better preservation of the myofibrillar architecture and glycogen stores in the DN group.

Fine-tuning of macrophage activation using synthetic rocaglate derivatives.

Drug-resistant bacteria represent a significant global threat. Given the dearth of new antibiotics, host-directed therapies (HDTs) are especially desirable. As IFN-gamma (IFNγ) plays a central role in host resistance to intracellular bacteria, including Mycobacterium tuberculosis, we searched for small molecules to augment the IFNγ response in macrophages. Using an interferon-inducible nuclear protein Ipr1 as a biomarker of macrophage activation, we performed a high-throughput screen and identified molecules that synergized with low concentration of IFNγ. Several active compounds belonged to the flavagline (rocaglate) family. In primary macrophages a subset of rocaglates 1) synergized with low concentrations of IFNγ in stimulating expression of a subset of IFN-inducible genes, including a key regulator of the IFNγ network, Irf1; 2) suppressed the expression of inducible nitric oxide synthase and type I IFN and 3) induced autophagy. These compounds may represent a basis for macrophage-directed therapies that fine-tune macrophage effector functions to combat intracellular pathogens and reduce inflammatory tissue damage. These therapies would be especially relevant to fighting drug-resistant pathogens, where improving host immunity may prove to be the ultimate resource.

Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures.

Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.

FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells.

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.

Note: Neutron shutter for Kolkata superconducting cyclotron.

In particle accelerator facilities, experimental areas are isolated from active accelerator area with thick concrete walls. A neutron shutter is used to isolate the experimental areas from the active accelerator area in the beam line. These shutters are provided primarily to reduce the secondary radiations like neutrons in the experimental areas to permissible limit when the primary beam is blocked in the accelerator area. The reduced radiation level in the experimental areas makes the experimental areas accessible. The shutters should allow the primary beam to pass to the experimental caves when they retracted from the beam line. A new neutron shutter has been designed and fabricated. This shutter incorporates compact features with considerable reduction in length, surface area and volume. The attenuation of secondary radiations is evaluated using Monte-Carlo radiation transport code FLUKA. It is found that the features of the shutter are sufficiently good enough to reduce the diffused secondary radiations well within the permissible levels.

Interaction of bacteriophage l with its E. coli receptor, LamB.

The initial step of viral infection is the binding of a virus onto the host cell surface. This first viral-host interaction would determine subsequent infection steps and the fate of the entire infection process. A basic understating of the underlining mechanism of initial virus-host binding is a prerequisite for establishing the nature of viral infection. Bacteriophage λ and its host Escherichia coli serve as an excellent paradigm for this purpose. λ phages bind to specific receptors, LamB, on the host cell surface during the infection process. The interaction of bacteriophage λ with the LamB receptor has been the topic of many studies, resulting in wealth of information on the structure, biochemical properties and molecular biology of this system. Recently, imaging studies using fluorescently labeled phages and its receptor unveil the role of spatiotemporal dynamics and divulge the importance of stochasticity from hidden variables in the infection outcomes. The scope of this article is to review the present state of research on the interaction of bacteriophage λ and its E. coli receptor, LamB.

Structural basis of transcription initiation.

During transcription initiation, RNA polymerase (RNAP) binds and unwinds promoter DNA to form an RNAP-promoter open complex. We have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, σ(A), and a promoter DNA fragment corresponding to the transcription bubble and downstream double-stranded DNA of the RNAP-promoter open complex. The structures show that σ recognizes the -10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases and that RNAP recognizes the -4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. The structures further show that interactions between σ and template-strand single-stranded DNA (ssDNA) preorganize template-strand ssDNA to engage the RNAP active center.

Evolutionary link between the mycobacterial plasmid pAL5000 replication protein RepB and the extracytoplasmic function family of σ factors.

Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the Actinobacteria. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10(-3)) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) σ factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of σ factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C- and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules.