Evolution of drug-resistant viral populations during interruption of antiretroviral therapy

Analysis of a large number of HIV-1 genomes at multiple time points after antiretroviral treatment (ART) interruption allows determination of the evolution of drug-resistant viruses and viral fitness in vivo in the absence of drug selection pressure. Using a parallel allele-specific sequencing (PASS) assay, potential primary drug-resistant mutations in five individual patients were studied by analyzing over 18,000 viral genomes. A three-phase evolution of drug-resistant viruses was observed after termination of ART. In the first phase, viruses carrying various combinations of multiple-drug-resistant (MDR) mutations predominated with each mutation persisting in relatively stable proportions while the overall number of resistant viruses gradually increased. In the second phase, viruses with linked MDR mutations rapidly became undetectable and single-drug-resistant (SDR) viruses emerged as minority populations while wild-type viruses quickly predominated. In the third phase, low-frequency SDR viruses remained detectable as long as 59 weeks after treatment interruption. Mathematical modeling showed that the loss in relative fitness increased with the number of mutations in each viral genome and that viruses with MDR mutations had lower fitness than viruses with SDR mutations. No single viral genome had seven or more drug resistance mutations, suggesting that such severely mutated viruses were too unfit to be detected or that the resistance gain offered by the seventh mutation did not outweigh its contribution to the overall fitness loss of the virus. These data provide a more comprehensive understanding of evolution and fitness of drug-resistant viruses in vivo and may lead to improved treatment strategies for ART-experienced patients.     

Molecular Evidence of Increased Resistance to Anti-Folate Drugs in Plasmodium falciparum in North-East India: A Signal for Potential Failure of Artemisinin Plus Sulphadoxine-Pyrimethamine Combination Therapy

North-east India, being a corridor to South-east Asia, is believed to play an important role in transmitting drug resistant Plasmodium falciparum malaria to India and South Asia. North-east India was the first place in India to record the emergence of drug resistance to chloroquine as well as sulphadoxine/pyrimethamine. Presently chloroquine resistance is widespread all over the North-east India and resistance to other anti-malarials is increasing. In this study both in vivo therapeutic efficacy and molecular assays were used to screen the spectrum of drug resistance to chloroquine and sulphadoxine/pyrimethamine in the circulating P. falciparum strains. A total of 220 P. falciparum positives subjects were enrolled in the study for therapeutic assessment of chloroquine and sulphadoxine/pyrimethamine and assessment of point mutations conferring resistances to these drugs were carried out by genotyping the isolates following standard methods. Overall clinical failures in sulphadoxine/pyrimethamine and chloroquine were found 12.6 and 69.5% respectively, while overall treatment failures recorded were 13.7 and 81.5% in the two arms. Nearly all (99.0%) the isolates had mutant pfcrt genotype (76T), while 68% had mutant pfmdr-1 genotype (86Y). Mutation in dhps 437 codon was the most prevalent one while dhfr codon 108 showed 100% mutation. A total of 23 unique haplotypes at the dhps locus and 7 at dhfr locus were found while dhps-dhfr combined loci revealed 49 unique haplotypes. Prevalence of double, triple and quadruple mutations were common while 1 haplotype was found with all five mutated codons (F/AGEGS/T) at dhps locus. Detection of quadruple mutants (51I/59R/108N/164L) in the present study, earlier recorded from Car Nicobar Island, India only, indicates the presence of high levels of resistance to sulphadoxine/pyrimethamine in north-east India. Associations between resistant haplotypes and the clinical outcomes and emerging resistance in sulphadoxine/pyrimethamine in relation to the efficacy of the currently used artemisinin combination therapy are discussed.     

A Highlights from MBoC Selection: Role of the loop L4,5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation

Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L_4,5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L_4,5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L_4,5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein–bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.     

Suggestive evidence of association of C-159T functional polymorphism of the <Emphasis Type="Italic">CD14</Emphasis> gene with atopic asthma in northern and northwestern Indian populations

CD14 is a lipopolysaccharide receptor known to be an important modulator of Th1–Th2 response during early childhood. Genetic association studies of the CD14 gene with asthma and atopic disorders have shown positive as well as negative results in different ethnic populations. The aim of this study was to test for association of C-159T functional promoter polymorphism with atopic asthma and serum IgE levels in northern and northwestern Indian populations. DNA was assayed for the CD14 C-159T polymorphism in a case-control study involving atopic asthmatics (n=187) and healthy normal controls (n=227), and in a family-based association study of 106 trios. The case-control study showed an association at the genotypic (P=0.0146) as well as the allelic level (P=0.0048). Moreover, we observed a deviation of allelic transmission from random proportions (P=0.024) in the transmission disequilibrium test analysis. When we analyzed our results for serum total IgE levels, against this polymorphism, we observed a difference at the genotypic (P=0.0026) as well as at the allelic level (P=0.0016) in a case-control study, whereas no association in the quantitative transmission disequilibrium test analysis was obtained. These findings provide suggestive evidence of association of the CD14 gene locus with atopic asthma in northern and northwestern Indian populations.     

Bio-control activity of Purpureocillium lilacinum strains in managing root-knot disease of tomato caused by Meloidogyne incognita

The potential of 24 indigenous isolates of Purpureocillium lilacinum (Paecilomyces lilacinus) (Thom) Samson collected from different agro-climatic zones of India was investigated against the root-knot nematode, Meloidogyne incognita. The studies were conducted in vitro (larvicidal, ovicidal and egg-parasitising capacity) and under naturally infested field conditions with selected strains. Repeated field trials were conducted with talc-based preparations of fungal strains at 10 kg ha^?1, which were applied mixed in farm yard manure (FYM) at 1.5 t ha^?1. Results (in vitro) showed that all tested isolates were capable to parasitise eggs, inhibit egg hatching and cause juvenile mortality of M. incognita at various levels. Based on the performance under in vitro studies, eight isolates (NDPL-01, ANDPL-02, SHGPL-03, HYBPL-04, AHDPL-05, PTNPL-06, SNGPL-07 and VNSPL-08) were re-tested to confirm the results. HYBDPL-04 was found causing highest mortality (80%), inhibition of egg hatching (90%) as well as parasitisation of M. incognita eggs (75%). Under field trials also, the best protection of root-knot disease of tomato (Lycopersicon esculentum L.), in terms of reduction of galls (61%) and reproductive factor (P_f/P_i (RF) = 0.2) was achieved through application of HYBDPL-04 + FYM compared to control and other tested isolates. It also enhanced marketable yield of tomato up to 43%. It is concluded that the HYBDPL-04 strain of P. lilacinum is highly effective for management of root-knot disease of tomato under naturally infested field conditions. It is the isolate which produced the maximum number of metabolites which were extracted through high pressure liquid chromatography.     

Sustaining immunity during starvation in bivalve mollusc: A costly affair

Complete or partial depletion of resource in a freshwater habitat is a common phenomenon. As a consequence, aquatic fauna including bivalve molluscs may be exposed to dietary stress on a seasonal basis. Haemocyte based innate immune profile of the freshwater mollusc Lamellidens marginalis (Bivalvia: Eulamellibranchiata) was evaluated under starvation induced stress for a maximum period of 32 days in a controlled laboratory condition. During starvation, the bivalve haemocytes maintained a homeostasis in phagocytic efficacy and nitric oxide generation ability with respect to the control. The mollusc maintained a significantly high protein content in its haemolymph and tissues under the nutritional stress with respect to the control. The dietary stress had no significant impact on the activity of digestive tissue derived α-amylase till sixteenth day but by 32 days the enzyme activity went down significantly. The histopathological profile revealed that the bivalve was adapted to maintain a steady immune profile by incurring degeneration of its own tissue structure. The total haemocyte count surged significantly till 16 days but differed insignificantly with respect to the control at 32 days implying probable haematopoietic exhaustion. The study reflects the instinctive urge of the bivalve to maintain immune physiology at heavy metabolic cost under nutrient limited condition.     

SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain

Previously a distinct D1-like dopamine receptor (DAR) that selectively couples to phospholipase C/phosphatidylinositol (PLC/PI) was proposed. However, lack of a selective agonist has limited efforts aimed at characterizing this receptor. We characterized the in vitro and in vivo effects of SKF83959 in regulating PI metabolism. SKF83959 stimulates (EC50, 8 micro m) phosphatidylinositol 4,5-biphosphate hydrolysis in membranes of frontal cortex (FC) but not in membranes from PC12 cells expressing classical D1A DARs. Stimulation of FC PI metabolism was attenuated by the D1 antagonist, SCH23390, indicating that SKF83959 activates a D1-like DAR. The PI-linked DAR is located in hippocampus, cerebellum, striatum and FC. Most significantly, administration of SKF83959 induced accumulations of IP3 in striatum and hippocampus. In contrast to other D1 DAR agonists, SKF83959 did not increase cAMP production in brain or in D1A DAR-expressing PC12 cell membranes. However, SKF83959 inhibited cAMP elevation elicited by the D1A DAR agonist, SKF81297, indicating that the compound is an antagonist of the classical D1A DAR. Lastly, we demonstrated that SKF83959 enhances [35S]guanosine 5'-O-(3-thiotriphosphate) binding to membrane Galphaq and Galphai proteins, suggesting that PI stimulation is mediated by activation of these guanine nucleotide-binding regulatory proteins. Results indicate that SKF83959 is a selective agonist for the PI-linked D1-like DAR, providing a unique tool for investigating the functions of this brain D1 DAR subtype.     

Hsp90‐mediated regulation of DYRK3 couples stress granule disassembly and growth via mTORC1 signaling

Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual‐specificity tyrosine‐phosphorylation‐regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.