Increasing the carbon efficiency of citric acid production

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Characterization of Partial and Near Full-Length Genomes of HIV-1 Strains Sampled from Recently Infected Individuals in São Paulo, Brazil

Background: Genetic variability is a major feature of human immunodeficiency virus type 1 (HIV-1) and is considered the key factor frustrating efforts to halt the HIV epidemic. A proper understanding of HIV-1 genomic diversity is a fundamental prerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines design. Here, we report on the partial and near full-length genomic (NFLG) variability of HIV-1 isolates from a well-characterized cohort of recently infected patients in Sao Paul, Brazil.Methodology: HIV-1 proviral DNA was extracted from the peripheral blood mononuclear cells of 113 participants. the NFLG and partial fragments were determined by overlapping nested PCR and direct sequencing. the data were phylogenetically analyzed.Results: of the 113 samples (90.3% male; median age 31 years; 79.6% homosexual men) studied, 77 (68.1%) NFLGs and 32 (29.3%) partial fragments were successfully subtyped. of the successfully subtyped sequences, 88 (80.7%) were subtype B sequences, 12 (11%) BF1 recombinants, 3 (2.8%) subtype C sequences, 2 (1.8%) BC recombinants and subclade F1 each, 1 (0.9%) CRF02 AG, and 1 (0.9%) CRF31 BC. Primary drug resistance mutations were observed in 14/101 (13.9%) of samples, with 5.9% being resistant to protease inhibitors and nucleoside reverse transcriptase inhibitors (NRTI) and 4.9% resistant to non-NRTIs. Predictions of viral tropism were determined for 86 individuals. X4 or X4 dual or mixed-tropic viruses (X4/DM) were seen in 26 (30.2%) of subjects. the proportion of X4 viruses in homosexuals was detected in 19/69 (27.5%).Conclusions: Our results confirm the existence of various HIV-1 subtypes circulating in São Paulo, and indicate that subtype B account for the majority of infections. Antiretroviral (ARV) drug resistance is relatively common among recently infected patients. the proportion of X4 viruses in homosexuals was significantly higher than the proportion seen in other study populations.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Fac Med, Div Clin Immunol & Allergy, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilFundacao Pro Sangue, Blood Ctr Sau Paulo, São Paulo, BrazilUniv São Paulo, Dept Infect Dis, São Paulo, BrazilPubl Hlth Dept São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilFAPESP: 04/15856-9FAPESP: 2006/50096-0Web of Scienc

Salinity variations in the northern Coorong Lagoon, South Australia: Significant changes in the ecosystem following human alteration to the natural water regime

European settlement and drought have significantly impacted the hydrology of the Coorong, a shallow coastal lagoon complex in South Australia, which is part of a terminal wetland at the mouth of the River Murray. An increased salinity associated with lower water levels and progressive isolation from ocean flushes contributed to a severe decline in ecological diversity over the past decades. Here we have conducted a molecular and stable isotopic study of a sedimentary core from the northern Coorong Lagoon spanning more than 5000 years to investigate the recent palaeoenvironmental history of the ecosystem. Major alterations were evident in many biogeochemical parameters in sediments deposited after the 1950s coinciding with the beginning of intensified water regulations. The most prominent shift occurred in δ13C profiles of C21–C33n-alkanes from average values of −23.5‰ to an average of −28.2‰.Further changes included decreases in carbon preference index (CPI) and average chain length (ACL) of the n-alkane series as well as significant increases in algal (e.g. C20 HBI, long chain alkenes and C29-alkadiene) and bacterial (e.g. 13C depleted short chain n-alkanes and hopanoids, δ13C: −35.9‰ to −30.1‰) derived hydrocarbons. Long chain n-alkanes with a strong odd/even predominance as observed here are typically attributed to terrigenous plants. In the Coorong however, terrigenous input to sedimentary OM is only minor. Therefore changes in the before mentioned parameters were attributed to a source transition from a major contribution of macrophytes towards predominantly microalgae and bacteria.δD values of C21–C33n-alkanes showed a general trend towards more enriched values in younger sediments, indicating an overall rising salinity. However, the most pronounced positive shift in these profiles again occurred after the 1950s. Altogether this study demonstrates that the recent human induced changes of the Coorong hydrology, compounded by a severe drought led to an increase in salinity and alterations of primary production which have been much more significant than natural variations occurring throughout the Holocene over several thousands of years

STIL binding to Polo-box 3 of PLK4 regulates centriole duplication

Polo-like kinases (PLK) are eukaryotic regulators of cell cycle progression, mitosis and cytokinesis; PLK4 is a master regulator of centriole duplication. Here, we demonstrate that the SCL/TAL1 interrupting locus (STIL) protein interacts via its coiled-coil region (STIL-CC) with PLK4 in vivo. STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region. Structure determination of free PLK4-PB3 and its STIL-CC complex via NMR and crystallography reveals a novel mode of Polo-box-peptide interaction mimicking coiled-coil formation. In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding. We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication

Structural basis for RNA 3′-end recognition by Hfq

The homohexameric (L)Sm protein Hfq is a central mediator of small RNA-based gene regulation in bacteria. Hfq recognizes small regulatory RNAs (sRNAs) specifically, despite their structural diversity. This specificity could not be explained by previously described RNA-binding modes of Hfq. Here we present a distinct and preferred mode of Hfq–RNA interaction that involves the direct recognition of a uridine-rich RNA 3′ end. This feature is common in bacterial RNA transcripts as a consequence of Rho-independent transcription termination and hence likely contributes significantly to the general recognition of sRNAs by Hfq. Isothermal titration calorimetry shows nanomolar affinity between Salmonella typhimurium Hfq and a hexauridine substrate. We determined a crystal structure of the complex that reveals a constricted RNA backbone conformation in the proximal RNA-binding site of Hfq, allowing for a direct protein contact of the 3′ hydroxyl group. A free 3′ hydroxyl group is crucial for the high-affinity interaction with Hfq also in the context of a full-length sRNA substrate, RybB. The capacity of Hfq to occupy and sequester the RNA 3′ end has important implications for the mechanisms by which Hfq is thought to affect sRNA stability, turnover, and regulation

Co-crystallization with conformation-specific designed ankyrin repeat proteins explains the conformational flexibility of BCL-W

BCL-W is a member of the BCL-2 family of anti-apoptotic proteins. A key event in the regulation of apoptosis is the heterodimerization between anti-apoptotic and pro-apoptotic family members, which involves a conserved surface-exposed groove on the anti-apoptotic proteins. Crystal structures of the ligand binding-competent conformation exist for all anti-apoptotic family members, with the exception of BCL-W, due to the flexibility of the BCL-W groove region. Existing structures had suggested major deviations of the BCL-W groove region from the otherwise structurally highly related remaining anti-apoptotic family members. To capture its ligand binding-competent conformation by counteracting the conformational flexibility of the BCL-W groove, we had selected high-affinity groove-binding designed ankyrin repeat proteins (DARPins) using ribosome display. We now determined two high-resolution crystal structures of human BCL-W in complex with different DARPins at resolutions 1.5 and 1.85Å, in which the structure of BCL-W is virtually identical, and BCL-W adopts a conformation extremely similar to the ligand-free conformation of its closest relative BCL-XL in both structures. However, distinct differences to all previous BCL-W structures are evident, notably in the ligand-binding region. We provide the first structural explanation for the conformational flexibility of the BCL-W groove region in comparison to other BCL-2 family members. Due to the importance of the anti-apoptotic BCL-2 family as drug targets, the presented crystal structure of ligand binding-competent BCL-W may serve as a valuable basis for structure-based drug design in the future and provides a missing piece for the structural characterization of this protein family

Conserved sequence motifs and the structure of the mTOR kinase domain

The atypical serine/threonine kinase mTOR (mammalian target of rapamycin) is a central regulator of cell growth and metabolism. mTOR is part of two multisubunit signalling complexes, mTORC1 and mTORC2. Although many aspects of mTOR signalling are understood, the lack of high-resolution structures impairs a detailed understanding of complex assembly, function and regulation. The structure of the kinase domain is of special interest for the development of mTOR inhibitors as anti-cancer agents. A homology model of the mTOR kinase domain was derived from the structure of PI3Ks (phosphoinositide 3-kinases). More recently, the crystal structure of the catalytic domain of human mTOR was determined, providing long-awaited structural insight into the architecture of mTOR. Interestingly, the homology model predicted several aspects of the crystal structure. In the present paper, we revisit the homology model in the context of the now available crystal structure of the mTOR kinase domain

Architecture of human mTOR complex 1

Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer and neurodegeneration. We resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)-rapamycin, by combining cryo-electron microscopy at 5.9 Å resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 Å resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved N-terminal domain of Raptor is juxtaposed with the kinase active site