Variation in HIV-1 Nef function within and among viral subtypes reveals genetically separable antagonism of SERINC3 and SERINC5

HIV Nef counteracts cellular host restriction factors SERINC3 and SERINC5, but our understanding of how naturally occurring global Nef sequence diversity impacts these activities is limited. Here, we quantify SERINC3 and SERINC5 internalization function for 339 Nef clones, representing the major pandemic HIV-1 group M subtypes A, B, C and D. We describe distinct subtype-associated hierarchies for Nef-mediated internalization of SERINC5, for which subtype B clones display the highest activities on average, and of SERINC3, for which subtype B clones display the lowest activities on average. We further identify Nef polymorphisms that modulate its ability to counteract SERINC proteins, including substitutions in the N-terminal domain that selectively impair SERINC3 internalization. Our findings demonstrate that the SERINC antagonism activities of HIV Nef differ markedly among major viral subtypes and between individual isolates within a subtype, suggesting that variation in these functions may contribute to global differences in viral pathogenesis

Coincident Activity of Converging Pathways Enables Simultaneous Long-Term Potentiation and Long-Term Depression in Hippocampal CA1 Network In Vivo

Memory is believed to depend on activity-dependent changes in the strength of synapses, e.g. long-term potentiation (LTP) and long-term depression (LTD), which can be determined by the sequence of coincident pre- and postsynaptic activity, respectively. It remains unclear, however, whether and how coincident activity of converging efferent pathways can enable LTP and LTD in the pathways simultaneously. Here, we report that, in pentobarbital-anesthetized rats, stimulation (600 pulses, 5 Hz) to Schaffer preceding to commissural pathway within a 40-ms timing window induced similar magnitudes of LTP in both pathways onto synapses of CA1 neurons, with varied LTP magnitudes after reversal of the stimulation sequence. In contrast, in urethane-anesthetized or freely-moving rats, the stimulation to Schaffer preceding to commissural pathway induced Schaffer LTP and commissural LTD simultaneously within a 40-ms timing window, without affecting synaptic efficacy in the reversed stimulation sequence. Coincident activity of Schaffer pathways confirmed the above findings under pentobarbital and urethane anesthesia. Thus, coincident activity of converging afferent pathways tends to switch the pathways to be LTP only or LTP/LTD depending on the activity states of the hippocampus. This network rule strengthens the view that activity-dependent synaptic plasticity may well contribute to memory process of the hippocampal network with flexibility or stability from one state to another

Identification and Characterization of RBM44 as a Novel Intercellular Bridge Protein

Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14

Genomotyping of Coxiella burnetii Using Microarrays Reveals a Conserved Genomotype for Hard Tick Isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype

Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

BACKGROUND:Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. METHODOLOGY/PRINCIPAL FINDINGS:The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+) ([Ca(2+)](c)) and cAMP ([cAMP](c)) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+)](c). The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+)](c) response. The effect of sucralose on [Ca(2+)](c) was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q) inhibitor. Sucralose also induced sustained elevation of [cAMP](c), which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. CONCLUSIONS:Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+) and cAMP-dependent mechanisms

Interplay between Kinase Domain Autophosphorylation and F-Actin Binding Domain in Regulating Imatinib Sensitivity and Nuclear Import of BCR-ABL

BACKGROUND: The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood. METHODOLOGY/PRINCIPAL FINDINGS: By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib. CONCLUSIONS/SIGNIFICANCE: Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML

The individual environment, not the family is the most important influence on preferences for common non-alcoholic beverages in adolescence

Beverage preferences are an important driver of consumption, and strong liking for beverages high in energy (e.g. sugar-sweetened beverages [SSBs]) and dislike for beverages low in energy (e.g. non-nutritive sweetened beverages [NNSBs]) are potentially modifiable risk factors contributing to variation in intake. Twin studies have established that both genes and environment play important roles in shaping food preferences; but the aetiology of variation in non-alcoholic beverage preferences is unknown. 2865 adolescent twins (18–19-years old) from the Twins Early Development Study were used to quantify genetic and environmental influence on variation in liking for seven non-alcoholic beverages: SSBs; NNSBs; fruit cordials, orange juice, milk, coffee, and tea. Maximum Likelihood Structural Equation Modelling established that beverage preferences have a moderate to low genetic basis; from 18% (95% CI: 10%, 25%) for orange juice to 42% (36%, 43%) for fruit cordials. Aspects of the environment that are not shared by twin pairs explained all remaining variance in drink preferences. The sizeable unique environmental influence on beverage preferences highlights the potential for environmental modification. Policies and guidelines to change preferences for unhealthy beverages may therefore be best directed at the wider environment

An Ultra-High Discrimination Y Chromosome Short Tandem Repeat Multiplex DNA Typing System

In forensic casework, Y chromosome short tandem repeat markers (Y-STRs) are often used to identify a male donor DNA profile in the presence of excess quantities of female DNA, such as is found in many sexual assault investigations. Commercially available Y-STR multiplexes incorporating 12–17 loci are currently used in forensic casework (Promega's PowerPlex® Y and Applied Biosystems' AmpFlSTR® Yfiler®). Despite the robustness of these commercial multiplex Y-STR systems and the ability to discriminate two male individuals in most cases, the coincidence match probabilities between unrelated males are modest compared with the standard set of autosomal STR markers. Hence there is still a need to develop new multiplex systems to supplement these for those cases where additional discriminatory power is desired or where there is a coincidental Y-STR match between potential male participants. Over 400 Y-STR loci have been identified on the Y chromosome. While these have the potential to increase the discrimination potential afforded by the commercially available kits, many have not been well characterized. In the present work, 91 loci were tested for their relative ability to increase the discrimination potential of the commonly used ‘core’ Y-STR loci. The result of this extensive evaluation was the development of an ultra high discrimination (UHD) multiplex DNA typing system that allows for the robust co-amplification of 14 non-core Y-STR loci. Population studies with a mixed African American and American Caucasian sample set (n = 572) indicated that the overall discriminatory potential of the UHD multiplex was superior to all commercial kits tested. The combined use of the UHD multiplex and the Applied Biosystems' AmpFlSTR® Yfiler® kit resulted in 100% discrimination of all individuals within the sample set, which presages its potential to maximally augment currently available forensic casework markers. It could also find applications in human evolutionary genetics and genetic genealogy

Interleukin-10 Mediated Autoregulation of Murine B-1 B-Cells and Its Role in Borrelia hermsii Infection

B cells are typically characterized as positive regulators of the immune response, primarily by producing antibodies. However, recent studies indicate that various subsets of B cells can perform regulatory functions mainly through IL-10 secretion. Here we discovered that peritoneal B-1 (B-1P) cells produce high levels of IL-10 upon stimulation with several Toll-like receptor (TLR) ligands. High levels of IL-10 suppressed B-1P cell proliferation and differentiation response to all TLR ligands studied in an autocrine manner in vitro and in vivo. IL-10 that accumulated in cultures inhibited B-1P cells at second and subsequent cell divisions mainly at the G1/S interphase. IL-10 inhibits TLR induced B-1P cell activation by blocking the classical NF-κB pathway. Co-stimulation with CD40 or BAFF abrogated the IL-10 inhibitory effect on B-1P cells during TLR stimulation. Finally, B-1P cells adoptively transferred from the peritoneal cavity of IL-10−/− mice showed better clearance of Borrelia hermsii than wild-type B-1P cells. This study described a novel autoregulatory property of B-1P cells mediated by B-1P cell derived IL-10, which may affect the function of B-1P cells in infection and autoimmunity