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Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode Caenorhabditis elegans can selectively impair the silencing of some genes. Here, we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changing cis -regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells, and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, are enriched within 'pUG zones' matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs., Competing Interests: DK, PR, FE, LD, AJ No competing interests declared, (© 2024, Knudsen-Palmer et al.)

The mechanosensitive ion channels Transient Receptor Potential Vanilloid 4 (TRPV4) and PIEZO1 transduce physiologic and supraphysiologic magnitudes of mechanical signals in the chondrocyte, respectively. TRPV4 activation promotes chondrogenesis, while PIEZO1 activation by supraphysiologic deformations drives cell death. The mechanisms by which activation of these channels discretely drives changes in gene expression to alter cell behavior remain to be determined. To date, no studies have contrasted the transcriptomic response to activation of these channels nor has any published data attempted to correlate these transcriptomes to alterations in cellular function. This study used RNA sequencing to comprehensively investigate the transcriptomes associated with activation of TRPV4 or PIEZO1, revealing that TRPV4 and PIEZO drive distinct transcriptomes and also exhibit unique co-regulated clusters of genes. Notably, activation of PIEZO1 through supraphysiologic deformation induced a transient inflammatory profile that overlapped with the interleukin (IL)-1-responsive transcriptome and contained genes associated with cartilage degradation and osteoarthritis progression. However, both TRPV4 and PIEZO1 were also shown to elicit anabolic effects. PIEZO1 expression promoted a pro-chondrogenic transcriptome under unloaded conditions, and daily treatment with PIEZO1 agonist Yoda1 significantly increased sulfated glycosaminoglycan deposition in vitro. These findings emphasize the presence of a broad "mechanome" with distinct effects of TRPV4 and PIEZO1 activation in chondrocytes, suggesting complex roles for PIEZO1 in both the physiologic and pathologic responses of chondrocytes. The identification of transcriptomic profiles unique to or shared by PIEZO1 and TRPV4 (distinct from IL-1-induced inflammation) could inform future therapeutic designs targeting these channels for the management and treatment of osteoarthritis., (© 2024 Federation of American Societies for Experimental Biology.)

Nuclear factor-erythroid 2-related factor-2 (Nrf2) is a transcription factor that serves as a master regulator of anti-inflammatory agents, phase I xenobiotic, and phase II antioxidant enzymes, all of which provide a cytoprotective role during disease progression. We hypothesized that oral administration of a purported phytochemical Nrf2-activator, PB125 ® , would increase long bone strength in aging Hartley guinea pigs, a model prone to musculoskeletal decline. Male (N = 56) and female (N = 56) guinea pigs were randomly assigned to receive daily oral treatment with either PB125 ® or vehicle control. Animals were treated for a consecutive 3-months (starting at 2-months of age) or 10-months (starting at 5-months of age) and sacrificed at 5-months or 15-months of age, respectively. Outcome measures included: (1) ANY-maze™ enclosure monitoring, (2) quantitative microcomputed tomography, and (3) biomechanical testing. Treatment with PB125 ® for 10 months resulted in increased long bone strength as determined by ultimate bending stress in female Hartley guinea pigs. In control groups, increasing age resulted in significant effects on geometric and structural properties of long bones, as well as a trending increase in ultimate bending stress. Furthermore, both age and sex had a significant effect on the geometric properties of both cortical and trabecular bone. Collectively, this work suggests that this nutraceutical may serve as a promising target and preventive measure in managing the decline in bone mass and quality documented in aging patients. Auxiliary to this main goal, this work also capitalized upon 5 and 15-month-old male and female animals in the control group to characterize age- and sex-specific differences on long bone geometric, structural, and material properties in this animal model., (© 2023. The Author(s) under exclusive licence to Biomedical Engineering Society.)

The development of modeling concepts with the 2001 NRC (National Research Council Nutrient Requirements of Dairy Cattle represented a big step toward understanding and applying the underlying mechanisms associated with young calf growth. Factors such as the plane of nutrition being provided (protein and energy), breed and environmental stressors impact calf growth. Investigation into delivering the proper amounts of energy and protein through the liquid and dry feeds to optimize growth needs to continue as well as further defining the most effective means to transition from the nonruminant to ruminant phase while minimizing postweaning lag., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to evaluate the reliability and validity of the updated 2019 CDC Worksite Health ScoreCard (CDC ScoreCard), which includes four new modules., Methods: We pilot tested the updated instrument at 93 worksites, examining question response concurrence between two representatives from each worksite. We conducted cognitive interviews and site visits to evaluate face validity, and refined the instrument for public distribution., Results: The mean question concurrence rate was 73.4%. Respondents reported the tool to be useful for assessing current workplace programs and planning future initiatives. On average, 43% of possible interventions included in the CDC ScoreCard were in place at the pilot sites., Conclusion: The updated CDC ScoreCard is a valid and reliable tool for assessing worksite health promotion policies, educational and lifestyle counseling programs, environmental supports, and health benefits.

Objective: To evaluate absenteeism, presenteeism, and total lost productive time (LPT) associated with multimorbidity., Methods: Cross-sectional data from 3228 state-government employees from Tasmania were collected in 2013. The validated measures of absenteeism, presenteeism, and LPT were obtained from employees' self-reported data over a 28-day period. Analyses were stratified by sex. Negative binomial models were used to estimate the associations between multimorbidity and LPT., Results: The average health-related total LPT was 1.2 (standard deviation [SD] = 2.4) and 1.7 (SD = 3.5) days for men and women with multimorbidity, respectively. Women (rate ratio [RR] = 2.9, 95% confidence interval [CI] 1.8 to 4.9) and men (RR = 4.4, 95%CI 3.0 to 6.2) with 4+ chronic conditions were significantly more likely to report LPT compared with those without any chronic conditions., Conclusion: We found multimorbidity is of concern within the workforce, with a positive association of multimorbidity and LPT observed, and significant differences in LPT between men and women reporting multimorbidity.

Glucose-6-phosphate 3-dehydrogenase (NtdC) is an NAD-dependent oxidoreductase encoded in the NTD operon of Bacillus subtilis. The oxidation of glucose 6-phosphate by NtdC is the first step in kanosamine biosynthesis. The product, 3-oxo-d-glucose 6-phosphate (3oG6P), has never been synthesized or isolated. The NtdC-catalyzed reaction is very slow at low and neutral pH, and its rate increases to a maximum near pH 9.5. However, under alkaline conditions, the product is not stable because of ring opening followed by deprotonation of the 1,3-dicarbonyl compound. The absorbance band due to this enolate at 310 nm overlaps with that of the other enzymatic product, NADH, complicating kinetic measurements. We report the deconvolution of the resulting spectra of the reaction to determine the rate constants and likely kinetic mechanism. In doing so, we were able to determine the extinction coefficient of the enolate of 3oG6P (23000 M -1 cm -1 ), which allowed the measurement of the first-order rate constant (5.51 × 10 -3 s -1 ) and activation energy (93 kJ mol -1 ) of nonenzymatic enolate formation. Using deuterium-labeled substrates, we show that hydride transfer from carbon 3 is partially rate-limiting in the enzymatic reaction, and deuterium substitution on carbon 2 has no significant effect on the enzymatic reaction but lowers the rate of deprotonation of 3oG6P 4-fold. These experiments clearly establish the regiochemistry of the reactions. Coupling of the NtdC reaction with the subsequent step in the pathway, NtdA-catalyzed glutamate-dependent amino transfer, has a small but significant effect on the rate of NAD reduction, consistent with these enzymes working together to process the unstable metabolite.

Dihydrodipicolinate synthase is a tetrameric enzyme of the diaminopimelate pathway in bacteria and plants. The protein catalyzes the condensation of pyruvate (Pyr) and aspartate semialdehyde en route to the end product lysine (Lys). Dihydrodipicolinate synthase from Campylobacter jejuni (CjDHDPS) is allosterically inhibited by Lys. CjDHDPS is a promising antibiotic target, as highlighted by the recent development of a potent bis-lysine (bisLys) inhibitor. The mechanism whereby Lys and bisLys allosterically inhibit CjDHDPS remains poorly understood. In contrast to the case for other allosteric enzymes, crystallographically detectable conformational changes in CjDHDPS upon inhibitor binding are very minor. Also, it is difficult to envision how Pyr can access the active site; the available X-ray data seemingly imply that each turnover step requires diffusion-based mass transfer through a narrow access channel. This study employs hydrogen/deuterium exchange mass spectrometry for probing the structure and dynamics of CjDHDPS in a native solution environment. The deuteration kinetics reveal that the most dynamic protein regions are in the direct vicinity of the substrate access channel. This finding is consistent with the view that transient opening/closing fluctuations facilitate access of the substrate to the active site. Under saturating conditions, both Lys and bisLys cause dramatically reduced dynamics in the inhibitor binding region. In addition, rigidification extends to regions close to the substrate access channel. This finding strongly suggests that allosteric inhibitors interfere with conformational fluctuations that are required for CjDHDPS substrate turnover. In particular, our data imply that Lys and bisLys suppress opening/closing events of the access channel, thereby impeding diffusion of the substrate into the active site. Overall, this work illustrates why allosteric control does not have to be associated with crystallographically detectable large-scale transitions. Our experiments provide evidence that in CjDHDPS allostery is mediated by changes in the extent of thermally activated conformational fluctuations.

Dihydrodipicolinate synthase (DHDPS), an enzyme required for bacterial peptidoglycan biosynthesis, catalyzes the condensation of pyruvate and β-aspartate semialdehyde (ASA) to form a cyclic product which dehydrates to form dihydrodipicolinate. DHDPS has, for several years, been considered a putative target for novel antibiotics. We have designed the first potent inhibitor of this enzyme by mimicking its natural allosteric regulation by lysine, and obtained a crystal structure of the protein-inhibitor complex at 2.2 Å resolution. This novel inhibitor, which we named "bislysine", resembles two lysine molecules linked by an ethylene bridge between the α-carbon atoms. Bislysine is a mixed partial inhibitor with respect to the first substrate, pyruvate, and a noncompetitive partial inhibitor with respect to ASA, and binds to all forms of the enzyme with a Ki near 200 nM, more than 300 times more tightly than lysine. Hill plots show that the inhibition is cooperative, indicating that the allosteric sites are not independent despite being located on opposite sides of the protein tetramer, separated by approximately 50 Å. A mutant enzyme resistant to lysine inhibition, Y110F, is strongly inhibited by this novel inhibitor, suggesting this may be a promising strategy for antibiotic development.

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

Cps2L, a thymidylytransferase, is the first enzyme in Streptococcus pneumoniae L-rhamnose biosynthesis and an antibacterial target. We herein report the evaluation of six sugar phosphate analogues selected to further probe Cps2L substrate tolerance. A modified continuous spectrophotometric assay was employed for facile detection of pyrophosphate (PPi) released from nucleotidylyltransfase-catalysed condensation of sugar 1-phosphates and nucleoside triphosphates to produce sugar nucleotides. Additionally, experiments using waterLOGSY NMR spectroscopy were investigated as a complimentary method to evaluate binding affinity to Cps2L.

Dihydrodipicolinate synthase (DHDPS), an enzyme found in most bacteria and plants, controls a critical step in the biosynthesis of l-lysine and meso-diaminopimelate, necessary components for bacterial cell wall biosynthesis. DHDPS catalyzes the condensation of pyruvate and (S)-aspartate-β-semialdehyde, forming an unstable product that is dehydrated to dihydrodipicolinate. The tetrameric enzyme is allosterically inhibited by l-lysine, and a better understanding of the allosteric inhibition mechanism is necessary for the design of potent antibacterial therapeutics. Here we describe the high-resolution crystal structures of DHDPS from Campylobacter jejuni with and without its inhibitor bound to the allosteric sites. These structures reveal a role for Y110 in the regulation of the allosteric inhibition by lysine. Mutation of Y110 to phenylalanine results in insensitivity to lysine inhibition, although the mutant crystal structure reveals that lysine does bind in the allosteric site. Comparison of the lysine-bound Y110F structure with wild-type structures reveals that key structural changes due to lysine binding are absent in this mutant.

Lactobacillus casei BL23 contains two genes, iolG1 and iolG2, homologous with inositol dehydrogenase encoding genes from many bacteria. Inositol dehydrogenase catalyzes the oxidation of inositol with concomitant reduction of NAD+. The protein encoded by iolG2, LcIDH2, has been purified to homogeneity, crystallized and cryoprotected for diffraction at 77 K. The crystals had a high mosaicity and poor processing statistics. Subsequent diffraction measurements were performed without cryoprotectant at room temperature. These crystals were radiation-resistant and a full diffraction data set was collected at room temperature to 1.6 Å resolution.

myo-Inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis converts myo-inositol to scyllo-inosose and is strictly dependent on NAD for activity. We sought to alter the coenzyme specificity to generate an NADP-dependent enzyme in order to enhance our understanding of coenzyme selectivity and to create an enzyme capable of recycling NADP in biocatalytic processes. Examination of available structural information related to the GFO/MocA/IDH family of dehydrogenases and precedents for altering coenzyme selectivity allowed us to select residues for substitution, and nine single, double, and triple mutants were constructed. Mutagenesis experiments with B. subtilis IDH proved extremely successful; the double mutant D35S/V36R preferred NADP to NAD by a factor of 5. This mutant is an excellent catalyst with a second-order rate constant with respect to NADP of 370 000 s⁻¹ M⁻¹, and the triple mutant A12K/D35S/V36R had a value of 570 000 s⁻¹ M⁻¹, higher than that of the wild-type IDH with NAD. The high-resolution X-ray crystal structure of the double mutant A12K/D35S was solved in complex with NADP. Surprisingly, the binding of the coenzyme is altered such that although the nicotinamide ring maintains the required position for catalysis, the coenzyme has twisted by nearly 90°, so the adenine moiety no longer binds to a hydrophobic cleft in the Rossmann fold as in the wild-type enzyme. This change in binding conformation has not previously been observed in mutated dehydrogenases.

Dihydrodipicolinate synthase (DHDPS), an enzyme of the meso-diaminopimelate pathway of lysine biosynthesis, is essential for bacterial growth and is considered a target for novel antibiotics. We have studied DHDPS from Campylobacter jejuni for the first time, determining the kinetic mechanism of catalysis and inhibition with its natural allosteric feedback inhibitor (S)-lysine. The tetrameric enzyme is known to have two allosteric sites, each of which binds two molecules of lysine. The results suggest that lysine binds highly cooperatively, and primarily to the F form of the enzyme during the ping-pong mechanism. By applying graphical methods and nonlinear regression, we have discriminated between the possible kinetic models and determined the kinetic and inhibition constants and Hill coefficients. We conclude that (S)-lysine is an uncompetitive partial inhibitor with respect to its first substrate, pyruvate, and a mixed partial inhibitor with respect to its second substrate, (S)-aspartate-β-semialdehyde (ASA), which differs from the kinetic models for inhibition reported for DHDPS from other sources. The Hill coefficients for the binding of lysine to different forms of the enzyme are all greater than 2, suggesting that the two allosteric sites are not independent. It has been found that ASA binds cooperatively in the presence of (S)-lysine, and the cooperativity of binding increases at near-KM concentrations of pyruvate. The incorporation of Hill coefficients into the kinetic equations was crucial for determining the kinetic model for this enzyme.

The ntd operon in Bacillus subtilis is essential for biosynthesis of 3,3'-neotrehalosadiamine (NTD), an unusual nonreducing disaccharide reported to have antibiotic properties. It has been proposed that the three enzymes encoded within this operon, NtdA, NtdB, and NtdC, constitute a complete set of enzymes required for NTD synthesis, although their functions have never been demonstrated in vitro. We now report that these enzymes catalyze the biosynthesis of kanosamine from glucose-6-phosphate: NtdC is a glucose-6-phosphate 3-dehydrogenase, NtdA is a pyridoxal phosphate-dependent 3-oxo-glucose-6-phosphate:glutamate aminotransferase, and NtdB is a kanosamine-6-phosphate phosphatase. None of these enzymatic reactions have been reported before. This pathway represents an alternate route to the previously reported pathway from Amycolatopsis mediterranei which derives kanosamine from UDP-glucose.

The LYS12 gene from Candida albicans, coding for homoisocitrate dehydrogenase was cloned and expressed as a His-tagged protein in Escherichia coli. The purified gene product catalyzes the Mg(2+)- and K(+)-dependent oxidative decarboxylation of homoisocitrate to α-ketoadipate. The recombinant enzyme demonstrates strict specificity for homoisocitrate. SDS-PAGE of CaHIcDH revealed its molecular mass of 42.6 ± 1 kDa, whereas in size-exclusion chromatography, the enzyme eluted in a single peak corresponding to a molecular mass of 158 ± 3 kDa. Native electrophoresis showed that CaHIcDH may exist as a monomer and as a tetramer and the latter form is favored by homoisocitrate binding. CaHIcDH is an hysteretic enzyme. The K(M) values of the purified His-tagged enzyme for NAD(+) and homoisocitrate were 1.09 mM and 73.7 μM, respectively, and k(cat) was 0.38 s(-1). Kinetic parameters determined for the wild-type CaHIcDH were very similar. The enzyme activity was inhibited by (2R,3S)-3-(p-carboxybenzyl)malate (CBMA), with IC(50) = 3.78 mM. CBMA demonstrated some moderate antifungal activity in minimal media that could be enhanced upon conversion of the enzyme inhibitor into its trimethyl ester derivative (TMCBMA). TMCBMA is the first reported antifungal for which an enzyme of the AAP was identified as a molecular target., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

MenD catalyzes the thiamin diphosphate-dependent decarboxylative carboligation of α-ketoglutarate and isochorismate. The enzyme is essential for menaquinone biosynthesis in many bacteria and has been proposed to be an antibiotic target. The kinetic mechanism of this enzyme has not previously been demonstrated because of the limitations of the UV-based kinetic assay. We have reported the synthesis of an isochorismate analogue that acts as a substrate for MenD. The apparent weaker binding of this analogue is advantageous in that it allows accurate kinetic experiments at substrate concentrations near K(m). Using this substrate in concert with the dead-end inhibitor methyl succinylphosphonate, an analogue of α-ketoglutarate, we show that MenD follows a ping-pong kinetic mechanism. Using both the natural and synthetic substrates, we have measured the effects of 12 mutations of residues at the active site. The results give experimental support to previous models and hypotheses and allow observations unavailable using only the natural substrate.

Inositol dehydrogenase from Bacillus subtilis (BsIDH) is a NAD+-dependent enzyme that catalyses the oxidation of the axial hydroxy group of myo-inositol to form scyllo-inosose. We have determined the crystal structures of wild-type BsIDH and of the inactive K97V mutant in apo-, holo- and ternary complexes with inositol and inosose. BsIDH is a tetramer, with a novel arrangement consisting of two long continuous β-sheets, formed from all four monomers, in which the two central strands are crossed over to form the core of the tetramer. Each subunit in the tetramer consists of two domains: an N-terminal Rossmann fold domain containing the cofactor-binding site, and a C-terminal domain containing the inositol-binding site. Structural analysis allowed us to determine residues important in cofactor and substrate binding. Lys97, Asp172 and His176 are the catalytic triad involved in the catalytic mechanism of BsIDH, similar to what has been proposed for related enzymes and short-chain dehydrogenases. Furthermore, a conformational change in the nicotinamide ring was observed in some ternary complexes, suggesting hydride transfer to the si-face of NAD+. Finally, comparison of the structure and sequence of BsIDH with other putative inositol dehydrogenases allowed us to differentiate these enzymes into four subfamilies based on six consensus sequence motifs defining the cofactor- and substrate-binding sites.

A novel analog of isochorismate, in which the enolpyruvyl substituent is replaced with a carboxymethoxyl group, has been synthesized in four steps from a known intermediate. This analog is more stable than the natural product, but still acts as a good substrate for the enzyme MenD (SEPHCHC synthase). The enzyme consumes the (+)-enantiomer only, with an apparent turnover similar to that of the natural substrate, and an apparent Michaelis constant conveniently higher than that of isochorismate. This analog will be useful in the study of any isochorismate-utilizing enzyme., (Copyright 2010 Elsevier Ltd. All rights reserved.)

MenD is a thiamin diphosphate-dependent enzyme catalyzing the first unique step in menaquinone biosynthesis in bacteria. We have synthesized acylphosphonate ester analogues of alpha-ketoglutarate, a substrate of MenD. These compounds are competitive inhibitors of MenD, with K(i) values as low as 700 nM. Observed structure-activity relationships are in notable contrast to those reported previously for succinylphosphonate inhibition of the alpha-ketoglutarate dehydrogenase complex, despite the apparent homology of these enzymes, and the identical decarboxylation reactions catalyzed. Inhibiting menaquinone biosynthesis is a proposed approach to inhibiting Mycobacterium tuberculosis growth. These inhibitors showed no significant inhibition of M. tuberculosis growth in vitro under aerobic and hypoxic conditions but give new information about the binding characteristics of the MenD active site. Site-directed mutation of Ser391 to alanine had only a minor effect on catalysis, but even the conservative mutation of Arg395 to lysine had a significant effect on the catalytic processing of isochorismate.

Eighteen dengue viruses (DENVs) representing all four serotypes, isolated from pediatric patients at children's hospital, Queen Sirikit National Institute of Child Health, Bangkok, Thailand exhibiting a diverse spectrum of disease ranging from uncomplicated dengue fever (DF) to severe dengue hemorrhagic fever (DHF), were tested for their ability to attach to host cells, replicate and interfere with the IFNalpha signaling pathway by interfering with signal transducer and activator of transcription 1 (STAT-1) function. Although most isolates suppressed IFNalpha-induced STAT-1 phosphorylation, our results showed no difference between DENV strains associated with DF and those associated with DHF. However, the DHF isolates tended replicate to higher titers in dendritic cells (DCs) than the DF isolates, but this ability was independent of their cell-binding capability. Our results suggest that the emergence early in infection of viruses with a high degree of replication fitness may play an important role in DENV pathogenesis.

High mobility group box 1 (HMGB1) protein functions in regulation of transcription, cellular activation and pro-inflammatory responses. However, the potential role of HMGB1 during viral infection has not been investigated. This study attempted to elucidate whether the HMGB1-mediated inflammatory response contributes to the pathogenesis of dengue virus (DENV) infection. Our data showed that HMGB1 was released at low DENV infection levels (m.o.i. of 1) under non-necrotic conditions by human dendritic cells (DCs). When DENV-infected DCs were co-cultured with autologous T cells, there was increased production of HMGB1 by both cell types. HMGB1 regulated tumour necrosis factor alpha, interleukin (IL)-6, IL-8 and alpha interferon secretion in DENV-infected DCs. Additionally, increased HMGB1 production was associated with reduced DENV replication titres in DCs. These results suggest that HMGB1 production influences DENV infection in susceptible hosts.

Studies of acoustic remote sensing of the plumes that result from the injection of particulate matter in the ocean, either naturally or by dumping or dredging activities, have assumed the scattering is incoherent. These plumes are always turbulent, however. The particle density is a passive scalar that is advected by the turbulent velocity flow. The possibility exists, therefore, that the scattered waves from a significant number of particles add coherently as a result of Bragg scattering. In this paper, we investigate this possibility. We derive an expression for the ratio of the coherent intensity to the incoherent one in terms of the turbulent spectrum and the properties of the particles that make up the plume. The sonar is modeled as a high-Q, monostatic, pulsed sonar with arbitrary pulse envelope and arbitrary, but narrow, beam pattern. We apply the formalism to acoustic remote sensing of black smoker hydrothermal plumes. We find that, at most, the coherent intensity is less than 1% of the incoherent one. The implications are that Bragg scattering does not lead to a significant coherent component and in analyses of scattering from this type of plume, one can ignore the complications of turbulence altogether.

NtdA is a putative sugar aminotransferase that is required for the synthesis of 3,3'-neotrehalosadiamine. The enzyme was purified to homogeneity by means of Ni(2+)-affinity chromatography and was crystallized using the microbatch method. X-ray diffraction data were collected from a single crystal to 2.3 A resolution at the Canadian Light Source (CLS). The crystals belonged to space group P2(1), with unit-cell parameters a = 50.3, b = 106.7, c = 96.7 A, beta = 96.2 degrees, and contained two molecules per asymmetric unit.

Myeloid and plasmacytoid dendritic cells (mDC and pDC) are naturally distinctive subsets. We exposed both subsets to dengue virus (DV) in vitro and investigated their functional characteristics. High levels of DV replication in mDC were found to correlate with DC-SIGN expression. Production of inflammatory cytokines by mDC increased gradually after DV-infection, which was dependent on DV replication. Co-stimulatory markers were upregulated on mDC upon DV-infection. On the contrary, lower levels of DV-replication were observed in pDC, but the cytokine production in pDC was quicker and stronger. This cytokine response was not dependent on viral replication, but dependent on cell endosomal activity and TLR7, and could be also induced by purified DV genome RNA. These results clearly suggested functional differences between mDC and pDC in response to DV infection. Additionally, the TLR7-mediated recognition of DV RNA may be involved in pDC functional activation.

Homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis. Despite the homology of this iron-sulfur protein to aconitase, previously studied homoaconitases catalyze only the hydration of cis-homoaconitate to form homoisocitrate rather than the complete isomerization of homocitrate to homoisocitrate. The MJ1003 and MJ1271 proteins from the methanogen Methanocaldococcus jannaschii formed the first homoaconitase shown to catalyze both the dehydration of (R)-homocitrate to form cis-homoaconitate, and its hydration is shown to produce homoisocitrate. This heterotetrameric enzyme also used the analogous longer chain substrates cis-(homo)(2)aconitate, cis-(homo)(3)aconitate, and cis-(homo)(4)aconitate, all with similar specificities. A combination of the homoaconitase with the M. jannaschii homoisocitrate dehydrogenase catalyzed all of the isomerization and oxidative decarboxylation reactions required to form 2-oxoadipate, 2-oxopimelate, and 2-oxosuberate, completing three iterations of the 2-oxoacid elongation pathway. Methanogenic archaeal homoaconitases and fungal homoaconitases evolved in parallel in the aconitase superfamily. The archaeal homoaconitases share a common ancestor with isopropylmalate isomerases, and both enzymes catalyzed the hydration of the minimal substrate maleate to form d-malate. The variation in substrate specificity among these enzymes correlated with the amino acid sequences of a flexible loop in the small subunits.

MosA, a dihydrodipicolinate synthase (DHDPS) from Sinorhizobium meliloti L5-30, catalyzes a class I aldolase reaction that is allosterically inhibited by (S)-lysine. The thermodynamics of (S)-lysine binding to apoenzyme, and to enzyme saturated with pyruvate or with 2-oxobutyrate, are evaluated here using isothermal titration microcalorimetry. Results unambiguously support a noncompetitive mechanism, with substrate-dependent differences in the energetics of inhibitor binding. Inhibition is strikingly cooperative: a second molecule of (S)-lysine binds 10(5) times more tightly than the first.

MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium that forms a symbiotic relationship with leguminous plants. MosA was proposed to catalyze the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine (compounds known as rhizopines), despite the MosA sequence showing a strong resemblance to dihydrodipicolinate synthase (DHDPS) sequences rather than to methyltransferases. Our laboratory has already shown that MosA is an efficient catalyst of the DHDPS reaction. Here we report the structure of MosA, solved to 1.95 A resolution, which resembles previously reported DHDPS structures. In this structure Lys161 forms a Schiff base adduct with pyruvate, consistent with the DHDPS mechanism. We have synthesized both known rhizopines and investigated their ability to interact with MosA in the presence and absence of methyl donors. No MosA-catalyzed methyltransferase activity is observed in the presence of scyllo-inosamine and S-adenosylmethionine (SAM). 2-Oxobutyrate can form a Schiff base with MosA, acting as a competitive inhibitor of MosA-catalyzed dihydrodipicolinate synthesis. It can be trapped on the enzyme by reaction with sodium borohydride, but does not act as a methyl donor. The presence of rhizopines does not affect the kinetics of dihydrodipicolinate synthesis. Isothermal titration calorimetry (ITC) shows no apparent interaction of MosA with rhizopines and SAM. Similar experiments with pyruvate as titrant demonstrate that the reversible Schiff base formation is largely entropically driven. This is the first use of ITC to study Schiff base formation between an enzyme and its substrate.