Reversion and T Cell Escape Mutations Compensate the Fitness Loss of a CD8+ T Cell Escape Mutant in Their Cognate Transmitted/Founder Virus

Immune escape mutations that revert back to the consensus sequence frequently occur in newly HIV-1-infected individuals and have been thought to render the viruses more fit. However, their impact on viral fitness and their interaction with other immune escape mutations have not been evaluated in the background of their cognate transmitted/founder (T/F) viral genomes. To precisely determine the role of reversion mutations, we introduced reversion mutations alone or together with CD8⁺ T cell escape mutations in their unmodified cognate T/F viral genome and determined their impact on viral fitness in primary CD4⁺ T cells. Two reversion mutations, V247I and I64T, were identified in Gag and Tat, respectively, but neither had measurable effect on the fitness of their cognate T/F virus. The V247I and G248A mutations that were detected before and concurrently with the potent T cell escape mutation T242N, respectively, were selected by early T cell responses. The V247I or the G248A mutation alone partially restored the fitness loss caused by the T242N mutation. Together they could fully restore the fitness of the T242N mutant to the T/F level. These results demonstrate that the fitness loss caused by a T cell escape mutation could be compensated by preexisting or concurrent reversion and other T cell escape mutations. Our findings indicate that the overall viral fitness is modulated by the complex interplay among T cell escape, compensatory and reversion mutations to maintain the balance between immune escape and viral replication capacity.     

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

Pathogenicity of Mycobacterium tuberculosis Is Expressed by Regulating Metabolic Thresholds of the Host Macrophage

The success of Mycobacterium tuberculosis as a pathogen derives from its facile adaptation to the intracellular milieu of human macrophages. To explore this process, we asked whether adaptation also required interference with the metabolic machinery of the host cell. Temporal profiling of the metabolic flux, in cells infected with differently virulent mycobacterial strains, confirmed that this was indeed the case. Subsequent analysis identified the core subset of host reactions that were targeted. It also elucidated that the goal of regulation was to integrate pathways facilitating macrophage survival, with those promoting mycobacterial sustenance. Intriguingly, this synthesis then provided an axis where both host- and pathogen-derived factors converged to define determinants of pathogenicity. Consequently, whereas the requirement for macrophage survival sensitized TB susceptibility to the glycemic status of the individual, mediation by pathogen ensured that the virulence properties of the infecting strain also contributed towards the resulting pathology.     

High‐resolution crystal structure of human Mapkap kinase 3 in complex with a high affinity ligand

The Mapkap kinases 2 and 3 (MK2 and MK3) have been implicated in intracellular signaling pathways leading to the production of the pro‐inflammatory cytokine tumor necrosis factor alpha. MK2 has been pursued by the biopharmaceutical industry for many years for the development of a small molecule anti‐inflammatory treatment and drug‐like inhibitors have been described. The development of some of these compounds, however, has been slowed by the absence of a high‐resolution crystal structure of MK2. Herein we present a high‐resolution (1.9 Å) crystal structure of the highly homologous MK3 in complex with a pharmaceutical lead compound. While all of the canonical features of Ser/Thr kinases in general and MK2 in particular are recapitulated in MK3, the detailed analysis of the binding interaction of the drug‐like ligand within the adenine binding pocket allows relevant conclusions to be drawn for the further design of potent and selective drug candidates.     

Development and application of genomic resources for comparative and translational genomics in legumes through leveraging genomic sequence of <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The expressed sequence tags (ESTs) of common bean were BLAST aligned with barred medic genome sequence and developed 1196 conserved intron spanning primers (CISPs) to facilitate genetic studies in legumes. Randomly selected 288 CISPs, representing loci on barrel medic genome, were tested on 10 selected members of legume family. On the source taxa, the highest single copy amplification success rates of 61.8% (barrel medic) and 56.2% (common bean) was obtained. The success rate of markers was 54.5% in cowpea followed by 53.5% in pigeonpea and chickpea, signifying cross taxon amplification and their potential use in comparative genomics. However, relatively low percentages of primer set amplified (40–43%) in soybean, urdbean and peanut. Further, these primers were tested on different varieties of chickpea, pigeonpea and cowpea. The PCR products were sequenced and aligned which resulted in detection of 26 SNPs and eight INDeLs in cowpea, seven SNPs and two INDeLs in chickpea and 27 SNPs and 14 INDeLs in pigeonpea. These SNPs were successfully converted in to size variation for gel-based genotyping. The CISP markers developed in this study are expected to aid in map saturation of legumes and in marker-assisted selection for accelerated crop improvement.     

Extracellular Vesicles in Brain Tumor Progression

Brain tumors can be viewed as multicellular ‘ecosystems’ with increasingly recognized cellular complexity and systemic impact. While the emerging diversity of malignant disease entities affecting brain tissues is often described in reference to their signature alterations within the cellular genome and epigenome, arguably these cell-intrinsic changes can be regarded as hardwired adaptations to a variety of cell-extrinsic microenvironmental circumstances. Conversely, oncogenic events influence the microenvironment through their impact on the cellular secretome, including emission of membranous structures known as extracellular vesicles (EVs). EVs serve as unique carriers of bioactive lipids, secretable and non-secretable proteins, mRNA, non-coding RNA, and DNA and constitute pathway(s) of extracellular exit of molecules into the intercellular space, biofluids, and blood. EVs are also highly heterogeneous as reflected in their nomenclature (exosomes, microvesicles, microparticles) attempting to capture their diverse origin, as well as structural, molecular, and functional properties. While EVs may act as a mechanism of molecular expulsion, their non-random uptake by heterologous cellular recipients defines their unique roles in the intercellular communication, horizontal molecular transfer, and biological activity. In the central nervous system, EVs have been implicated as mediators of homeostasis and repair, while in cancer they may act as regulators of cell growth, clonogenicity, angiogenesis, thrombosis, and reciprocal tumor-stromal interactions. EVs produced by specific brain tumor cell types may contain the corresponding oncogenic drivers, such as epidermal growth factor receptor variant III (EGFRvIII) in glioblastoma (and hence are often referred to as ‘oncosomes’). Through this mechanism, mutant oncoproteins and nucleic acids may be transferred horizontally between cellular populations altering their individual and collective phenotypes. Oncogenic pathways also impact the emission rates, types, cargo, and biogenesis of EVs, as reflected by preliminary analyses pointing to differences in profiles of EV-regulating genes (vesiculome) between molecular subtypes of glioblastoma, and in other brain tumors. Molecular regulators of vesiculation can also act as oncogenes. These intimate connections suggest the context-specific roles of different EV subsets in the progression of specific brain tumors. Advanced efforts are underway to capture these events through the use of EVs circulating in biofluids as biomarker reservoirs and to guide diagnostic and therapeutic decisions.     

Design,synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor

An absolute or relative deficiency of pancreatic β-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect β-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces β-cell replication and attenuate β-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic β-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional β-cell mass.