Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 2011¹.For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)². This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients'' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL<1000 copies/μl roughly increased in the last years (Fig. 3).The main steps for tropism testing (Fig. 4) demonstrated in this video: 1. Collection of a blood sample 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 3. Amplification of the env region 4. Amplification of the V3 region 5. Sequence reaction of the V3 amplicon 6. Purification of the sequencing samples 7. Sequencing the purified samples 8. Sequence editing 9. Sequencing data interpretation and tropism prediction     

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin Lüscher used the term pheromone for the first time in 1959¹ to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules² secreted and/or contained in biological fluids^3,4, such as urine, a liquid known to be a main source of pheromones³. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions^5,6, hierarchical organisations³ and sexual interactions^7,8 and are consequently directly correlated with the survival of a given species^9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)^10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen^13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)¹⁵. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection¹⁶. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize^17,18. Two main vomeronasal receptor families, V1Rs and V2Rs^19,20,21,22, are composed respectively by 240²³ and 120²⁴ members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)²⁵ and formyl peptide receptors (FPRs)^26,27 are also expressed in VSNs.Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons²⁸ TRPC2 independent transduction channels have been suggested^6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line³⁰, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.     

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses¹. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation². This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency⁴. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively⁵. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.Contraction of smooth muscle is generally regulated by the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) plus sensitivity to Ca^2+, of the contractile elements in response to changes in the environment surrounding the cell⁶. [Ca²⁺]_i is determined by the combination of the movement of Ca²⁺ through plasma membrane ligand or voltage gated Ca²⁺ channels and the release and uptake of Ca²⁺ from internal stores. Cytosolic Ca²⁺ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction⁸. However, the sensitivity of this pathway to Ca²⁺ can be regulated by the MLC phosphatase (MLCP)⁹. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.Here, we present a method to evaluate changes in [Ca²⁺]_i over time in isolated, perfused lymphatics in order to study Ca²⁺-dependent and Ca²⁺-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca²⁺]_i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca²⁺]_i was determined by loading lymphatics with the ratiometric, Ca²⁺-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.     

G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase

The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL), which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 switch gene-2 (G0S2). CGI-58 activates and G0S2 inhibits ATGL activity. In contrast to mice, the functional role of G0S2 in human adipocyte lipolysis is poorly characterized. Here we show that overexpression or silencing of G0S2 in human SGBS adipocytes decreases and increases lipolysis, respectively. Human G0S2 is upregulated during adipocyte differentiation and inhibits ATGL activity in a dose-dependent manner. Interestingly, C-terminally truncated ATGL mutants, which fail to localize to lipid droplets, translocate to the lipid droplet upon coexpression with G0S2, suggesting that G0S2 anchors ATGL to lipid droplets independent of ATGL''s C-terminal lipid binding domain. Taken together, our results indicate that G0S2 also regulates human lipolysis by affecting enzyme activity and intracellular localization of ATGL. Increased lipolysis is known to contribute to the pathogenesis of insulin resistance, and G0S2 expression has been shown to be reduced in poorly controlled type 2 diabetic patients. Our data indicate that downregulation of G0S2 in adipose tissue could represent one of the underlying causes leading to increased lipolysis in the insulin-resistant state.     

The Usage of Plasmalemmal Vesicles Inverted by Brij 58 Treatment for Studying Processes,which Occur on the Cytosolic Membrane Surface

We studied the possible use of the detergent Brij 58 in physiological experiments for the reorientation of right-side-out plasmalemmal vesicles, which were isolated from wheat (Triticum aestivum L.) coleoptiles. The activities of K⁺, Mg²⁺-ATPase and the ATP-dependent H⁺-potential were higher in Brij 58-treated vesicles, whereas membrane permeability for K⁺ and Na⁺ remained unchanged. Brij 58 did not suppress the ATP-dependent IAA transport into vesicles and RNA polymerase II activation by IAA–protein plasmalemmal complexes in the system of isolated nuclei. The conclusion was that, using Brij 58, we could obtain the plasmalemmal fraction, which consisted almost completely of closed inverted vesicles. These vesicles can be applied for the in vitro study of the processes, which occur on the cytosolic plasmalemmal surface.     

Shedding new light on lipid functions with CARS and SRS microscopy

Modern optical microscopy has granted biomedical scientists unprecedented access to the inner workings of a cell, and revolutionized our understanding of the molecular mechanisms underlying physiological and disease states. In spite of these advances, however, visualization of certain classes of molecules (e.g. lipids) at the sub-cellular level has remained elusive. Recently developed chemical imaging modalities – Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy – have helped bridge this gap. By selectively imaging the vibration of a specific chemical group, these non-invasive techniques allow high-resolution imaging of individual molecules in vivo, and circumvent the need for potentially perturbative extrinsic labels. These tools have already been applied to the study of fat metabolism, helping uncover novel regulators of lipid storage. Here we review the underlying principle of CARS and SRS microscopy, and discuss the advantages and caveats of each technique. We also review recent applications of these tools in the study of lipids as well as other biomolecules, and conclude with a brief guide for interested researchers to build and use CARS/SRS systems for their own research. This article is part of a Special Issue entitled Tools to study lipid functions.     

Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58

Glucose-regulated GRP58 has shown clinical applications to endoplasmic reticulum (ER) stress and cancer. GRP58 is localized in the cytosol, endoplasmic reticulum (ER) and nucleus. Twenty-four amino acids at the N-terminal hydrophobic region are known to target GRP58 to ER for synthesis at the ER membrane and translocation into the ER lumen. In addition, GRP58 contains putative nuclear localization (494KPKKKKK500) and ER retention (502QEDL505) signals. However, the role of these signals in nuclear import and ER retention of GRP58 remains unknown. Present studies investigated the signals that control nuclear localization and ER retention of GRP58. Deletion/mutation of nuclear localization signal (NLS) abrogated nuclear import of GRP58. NLS attached to EGFP localized EGFP in the nucleus. However, deletion/mutation of putative ER retention signal alone did not alter ER retention of GRP58. Interestingly, a combined deletion/mutation of NLS and ER retention signals blocked the GRP58 retention in the ER. These results concluded that overlapping NLS and ER retention signal sequences regulate nuclear localization and ER retention of GRP58.     

Phylogeographic analysis of human papillomavirus 58

Human papillomavirus 58 (HPV58) is one type of HPV with high risk of causing cervical cancer. Unusually high prevalence of HPV58 has been reported in Asia, Africa and some other areas. However, due to the scattered distribution of global data, in addition to the lack of data of some HPV58 high-incidence nations and regions, like Mainland China, a comprehensive analysis of the global geographical distribution of HPV58 remains blank so far. In this study, HPV58 from the human cervical cancer tissue was detected in Mainland China, and 14 new HPV58-E6/L1 gene sequences were obtained. Moreover, phylogeographic analysis has been conducted combining the HPV58 sequences that have been deposited in GenBank since 1985. The study result shows that the sequences detected from the Shanghai, Jiangsu and Sichuan areas are homologous with those found in the past from Hong Kong and Xi’an, China, as well as Japan and other Southeast Asian areas. Furthermore, Western Africa is considered to be the “root” source of the HPV58 variant, while Mainland China and Southeast Asia are “transit points” and the new sources of HPV58 after receiving the isolates from the “root” source; like HPV16 and HPV18, the HPV58 might also be one of the major HPV types associated with the development and spread of cervical cancer.     

广适性光温敏不育系Y58S幼穗分化期耐冷性表现及生理机制

为了阐明水稻光温敏不育系幼穗分化期耐冷的形态生理机制,以耐冷不育系Y58S和4个生产上常用光温敏不育系为试验材料,研究了低温胁迫(17.5℃,10 d)下结实率、穗部形态、株高、光合特性以及抗氧化物酶系统等的变化。结果表明,低温胁迫下Y58S的幼穗分化期耐冷性在5个不育系中最强,与敏感的C815S和株1S相比,Y58S表现为株高和穗长降低幅度较小,保持较高结实率;光合作用受低温影响不显著,SPAD值、光合速率等光合指标无显著变化;SOD、POD活性降低幅度较小,MDA含量、相对电导率增幅较小。     

Red blood cells promote survival and cell cycle progression of human peripheral blood T cells independently of CD58/LFA-3 and heme compounds

Red blood cells (RBC) are known to modulate T cell proliferation and function possibly through downregulation of oxidative stress. By examining parameters of activation, division, and cell death in vitro, we show evidence that the increase in survival afforded by RBC is due to the maintenance of the proliferative capacity of the activated T cells. We also show that the CD3+CD8+ T cell subset was preferentially expanded and rescued from apoptosis both in bulk peripheral blood lymphocyte cultures and with highly purified CD8+ T cells. The ability of RBC to induce survival of dividing T cells was not affected by blocking the CD58/CD2 interaction. Moreover, addition of hemoglobin, heme or protoporphyrin IX to cultures of activated T cells did not reproduce the effect of intact RBC. Considering that RBC circulate throughout the body, they could play a biological role in the modulation of T cell differentiation and survival in places of active cell division. Neither CD58 nor the heme compounds studied seem to play a direct relevant role in the modulation of T cell survival.