An insight into the role of branched-chain α-keto acid dehydrogenase (BKD) complex in branched-chain fatty acid biosynthesis and virulence of Listeria monocytogenes.

Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis. Positive regulatory factor A (PrfA) is a pleiotropic master activator of virulence genes of L. monocytogenes that becomes active upon the entry of the bacterium into the cytosol of infected cells. L. monocytogenes can survive and multiply at low temperatures; this is accomplished through the maintenance of appropriate membrane fluidity via branched-chain fatty acid (BCFA) synthesis. Branched-chain α-keto acid dehydrogenase (BKD), which is composed of four polypeptides encoded by lpd, bkdA1, bkdA2, and bkdB, is known to play a vital role in BCFA biosynthesis. Here, we constructed BKD-deficient Listeria strains by in-frame deletion of lpd, bkdA1, bkdA2, and bkdB genes. To determine the role in in vivo and in vitro, mouse model challenges, plaque assay in murine L2 fibroblast, and intracellular replication in J744A.1 macrophage were conducted. BKD-deficient strains exhibited defects in BCFA composition, virulence, and PrfA-regulon function within the host cells. Transcriptomics analysis revealed that the transcript level of the PrfA-regulon was lower in ΔbkdA1 strain than those in the wild-type. This study demonstrates that L. monocytogenes strains lacking BKD complex components were defective in PrfA-regulon function, and full activation of wild-type prfA may not occur within host cells in the absence of BKD. Further study will investigate the consequences of BKD deletion on PrfA function through altering BCFA catabolism.IMPORTANCEListeria monocytogenes is the causative agent of listeriosis, a disease with a high mortality rate. In this study, we have shown that the deletion of BKD can impact the function of PrfA and the PrfA-regulon. The production of virulence proteins within host cells is necessary for L. monocytogenes to promote its intracellular survival and is likely dependent on membrane integrity. We thus report a link between L. monocytogenes membrane integrity and the function of PrfA. This knowledge will increase our understanding of L. monocytogenes pathogenesis, which may provide insight into the development of antimicrobial agents.

Catabolite control protein C contributes to virulence and hydrogen peroxide-induced oxidative stress responses in Listeria monocytogenes.

Listeria monocytogenes causes listeriosis, an infectious and potentially fatal disease of animals and humans. A diverse network of transcriptional regulators, including LysR-type catabolite control protein C (CcpC), is critical for the survival of L. monocytogenes and its ability to transition into the host environment. In this study, we explored the physiological and genetic consequences of deleting ccpC and the effects of such deletion on the ability of L. monocytogenes to cause disease. We found that ccpC deletion did not impact hemolytic activity, whereas it resulted in significant reductions in phospholipase activities. Western blotting revealed that the ΔccpC strain produced significantly reduced levels of the cholesterol-dependent cytolysin LLO relative to the wildtype F2365 strain. However, the ΔccpC mutant displayed no significant intracellular growth defect in macrophages. Furthermore, ΔccpC strain exhibited reduction in plaque numbers in fibroblasts compared to F2365, but plaque size was not significantly affected by ccpC deletion. In a murine model system, the ΔccpC strain exhibited a significantly reduced bacterial burden in the liver and spleen compared to the wildtype F2365 strain. Interestingly, the deletion of this gene also enhanced the survival of L. monocytogenes under conditions of H2O2-induced oxidative stress. Transcriptomic analyses performed under H2O2-induced oxidative stress conditions revealed that DNA repair, cellular responses to DNA damage and stress, metalloregulatory proteins, and genes involved in the biosynthesis of peptidoglycan and teichoic acids were significantly induced in the ccpC deletion strain relative to F2365. In contrast, genes encoding internalin, 1-phosphatidylinositol phosphodiesterase, and genes associated with sugar-specific phosphotransferase system components, porphyrin, branched-chain amino acids, and pentose phosphate pathway were significantly downregulated in the ccpC deletion strain relative to F2365. This finding highlights CcpC as a key factor that regulates L. monocytogenes physiology and responses to oxidative stress by controlling the expression of important metabolic pathways.

The re-emergence and transmission of Monkeypox virus in Nigeria: the role of one health.

The Monkeypox virus, commonly abbreviated as mpox, is a viral zoonosis that is experiencing a resurgence in prevalence. It is endemic to regions of West and Central Africa that are characterized by dense forested areas. Various measures pertaining to animals, humans, and the environment have been recognized as potential factors and catalysts for the spread of the disease throughout the impacted regions of Africa. This study examines the various factors contributing to the transmission of the virus in Nigeria, with a particular focus on the animal-human and inter-human modes of transmission in rural communities and healthcare facilities. The One Health approach was emphasized as crucial in the prevention and management of this issue. Literature suggests that preventing repeated zoonotic introductions could potentially halt the transmission of the mpox virus from animal to human hosts, leading to a potential decrease in human infections.

Increased Prevalence of Lassa Fever Virus-Positive Rodents and Diversity of Infected Species Found during Human Lassa Fever Epidemics in Nigeria.

The dynamics of Lassa virus (LASV) infections in rodent reservoirs and their endemic human caseloads remain poorly understood. During the endemic period, human infections are believed to be associated with the seasonal migration of Mastomys natalensis, thought to be the primary reservoir that triggers multiple spillovers of LASV to humans. It has become imperative to improve LASV diagnosis in rodents while updating their prevalence in two regions of Lassa fever endemicity in Nigeria. Rodents (total, 942) were trapped in Ondo (531) and Ebonyi (411) states between October 2018 and April 2020 for detection of LASV using various tissues. Overall, the LASV prevalence was 53.6%. The outbreak area sampled in Ondo had three and two times higher capture success and LASV prevalence, respectively, than Ebonyi State. This correlated with the higher number of annual cases of Lassa fever (LF) in Ondo State versus Ebonyi State. All rodent genera (Mastomys, Rattus, Crocidura, Mus, and Tatera) captured in both states showed slightly variable LASV positivity, with Rattus spp. being the most predominantly infected (77.3%) rodents in Ondo State versus Mastomys spp. (41.6%) in Ebonyi State. The tissues with the highest LASV positivity were the kidneys, spleen, and testes. The finding of a relatively high LASV prevalence in all of the rodent genera captured highlights the complex interspecies transmission dynamics of LASV infections in the reservoirs and their potential association with increased environmental contact, as well as the risk of zoonotic spillover in these communities, which have the highest prevalence of Lassa fever in Nigeria. IMPORTANCE Our findings show the highest LASV positivity in small rodents ever recorded and the first direct detection of LASV in Tatera spp. Our findings also indicate the abundance of LASV-infected small rodents in houses, with probable interspecies transmission through vertical and horizontal coitus routes. Consequently, we suggest that the abundance of different reservoir species for LASV may fuel the epizootic outbreaks of LF in affected human communities. The high prevalence of LASV with the diversity of affected rodents has direct implications for our understanding of the transmission risk, mitigation, and ultimately, the prevention of LF in humans. Optimal tissues for LASV detection in rodents are also presented.

Reducing risk in basin scale CO2 sequestration: a framework for integrated monitoring design.

Injection of CO(2) into geological structures is a key technology for sequestering CO(2) emissions captured from the combustion of fossil fuels. Current projects inject volumes on the order of megatonnes per year. However, injection volumes must be increased by several orders of magnitude for material reductions in ambient concentrations. A number of questions surrounding safety and security of injection have been raised about the large scale deployment of geological CO(2) sequestration. They are site specific and require an effective monitoring strategy to mitigate risks of concern to stakeholders. This paper presents a model-based framework for monitoring design that can provide a quantitative understanding of the trade-offs between operational decisions of cost, footprint size, and uncertainty in monitoring strategies. Potential risks and challenges of monitoring large scale CO(2) injection are discussed, and research areas needed to address uncertainties are identified. Lack of clear guidance surrounding monitoring has contributed to hampering the development of policies to promote the deployment of large scale sequestration projects. Modeling provides an understanding of site specific processes and allows insights into the complexity of these systems, facilitating the calibration of an appropriate plan to manage risk. An integrated policy for risk-based monitoring design, prior to large scale deployment of sequestration will ensure safe and secure storage through an understanding of the real risks associated with large scale injection.

The pharmacokinetics and disposition of MK-0524, a Prosglandin D2 Receptor 1 antagonist, in rats, dogs and monkeys.

MK-0524 is a potent, selective and orally active Prosglandin D(2) Receptor 1 (DP(1)) antagonist currently under clinical development for the treatment of niacin-induced flushing. Experiments to study the pharmacokinetics, metabolism and excretion of MK-0524 were conducted in rats, dogs and monkeys. MK-0524 displayed linear kinetics and rapid absorption following an oral dose. Following intravenous (i.v.) administration of MK-0524 to rats and dogs (1 and 5 mg/kg), the mean Cl(p) was approximately 2 and approximately 6 ml/min/kg, the T(1/2) was approximately 7 and approximately 13 h and the Vd(ss) was approximately 1 and approximately 5 L/kg, respectively. In monkeys dosed i.v. at 3 mg/kg, the corresponding values were 8 ml/min/kg, 3 h and 1 L/kg, respectively. Following oral dosing of MK-0524 to rats (5, 25 and 100 mg/kg), dogs (5 mg/kg) and monkeys (3 mg/kg), the absorption was rapid with the mean C(max) occurring between 1 and 4 h. Absolute oral bioavailability values in rats, dogs and monkeys were 50, 70 and 8%, respectively. The major circulating metabolite was the acyl glucuronide of MK-0524 (M2), with ratios of glucuronide to the parent aglycone being highest in the monkey followed by dog and rat. In bile duct-cannulated rats and dogs, MK-0524 was eliminated primarily via acyl glucuronidation followed by biliary excretion of the acyl glucuronide, M2, the major drug-related entity in bile.

Radiofrequency ablation as a salvage procedure for ruptured hepatocellular carcinoma.

Spontaneous rupture of hepatocellular carcinoma is a distinct presentation causing acute abdomen. Different treatment approaches have been advocated including emergency hepatectomy, initial hemostasis by hepatic artery ligation or transarterial embolization and second-stage hepatectomy. Unrecognized ruptured hepatocellular carcinoma during laparotomy is often encountered in countries where the incidence of hepatocellular carcinoma is low. Radiofrequency ablation is a new localized thermal ablative technique for the treatment of unresectable hepatic tumors including hepatocellular carcinoma. We report a case where radiofrequency ablation was used as a salvage procedure for ruptured hepatocellular carcinoma during emergency laparotomy.

alpha-Ketocarboxylic acid-based inhibitors of protein tyrosine phosphatases.

A series of aryl alpha-ketocarboxylic acids was synthesized and investigated as inhibitors for the protein tyrosine phosphatase from Yersinia enterocolitica. IC(50) values for these compounds range from 79 to 2700 microM. Larger aromatic groups, and aromatic groups with high electron density, lead to more potent inhibitors. In general, the related aryl alpha-hydroxycarboxylic acids show lower activity.

Effects of ionization mode on charge-site-remote and related fragmentation reactions of long-chain quaternary ammonium ions.

Comparison of collisionally activated fragment spectra of long-chain quaternary ammonium ions, formed by liquid-assisted secondary ion mass spectrometry (LSIMS) and electrospray ionization (ESI), shows the latter are dominated by radical cations while the former yield mainly even-electron charge-site-remote (CSR) fragments, similar to the report for different precursors by Cheng et al., J. Am. Soc. Mass Spectrom. 1998, 9, 840. Here, mixed-site fragmentation products (formal loss of a radical directly bonded to the nitrogen plus a radical derived from the long chain) are of comparable importance for both ionization techniques. These observations are difficult to understand if the CSR ions are formed by a concerted rearrangement-elimination reaction, since precollision internal energies of the ESI ions are much lower than those of the ions from LSIMS. Alternatively, if one discards the concerted mechanism for high-energy CA, and assumes that the even-electron fragments are predominantly formed via homolytic bond cleavage, the colder radical cations from ESI survive to the detector while the more energized counterparts from LSIMS preferentially lose a hydrogen atom to yield the CSR ions, as proposed by Wysocki and Ross (Int. J. Mass Spectrom. Ion Processes 1991, 104, 179). The present work also attempts to reconcile discrepancies involving critical energies and known structures for neutral fragments.

5-Alkyltryptamine derivatives as highly selective and potent 5-HT1D receptor agonists.

A series of 5-alkyltryptamines (6) and the corresponding conformationally constrained analogues (8) have been synthesized. The structure activity relationships (SAR) at the 5-position of the indole skeleton and the ethylamine side chain have been studied. Functional activities were assessed using isolated rabbit saphenous vein. Potent, selective ligands were found (6e, Ki 2.5 nM, 5-HT1B/5-HT1D 125-fold) that have potential for treating acute migraine.

Charge-remote fragmentation characteristics of functionalized alkanes in high-energy collision-induced dissociation

In this study we report on high-energy, collision-induced dissociation processes leading to charge-remote fragmentations, using three alkyl cations, namely n-hexadecylpyridinium, n-hexadecyltriphenylphosphonium and n-hexadecyltriethylammonium, each with and without (2)H(2)-labelling at the C(9) position of the hexadecyl chain. The characteristic patterns corresponding to the formal elimination of alkane elements were observed, and the (2)H(2)-labelling at C(9) clearly affected only one charge-remote fragment ion of the homologous series. However, in addition to the expected fragment ion containing only one deuterium atom, a significant ion retaining two deuterium atoms was observed. MS/MS/MS experiments demonstrated clearly that the latter ion showed partial deuteration around the charge site, the level of deuteration depending on the structure of the original precursor cation. These results can be interpreted in terms of two novel, distinct mechanisms, one of which involves an excited state in an aromatic ring. Mixed-site fragmentation (MSF) ions were also observed from the phosphonium and ammonium ion precursors. We believe that the observation of the MSF process occurring at an sp(2)-hybridized center in the phosphonium series has not been reported previously. It thus becomes apparent that high-energy collisions leading to charge-remote reactions in fact lead to a broad range of pathways. Copyright 2000 John Wiley & Sons, Ltd.

Biotinylation of substituted cysteines in the nicotinic acetylcholine receptor reveals distinct binding modes for alpha-bungarotoxin and erabutoxin a.

Although previous results indicate that alpha-subunit residues Trp(187), Val(188), Phe(189), Tyr(190), and Pro(194) of the mouse nicotinic acetylcholine receptor are solvent-accessible and are in a position to contribute to the alpha-bungarotoxin (alpha-Bgtx) binding site (Spura, A., Russin, T. S., Freedman, N. D., Grant, M., McLaughlin, J. T., and Hawrot, E. (1999) Biochemistry 38, 4912-4921), little is known about the accessibility of other residues within this region. By determining second-order rate constants for the reaction of cysteine mutants at alpha184-alpha197 with the thiol-specific biotin derivative (+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine , we now show that only very subtle differences in reactivity (approximately 10-fold) are detectable, arguing that the entire region is solvent-exposed. Importantly, biotinylation in the presence of saturating concentrations of the long neurotoxin alpha-Bgtx is significantly retarded for positions alphaW187C, alphaF189C, and reduced wild-type receptors (alphaCys(192) and alphaCys(193)), further emphasizing their major contribution to the alpha-Bgtx binding site. Interestingly, although biotinylation of position alphaV188C is not affected by the presence of alpha-Bgtx, erabutoxin a, which is a member of the short neurotoxin family, inhibits biotinylation at position alphaV188C, but not at alphaW187C or alphaF189C. Taken together, these results indicate that short and long neurotoxins establish interactions with distinct amino acids on the nicotinic acetylcholine receptor.

Combinatorial library of serine and cysteine protease inhibitors that interact with both the S and S' binding sites.

A combinatorial library of 400 inhibitors has been synthesized and screened against several serine and cysteine proteases including plasmin, cathepsin B, and papain. The inhibitors are based upon a cyclohexanone nucleus and are designed to probe binding interactions in the S2 and S2' binding sites. This methodology has led to the discovery of inhibitor 15A, which incorporates Trp at both the P2 and P2' positions and has an inhibition constant against plasmin of 5 microM. Data from screening of the library shows that plasmin has a strong specificity for Trp at the S2 subsite and prefers to bind hydrophobic and aromatic amino acids such as Ile, Phe, Trp, and Tyr at the S2' subsite. In contrast, the S2' subsites of cathepsin B and papain do not show a strong preference for any particular amino acid.

4-Heterocyclohexanone-based inhibitors of the serine protease plasmin.

Three inhibitors that are based upon a 4-heterocyclohexanone nucleus were synthesized and evaluated for activity against the serine protease plasmin. Inhibitors of plasmin have potential as cancer chemotherapeutic agents that act by blocking both angiogenesis and metastasis. Inhibitor 1 has moderate activity against plasmin but shows good selectivity for this enzyme compared to other serine proteases including trypsin, thrombin, and kallikrein. Inhibitor 2 shows both good activity and selectivity for plasmin. Inhibitor 3, which does not incorporate an aminohexyl group that can interact with the S1 subsite, has poor activity. These results, along with previous work, demonstrate that the 4-heterocyclohexanone nucleus can effectively serve as the basis for designing inhibitors of both serine and cysteine proteases.

[A case report of urinary tract infection and meningitis caused by methicillin-resistant Staphylococcus aureus (MRSA) after transurethral resection of the prostate].

We present a case of meningitis that developed following a urinary tract infection caused by methicillin-resistant Staphylococcus aureus (MRSA) after transurethral resection of the prostate. The patient, a 69-year-old man with diabetes mellitis, underwent transurethral resection of the prostate following a diagnosis of benign prostatic hypertrophy. On the 4 th day after surgery, high fever occurred immediately after the removal of the indwelling urethral catheter. Cultures of urine and blood revealed MRSA. On the 6 th day after surgery, severe lumbago was evident and MRSA was isolated from cerebrospinal fluid. Separate administration of arbekacin or vancomycin, to which the isolated MRSA was sensitive, was not effective. Combined therapy with fosfomycin, vancomycin and human immunoglobulin effectively relieved the inflammation. Although it is generally reported that the pathogenicity of MRSA is low in the urinary tract, this case suggests that a urinary tract infection caused by MRSA can advance to sepsis and meningitis.

Inhibition of phosphatase activity by positively-charged cyclodextrins.

[formula: see text] Aminocyclodextrins are known to bind phosphate esters such as phosphotyrosine and p-nitrophenyl phosphate. This paper describes the inhibition of phosphate ester hydrolysis, as catalyzed by lambda-protein phosphatase and acid phosphatase, that is caused by such binding interactions. ROESY studies provide structural information about the cyclodextrin-aryl phosphate complexes. In addition, these experiments are used to generate approximations of the rates of dissociation of the noncovalent complexes.

Abnormal cell morphology and cytotoxic effect are induced by 6-0-palmitol-ascorbate-2-0-phosphate, but not by ascorbic acid or hyperthermia alone.

A new derivative of ascorbic acid (AsA), 6-0-palmitoyl-ascorbate 2-0-phosphate (Asc2P6Pal) was developed to enhance the antitumor activity of AsA. When Ehrlich ascites tumor cells were treated with 50 microM Asc2P6Pal at 37 degrees C or 42 degrees C for 1 h and then cultured for 20 h, most of cells exhibited some morphological abnormalities, including exudation of intracellular granules together with other contents on the cell membrane surface, resulting in cell fragmentation. The abnormal features were further enhanced by a long term culture for 96 h and heat treatment at 42 degrees C. In contrast, no abnormality was detected for untreated cells or cells treated with AsA (free acid) at 37 degrees C or 42 degrees C. Cells cultured for 96 h after the treatment suffered from inhibition of DNA synthesis and proliferation. This inhibition was markedly enhanced by combination with the hyperthermic treatment at 42 degrees C, but not for a short-term culture of 20 h after the treatment. No effects were seen upon similar treatment with AsA. The abnormal cells produced during culture for 20 h after the treatment were evaluated to be viable, because they failed to be stained with trypan blue and retained most of the DNA synthesizing ability of Asc2P6Pal-untreated cells. However, they appeared and died after a continuous 76 h of culture (96 h).

The leuO gene product has a latent ability to relieve bgl silencing in Escherichia coli.

The Escherichia coli bgl operon is of interest, since its expression is silent (phenotypically Bgl-), at least under standard laboratory conditions. Here we attempted to identify a trans-acting factor(s) that is presumably relevant to the regulation of bgl by a random insertion mutagenesis with mini-Tn10. These collected mutations, conferring the phenotype of Bgl+, were localized in three loci on the genetic map, two of which appeared to be hns and bglJ, which were previously implicated as the factors affecting the Bgl phenotype. The other locus at 1 to 2 min on the genetic map appeared to be a new one. In this case, the insertion mutation was found to be just in front of the leuO gene encoding a putative LysR-like DNA-binding protein. Genetic analyses revealed that overproduction of LeuO in the wild-type cells causes the phenotype of Bgl+. A leuO deletion mutant was also characterized in terms of expression of bgl. From these results, the possible function of LeuO in bgl expression will be discussed from an evolutionary and/or ecological point of view.