Multiple phases of human occupation in Southeast Arabia between 210,000 and 120,000 years ago.

Changing climatic conditions are thought to be a major control of human presence in Arabia during the Paleolithic. Whilst the Pleistocene archaeological record shows that periods of increased monsoon rainfall attracted human occupation and led to increased population densities, the impact of arid conditions on human populations in Arabia remains largely speculative. Here, we present data from Jebel Faya in Southeast (SE) Arabia, which document four periods of human occupation between c. 210,000 and 120,000 years ago. The Jebel Faya record indicates that human occupation of SE Arabia was more regular and not exclusively linked to major humid periods. Our data show that brief phases of increased rainfall additionally enabled human settlement in the Faya region. These results imply that the mosaic environments in SE Arabia have likely formed a population refugia at the end of the Middle and the beginning of the Late Pleistocene.

Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates.

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.

Distinct classes of misfolded proteins differentially affect the growth of yeast compromised for proteasome function.

Maintenance of the proteome (proteostasis) is essential for cellular homeostasis and prevents cytotoxic stress responses that arise from protein misfolding. However, little is known about how different types of misfolded proteins impact homeostasis, especially when protein degradation pathways are compromised. We examined the effects of misfolded protein expression on yeast growth by characterizing a suite of substrates possessing the same aggregation-prone domain but engaging different quality control pathways. We discovered that treatment with a proteasome inhibitor was more toxic in yeast expressing misfolded membrane proteins, and this growth defect was mirrored in yeast lacking a proteasome-specific transcription factor, Rpn4p. These results highlight weaknesses in the proteostasis network's ability to handle the stress arising from an accumulation of misfolded membrane proteins.

Deletion of Two Genes in Burkholderia pseudomallei MSHR668 That Target Essential Amino Acids Protect Acutely Infected BALB/c Mice and Promote Long Term Survival.

Melioidosis is an emerging disease that is caused by the facultative intracellular pathogen Burkholderia pseudomallei. It is intrinsically resistant to many antibiotics and host risk factors play a major role in susceptibility to infection. Currently, there is no human or animal vaccine against melioidosis. In this study, multiple B. pseudomallei MSHR668 deletion mutants were evaluated as live attenuated vaccines in the sensitive BALB/c mouse model of melioidosis. The most efficacious vaccines after an intraperitoneal challenge with 50-fold over the 50% median lethal dose (MLD50) with B. pseudomallei K96243 were 668 ΔhisF and 668 ΔilvI. Both vaccines completely protected mice in the acute phase of infection and showed significant protection (50% survivors) during the chronic phase of infection. The spleens of the survivors that were examined were sterile. Splenocytes from mice vaccinated with 668 ΔhisF and 668 ΔilvI expressed higher amounts of IFN-γ after stimulation with B. pseudomallei antigens than splenocytes from mice vaccinated with less protective candidates. Finally, we demonstrate that 668 ΔhisF is nonlethal in immunocompromised NOD/SCID mice. Our results show that 668 ΔhisF and 668 ΔilvI provide protective cell-mediated immune responses in the acute phase of infection and promote long term survival in the sensitive BALB/c mouse model of melioidosis.

Suppressed-Doppler slit jet infrared spectroscopy of astrochemically relevant cations: ν1 and ν4 NH stretching modes in NH3D.

A suppressed-Doppler (Δν = 180 MHz) infrared spectrum of monodeuterated ammonium ions (NH3D+) has been obtained for the ν1 (symmetric) and ν4 (degenerate) N-H stretch bands via direct absorption high resolution IR laser spectroscopy in a planar slit jet discharge expansion. The ion is efficiently generated by H3 + protonation of NH2D in a discharge mixture of H2/NH2D, with the resulting expansion rapidly cooling the molecular ions into low rotational states. The first high-resolution infrared spectrum of ν1 is reported, as well as many previously unobserved transitions in the ν4 rovibrational manifold. Simultaneous observation of both ν1 and ν4 permits elucidation of both the vibrational ground and excited state properties of the ion, including rigorous benchmarking of band origins against high-level anharmonic ab initio theory as well as determination of the ν1:ν4 intensity ratio for comparison with bond-dipole model predictions. Ground-state combination differences from this work and earlier studies permit the rotational constants of NH3D+ to be determined to unprecedented accuracy, the results of which support previous laboratory and astronomical assignment of the 10-00 pure rotational transition and should aid future searches for other rotational transitions as well.

Glucopyranosyl lipid adjuvant enhances immune response to Ebola virus-like particle vaccine in mice.

The identification of adjuvants that promote lasting antigen-specific immunity and augment vaccine efficacy are integral to the development of new protein-based vaccines. The Ebola virus-like particle (VLP) vaccine expressing Ebola virus glycoprotein (GP) and matrix protein (VP40) was used in this study to evaluate the ability of TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a stable emulsion (SE) to enhance immunogenicity and promote durable protection against mouse-adapted Ebola virus (ma-EBOV). Antibody responses and Ebola-specific T cell responses were evaluated post vaccination. Survival analysis after lethal ma-EBOV challenge was performed 4 weeks and 22 weeks following final vaccination. GLA-SE enhanced EBOV-specific immunity and resulted in long-term protection against challenge with ma-EBOV infection in a mouse model. Specifically, GLA-SE elicited Th1-skewed antibodies and promoted the generation of EBOV GP-specific polyfunctional T cells. These results provide further support for the utility of TLR4 activating GLA-SE-adjuvanted vaccines.

Exploring the Functional Consequences of Protein Backbone Alteration in Ubiquitin through Native Chemical Ligation.

Ubiquitin (Ub) plays critical roles in myriad protein degradation and signaling networks in the cell. We report herein Ub mimetics based on backbones that blend natural and artificial amino acid units. The variants were prepared by a modular route based on native chemical ligation. Biological assays show that some are enzymatically polymerized onto protein substrates, and that the resulting Ub tags are recognized for downstream pathways. These results advance the size and complexity of folded proteins mimicked by artificial backbones and expand the functional scope of such agents.

Hsp104 facilitates the endoplasmic-reticulum-associated degradation of disease-associated and aggregation-prone substrates.

Misfolded proteins in the endoplasmic reticulum (ER) are selected for ER-associated degradation (ERAD). More than 60 disease-associated proteins are substrates for the ERAD pathway due to the presence of missense or nonsense mutations. In yeast, the Hsp104 molecular chaperone disaggregates detergent-insoluble ERAD substrates, but the spectrum of disease-associated ERAD substrates that may be aggregation prone is unknown. To determine if Hsp104 recognizes aggregation-prone ERAD substrates associated with human diseases, we developed yeast expression systems for a hydrophobic lipid-binding protein, apolipoprotein B (ApoB), along with a chimeric protein harboring a nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator (CFTR) into which disease-causing mutations were introduced. We discovered that Hsp104 facilitates the degradation of ER-associated ApoB as well as a truncated CFTR chimera in which a premature stop codon corresponds to a disease-causing mutation. Chimeras containing a wild-type version of the CFTR domain or a different mutation were stable and thus Hsp104 independent. We also discovered that the detergent solubility of the unstable chimera was lower than the stable chimeras, and Hsp104 helped retrotranslocate the unstable chimera from the ER, consistent with disaggregase activity. To determine why the truncated chimera was unstable, we next performed molecular dynamics simulations and noted significant unraveling of the CFTR nucleotide-binding domain. Because human cells lack Hsp104, these data indicate that an alternate disaggregase or mechanism facilitates the removal of aggregation-prone, disease-causing ERAD substrates in their native environments.

Sub-Doppler slit jet infrared spectroscopy of astrochemically relevant cations: The NH stretching mode in ND3H.

High-resolution rotationally resolved spectra of the N-H stretch vibrational mode (ν 1) of jet-cooled ND3H+ ions are collected and analyzed in a sub-Doppler slit-jet infrared spectrometer. The isotopomeric ammonium ions are generated by proton transfer from H3 + to ND3 in a discharge of an ND3/H2 gas mixture, whereby the slit jet expansion cools the nascent ND3H+ ions into lower rotational states. Rotational assignments are confirmed by four-line combination differences that agree to within the spectrometer precision (9 MHz). Based on precision two-line ground-state combination differences and a symmetric top Hamiltonian, the B, D J , and D JK rotational constants for the ground vibrational state of ND3H+ are determined with high precision for the first time. Approximate rotational constants for the ν 1 excited state are also determined, with a band origin at 3316.8425(19) cm-1 and in remarkable (∼0.1 cm-1) agreement with high level anharmonic theoretical predictions by Guo and co-workers [J. Phys. Chem. A, 120, 2185 (2016)]. Our results allow us to predict several low-J pure rotational transitions of ND3H+, which we hope will support future studies of this important ion in laboratory and astronomical rotational spectroscopy.

Using reduced density matrix techniques to capture static and dynamic correlation in the energy landscape for the decomposition of the CH2CH2ONO radical and support a non-IRC pathway.

The unexpected abundance of HNO in the photodecomposition of the radical 2-nitrosooxy ethyl (CH2CH2ONO) is investigated through calculations of the potential energy surface by the anti-Hermitian contracted Schrödinger equation (ACSE) method, which directly generates the 2-electron reduced density matrix. The ACSE, which is able to balance single-reference (dynamic) and multi-reference (static) correlation effects, reveals some subtle correlation effects along the intrinsic reaction coordinate (IRC) en route to NO + oxirane, an IRC which offers a potential bifurcation to the HNO + vinoxy product channel. These effects were not fully captured by either single-reference techniques, such as coupled cluster, or multi-reference techniques, such as second-order multi-reference perturbation theory. These correlation effects reveal small to moderate energy changes in key transition states, which have implications for the reaction mechanism as related to the production of HNO.

Drug induced thrombotic microangiopathy caused by levofloxacin.

Thrombotic microangiopathy is a rare but serious manifestation of a variety of diseases. The key features are microangiopathic haemolysis, thrombocytopaenia, renal dysfunction and neurological symptoms. Here we discuss the case of a previously well male presenting with community-acquired pneumonia who developed thrombotic microangiopathy during admission. This case illustrates the difficulties in the differential diagnosis and reminds us of the importance of the peripheral blood film in identifying the cause of thrombocytopaenia. One life-threatening cause of thrombotic microangiopathy is thrombotic thrombocytopaenia purpura and when that diagnosis is suspected emergency plasma exchange is essential. Many drugs can cause thrombotic microangiopathy and here we highlight the commonly-prescribed antibiotic levofloxacin as the culprit.

Substrate Insolubility Dictates Hsp104-Dependent Endoplasmic-Reticulum-Associated Degradation.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by ER-associated degradation (ERAD). Although the retrotranslocation of misfolded proteins from the ER has been reconstituted, how a polypeptide is initially selected for ERAD remains poorly defined. To address this question while controlling for the diverse nature of ERAD substrates, we constructed a series of truncations in a single ER-tethered domain. We observed that the truncated proteins exhibited variable degradation rates and discovered a positive correlation between ERAD substrate instability and detergent insolubility, which demonstrates that aggregation-prone species can be selected for ERAD. Further, Hsp104 facilitated degradation of an insoluble species, consistent with the chaperone's disaggregase activity. We also show that retrotranslocation of the ubiquitinated substrate from the ER was inhibited in the absence of Hsp104. Therefore, chaperone-mediated selection frees the ER membrane of potentially toxic, aggregation-prone species.

Primary Product Branching in the Photodissociation of Chloroacetaldehyde at 157 nm.

We used crossed laser-molecular beam scattering to study the primary photodissociation channels of chloroacetaldehyde (CH2ClCHO) at 157 nm. In addition to the C-Cl bond fission primary photodissociation channel, the data evidence two other photodissociation channels: HCl photoelimination and C-C bond fission. This is the first direct evidence of the C-C bond fission channel in chloroacetaldehyde, and we found that it significantly competes with the C-Cl bond fission channel. We determined the total primary photodissociation branching fractions for C-Cl fission:HCl elimination:C-C fission to be 0.65:0.07:0.28. The branching between the primary channels suggests the presence of interesting excited state dynamics in chloroacetaldehyde. Some of the vinoxy radicals from C-Cl photofission and most of the ketene cofragments formed in HCl photoelimination have enough internal energy to undergo secondary dissociation. While our previous velocity map imaging study on the photodissociation of chloroacetaldehyde at 157 nm focused on the barrier for the unimolecular dissociation of vinoxy to H + ketene, this work shows that the HCl elimination channel contributed to the high kinetic energy portion of the m/z = 42 signal in that study.

Dissociative Photoionization of the Elusive Vinoxy Radical.

These experiments report the dissociative photoionization of vinoxy radicals to m/z = 15 and 29. In a crossed laser-molecular beam scattering apparatus, we induce C-Cl bond fission in 2-chloroacetaldehyde by photoexcitation at 157 nm. Our velocity measurements, combined with conservation of angular momentum, show that 21% of the C-Cl photofission events form vinoxy radicals that are stable to subsequent dissociation to CH3 + CO or H + ketene. Photoionization of these stable vinoxy radicals, identified by their velocities, which are momentum-matched with the higher-kinetic-energy Cl atom photofragments, shows that the vinoxy radicals dissociatively photoionize to give signal at m/z = 15 and 29. We calibrated the partial photoionization cross section of vinoxy to CH3+ relative to the bandwidth-averaged photoionization cross section of the Cl atom at 13.68 eV to put the partial photoionization cross sections on an absolute scale. The resulting bandwidth-averaged partial cross sections are 0.63 and 1.3 Mb at 10.5 and 11.44 eV, respectively. These values are consistent with the upper limit to the cross section estimated from a study by Savee et al. on the O(3P) + propene bimolecular reaction. We note that the uncertainty in these values is primarily dependent on the signal attributed to C-Cl primary photofission in the m/z = 35 (Cl+) time-of-flight data. While the value is a rough estimate, the bandwidth-averaged partial photoionization cross section of vinoxy to HCO+ calculated from the signal at m/z = 29 at 11.53 eV is approximately half that of vinoxy to CH3+. We also present critical points on the potential energy surface of the vinoxy cation calculated at the G4//B3LYP/6-311++G(3df,2p) level of theory to support the observation of dissociative ionization of vinoxy to both CH3+ and HCO+.

Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates.

Integral membrane proteins fold inefficiently and are susceptible to turnover via the endoplasmic reticulum-associated degradation (ERAD) pathway. During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated to the cytoplasm for proteasomal degradation. Although many aspects of this pathway are defined, how transmembrane helices (TMHs) are removed from the membrane and into the cytoplasm before degradation is poorly understood. In this study, we asked whether the hydrophobic character of a TMH acts as an energetic barrier to retrotranslocation. To this end, we designed a dual-pass model ERAD substrate, Chimera A*, which contains the cytoplasmic misfolded domain from a characterized ERAD substrate, Sterile 6* (Ste6p*). We found that the degradation requirements for Chimera A* and Ste6p* are similar, but Chimera A* was retrotranslocated more efficiently than Ste6p* in an in vitro assay in which retrotranslocation can be quantified. We then constructed a series of Chimera A* variants containing synthetic TMHs with a range of ΔG values for membrane insertion. TMH hydrophobicity correlated inversely with retrotranslocation efficiency, and in all cases, retrotranslocation remained Cdc48p dependent. These findings provide insight into the energetic restrictions on the retrotranslocation reaction, as well as a new computational approach to predict retrotranslocation efficiency.

The evolving role of ubiquitin modification in endoplasmic reticulum-associated degradation.

The endoplasmic reticulum (ER) serves as a warehouse for factors that augment and control the biogenesis of nascent proteins entering the secretory pathway. In turn, this compartment also harbors the machinery that responds to the presence of misfolded proteins by targeting them for proteolysis via a process known as ER-associated degradation (ERAD). During ERAD, substrates are selected, modified with ubiquitin, removed from the ER, and then degraded by the cytoplasmic 26S proteasome. While integral membrane proteins can directly access the ubiquitination machinery that resides in the cytoplasm or on the cytoplasmic face of the ER membrane, soluble ERAD substrates within the lumen must be retrotranslocated from this compartment. In either case, nearly all ERAD substrates are tagged with a polyubiquitin chain, a modification that represents a commitment step to degrade aberrant proteins. However, increasing evidence indicates that the polyubiquitin chain on ERAD substrates can be further modified, serves to recruit ERAD-requiring factors, and may regulate the ERAD machinery. Amino acid side chains other than lysine on ERAD substrates can also be modified with ubiquitin, and post-translational modifications that affect substrate ubiquitination have been observed. Here, we summarize these data and provide an overview of questions driving this field of research.

Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein.

In the kidney, the epithelial sodium channel (ENaC) regulates blood pressure through control of sodium and volume homeostasis, and in the lung, ENaC regulates the volume of airway and alveolar fluids. ENaC is a heterotrimer of homologous α-, ß- and γ-subunits, and assembles in the endoplasmic reticulum (ER) before it traffics to and functions at the plasma membrane. Improperly folded or orphaned ENaC subunits are subject to ER quality control and targeted for ER-associated degradation (ERAD). We previously established that a conserved, ER lumenal, molecular chaperone, Lhs1/GRP170, selects αENaC, but not ß- or γ-ENaC, for degradation when the ENaC subunits were individually expressed. We now find that when all three subunits are co-expressed, Lhs1-facilitated ERAD was blocked. To determine which domain-domain interactions between the ENaC subunits are critical for chaperone-dependent quality control, we employed a yeast model and expressed chimeric α/ßENaC constructs in the context of the ENaC heterotrimer. We discovered that the ßENaC transmembrane domain was sufficient to prevent the Lhs1-dependent degradation of the α-subunit in the context of the ENaC heterotrimer. Our work also found that Lhs1 delivers αENaC for proteasome-mediated degradation after the protein has become polyubiquitinated. These data indicate that the Lhs1 chaperone selectively recognizes an immature form of αENaC, one which has failed to correctly assemble with the other channel subunits via its transmembrane domain.

GABA (γ-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000.

The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Local adaptation is associated with zinc tolerance in Pseudomonas endophytes of the metal-hyperaccumulator plant Noccaea caerulescens.

Metal-hyperaccumulating plants, which are hypothesized to use metals for defence against pests and pathogens, provide a unique context in which to study plant-pathogen coevolution. Previously, we demonstrated that the high concentrations of zinc found in leaves of the hyperaccumulator Noccaea caerulescens provide protection against bacterial pathogens, with a potential trade-off between metal-based and pathogen-induced defences. We speculated that an evolutionary arms race between zinc-based defences in N. caerulescens and zinc tolerance in pathogens might have driven the development of the hyperaccumulation phenotype. Here, we investigate the possibility of local adaptation by bacteria to the zinc-rich environment of N. caerulescens leaves and show that leaves sampled from the contaminated surroundings of a former mine site harboured endophytes with greater zinc tolerance than those within plants of an artificially created hyperaccumulating population. Experimental manipulation of zinc concentrations in plants of this artificial population influenced the zinc tolerance of recovered endophytes. In laboratory experiments, only endophytic bacteria isolated from plants of the natural population were able to grow to high population densities in any N. caerulescens plants. These findings suggest that long-term coexistence with zinc-hyperaccumulating plants leads to local adaptation by endophytic bacteria to the environment within their leaves.

Dissociation Pathways of the CH2CH2ONO Radical: NO2 + Ethene, NO + Oxirane, and a Non-Intrinsic Reaction Coordinate HNO + Vinoxy Pathway.

We first characterize the dissociation pathways of BrCH2CH2ONO, a substituted alkyl nitrite, upon photoexcitation at 193 nm under collision-free conditions, in a crossed laser-molecular beam scattering apparatus using vacuum ultraviolet photoionization detection. Three primary photodissociation pathways occur: photoelimination of HNO, leading to the products HNO + BrCH2CHO; C-Br bond photofission, leading to Br + CH2CH2ONO; and O-NO bond photofission, leading to NO + BrCH2CH2O. The data show that alkyl nitrites can eliminate HNO via a unimolecular mechanism in addition to the commonly accepted bulk disproportionation mechanism. Some of the products from the primary photodissociation pathways are highly vibrationally excited, so we then probe the product branching from the unimolecular dissociation of these unstable intermediates. Notably, the vibrationally excited CH2CH2ONO radicals undergo two channels predicted by statistical transition-state theory, and an additional non-intrinsic reaction coordinate channel, HNO elimination. CH2CH2ONO is formed with high rotational energy; by employing rotational models based on conservation of angular momentum, we predict, and verify experimentally, the kinetic energies of stable CH2CH2ONO radicals and the angular distribution of dissociation products. The major dissociation pathway of CH2CH2ONO is NO2 + ethene, and some of the NO2 is formed with sufficient internal energy to undergo further photodissociation. Nascent BrCH2CHO and CH2Br are also photodissociated upon absorption of a second 193 nm photon; we derive the kinetic energy release of these dissociations based on our data, noting similarities to the analogous photodissociation of ClCH2CHO and CH2Cl.