Lentiviral Vpx Accessory Factor Targets VprBP/DCAF1 Substrate Adaptor for Cullin 4 E3 Ubiquitin Ligase to Enable Macrophage Infection

Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4–based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism

Brachyury and Related Tbx Proteins Interact with the Mixl1 Homeodomain Protein and Negatively Regulate Mixl1 Transcriptional Activity

Mixl1 is a homeodomain transcription factor required for mesoderm and endoderm patterning during mammalian embryogenesis. Despite its crucial function in development, co-factors that modulate the activity of Mixl1 remain poorly defined. Here we report that Mixl1 interacts physically and functionally with the T-box protein Brachyury and related members of the T-box family of transcription factors. Transcriptional and protein analyses demonstrated overlapping expression of Mixl1 and Brachyury during embryonic stem cell differentiation. In vitro protein interaction studies showed that the Mixl1 with Brachyury associated via their DNA-binding domains and gel shift assays revealed that the Brachyury T-box domain bound to Mixl1-DNA complexes. Furthermore, luciferase reporter experiments indicated that association of Mixl1 with Brachyury and related T-box factors inhibited the transactivating potential of Mixl1 on the Gsc and Pdgfrα promoters. Our results indicate that the activity of Mixl1 can be modulated by protein-protein interactions and that T-box factors can function as negative regulators of Mixl1 activity

A Defined, Feeder-Free, Serum-Free System to Generate In Vitro Hematopoietic Progenitors and Differentiated Blood Cells from hESCs and hiPSCs

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimentional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells

Neurochemical Architecture of the Central Complex Related to Its Function in the Control of Grasshopper Acoustic Communication

The central complex selects and coordinates the species- and situation-specific song production in acoustically communicating grasshoppers. Control of sound production is mediated by several neurotransmitters and modulators, their receptors and intracellular signaling pathways. It has previously been shown that muscarinic cholinergic excitation in the central complex promotes sound production whereas both GABA and nitric oxide/cyclic GMP signaling suppress its performance. The present immunocytochemical and pharmacological study investigates the question whether GABA and nitric oxide mediate inhibition of sound production independently. Muscarinic ACh receptors are expressed by columnar output neurons of the central complex that innervate the lower division of the central body and terminate in the lateral accessory lobes. GABAergic tangential neurons that innervate the lower division of the central body arborize in close proximity of columnar neurons and thus may directly inhibit these central complex output neurons. A subset of these GABAergic tangential neurons accumulates cyclic GMP following the release of nitric oxide from neurites in the upper division of the central body. While sound production stimulated by muscarine injection into the central complex is suppressed by co-application of sodium nitroprusside, picrotoxin-stimulated singing was not affected by co-application of this nitric oxide donor, indicating that nitric oxide mediated inhibition requires functional GABA signaling. Hence, grasshopper sound production is controlled by processing of information in the lower division of the central body which is subject to modulation by nitric oxide released from neurons in the upper division

The Comparative Osteology of the Petrotympanic Complex (Ear Region) of Extant Baleen Whales (Cetacea: Mysticeti)

Anatomical comparisons of the ear region of baleen whales (Mysticeti) are provided through detailed osteological descriptions and high-resolution photographs of the petrotympanic complex (tympanic bulla and petrosal bone) of all extant species of mysticete cetaceans. Salient morphological features are illustrated and identified, including overall shape of the bulla, size of the conical process of the bulla, morphology of the promontorium, and the size and shape of the anterior process of the petrosal. We place our comparative osteological observations into a phylogenetic context in order to initiate an exploration into petrotympanic evolution within Mysticeti.The morphology of the petrotympanic complex is diagnostic for individual species of baleen whale (e.g., sigmoid and conical processes positioned at midline of bulla in Balaenoptera musculus; confluence of fenestra cochleae and perilymphatic foramen in Eschrichtius robustus), and several mysticete clades are united by derived characteristics. Balaenids and neobalaenids share derived features of the bulla, such as a rhomboid shape and a reduced anterior lobe (swelling) in ventral aspect, and eschrichtiids share derived morphologies of the petrosal with balaenopterids, including loss of a medial promontory groove and dorsomedial elongation of the promontorium. Monophyly of Balaenoidea (Balaenidae and Neobalaenidae) and Balaenopteroidea (Balaenopteridae and Eschrichtiidae) was recovered in phylogenetic analyses utilizing data exclusively from the petrotympanic complex.This study fills a major gap in our knowledge of the complex structures of the mysticete petrotympanic complex, which is an important anatomical region for the interpretation of the evolutionary history of mammals. In addition, we introduce a novel body of phylogenetically informative characters from the ear region of mysticetes. Our detailed anatomical descriptions, illustrations, and comparisons provide valuable data for current and future studies on the phylogenetic relationships, evolution, and auditory physiology of mysticetes and other cetaceans throughout Earth's history

ER stress in rodent islets of langerhans is concomitant with obesity and β-cell compensation but not with β-cell dysfunction and diabetes

Objective: The objective of this study was to determine whether ER stress correlates with β-cell dysfunction in obesity-associated diabetes. Methods: Quantitative RT-PCR and western blot analysis were used to investigate changes in the expression of markers of ER stress, the unfolded protein response (UPR) and β-cell function in islets isolated from (1) non-diabetic Zucker obese (ZO) and obese female Zucker diabetic fatty (fZDF) rats compared with their lean littermates and from (2) high-fat-diet-fed fZDF rats (HF-fZDF), to induce diabetes, compared with age-matched non-diabetic obese fZDF rats. Results: Markers of an adaptive ER stress/UPR and β-cell function are elevated in islets isolated from ZO and fZDF rats compared with their lean littermates. In islets isolated from HF-fZDF rats, there was no significant change in the expression of markers of ER stress compared with age matched, obese, non-diabetic fZDF rats. Conclusions: These results provide evidence that obesity-induced activation of the UPR is an adaptive response for increasing the ER folding capacity to meet the increased demand for insulin. As ER stress is not exacerbated in high-fat-diet-induced diabetes, we suggest that failure of the islet to mount an effective adaptive UPR in response to an additional increase in insulin demand, rather than chronic ER stress, may ultimately lead to β-cell failure and hence diabetes

Circulating microRNAs in sera correlate with soluble biomarkers of immune activation but do not predict mortality in ART treated individuals with HIV-1 infection: A case control study

Introduction: The use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals. Materials and Methods: A set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed. Results: None of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR- 145 correlated with nadir CD4+ T cell count. Discussion: No associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection