HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism

Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop-CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.     

Investigation of the potential for triterpene synthesis in rice through genome mining and metabolic engineering

The first committed step in sterol biosynthesis in plants involves the cyclization of 2,3-oxidosqualene by the oxidosqualene cyclase (OSC) enzyme cycloartenol synthase. 2,3-Oxidosqualene is also a precursor for triterpene synthesis. Antimicrobial triterpenes are common in dicots, but seldom found in monocots, with the notable exception of oat. Here, through genome mining and metabolic engineering, we investigate the potential for triterpene synthesis in rice. The first two steps in the oat triterpene pathway are catalysed by a divergent OSC (AsbAS1) and a cytochrome P450 (CYP51). The genes for these enzymes form part of a metabolic gene cluster. To investigate the origins of triterpene synthesis in monocots, we analysed systematically the OSC and CYP51 gene families in rice. We also engineered rice for elevated triterpene content. We discovered a total of 12 OSC and 12 CYP51 genes in rice and uncovered key events in the evolution of triterpene synthesis. We further showed that the expression of AsbAS1 in rice leads to the accumulation of the simple triterpene, β-amyrin. These findings provide new insights into the evolution of triterpene synthesis in monocots and open up opportunities for metabolic engineering for disease resistance in rice and other cereals.     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Combination of Immune and Viral Factors Distinguishes Low-Risk versus High-Risk HIV-1 Disease Progression in HLA-B*5701 Subjects

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1 in vivo evolution and epitope-specific CD8(+) T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4(+) T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8(+) T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8(+) T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.     

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma

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6-Bromoindolglyoxylamido derivatives as antimicrobial agents and antibiotic enhancers

The combination of increased incidence of drug-resistant strains of bacteria and a lack of novel drugs in development creates an urgency for the search for new antimicrobials. Initial screening of compounds from an in-house library identified two 6-bromoindolglyoxylamide polyamine derivatives (3 and 4) that exhibited intrinsic antimicrobial activity towards Gram-positive bacteria, Staphylococcus aureus and S. intermedius with polyamine 3 also displaying in vitro antibiotic enhancing properties against the resistant Gram-negative bacterium Pseudomonas aeruginosa. A series of 6-bromo derivatives (5–15) were prepared and biologically evaluated, identifying analogues with enhanced antibacterial activity towards Escherichia coli and with moderate to excellent antifungal properties. Polyamine 3, which includes a spermine chain, was the most potent of the series – its mechanism of action was attributed to rapid membrane permeabilization and depolarization in both Gram-positive and Gram-negative bacteria.     

Literature sources for primary environmental science teachers

With school days seemingly getting shorter and packed with more and more obligations, teachers integrate core subjects like science and reading in the hopes of delivering a curriculum that is meaningful and content rich. Using trade books, primary teachers can develop and deliver science content designed to introduce students to proenvironmental behaviors built around the Next Generation Science Earth and Human Activity Standard, Human Impacts on Earth Systems, ESS3-C. The Next Generation Science standards call for integrating literacy skills and science to develop knowledge of scientific topics. This article shares ideas on how primary teachers can use a variety of children’s literature as a foundation to inspire young students to take small steps toward developing proenvironmental behaviors. Under an umbrella of three environmental themes, proenvironmental behavior, trash, and water pollution, the article describes key concepts, vocabulary, and activities designed to develop a mindfulness of proenvironmental behaviors for each selected text. While texts and learning experiences are intentionally focused on young learners (Kindergarten/first grade), they can be adapted and used in all primary grades.     

Spatial Pattern Dynamics of 3D Stem Cell Loss of Pluripotency via Rules-Based Computational Modeling

Pluripotent embryonic stem cells (ESCs) have the unique ability to differentiate into cells from all germ lineages, making them a potentially robust cell source for regenerative medicine therapies, but difficulties in predicting and controlling ESC differentiation currently limit the development of therapies and applications from such cells. A common approach to induce the differentiation of ESCs in vitro is via the formation of multicellular aggregates known as embryoid bodies (EBs), yet cell fate specification within EBs is generally considered an ill-defined and poorly controlled process. Thus, the objective of this study was to use rules-based cellular modeling to provide insight into which processes influence initial cell fate transitions in 3-dimensional microenvironments. Mouse embryonic stem cells (D3 cell line) were differentiated to examine the temporal and spatial patterns associated with loss of pluripotency as measured through Oct4 expression. Global properties of the multicellular aggregates were accurately recapitulated by a physics-based aggregation simulation when compared to experimentally measured physical parameters of EBs. Oct4 expression patterns were analyzed by confocal microscopy over time and compared to simulated trajectories of EB patterns. The simulations demonstrated that loss of Oct4 can be modeled as a binary process, and that associated patterns can be explained by a set of simple rules that combine baseline stochasticity with intercellular communication. Competing influences between Oct4+ and Oct4− neighbors result in the observed patterns of pluripotency loss within EBs, establishing the utility of rules-based modeling for hypothesis generation of underlying ESC differentiation processes. Importantly, the results indicate that the rules dominate the emergence of patterns independent of EB structure, size, or cell division. In combination with strategies to engineer cellular microenvironments, this type of modeling approach is a powerful tool to predict stem cell behavior under a number of culture conditions that emulate characteristics of 3D stem cell niches.