Thermostability-based binding assays reveal complex interplay of cation, substrate and lipid binding in the bacterial DASS transporter, VcINDY

The divalent anion sodium symporter (DASS) family of transporters (SLC13 family in humans) are key regulators of metabolic homeostasis, disruption of which results in pro- tection from diabetes and obesity, and inhibition of liver cancer cell proliferation. Thus, DASS transporter inhibitors are attractive targets in the treatment of chronic, age-related metabolic diseases. The characterisation of several DASS transporters has revealed vari- ation in the substrate selectivity and flexibility in the coupling ion used to power transport. Here, using the model DASS co-transporter, VcINDY from Vibrio cholerae, we have exam- ined the interplay of the three major interactions that occur during transport: the coupling ion, the substrate, and the lipid environment. Using a series of high-throughput thermo- stability-based interaction assays, we have shown that substrate binding is Na+-depend- ent; a requirement that is orchestrated through a combination of electrostatic attraction and Na+-induced priming of the binding site architecture. We have identified novel DASS ligands and revealed that ligand binding is dominated by the requirement of two carb- oxylate groups in the ligand that are precisely distanced to satisfy carboxylate interaction regions of the substrate-binding site. We have also identified a complex relationship between substrate and lipid interactions, which suggests a dynamic, regulatory role for lipids in VcINDY’s transport cycle

Solvent accessibility changes in a Na+-dependent C4-dicarboxylate transporter suggest differential substrate effects in a multistep mechanism

The divalent anion sodium symporter (DASS) family (SLC13) play critical roles in metabolic homeostasis, influencing many processes including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyse the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested are modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate binding site-associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structural-characterised representative of the entire DASS family

Trends in Weekly Reported Net use by Children During and after Rainy Season in Central Tanzania.

The use of long-lasting insecticidal nets (LLINs) is one of the principal interventions to prevent malaria in young children, reducing episodes of malaria by 50% and child deaths by one fifth. Prioritizing young children for net use is important to achieve mortality reductions, particularly during transmission seasons. Households were followed up weekly from January through June 2009 to track net use among children under seven under as well as caretakers. Net use rates for children and caretakers in net-owning households were calculated by dividing the number of person-weeks of net use by the number of person-weeks of follow-up. Use was stratified by age of the child or caretaker status. Determinants of ownership and of use were assessed using multivariate models. Overall, 60.1% of the households reported owning a bed net at least once during the study period. Among net owners, use rates remained high during and after the rainy season. Rates of use per person-week decreased as the age of the child rose from 0 to six years old; at ages 0-23 months and 24-35 months use rates per person-week were 0.93 and 0.92 respectively during the study period, while for children ages 3 and 4 use rates per person-week were 0.86 and 0.80. For children ages 5-6 person-week ratios dropped to 0.55. This represents an incidence rate ratio of 1.67 for children ages 0-23 months compared to children aged 5-6. Caretakers had use rates similar to those of children age 0-35 months. Having fewer children under age seven in the household also appeared to positively impact net use rates for individual children. In this area of Tanzania, net use is very high among net-owning households, with no variability either at the beginning or end of the rainy season high transmission period. The youngest children are prioritized for sleeping under the net and caretakers also have high rates of use. Given the high use rates, increasing the number of nets available in the household is likely to boost use rates by older children

TMEM27 Suppresses Tumor Development by Promoting Ret Ubiquitination, Positioning, and Degradation

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma

Less-than-lethal "flashbang" diversionary device.

Diversionary devices such as flashbang grenades are used in a wide variety of military and law-enforcement operations. They function to distract and/or incapacitate adversaries in scenarios ranging from hostage rescue to covert strategic paralysis operations. There are a number of disadvantages associated with currently available diversionary devices. Serious injuries and fatalities have resulted from their use both operationally and in training. Because safety is of paramount importance, desired improvements to these devices include protection against inadvertent initiation, the elimination of the production of high-velocity fragments, less damaging decibel output and increased light output. Sandia National Laboratories has developed a next-generation diversionary flash-bang device that will provide the end user with these enhanced safety features

Comparative Genomics of Emerging Human Ehrlichiosis Agents

Anaplasma (formerly Ehrlichia) phagocytophilum, Ehrlichia chaffeensis, and Neorickettsia (formerly Ehrlichia) sennetsu are intracellular vector-borne pathogens that cause human ehrlichiosis, an emerging infectious disease. We present the complete genome sequences of these organisms along with comparisons to other organisms in the Rickettsiales order. Ehrlichia spp. and Anaplasma spp. display a unique large expansion of immunodominant outer membrane proteins facilitating antigenic variation. All Rickettsiales have a diminished ability to synthesize amino acids compared to their closest free-living relatives. Unlike members of the Rickettsiaceae family, these pathogenic Anaplasmataceae are capable of making all major vitamins, cofactors, and nucleotides, which could confer a beneficial role in the invertebrate vector or the vertebrate host. Further analysis identified proteins potentially involved in vacuole confinement of the Anaplasmataceae, a life cycle involving a hematophagous vector, vertebrate pathogenesis, human pathogenesis, and lack of transovarial transmission. These discoveries provide significant insights into the biology of these obligate intracellular pathogens

Iron isotope fractionation in subterranean estuaries

Author Posting. © Elsevier B.V., 2008. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 72 (2008): 3413-3430, doi:10.1016/j.gca.2008.05.001.Dissolved Fe concentrations in subterranean estuaries, like their river-seawater counterparts, are strongly controlled by non-conservative behavior during mixing of groundwater and seawater in coastal aquifers. Previous studies at a subterranean estuary of Waquoit Bay on Cape Cod, USA demonstrate extensive precipitation of groundwater-borne dissolved ferrous iron and subsequent accumulation of iron oxides onto subsurface sands. Waquoit Bay is thus an excellent natural laboratory to assess the mechanisms of Fe-isotope fractionation in redoxstratified environments and determine potential Fe-isotope signatures of groundwater sources to coastal seawater. Here, we report Fe isotope compositions of iron-coated sands and porewaters beneath the intertidal zone of Waquoit Bay. The distribution of pore water Fe shows two distinct sources of Fe: one residing in the upward rising plume of Fe-rich groundwater and the second in the salt-wedge zone of pore water. The groundwater source has high Fe(II) concentration consistent with anoxic conditions and yield δ56Fe values between 0.3 and –1.3‰. In contrast, sediment porewaters occurring in the mixing zone of the subterranean estuary have very low δ56Fe values down to –5‰. These low δ56Fe values reflect Fe-redox cycling and result from the preferential retention of heavy Fe-isotopes onto newly formed Fe-oxyhydroxides. Analysis of Feoxides precipitated onto subsurface sands in two cores from the subterranean estuary revealed strong δ56Fe and Fe concentration gradients over less than 2m, yielding an overall range of δ56Fe values between –2 and 1.5‰. The relationship between Fe concentration and δ56Fe of Fe-rich sands can be modeled by the progressive precipitation of Fe-oxides along fluid flow through the subterranean estuary. These results demonstrate that large-scale Fe isotope fractionation (up to 5‰) can occur in subterranean estuaries, which could lead to coastal seawater characterized by very low δ56Fe values relative to river values.This study was supported by the National Science Foundation (OCE 0550066) to OR and ES , (OCE 0095384) to MC and ES and NASA Astrobiology Institute - Cycle 3 CAN-02-OSS-02 to KJE