Systematic Analysis of the Effect of Genomic Knock-Out of Non-Essential Promiscuous HAD-Like Phosphatases YcsE, YitU and YwtE on Flavin and Adenylate Content in Bacillus Subtilis.

Studying the metabolic role of non-essential promiscuous enzymes is a challenging task, as genetic manipulations usually do not reveal at which point(s) of the metabolic network the enzymatic activity of such protein is beneficial for the organism. Each of the HAD-like phosphatases YcsE, YitU and YwtE of Bacillus subtilis catalyzes the dephosphorylation of 5-amino-6-ribitylamino-uracil 5'-phosphate, which is essential in the biosynthesis of riboflavin. Using CRISPR technology, we have found that the deletion of these genes, individually or in all possible combinations failed to cause riboflavin auxotrophy and did not result in significant growth changes. Analysis of flavin and adenylate content in B. subtilis knockout mutants showed that (i) there must be one or several still unidentified phosphatases that can replace the deleted proteins; (ii) such replacements, however, cannot fully restore the intracellular content of any of three flavins studied (riboflavin, FMN, FAD); (iii) whereas bacterial fitness was not significantly compromised by mutations, the intracellular balance of flavins and adenylates did show some significant changes.

Diversification in the inositol tris/tetrakisphosphate kinase (ITPK) family: crystal structure and enzymology of the outlier AtITPK4.

Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Šresolution that, along with a description of the enantiospecificity of the enzyme, affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a KM for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologues in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites.

The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target.

Betaine regulates adipogenic and osteogenic differentiation of hAD-MSCs.

BACKGROUND: With an ageing population, the incidence of bone loss and obesity are increasing. Numerous studies emphasized the multidirectional differentiation ability of mesenchymal stem cells (MSCs), and reported betaine modulated the osteogenic differentiation and adipogenic differentiation of MSCs in vitro. We wondered how betaine affected the differentiation of hAD-MSCs and hUC-MSCs. METHODS AND RESULTS: ALP staining and alizarin red S (ARS) staining were proved 10 mM betaine significantly increased the number of ALP-positive cells and plaque calcified extracellular matrices, accompanying by the up-regulation of OPN, Runx-2 and OCN. Oil red O staining demonstrated the number and size of lipid droplets were reduced, the expression of adipogenic master genes such as PPARγ, CEBPα and FASN were down-regulated simultaneously. For further investigating the mechanism of betaine on hAD-MSCs, RNA-seq was performed in none-differentiation medium. The Gene Ontology (GO) analysis showed fat cell differentiation and bone mineralization function terms were enriched, and KEGG showed PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction and ECM-receptor interaction pathways were enriched in betaine treated hAD-MSCs, demonstrated betaine had a positive inducing effect on osteogenic of hAD-MSCs in the non-differentiation medium in vitro, which is opposite to the effect on adipogenic differentiation. CONCLUSIONS: Our study demonstrated that betaine promoted osteogenic and compromised adipogenic differentiation of hUC-MSCs and hAD-MSCs upon low concentration administration. PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction and ECM-receptor interaction were significantly enriched under betaine-treated. We showed hAD-MSCs were more sensitive to betaine stimulation and have a better differentiation ability than hUC-MSCs. Our results contributed to the exploration of betaine as an aiding agent for MSCs therapy.

[Nursing and end-of-life care at home in a health crisis]. / Soins infirmiers et fin de vie à domicile en période de crise sanitaire.

The Covid-19 pandemic led healthcare professionals to reconsider their work organization and some of their practices, in order to respond to the health emergency and the importance of care needs. While hospital teams managed the most serious and complex health situations, home care workers also made significant efforts to redeploy their rounds, to take care of and accompany patients at the end of their lives and their loved ones while managing hygiene constraints. A nurse looks back on one of these cases and the questions it raised.

Epidemiologic Features and Control Measures during Monkeypox Outbreak, Spain, June 2022.

During June 2022, Spain was one of the countries most affected worldwide by a multicountry monkeypox outbreak with chains of transmission without identified links to disease-endemic countries. We provide epidemiologic features of cases reported in Spain and the coordinated measures taken to respond to this outbreak.

As the virus evolves, so too must we: a drug developer's perspective : We need a new paradigm in searching for next-generation countermeasures.

The SARS-CoV-2 virus has been raging globally for over 2 years with no end in sight. It has become clear that this virus possesses enormous genetic plasticity, and it will not be eradicated. Under increasing selective pressure from population immunity, the evolution of SARS-CoV-2 has driven it towards greater infectivity, and evasion of humoral and cellular immunity. Omicron and its expanding army of subvariants and recombinants have impaired vaccine protection and made most antibody drugs obsolete. Antiviral drugs, though presently effective, may select for more resistant strains over time. It may be inevitable, then, that future SARS-CoV-2 variants will be immune to our current virus-directed countermeasures. Thus, to gain control over the virus, we need to adopt a new paradigm in searching for next-generation countermeasures and develop host-directed therapeutics (HDTx) and host-directed antivirals (HDA). Different from the virus-directed countermeasures, HDTx and HDA may offer variant agnostic treatment to reduce the risk and severity of infections. In addition, they may exert more uniform effects against the genetically diverse SARS-CoV-2 quasispecies, thereby diminishing the risk of selecting resistant variants. Some promising HDTx and HDA approaches are summarized here.

Headache provocation by nitric oxide in men who have never experienced a headache.

INTRODUCTION: In the general population 4% have never experienced a headache. Freedom from headache could be due to distinctive protective mechanisms or a lack of environmental risk factors for headache. Isosorbide-5-mononitrate is an organic nitrate which in the body is metabolised to nitric oxide. The nitric oxide pathway plays a crucial role in the primary headaches. We hypothesized that people who are free from headache are protected by distinctive mechanisms in the nitric oxide pathway. METHODS: We performed an observer blinded case-control study using nitric oxide to provoke a headache. 32 headache free male participants and 26 randomly selected male controls received 60 mg Isosorbide-5-mononitrate orally on the study day. Participants fill out a headache diary with headache intensity and characteristics until 12 hours after administration of Isosorbide-5-mononitrate. Primary endpoint were areas under the curve of headache intensity score. RESULTS: All 58 participants completed the study. There was no significant difference in headache incidence, headache intensity score or migraine-like attack between headache free participants and controls. CONCLUSION: We show that men who have never experienced a headache develop a headache when provoked with Isosorbide-5-mononitrate. This indicates that freedom from headache in men is not related to the nitric oxide pathway which is involved in the primary headache disorders.

The Repeatable Battery for the Assessment of Neuropsychological Status as a screening strategy for HIV-Associated Neurocognitive Disorders.

HIV-infected people are at risk for neurocognitive impairment (HIV-Associated Neurocognitive Disorders - HAND). To evaluate whether the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), a widely used neurocognitive screening tool, could be a valid instrument for HAND identification, we evaluated 166 HIV-infected subjects. Our results showed that 96 (57.8%) HIV-infected scored RBANS Total Index Score <85 (at least one SD below the normal), 12 (7.2%) of them scored RBANS Total Index Score <70 (at least 2 SD below the normal, indicating a possible HIV-Associated Dementia). The more compromised areas were Immediate and Delayed Memory, and Attention. In the group with RBANS Total Index Score <85, there were significantly lower scores of Mini Mental State Examination (P = 0.0008), Clock Drawing Test (P = 0.0015) and higher score of Geriatric Depression Scale (P = 0.02) compared to the RBANS Total Index Score ≥85 group. Using a stepwise logistic regression, considering RBANS Total Index Score as dependent variable, we found a positive interaction with tenofovir/emtricitabine assumption (P = 0.027), Clock Drawing Test (P = 0.0125) and educational level (P = 0.0054). Being the viro-immunological markers not capable of predicting cognitive decline in HIV-infected individuals, our data suggest that RBANS may be a valid tool for the early identification of HIV-related cognitive impairment.

Diverse Functions of Tim50, a Component of the Mitochondrial Inner Membrane Protein Translocase.

Mitochondria are essential in eukaryotes. Besides producing 80% of total cellular ATP, mitochondria are involved in various cellular functions such as apoptosis, inflammation, innate immunity, stress tolerance, and Ca2+ homeostasis. Mitochondria are also the site for many critical metabolic pathways and are integrated into the signaling network to maintain cellular homeostasis under stress. Mitochondria require hundreds of proteins to perform all these functions. Since the mitochondrial genome only encodes a handful of proteins, most mitochondrial proteins are imported from the cytosol via receptor/translocase complexes on the mitochondrial outer and inner membranes known as TOMs and TIMs. Many of the subunits of these protein complexes are essential for cell survival in model yeast and other unicellular eukaryotes. Defects in the mitochondrial import machineries are also associated with various metabolic, developmental, and neurodegenerative disorders in multicellular organisms. In addition to their canonical functions, these protein translocases also help maintain mitochondrial structure and dynamics, lipid metabolism, and stress response. This review focuses on the role of Tim50, the receptor component of one of the TIM complexes, in different cellular functions, with an emphasis on the Tim50 homologue in parasitic protozoan Trypanosoma brucei.

The Eyes Absent Proteins: Unusual HAD Family Tyrosine Phosphatases.

Here, we review the haloacid dehalogenase (HAD) class of protein phosphatases, with a particular emphasis on an unusual group of enzymes, the eyes absent (EYA) family. EYA proteins have the unique distinction of being structurally and mechanistically classified as HAD enzymes, yet, unlike other HAD phosphatases, they are protein tyrosine phosphatases (PTPs). Further, the EYA proteins are unique among the 107 classical PTPs in the human genome because they do not use a Cysteine residue as a nucleophile in the dephosphorylation reaction. We will provide an overview of HAD phosphatase structure-function, describe unique features of the EYA family and their tyrosine phosphatase activity, provide a brief summary of the known substrates and cellular functions of the EYA proteins, and speculate about the evolutionary origins of the EYA family of proteins.

NLIP and HAD-like Domains of Pah1 and Lipin 1 Phosphatidate Phosphatases Are Essential for Their Catalytic Activities.

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, controlling phospholipids and triacylglycerol metabolisms. Pah1 and human Lipin 1 are intrinsically disordered proteins with 56% and 43% unfolded regions, respectively. Truncation analysis of the conserved and non-conserved regions showed that N- and C-conserved regions are essential for the catalytic activity of Pah1. PAP activities can be detected in the conserved N-terminal Lipin (NLIP) domain and C-terminal Lipin (CLIP)/haloacid dehalogenase (HAD)-like domain of Pah1 and Lipin 1, suggesting that the evolutionarily conserved domains are essential for the catalytic activity. The removal of disordered hydrophilic regions drastically reduced the protein solubility of Pah1. Thioredoxin is an efficient fusion protein for production of soluble NLIP-HAD recombinant proteins in Escherichia coli.

Pain sensitivity in men who have never experienced a headache: an observer blinded case control study.

BACKGROUND: Headache affects 90-99% of the population. Based on the question "Do you think that you never ever in your whole life have had a headache?" 4% of the population say that they have never experienced a headache. The rarity of never having had a headache suggests that distinct biological and environmental factors may be at play. We hypothesized that people who have never experienced a headache had a lower general pain sensitivity than controls. METHODS: We included 99 male participants, 47 headache free participants and 52 controls, in an observer blinded nested case-control study. We investigated cold pain threshold and heat pain threshold using a standardized quantitative sensory testing protocol, pericranial tenderness with total tenderness score and pain tolerance with the cold pressor test. Differences between the two groups were assessed with the unpaired Student's t-test or Mann-Whitney U test as appropriate. RESULTS: There was no difference in age, weight or mean arterial pressure between headache free participants and controls. We found no difference in pain detection threshold, pericranial tenderness or pain tolerance between headache free participants and controls. CONCLUSION: Our study clearly shows that freedom from headache is not caused by a lower general pain sensitivity. The results support the hypothesis that headache is caused by specific mechanisms, which are present in the primary headache disorders, rather than by a decreased general sensitivity to painful stimuli. TRIAL REGISTRATION: Registered at ClinicalTrials.gov ( NCT04217616 ), 3rd January 2020, retrospectively registered.

Frequency of emotional disturbances among hostelites and day scholars medical students.

OBJECTIVE: This study was aimed to determine and compare the frequency of the emotional disturbances, anxiety and depression among the medical students on Hospital Anxiety Depression scale (HAD) score among the hostelites and day scholars. METHODS: This cross sectional study was conducted at a private medical college in Faisalabad, Pakistan, from March 2019 to April 2019, comprising of 105 male and female students. Validated Hospital Anxiety depression scale (HAD-A and HAD-D) was used to collect data from 1st year and 2nd year MBBS students in order to evaluate anxiety and depression among them. Data was analyzed on SPSS 21. Pearson's Chi-square was applied to compare the percentages of anxious and depressed subjects among the studied group. Independent sample t-test was applied for comparison of mean HAD scores between hostelites and day scholars. P value ≤ 0.05 was taken as significant. RESULTS: There were 105 students in the study with a mean age of 19.4±0.68 years. Overall, 82.7% students had anxiety, and 52.1% suffered from depression. Average HAD -A and HAD- D scores were 11.2±3.41 and 7.2±3.37 respectively. Greater number of hostelites was suffering from emotional disturbances as compared to day scholars. HAD -A scores was significantly higher in hostelites than day scholars with P value 0.003*(11.85±3.42 Vs 10.92 ±2.56). HAD- D scores were also higher in hostilities but difference was insignificant. (7.57±3.42 Vs 6.85 ±1.58). CONCLUSIONS: Emotional disturbance including anxiety and depression are found to be highly prevalent among medical students of a private medical college in Faisalabad. Hostelites are more prone to anxiety and depression than day scholars.

Aziridine Formation by a FeII /α-Ketoglutarate Dependent Oxygenase and 2-Aminoisobutyrate Biosynthesis in Fungi.

Aziridine is a characteristically reactive molecule with increased bioactivity due to its strained ring structure. Here, we investigated the biosynthesis of 2-aminoisobutyric acid (AIB) in Penicillium, and successfully reconstituted the three-step biosynthesis from L-Val to AIB in vitro. This previously unknown aziridine formation pathway proceeded with the non-heme iron and α-ketoglutarate-dependent (FeII /αKG) oxygenase TqaL, followed by aziridine ring opening by the haloalkanoic acid dehalogenase (HAD)-type hydrolase TqaF, and subsequent oxidative decarboxylation by the NovR/CloR-like non-heme iron oxygenase TqaM. Furthermore, the X-ray crystal structure of the C-N bond forming FeII /αKG oxygenase TqaL was solved at 2.0 Šresolution. This work presents the first molecular basis for aziridine biogenesis, thereby expanding the catalytic repertoire of the FeII /αKG oxygenases. We also report the unique aziridine ring opening by a HAD-type hydrolase and the remarkable oxidative decarboxylation by a non-heme iron oxygenase to produce AIB.

On-Surface Hydrogen/Deuterium Isotope Exchange in Polycyclic Aromatic Hydrocarbons.

Hydrogen plays important roles in the on-surface synthesis of carbon-based materials in ultra-high vacuum. The complex interplay between hydrogen and surface-adsorbed polycyclic aromatic hydrocarbons (PAHs) is tracked by in situ time-of-flight secondary ion mass spectrometry (ToF-SIMS) combined with isotope labeling. In situ deuterium labeling of prototypical PAHs, coronene (CR) and 7-armchair graphene nanoribbons (GNRs), on Au(111) is achieved by annealing either in D2 gas or in the vapor of perdeuterio-acenaphthene. By following the mass spectra of in situ deuterated CR mixed with hydrogen-CR, it is demonstrated that PAHs adsorbed at hot Au(111) surfaces continuously exchange hydrogen atoms. Also, D2 present during the Ullmann coupling step leads to incorporation of deuterium and to shorter GNRs.

[Prevalence of anxiety and depression in patients with cardiovascular disease during the COVID-19 pandemic]. / Prevalencia de ansiedad y depresión en pacientes con enfermedad cardiovascular durante la pandemia COVID-19.

INTRODUCTION: The COVID-19 pandemic and the sanitary measures implemented had an impact on the mental health of the most vulnerable populations. AIMS: To know the prevalence of anxiety and depression in patients with cardiovascular disease and/or vascular risk factors after the end of quarantine and compare it with the prevalence during quarantine and before the COVID-19 pandemic. METHODS: An online questionnaire was sent 150 days after the quarantine ended. The Hospital Anxiety Depression (HAD) scale was used and clinical and demographic data were recorded. The results were compared with samples obtained during quarantine and the EPICA study conducted in 2016 before the COVID-19 pandemic. RESULTS: The samples included 1076, 3542 and 1035 patients respectively. The prevalence of anxiety fluctuated but did not change (20.8%, 13.5% and 21% respectively). Depression increased its prevalence (9.8%, 16.7% and 19.7% respectively). Depression was associated with coronary heart disease and risk behaviors (cigarette smoking and sedentary lifestyle). CONCLUSION: Depression doubled its prevalence during the COVID-19 pandemic in patients with cardiovascular disease and/or vascular risk factors. The effects of the pandemic appear to extend beyond the duration of the quarantine.

Biochemical characterization of phosphoserine phosphatase SerB2 from Mycobacterium marinum.

SerB2 is an essential phosphoserine phosphatase (PSP) that has been shown to be involved in Mycobacterium tuberculosis (Mtb) immune evasion mechanisms, and a drug target for the development of new antitubercular agents. A highly similar (91.0%) orthologous enzyme exists in the surrogate organism Mycobacterium marinum (Mma) and could have acquired similar properties. By homology modeling, we show that the two PSPs are expected to exhibit almost identical architectures. MmaSerB2 folds into a homodimer formed by two intertwined subunits including two ACT regulatory domains followed by a catalytic core typical of HAD (haloacid dehalogenase) phosphatases. Their in vitro catalytic properties are closely related as MmaSerB2 also depends on Mg2+ for the dephosphorylation of its substrate, O-phospho-l-serine (PS), and is most active at neutral pH and temperatures around 40 °C. Moreover, an enzyme kinetics study revealed that the enzyme is inhibited by PS as well, but at lower concentrations than MtbSerB2. Substrate inhibition could occur through the binding of PS in the second active site and/or at the ACT domains interface. Finally, previously described beta-carboline MtbSerB2 inhibitors also decrease the phosphatase activity of MmaSerB2. Altogether, these results provide useful information when M.marinum is used as a model to study immune evasion in tuberculosis.

Engineering of Thermostable ß-Hydroxyacid Dehydrogenase for the Asymmetric Reduction of Imines.

The ß-hydroxyacid dehydrogenase from Thermocrinus albus (Ta-ßHAD), which catalyzes the NADP+ -dependent oxidation of ß-hydroxyacids, was engineered to accept imines as substrates. The catalytic activity of the proton-donor variant K189D was further increased by the introduction of two nonpolar flanking residues (N192 L, N193 L). Engineering the putative alternative proton donor (D258S) and the gate-keeping residue (F250 A) led to a switched substrate specificity as compared to the single and triple variants. The two most active Ta-ßHAD variants were applied to biocatalytic asymmetric reductions of imines at elevated temperatures and enabled enhanced product formation at a reaction temperature of 50 °C.

HIV-associated neurocognitive disorders at Moi teaching and referral hospital, Eldoret, Kenya.

BACKGROUND: Human Immunodeficiency Virus (HIV) infection causes a myriad of neurological complications including cognitive deficits referred to as HIV-Associated Neurocognitive Disorders (HAND). With the introduction of combination antiretroviral therapy, there has been an epidemiological shift in cognitive disorders with a decline in the more severe HIV-Associated Dementia (HAD) to an increase in the less severe HAND: Asymptomatic Neurocognitive Impairment (ANI) and HIV-associated Mild Neurocognitive Disorder (MND). Central Nervous System (CNS) involvement in HIV interferes with cognitively demanding activities of daily living and hence a worse quality of life. Early diagnosis is delayed until symptoms are overt. METHODS: We conducted a cross sectional analytical study of HIV infected persons on antiretroviral therapy attending HIV clinic. A systematic random sampling was done to select 360 patients. An interviewer administered structured questionnaire was used to collect socio-demographic data while the CD4 count and viral load were retrieved from the Academic Model Providing Access to Healthcare (AMPATH) database. Pearson's Chi Square test was used to compare proportions while independent sample t- test was used to compare continuous variables between the patients diagnosed with HAND and those without HAND. Logistic regression model was used to assess the factors associated with HAND. RESULTS: The mean age of the study participants was 40.2 years. The overall prevalence of HAND was (81.1%) N = 292. Mild HAND (ANI and MND) was present (78.6%) N = 283, Severe HAND (HAD) (2.5%) N = 9. The factors associated with HAND were older age OR: 1.06 (95% CI: 1.03, 1.10), male gender OR: 0.48 (95% CI: 0.24, 0.97), Advanced WHO clinical staging OR: 2.45 (95% CI: 1.20, 5.01) and a higher level of education; secondary/tertiary OR: 0.16 (95% CI: 0.07, 0.38); 0.11 (95% CI: 0.04, 0.35). CONCLUSION: The prevalence of HAND in this study population was found to be high (81.1%). Older age and advanced WHO clinical staging were associated with an increased risk of hand while higher level of education and male gender were protective.