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Håkan Bergh,1,2 Margit Albrektson,3 Clovis Kastberg,4 Amir Baigi,1,2 Lennart Hedström3 1School of Public Health and Community Medicine, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden; 2Department of Research & Development Unit, Hospital Varberg, Region Halland, Sweden; 3Public Dental Services, Västra Vall, Varberg, Sweden; 4Tre Tandläkare, Varberg, SwedenCorrespondence: Håkan Bergh, School of Public Health and Community Medicine, Institute of Medicine, P.O. Box 463, Gothenburg, 405 30, Sweden, Email hakan.e.bergh@gmail.com Lennart Hedström, Public Dental Service, Västra Vallgatan 14, Varberg, S-43244, Region Halland, Sweden, Email h.lennart.hedstrom@gmail.comObjective: To study the association between sublingual varices (SV) and cardiovascular (CVD) risk factors.Methods: A total of 989 consecutive dental patients aged 55– 84 years participated in the study, which applied a survey about risk factors, diseases and medications. Digital photos of the lateral borders of the tongue, height, weight, waist, hip and blood pressure were registered, and blood samples were analyzed. Those with SV were compared with those without SV (nSV).Results: Those with SV had more hypertension 41.8% vs 27.0% (p< 0.0001), a higher systolic blood pressure (BP) 139.5 (SD 18.6) mmHg vs 134.3 (SD 18.8) mmHg (95% CI − 7.73 &boxh; − 2.72), more diabetes type 2 (DM-2) 7.4% vs 3.8% (p=0.014), a higher fasting plasma glucose 5.9 (SD 1.5) mmol/L vs 5.7 (SD 1.0) mmol/L (95% CI − 0.42 &boxh; − 0.05), more dyslipidemia 24.1% vs 17.7% (p=0.018), lower HDL 1.6 vs 1.7 (p=0.003), a greater waist circumference 97.0 cm vs 93.9 cm (95% CI − 4.66 &boxh; − 1.46), a greater waist/hip ratio 0.92 cm/cm vs 0.90 cm/cm (95% CI − 0.03 &boxh; − 0.01), and a higher BMI 26.6 kg/m2 vs 26.0 kg/m2 (95% CI − 1.11 &boxh; − 0.03). The following associations with SV were found in multivariate analysis: hypertension OR=1.6 (95% CI 1.19 &boxh;2.13), a high systolic BP OR =1.5 (95% CI 1.11 &boxh;2.13), a high fP-glucose OR= 1.8 (95% CI 1.03 &boxh;3.21), a low HDL OR= 1.8 (95% CI 1.15 &boxh;2.92), a greater waist circumference OR= 1.68 (95% CI 1.10 &boxh;2.58), a greater waist/hip ratio OR=2.21 (95% CI 1.36 &boxh;3.58), and a higher BMI OR=1.05 (95% CI 1.02 &boxh;1.09).Conclusion: This study shows an association between SV and a high BP, a high fP-glucose, hypertension, diabetes mellitus type 2, dyslipidemia, abdominal obesity, older age and smoking.Keywords: sublingual varices, cardiovascular riskfactors

Looks at the role surface loops play in converting trypsin (Tr) to chymotrypsin (Ch). Similar tertiary structures; Cleavage residues preferred; Inability to transfer hydrolysis specificity from Tr to Ch; Description of loops; Catalytic rate and impaired substrate binding; Extended substrate binding and substrate discrimination.

Summary This is an account of research on spirituality being conducted by a group of professors and students at the Graduate School of Education and Psychology of Pepperdine University. The article presents a humanistic definition and description of spirituality based on the writings of Abraham Maslow, John Dewey, William James, Carl Jung, and others. A preliminary report is also made on the development of the Spiritual Orientation Inventory, a measure of spirituality based on the humanistic model and designed to assess the spirituality of those not affiliated with traditional religion. [ABSTRACT FROM AUTHOR]

BMS906024, a γ-secretase inhibitor that blocks Notch signaling, was previously shown to inhibit Cryptosporidium parvum growth in vitro. A structure-activity relationship (SAR) analysis of BMS906024 reported herein demonstrates the importance of the stereochemistry of the C-3 benzodiazepine and the succinyl β-substituent. However, concomitant removal of the succinyl α-substituent and switching the primary amide with secondary amides was tolerated. For example, 32 (SH287) inhibited C. parvum growth in HCT-8 host cells with an EC 50  = 6.4 nM and an EC 90  = 16 nM; however, blocking C. parvum growth with BMS906024 derivatives was correlative with inhibition of Notch signaling, highlighting that additional SAR analysis will be needed to separate these two activities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The emergence of drug resistant Mycobacterium tuberculosis, the causative agent of tuberculosis, demands the development of new drugs and new drug targets. We have recently reported that the d-phenylalanine benzoxazole Q112 has potent antibacterial activity against this pathogen with a distinct mechanism of action from other antimycobacterial agents. Q112 and previously reported derivatives were unstable in plasma and no free compound could be observed. Here we expand the structure-activity relationship for antimycobacterial activity and find nonhydrolyzable derivatives with decreased plasma binding. We also show that there is no correlation between antibacterial activity and inhibition of PanG, a putative target for these compounds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The dynamic interactions of enzymes and substrates underpins catalysis, yet few techniques can interrogate the dynamics of protein-bound ligands. Here we describe the use of field cycling NMR relaxometry to measure the dynamics of enzyme-bound substrates and cofactors in catalytically competent complexes of GMP reductase. These studies reveal new binding modes unanticipated by x-ray crystal structures and reaction-specific dynamic networks. Importantly, this work demonstrates that distal interactions not usually considered part of the reaction coordinate can play an active role in catalysis. The commercialization of shuttling apparatus will make field cycling relaxometry more accessible and expand its use to additional nuclei, promising more intriguing findings to come., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Roberts and Hedstrom.)

New drugs and new targets are urgently needed to treat tuberculosis. We discovered that d-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis ( Mtb ) in multiple media and in macrophage infections. A metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/coenzyme A biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs, since the enzyme annotated as PanE KPR is not essential in Mtb . The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum , but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an orthologue of PanG KPR and demonstrated that a purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112 -treated bacteria, indicating that Q112 has an additional target(s). Q112 -resistant strains contain loss-of-function mutations in the twin arginine translocase TatABC, further underscoring Q112 's unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.

Many bacterial pathogens, including Staphylococcus aureus , require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH ( Sa IMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. Five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro . Resistance resulted from substitutions in the cofactor/inhibitor binding site of Sa IMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive Sa IMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of Sa IMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

Inosine-5'-monophosphate dehydrogenase (IMPDH) is a potential target for microorganisms. However, identifying inhibitor design determinants for IMPDH orthologs continues to evolve. Herein, a series of mycophenolic anilide inhibitors of Cryptosporidium parvum and human IMPDHs are reported. Furthermore, molecular docking of 12 (e.g. SH-19; CpIMPDH K i,app  = 0.042 ± 0.015 µM, HsIMPDH2 K i,app  = 0.13 ± 0.05 µM) supports different binding modes with the two enzymes. For CpIMPDH the inhibitor extends into a pocket in an adjacent subunit. In contrast, docking suggests the inhibitor interacts with Ser276 in the NAD binding site in HsIMPDH2, as well as an adjacent pocket within the same subunit. These results provide further guidance for generating IMPDH inhibitors for enzymes found in an array of pathogenic microorganisms, including Mycobacterium tuberculosis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The remarkable power and specificity of enzyme catalysis rely on the dynamic alignment of the enzyme, substrates, and cofactors, yet the role of dynamics has usually been approached from the perspective of the protein. We have been using an underappreciated NMR technique, subtesla high-resolution field cycling 31 P NMR relaxometry, to investigate the dynamics of the enzyme-bound substrates and cofactor on guanosine-5'-monophosphate reductase (GMPR). GMPR forms two dead end, yet catalytically competent, complexes that mimic distinct steps in the catalytic cycle: E·IMP·NADP + undergoes a partial hydride transfer reaction, while E·GMP·NADP + undergoes a partial deamination reaction. A different cofactor conformation is required for each partial reaction. Here we report the effects of mutations designed to perturb cofactor conformation and ammonia binding with the goal of identifying the structural features that contribute to the distinct dynamic signatures of the hydride transfer and deamination complexes. These experiments suggest that Asp129 is a central cog in a dynamic network required for both hydride transfer and deamination. In contrast, Lys77 modulates the conformation and mobility of substrates and cofactors in a reaction-specific manner. Thr105 and Tyr318 are part of a deamination-specific dynamic network that includes the 2'-OH of GMP. These residues have comparatively little effect on the dynamic properties of the hydride transfer complex. These results further illustrate the potential of high-resolution field cycling NMR relaxometry for the investigation of ligand dynamics. In addition, exchange experiments indicate that NH 3 /NH 4 + has a high affinity for the deamination complex but a low affinity for the hydride transfer complex, suggesting that the movement of ammonia may gate the cofactor conformational change. Collectively, these experiments reinforce the view that the enzyme, substrates, and cofactor are linked in intricate, reaction-specific, dynamic networks and demonstrate that distal portions of the substrates and cofactors are critical features in these networks.

Mycophenolic acid (MPA) is a potent natural product inhibitor of fungal and other eukaryotic inosine 5'-monophosphate dehydrogenases (IMPDHs) originally isolated from spoiled corn silage. MPA is produced by the filamentous fungi Penicillium brevicompactum, which contains two IMPDHs, PbIMPDHA and PbIMPDHB, both of which are MPA-resistant. The MPA binding sites of these enzymes are identical to MPA-sensitive IMPDHs, so the structural determinants of resistance are unknown. Here we show that a single residue, Ser267, accounts for the MPA resistance of PbIMPDHA. Substitution of Ser267 with Ala, the residue most commonly found in this position in eukaryotic IMPDHs, makes PbIMPDHA sensitive to MPA. Conversely, Aspergillus nidulans IMPDH becomes MPA-resistant when the analogous Ala residue is substituted with Ser. These substitutions have little effect on the catalytic cycles of either enzyme, suggesting the fitness costs are negligible despite the strong conservation of Ala at this position. Intriguingly, while only 1% of fungal IMPDHs contain Ser or Thr at position 267, these residues are found in the IMPDHs from several Aspergillus species that grow at the low temperatures also favored by Penicillium. Perhaps Ser/Thr267 is an evolutionary signature of MPA exposure., (© 2019 The Protein Society.)

Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis, and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Although dominantly inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remains challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase-deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G>C variant in GMPR (NM&#95;006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G>C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells, and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the 19th locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes., (© 2019 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

A microbe's ecological niche and biotechnological utility are determined by its specific set of co-evolved metabolic pathways. The acquisition of new pathways, through horizontal gene transfer or genetic engineering, can have unpredictable consequences. Here we show that two different pathways for coumarate catabolism failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly acquired pathways and the modifications required to overcome these limitations. Both pathways required host mutations to enable effective growth with coumarate, but the necessary mutations differed. In one case, a pathway intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid replacements in IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. Molecular dynamics simulation of the wild type and a representative single-residue mutant provide insight into the structural and dynamic changes that relieve inhibition. These results demonstrate how deleterious interactions can limit pathway transfer, that these interactions can be traced to specific molecular interactions between host and pathway, and how evolution or engineering can alleviate these limitations., (© 2019 John Wiley & Sons Ltd.)

The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early-childhood mortality. Waterborne outbreaks occur frequently, even in countries with advanced water treatment capabilities, and there is currently no fully effective treatment. Nucleotide pathways are attractive targets for antimicrobial development, and several laboratories are designing inhibitors of these enzymes as potential treatment for Cryptosporidium infections. Here we take advantage of newly available molecular genetics for Cryptosporidium parvum to investigate nucleotide biosynthesis by directed gene ablation. Surprisingly, we found that the parasite tolerates the loss of classical targets including dihydrofolate reductase-thymidylate synthase (DHFR-TS) and inosine monophosphate dehydrogenase (IMPDH). We show that thymidine kinase provides a route to thymidine monophosphate in the absence of DHFR-TS. In contrast, only a single pathway has been identified for C. parvum purine nucleotide salvage. Nonetheless, multiple enzymes in the purine pathway, as well as the adenosine transporter, can be ablated. The resulting mutants are viable under normal conditions but are hypersensitive to inhibition of purine nucleotide synthesis in their host cell. Cryptosporidium might use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude that Cryptosporidium imports purine nucleotides from the host cell. The potential for ATP uptake from the host has significant impact on our understanding of parasite energy metabolism given that Cryptosporidium lacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-β-(d-ribofuranosyl)-3 H-imidazo-[4,5- d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of K i varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity.

Promiscuous inhibitors of tyrosine protein kinases, proteases and phosphatases are useful reagents for probing regulatory pathways and stabilizing lysates as well as starting points for the design of more selective agents. Ubiquitination regulates many critical cellular processes, and promiscuous inhibitors of deubiquitinases (DUBs) would be similarly valuable. The currently available promiscuous DUB inhibitors are highly reactive electrophilic compounds that can crosslink proteins. Herein we introduce diarylcarbonate esters as a novel class of promiscuous DUB inhibitors that do not have the liabilities associated with the previously reported compounds. Diarylcarbonates stabilize the high molecular weight ubiquitin pools in cells and lysates. They also elicit cellular phenotypes associated with DUB inhibition, demonstrating their utility in ubiquitin discovery. Diarylcarbonates may also be a useful scaffold for the development of specific DUB inhibitors., (Copyright © 2018. Published by Elsevier Ltd.)