A Curative Immune Profile One Week after Treatment of Indian Kala-Azar Patients Predicts Success with a Short-Course Liposomal Amphotericin B Therapy

Background

The present pilot study investigating the minimum dose for short-course single and double-dose treatment of kala-azar with an apparently new liposomal formulation of amphotericin B, Fungisome, led to identification of immunological components for early detection of success and/or failure to cure.

Methods

Patients were treated with 5, 7.5 (single-dose) and 10 mg/kg body weight (5 mg/kg double-dose) of Fungisome. Immunological investigations involving plasma cytokines and antigen-specific lymphoproliferation and cytokine responses from PBMCs were carried out before, 1 week after Fungisome treatment, at the time of relapse, and again after conventional amphotericin B treatment.

Results

At 1-month follow-up all the patients showed 100% initial cure. However, total doses of 5, 7.5 and 10 mg/kg Fungisome showed 60%, 50% and 90% cure, respectively, at 6-months posttreatment. Patients successfully cured demonstrated downregulation of IL-12 and IL-10 in plasma, and two-fold or more elevation of IFN-γ, IL-12 and TNF, and significant down-regulation of IL-10 and TGF-β in culture supernatants 1-week posttreatment irrespective of drug-dose. A differential immune profile, involving insignificant decline in IL-10 and IL-12 in plasma and negligible elevation of IFN-γ, IL-12 and TNF, and persistence of IL-10, despite decline in TGF-β in culture supernatants, in apparently cured individuals, corresponded with relapse within 6-months of treatment.

Conclusion

Immunological investigations revealed significant curative and non-curative immunomodulation 1-week posttreatment, correlating with successful cure and relapse, respectively. Although immune-correlation was dose-independent, almost consistent curative response in patients treated with the highest dose 10 mg/kg reflected a definitive impact of the higher-dose on the immune response.

Trial registration name and number

Clinical Trials Registry - India (CTRI) CTRI/2009/091/000764     

Recommendations for school-going students post CoVid-19 in Bangladesh

The CoVid-19 pandemic caused by SARS-CoV-2 has taken more lives than any other pandemic so far, with non-pharmacological interventions such as lockdown, school closures, and travel bans, especially social distance, abounding around the world. With limited resources, these interventions pose the ultimate challenge to the education system in developing countries like Bangladesh, especially in providing uninterrupted education for all children in rural areas, where a significant number of students are enrolled in this area. However, the initiative to close schools for an extended period has affected children physically, emotionally, socially, and in various ways. Noteworthy, it demands to reopen to protect the future of children. Schools have reopened in many countries around the world. It is of interest to document recommendations for school-going students post CoVid-19 in Bangladesh using evidence-based data, information, and knowledge. We document such data in the context of Bangladesh to take such policy initiatives.     

Naphthyridine-imidazole hybrid ligands for the construction of multinuclear architecture

Reaction of 2-imidazolyl-5,7-dimethyl-1,8-naphthyridine (L¹) with [Rh(COD)Cl]₂ (COD = 1,5-cyclooctadiene) affords the dinuclear complex [Rh(COD)Cl]₂(μ-L¹) (1). Elimination of chloride from the metal coordination sphere leads to a self-assembled tetranuclear macrocycle [Rh(COD)L¹]₄[ClO₄]₄ (2). A subtle alteration in the ligand framework results in the polymeric chain compound {Rh(COD)(L²)}_n(PF₆)_n (3) (L² = 2-imidazolyl-3-phenyl-1,8-naphthyridine). In all these complexes, the imidazole nitrogen and one of the naphthyridine nitrogen (away from the imidazole substituent) bind the metal. The ‘parallel’ and ‘perpendicular’ dispositions of nitrogens are observed in these compounds contributing to different Rh···Rh separations. The L¹ ligand adopts planar configuration, whereas the naphthyridine-imidazole rings deviate from planarity in L² yielding a polymeric structure. The extent of deviation is less in the polymeric structure {Mo₂(OAc)₄(L²)}_n (4) in which the ligand exhibits weak axial interactions to the metal.     

Stress tolerant virulent strains of Cronobacter sakazakii from food

Background

Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh.

Result

Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress.

Conclusion

Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-63) contains supplementary material, which is available to authorized users.     

Regulation of dynamin family proteins by post-translational modifications

Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agents involved in several biological processes such as endocytosis, maintenance of organelle morphology and viral resistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion or tubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulated by various post-translational modifications that affect their GTPase activity, multimerization or membrane association. Recently, several reports have demonstrated variety of such modifications providing a better understanding of the mechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In this review, we discuss major post-translational modifications along with their roles in the mechanism of dynamin functions and implications in various cellular processes.     

On the performance of adaptive preprocessing technique in analyzing high‐dimensional censored data

Preprocessing for high‐dimensional censored datasets, such as the microarray data, is generally considered as an important technique to gain further stability by reducing potential noise from the data. When variable selection including inference is carried out with high‐dimensional censored data the objective is to obtain a smaller subset of variables and then perform the inferential analysis using model estimates based on the selected subset of variables. This two stage inferential analysis is prone to circularity bias because of the noise that might still remain in the dataset. In this work, I propose an adaptive preprocessing technique that uses sure independence screening (SIS) idea to accomplish variable selection and reduces the circularity bias by some popularly known refined high‐dimensional methods such as the elastic net, adaptive elastic net, weighted elastic net, elastic net‐AFT, and two greedy variable selection methods known as TCS, PC‐simple all implemented with the accelerated lifetime models. The proposed technique addresses several features including the issue of collinearity between important and some unimportant covariates, which is often the case in high‐dimensional setting under variable selection framework, and different level of censoring. Simulation studies along with an empirical analysis with a real microarray data, mantle cell lymphoma, is carried out to demonstrate the performance of the adaptive pre‐processing technique.     

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal β/γ-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, ΔGRT1 ΔGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in ΔGRT1 ΔGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from ΔGRT1 ΔGRT2 cells appear less adhesive than those from the wild type.In eukaryotes, the directional transport of lumenal proteins throughout the network of membrane-bound organelles depends on reversible assembly of multisubunit protein complexes in the cytoplasm. For example, the assembly of a localized clathrin coat at a cell''s surface facilitates both the concentration of specific transmembrane receptors together with their bound ligands at that site and the invagination and budding of the plasma membrane, resulting in endocytosis (18). Similarly, other cytosolic coats assemble and direct traffic at the endoplasmic reticulum (ER) and Golgi apparatus (4). For one protein trafficking pathway in eukaryotic cells, however, the determinative protein self-assembly occurs not in the cytoplasm but within the lumen of the secretory pathway itself. Dense core granules (DCGs) are secretory vesicles whose lumenal cargo consists of a condensed polypeptide aggregate. This cargo is secreted when the vesicles fuse with the plasma membrane in response to a specific extracellular stimulus, an event called regulated exocytosis. The aggregation of the cargo occurs progressively within the secretory pathway, beginning in the trans-Golgi network (TGN), and may be promoted by multiple factors including compartment-specific proton and calcium levels (23). Aggregation facilitates the vesicular storage of concentrated secretory proteins but also serves as a sorting mechanism to segregate DCG proteins from proteins that are secreted via other pathways. Evidence for this mechanism includes in vitro experiments showing that some proteins released via constitutive exocytosis remain soluble under TGN-like conditions that promote DCG protein aggregation (10). In vivo, sorting would result if aggregated and soluble proteins exit the TGN in different carriers. Importantly, there is no evidence that sorting of DCG proteins at the TGN requires assembly of cytosolic coat complexes.While aggregative sorting represents an attractively simple mechanism, relatively little is known about the structure or dynamic properties of the aggregates themselves. This is an interesting issue, as illustrated by several phenomena. First, aggregates in some cell types, like those formed by proinsulin in pancreatic β cells, can become reordered as protein crystals during a multistage process called granule maturation (13). Second, Aplysia bag cells can sort different subsets of DCG proteins into distinct granules, suggesting that aggregation can be finely regulated and that different aggregates have different properties in vivo (20). Both of these phenomena have also been observed within the DCGs of unicellular ciliates (3, 14). In addition, ciliate DCGs demonstrate another degree of subtlety in DCG formation because the granule cores in many of these organisms are divided into distinct domains (25). The domain organization indicates that DCG proteins in these cells can segregate from one another even as they are sorted to the same vesicular destination. While the structures of DCGs in many ciliates have been captured by electron microscopy, molecular studies have advanced in two species, Tetrahymena thermophila and Paramecium tetraurelia (30, 33).In many ciliates, the individual DCGs are organized in at least two distinct domains within the lumen. First, the bulk of the cargo is organized as a core crystal that expands, spring-like, upon exocytosis (28). This expansion can drive rapid extrusion of the DCG contents, which may be essential for hunting or defensive behaviors (17). In addition, many ciliate DCGs possess a single polarized tip structure that is involved in DCG docking to the plasma membrane and exocytic fusion (25). These tip structures are also filled with condensed, highly organized proteins, which appear by both genetic and morphological criteria to be different from proteins making up the expansible core (1, 21). The proteins that form the distinct domains are beginning to be identified and analyzed. Those that constitute the expansible springs are encoded by homologous families of genes named GRL (granule lattice) in Tetrahymena and tmp (trichocyst matrix) in Paramecium (11, 12, 15). Assembly of Grl proteins begins in the ER with formation of heterooligomers. This is an obligatory step, as shown by the fact that deletion of individual Grl proteins by targeted gene disruption resulted in the ER retention of remaining Grl proteins (12). Further assembly of Grl proteins to form a crystal occurs during DCG maturation and is accompanied by site-specific proprotein processing (34). Upon exocytosis, the expansion of the crystalline core is controlled by calcium binding to the fully processed Grl proteins (34).In addition to the GRL family-encoded proteins, 13 other lumenal DCG proteins have been putatively or definitively identified in Tetrahymena, and homologous proteins are predicted in the Paramecium genome (6). The entire set belongs to a gene family that is defined by a carboxy-terminal β/γ-crystallin domain, which may function as a DCG-targeting motif (16). Studies of two different members of this family in Tetrahymena, IGR1 (induced during granule regeneration 1) and GRT1 (granule tip 1), suggested that these proteins are functionally distinct from the spring-forming Grl proteins. First, whereas gene disruption of any of the highly transcribed GRL genes resulted in grossly aberrant spring formation, no such defect was seen upon disruption of IGR1 (16). However, this could be explained by the fact that IGR1 encodes a relatively low-abundance protein in DCGs, and furthermore its function could be redundant with that of the highly related gene, IGR2.The second protein in the β/γ-crystallin domain family that has been investigated is the 80-kDa product of the GRT1 gene. Grt1p was first detected as one of the most abundant DCG components released during exocytosis (32). Biochemical analysis showed that Grt1p differs in its solubility from the Grl proteins and also that it is packaged intact in DCGs rather than undergoing proteolytic processing (31). Since processing is essential for Grl protein assembly and function, this difference appears highly significant. Second, Grt1p accumulates at a single pole of each DCG, corresponding to the tip of the organelle that docks and then fuses with the plasma membrane (5). Two Mendelian mutants with defects in DCG maturation show delocalized Grt1p, and these mutant DCGs can dock but do not appear to undergo exocytosis (5). These results suggested that Grt1p might be involved in forming a DCG tip domain that interacted with the plasma membrane.We have now investigated the trafficking and function of Grt1p. Our data provide both direct biochemical and cell-biological evidence that Grt1p and Grl proteins form distinct complexes during DCG biogenesis in Tetrahymena. Together with earlier results, our experiments provide genetic evidence that Grl and Grt complexes can be independently trafficked to DCGs. Cells lacking GRT1, together with the closely related GRT2, still show rapid and efficient release of DCG contents upon stimulation with secretagogues, but the released DCG contents are subtly different from those of the wild type, suggesting that Grt1p may primarily serve a postexocytic function.