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DFT calculations reveal mechanistic insights and different preferences in the substrate specificity of the dihydropyrimidinase from yeast and bacteria as well as the role of stereo-gate-loop residues in the stereospecificity of catalysis.

Summary Background Canonical and non-canonical WNT signaling are important for odontogenesis. WNT ligand secretion mediator (WLS; MIM611514) is required to transport lipid-modified WNT proteins from the Golgi to the cell membrane, where canonical and non-canonical WNT proteins are released into the extracellular milieu. Biallelic pathogenic variants in WLS are implicated in autosomal recessive Zaki syndrome (ZKS; MIM 619648), the only genetic condition known to be caused by pathogenic variants in WLS. Objective To investigate molecular etiology of dental anomalies in 250 patients with or without oral exostoses. Patients and methods Clinical and radiographic examination, and whole exome sequencing, were performed in the case of 250 patients with dental anomalies with or without oral exostoses. Results Four extremely rare heterozygous missense variants (p.Ile20Thr, p.Met46Leu, p.Ser453Ile and p.Leu516Phe) in WLS were identified in 11 patients with dental anomalies. In five of these patients, a torus palatinus or a torus mandibularis was observed. Conclusion We report for the first time the heterozygous WLS variants in patients with dental anomalies. Root maldevelopments in patients with WLS variants supports the role of canonical and non-canonical WNT signaling in root development. We also show that variants in WLS were implicated in torus palatinus and torus mandibularis. In addition, this is the first time that heterozygous carriers of WLS variants were found to manifest phenotypes. WLS variants were likely to have adverse effects on the concentration of WNT ligands delivered to the cell membrane, resulting in aberrant canonical and non-canonical WNT signaling, and subsequent phenotypes. Limitations of the study Patient’s positioning during the acquisition of panoramic radiography might have affected the appearance of the tooth structures. If we had all family members of each patient to study co-segregation between genotype and phenotype, it would have strengthened the association of WLS variants and the phenotypes.

Fatty acyl group utilized as both protection and migratory group for the synthesis of ceramides and glucosylceramides.

One of the most important steps in post-translational modifications of collagen type I chains is the hydroxylation of carbon-3 of proline residues by prolyl-3-hydroxylase-1 (P3H1). Genetic variants in P3H1 have been reported to cause autosomal recessive osteogenesis imperfecta (OI) type VIII. Clinical and radiographic examinations, whole-exome sequencing (WES), and bioinformatic analysis were performed in 11 Thai children of Karen descent affected by multiple bone fractures. Clinical and radiographic findings in these patients fit OI type VIII. Phenotypic variability is evident. WES identified an intronic homozygous variant (chr1:43212857A > G; NM_022356.4:c.2055 + 86A > G) in P3H1 in all patients, with parents in each patient being heterozygous for the variant. This variant is predicted to generate a new “CAG” splice acceptor sequence, resulting in the incorporation of an extra exon that leads to a frameshift in the final exon and subsequent non-functional P3H1 isoform a. Alternative splicing of P3H1 resulting in the absence of functional P3H1 caused OI type VIII in 11 Thai children of Karen descent. This variant appears to be specific to the Karen population. Our study emphasizes the significance of considering intronic variants.

Plants possess many glycoside hydrolase family 1 (GH1) β-glucosidases, which physiologically function in cell wall metabolism and activation of bioactive substances, but most remain uncharacterized. One GH1 isoenzyme AtBGlu42 in Arabidopsis thaliana has been identified to hydrolyze scopolin using the gene deficient plants, but no enzymatic properties were obtained. Its sequence similarity to another functionally characterized enzyme Os1BGlu4 in rice suggests that AtBGlu42 also acts on oligosaccharides. Here, we show that the recombinant AtBGlu42 possesses high kcat/Km not only on scopolin, but also on various β-glucosides, cellooligosaccharides, and laminarioligosaccharides. Of the cellooligosaccharides, cellotriose was the most preferred. The crystal structure, determined at 1.7 Å resolution, suggests that Arg342 gives unfavorable binding to cellooligosaccharides at subsite +3. The mutants R342Y and R342A showed the highest preference on cellotetraose or cellopentaose with increased affinities at subsite +3, indicating that the residues at this position have an important role for chain length specificity.

A mutation in DKK1 gene leads to inhibitory DKK1 function, over-activation of WNT/β-catenin signaling, disruptive development of dental epithelium, and subsequent mesiodens formation.

Mucopolysaccharidosis type I Hurler syndrome (MPS IH) is a severe lysosomal storage disorder caused by alpha-l-iduronidase (IDUA) deficiency. Premature truncation mutations (PTC) are the most common (50%-70%) type of IDUA mutations and correlate with MPS IH. Nonsense suppression therapy is a therapeutic approach that aims to induce stop codon readthrough. The different ability of gentamicin to bind mutant mRNA in readthrough is determined by nucleotide sequence (PTC context: UGA UAG UAA) and inserted amino acid including the nucleotide position +4 of the PTC, as well as the mRNA secondary structure. We used COS-7 cells to investigate the functional characteristics of p.Q500X and p.R619X, IDUA variants and the effects of gentamicin in inducing stop codon readthrough of seven IDUA variants including p.Q500X, p.R619X, p.Q70X, p.E299X, p.W312X, p.Q380X, and p.W402X. Moreover, we performed prediction of RNA secondary structure using the online tool RNAfold. We found that cells treated with gentamicin showed significantly enhanced full-length IDUA expression and restored IDUA activity, in a dose-dependent manner, only in cells expressing cDNA with W312X, Q380X, W402X, and R619X. Among the readthrough-responsive variants, we observed UGA PTC in W312X, W402X and R619X; and UAG PTC with C at nucleotide +4 in Q380X. Changes of RNA secondary structure were noted only in mutants with readthrough-responsive variants including W312X, Q380X, W402X, and R619X. Additional preclinical studies of selected PTCs with potential readthrough, using drugs with less oto-nephrotoxicity, in patient's skin fibroblasts and animal model are necessary for the premise of personalized medicine.

The endo-1,5-α-L-arabinanases, belonging to glycoside hydrolase family 43 (GH43), catalyse the hydrolysis of α-1,5-arabinofuranosidic bonds in arabinose-containing polysaccharides. These enzymes are proposed targets for industrial and medical applications. Here, molecular dynamics (MD), potential energy surface and free energy (potential of mean force) simulations are undertaken using hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to understand the active site dynamics, catalytic mechanism and the electrostatic influence of active site residues of the GH43 endo-arabinanase from G. stearothermophilus. The calculated results give support to the single-displacement mechanism proposed for the inverting GH43 enzymes: first a proton is transferred from the general acid E201 to the substrate, followed by a nucleophilic attack by water, activated by the general base D27, on the anomer carbon. A conformational change (2E ↔E3 ↔ 4E) in the −1 sugar ring is observed involving a transition state featuring an oxocarbenium ion character. Residues D87, K106, H271 are highlighted as potential targets for future mutation experiments in order to increase the efficiency of the reaction. To our knowledge, this is the first QM/MM study providing molecular insights into the glycosidic bond hydrolysis of a furanoside substrate by an inverting GH in solution. Communicated by Ramaswamy H. Sarma

Phenylketonuria (PKU) is an autosomal recessive amino acid metabolism disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH; EC1.14.16.1). This study aimed to assess the specific heterogeneity of PAH variants found in Thai population as well as evaluate enzyme activity and expression of novel variants. PAH gene from 13 patients was analyzed by PCR amplification and direct Sanger-sequencing of 13 exons of the coding region. The novel variants were transiently transfected in COS-7 cells for functional verification. Eleven different PAH variants were identified: all pathogenic variants were missense variants, of which the most frequent variant was p.R169L, accounting for 24% (6/25) of all identified alleles. Two novel variants p.R169L and p.Y317N and previously reported variants with mutated residues at the same positions (p.R169H and p.Y317H) were expressed in COS-7 cells. These showed mildly impaired residual activity levels (42.3–63.1% of wild type), while the protein levels were well expressed (82.8–110%), except for p.R169L, which showed decreased protein expression of 55.7% compared to the wild type enzyme. All subjects with p.R169L identified in at least one of pathogenic alleles (one case is homozygous) had a metabolic phenotype of mild hyperphenylalaninemia (HPA). Our data has expanded the information on the genetic heterogeneity of Thai patients with PAH deficiency. This finding emphasizes the importance of genotyping in patients with HPA, and in vitro studies can provide additional information for prediction of phenotype.

Nucleocidin 1 produced by Streptomyces calvus is one of five characterized natural products containing fluorine. It was discovered in 1956, but its biosynthesis is not yet completely resolved. Recently, the biosynthetic gene cluster of 1 was identified. The nucPNP gene, which was initially annotated as orf206 and encodes a putative purine nucleoside phosphorylase, is essential for nucleocidin production. In this study, we performed in vitro assays and showed NucPNP produced adenine 3 from methylthioadenosine (MTA) 2 and adenosine 4. We also showed the downstream enzyme, NucV annotated as adenine phosphoribosyltransferase (APRT), catalyzes AMP formation from adenine 3 and 5-phospho-α-D-ribose-1-diphosphate (PRPP) 5. However, the catalytic efficiency of NucV was much slower than its homolog ScAPRT involved in the biosynthesis of canonical purine nucleoside in the same strain. These results provide new insights in nucleocidin biosynthesis and could guide future research on organofluorine formation.

Harboyan syndrome or corneal dystrophy and progressive deafness (MIM #217400) is characterized by congenital hereditary endothelial dystrophy (CHED) and progressive, sensorineural hearing loss. Mutations in SLC4A11 are responsible for this rare genetic syndrome. Eight patients from seven unrelated families affected with Harboyan Syndrome with mean follow-up of 12.0 ± 0.9 years were thoroughly investigated for the ocular, hearing, and kidney function abnormalities and the outcome of penetrating keratoplasty (PK). Mutation analysis of SLC4A11 was performed. All patients presented with bilateral cloudy corneas since birth. Sensorineural hearing loss was detected in all patients. Seven patients (11 eyes) underwent PK with the median age at surgery of 10.1 years (7.1-22.9). The overall corneal graft survival rate after primary PK was 72.7% (8/11 eyes). The mean graft survival time was 94.6 months (95% CI 83.1-126.0). All patients had unremarkable kidney function. The c.2264G>A (p.Arg755Gln) mutation in SCL4A11 was detected in most patients (87.5%). All unrelated Karen tribe patients had p.Arg755Gln mutation, suggestive of founder effect. We found the allele frequency of this variant in the Karen population to be 0.01. The c.2263C>T (p.Arg755Trp) mutation was found in one patient with mild phenotype and the novel truncating protein mutation c.2127delG (p.Gly710fsx*25) in SCL4A11 was identified in two Thai sisters. Visual outcome and graft survival after PK were satisfactory. Our study shows that all studied patients with SLC4A11 mutations had CHED and sensorineural hearing loss, and SLC4A11 mutations were not related to the onset and severity of hearing loss or outcome of keratoplasty.

WNT/β-catenin and BMP signaling pathways play important roles in the process of tooth development. Dysregulation of WNT/β-catenin and BMP signaling is implicated in a number of human malformations, including dental anomalies. Whole exome and Sanger sequencing identified seven patients with LRP5 mutations (p.Asn1121Asp, p.Asp856Asn, p.Val1433Met, and p.Val1245Met) and six patients with BMP4 mutations (p.Asn150Lys, p.Gly168Arg, p.Arg269Gln, and p.Ala42Glu). All patients were affected with isolated dental anomalies (dental anomalies with no other structural defects), including mesiodens, tooth agenesis, unseparated roots, narrow roots, shortened and tapered roots, and taurodontism. Five patients with LRP5 and one with BMP4 mutations had oral exostoses. Protein models of LRP5 mutations indicate the possible functional effects of the mutations. Here we report for the first time that mutations in LRP5 are associated with dental anomalies. LRP5 appears to be the first gene related to pathogenesis of mesiodens. We also show for the first time that in addition to tooth agenesis, mutations in BMP4 are also implicated in root maldevelopment and torus mandibularis. Sharing of the phenotypes of the patients with LRP5 and BMP4 mutations, which include root maldevelopment, tooth agenesis, and torus mandibularis, implicates cross talks between the WNT/β-catenin and BMP signaling pathways, especially during root development.

The objective of this study was to investigate molecular etiologies of oral exostoses and dental anomalies in 14 patients from eight families.Oral and radiographic examinations were performed on every patient. Whole exome and Sanger sequencing were performed on DNA of the patients, the unaffected parents and unaffected siblings. LRP6 mutant proteins were modeled and analyzed.Five mutations in LRP6, including four missense (p.Glu72Lys, p.Lys82Asn, Tyr418His, and p.Ile773Val) and one nonsense mutation (p.Arg32Ter), were identified. These mutations have not been reported to be associated with dental anomalies or oral exostoses. Oral features included a variety of oral exostoses (7 of the 14 patients), root defects (6 of the 14 patients), and tooth agenesis (5 of the 14 patients). Less common dental anomalies included microdontia, tooth fusion, odontomas, and mesiodens. Analysis of the protein models of the five LRP6 mutations shed light on their likely impact on LRP6 protein structure and function.Fourteen patients with five LRP6 mutations, including two recurrent mutations and three novel ones, are reported. Our study shows for the first time that mutations in LRP6 are associated with mesiodens, fusion of teeth, odontomas, microdontia, long roots, molars with unseparated roots, and taurodontism.

Mutations in LTBP3 are associated with Dental Anomalies and Short Stature syndrome (DASS; MIM 601216), which is characterized by hypoplastic type amelogenesis imperfecta, hypodontia, underdeveloped maxilla, short stature, brachyolmia, aneurysm and dissection of the thoracic aorta. Here we report a novel (p.Arg545ProfsTer22) and a recurrent (c.3107-2A G) LTBP3 variants, in a Turkish family affected with DASS. The proband, who carried compound heterozygous variant c.3107-2A G, p.Arg545ProfsTer22, was most severely affected with DASS. The proband's father, who carried the heterozygous variant c.3107-2A G had short stature and prognathic mandible. The mother and brother of the proband carried the heterozygous variant p.Arg545ProfsTer22, but only the mother showed any DASS characteristics. The c.3107-2A G and the p.Arg545ProfsTer22 variants are expected to result in abnormal LTPB3 protein, failure of TGFβ-LAP-LTBP3 complex formation, and subsequent disruption of TGFβ secretion and activation. This is the first report of heterozygous carriers of LTBP3 variants showing phenotypes. The new findings of DASS found in this family include taurodontism, single-rooted molars, abnormal dentin, calcified dental pulp blood vessels, prognathic mandible, failure of mandibular tooth eruption, interatrial septal aneurysm, secundum atrial septal defect, tricuspid valve prolapse, and a recurrent glenohumeral joint dislocation.

WNT/β-catenin signaling is initiated by binding of a WNT protein to a Frizzled (FZD) receptor and a co-receptor, low-density lipoprotein (LDL) receptor-related protein 5 or 6 (LRP5/6). The objective of this study was to find the genetic variants responsible for dental anomalies found in 4 families.Clinical and radiographic examination and whole exome sequencing were performed on 5 patients affected with dental anomalies and the mutant proteins modeled.Five patients were heterozygous for the WNT10A variants, including c.877CT; p.Arg293Cys, c.874AG; p.Ser292Gly, c.1042CT; p.Arg348Cys, and c.1039GT; p.347GluX. The p.Arg293Cys and p.Ser292Gly mutations are located in the WNT10A N-terminal domain region with binding sites for FZD receptor, porcupine, WNTLESS, and extracellular binding proteins, so they are likely to have adverse effects on binding these proteins. The p.Arg348Cys mutation, which is located in the binding site of LRP5/6 co-receptors, is postulated to result in impaired binding to these co-receptors. The nonsense mutation p.347GluX is predicted to result in the truncation of most of the C-terminal domain, which is likely to disrupt the binding of WNT10A to WNTLESS, the membrane protein that binds lipid-acylated WNT proteins to carry them from the endoplasmic reticulum to the cell surface and FZD.Four novel mutations in WNT10A were identified in patients with isolated tooth agenesis. The mutations in the N-terminal domain and the interface between the N- and C-terminal domains of WNT10A in our patients are likely to disrupt its binding with FZD, LRP5/6, and various other proteins involved in WNT10A processing and transport, impair WNT and SHH signaling, and subsequently result in tooth agenesis, microdontia, and root maldevelopment.

Caspase-3 is a biochemical marker for cell apoptosis. Several studies focused on exploring caspase inhibitor potential in natural compounds. Hence, in this study investigated the anthocyanins as anti-apoptotic potential activity through caspase-3 using molecular docking. Six types of anthocyanin were retrieved from PubChem database and caspase-3 protein was downloaded from Protein Data Bank. Anthocyanins and caspase-3 protein were docked using HEX 8.0 program and visualized using Discovery Studio 4.1 software. The interaction among cyanidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargonidin-3-O-glucoside, peonidin-3-O-glucoside and petunidin-3-O-glucoside showed similar binding pattern on caspase-3 protein. All of them bind to BIR2 region and allosteric site of caspase-3, which are a crucial site for apoptosis regulation. Interestingly, malvidin-3-O-glucoside also interacted with caspase-3 in BIR1, BIR2 and BIR3 regions. In addition, anthocyanins-caspase-3 complex showed low energy and demonstrated several hydrogen bonds, hydrophobic interactions and van der Waals interactions, which indicated stable interaction. This study implies that all anthocyanins have potential as inhibitor of caspase-3 protein and might have potential as anti-apoptosis. Further in-vitro and in-vivo studies are need to confirm this experimental.

BackgroundPompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase (EC. 3.2.1.20) due to mutations in humanGAAgene. The objective of the present study was to examine clinical and molecular characteristics of infantile-onset Pompe disease (IOPD) in Thailand.MethodsTwelve patients with infantile-onset Pompe disease (IOPD) including 10 Thai and two other Asian ethnicities were enrolled. To examine the molecular characteristics of Pompe patients,GAAgene was analyzed by PCR amplification and direct Sanger-sequencing of 20 exons coding region. The novel mutations were transiently transfected in COS-7 cells for functional verification. The severity of the mutation was rated by study of the GAA enzyme activity detected in transfected cells and culture media, as well as the quantity and quality of the proper sized GAA protein demonstrated by western blot analysis. The GAA three dimensional structures were visualized by PyMol software tool.ResultsAll patients had hypertrophic cardiomyopathy, generalized muscle weakness, and undetectable or G (p.Tyr292X), c.1226insG (p.Asp409GlyfsX95), c.1538G > A (p.Asp513Gly), c.1895 T > G (p.Leu632Arg), and a previously reported rare allele of unknown significance: c.781G > A (p.Ala261Thr) were identified. The rating system ranked p.Tyr292X, p. Asp513Gly and p. Leu632Arg as class “B” and p. Ala261Thr as class “D” or “E”. These novel mutations were located in the N-terminal beta-sheet domain and the catalytic domain.ConclusionsThe present study provides useful information on the mutations ofGAAgene in the underrepresented population of Asia which are more diverse than previously described and showing the hotspots in exons 14 and 5, accounting for 62% of mutant alleles. Almost all mutations identified are in class A/B. These data can benefit rapid molecular diagnosis of IOPD and severity rating of the mutations can serve as a partial substitute for cross reactive immunological material (CRIM) study.

Glycoside hydrolase family1 β-glucosidases play a variety of roles in plants, but their in planta functions are largely unknown in rice (Oryza sativa). In this study, the biological function of Os12BGlu38, a rice β-glucosidase, expressed in bicellular to mature pollen, was examined. Genotype analysis of progeny of the self-fertilized heterozygous Os12BGlu38 T-DNA mutant, os12bglu38-1, found no homozygotes and a 1:1 ratio of wild type to heterozygotes. Reciprocal cross analysis demonstrated that Os12BGlu38 deficiency cannot be inherited through the male gamete. In cytological analysis, the mature mutant pollen appeared shrunken and empty. Histochemical staining and TEM showed that mutant pollen lacked intine cell wall, which was rescued by introduction of wild-type Os12BGlu38 genomic DNA. Metabolite profiling analysis revealed that cutin monomers and waxes, the components of the pollen exine layer, were increased in anthers carrying pollen of os12bglu38-1 compared with wild type and complemented lines. Os12BGlu38 fused with green fluorescent protein was localized to the plasma membrane in rice and tobacco. Recombinant Os12BGlu38 exhibited β-glucosidase activity on the universal substrate p-nitrophenyl β-d-glucoside and some oligosaccharides and glycosides. These findings provide evidence that function of a plasma membrane-associated β-glucosidase is necessary for proper intine development.

Conjugation of phytohormones with glucose is a means of modulating their activities, which can be rapidly reversed by the action of β-glucosidases. Evaluation of previously characterized recombinant rice β-glucosidases found that nearly all could hydrolyze abscisic acid glucose ester (ABA-GE). Os4BGlu12 and Os4BGlu13, which are known to act on other phytohormones, had the highest activity. We expressed Os4BGlu12, Os4BGlu13 and other members of a highly similar rice chromosome 4 gene cluster (Os4BGlu9, Os4BGlu10 and Os4BGlu11) in transgenic Arabidopsis. Extracts of transgenic lines expressing each of the five genes had higher β-glucosidase activities on ABA-GE and gibberellin A4 glucose ester (GA4-GE). The β-glucosidase expression lines exhibited longer root and shoot lengths than control plants in response to salt and drought stress. Fusions of each of these proteins with green fluorescent protein localized near the plasma membrane and in the apoplast in tobacco leaf epithelial cells. The action of these extracellular β-glucosidases on multiple phytohormones suggests they may modulate the interactions between these phytohormones.

Compared with normal cells, cancer cells usually exhibit an increase in glucose uptake as part of the Warburg effect. To take advantage of this hallmark of cancer, glucose transporters could be a good candidate for cancer targeting. Herein, we report novel glycoconjugate aza-BODIPY dyes (AZB-Glc and AZB-Glc-I) that contain two glucose moieties conjugated to near-infrared dyes via the azide-alkyne cycloaddition reaction. As anticipated, a higher level of AZB-Glc uptake was observed in breast cancer cells that overexpressed glucose transporters (GLUTs), especially GLUT-1, including the triple-negative breast cancer cell line (MDA-MB-231) and human breast adenocarcinoma cell line (MCF-7), compared to that of normal cells (human fetal lung fibroblasts, HFL1). The cellular uptake of AZB-Glc was in a dose- and time-dependent manner and also depended on GLUT, as evidenced by the decreased uptake of AZB-Glc in the presence of d-glucose or a glucose metabolism suppressor, combretastatin. In addition, light triggered cell death was also investigated through photodynamic therapy (PDT), since near-infrared (NIR) light is known to penetrate deeper tissue than light of shorter wavelengths. AZB-Glc-I, the analog of AZB-Glc containing iodine for enhanced singlet oxygen production upon NIR irradiation, was used for all treatment assays. AZB-Glc-I showed significant NIR light-induced cytotoxicity in cancer cells (IC50 = 1.4-1.6 μM under 1 min irradiation), which was about 20-times lower than that in normal cells (IC50 = 32 μM) under the same conditions, with negligible dark toxicity (IC50 > 100 μM) in all cell lines. Moreover, the singlet oxygen was detected inside the cancer cells after exposure to light in the presence of AZB-Glc-I. Therefore, our glucose conjugated systems proved to efficiently target cancer cells for enhanced photodynamic cancer therapy.

Adult-onset immunodeficiency syndrome (AOID) with anti-interferon (IFN)-γ autoantibodies is characterized by an AIDS-like illness with disruptive IFN-γ signaling. Patients generally present with recurrent and disseminated opportunistic infections along with neutrophilic dermatoses. Generalized pustular psoriasis (GPP; Online Mendelian Inheritance in Man #614204) is characterized by acute generalized erythema and scaling with numerous aseptic pustules. Mutations in SERPINA3 have been reported as predisposing risk factors for both AOID and GPP. Here, we report two unrelated patients, one with AOID and a pustular skin reaction and the other with GPP, who both carried the same heterozygous variant c.718G>A (p.Val240Met) in SERPINA1. Our observation of a shared mutation in SERPINA1 in AOID and GPP indicate possible pathobiological and disease mechanism similarities in these two disorders. Thus, variants in both SERPINA1, SERPINA3, and potentially other SERPIN family members may be associated with the etiology of GPP and AOID.

Black rice is considered to be functional food containing anthocyanins as bioactive compounds. This study examined the genomic and proteomic patterns in local black rice from Java Island, Indonesia, with attention to the mechanism of anthocyanin synthesis. Three kinds of black rice from Java Island, including black rice from East Java (BREJ), black rice from Central Java (BRCJ), and black rice from West Java (BRWJ), were studied in comparison to white rice (WREJ) and red rice (RREJ). Genomic profiling was done by simple sequence repeat (SSR) analysis, and sequencing of red coleoptile (Rc) and glycosyltransferase (GT) genes, followed by in silico analysis. Total anthocyanin was investigated by ultra-high performance liquid chromatography- diode array detector (UHPLC-DAD). The proteomic profiles were determined by liquid-chromatography and mass spectrometry of tryptic peptides. The SSR profiles showed a specific band in each black rice variant. The Rc gene exon-2 sequences were similar in the three black rice cultivars. The GT gene sequence was identified as a new variant that correlates with the purple stem, leaf, bran, and whole grain morphology seen exclusively in the BRWJ cultivar. The anthocyanin composition in Java black rice is diverse. The highest cyanidin level was seen in BRWJ and the highest level of peonidin-3-O-glucoside in BREJ. Proteomic profiling of the black rice cultivars demonstrated that the expression of proteins that might be related to the levels of anthocyanin synthesis varied. These studies conclude that the genomic, proteomic and anthocyanins composition of Java black rice cultivars may be used the improvement of their functional nutrition values.

Nucleocidin 1 produced by

Glucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. GlcCer is mainly degraded by two enzymes, lysosomal acid &beta, glucosidase (GBA) and nonlysosomal &beta, glucosidase (GBA2), which may have different isoforms because of alternative splicing. To understand which GBA2 isoforms are active and how they affect glycosphingolipid levels in cells, we expressed nine human GBA2 isoforms in COS-7 cells, confirmed their expression by qRT-PCR and Western blotting, and assayed their activity to hydrolyze 4-methylumbelliferyl-&beta, D-glucopyranoside (4MUG) in cell extracts. Human GBA2 isoform 1 showed high activity, while the other isoforms had activity similar to the background. Comparison of sphingolipid levels by ultra-high resolution/accurate mass spectrometry (UHRAMS) analysis showed that isoform 1 overexpression increased ceramide and decreased hexosylceramide levels. Comparison of ratios of glucosylceramides to the corresponding ceramides in the extracts indicated that GBA2 isoform 1 has broad specificity for the lipid component of glucosylceramide, suggesting that only one GBA2 isoform 1 is active and affects sphingolipid levels in the cell. Our study provides new insights into how increased breakdown of GlcCer affects cellular lipid metabolic networks.

Objective Our objective was to investigate the molecular etiology of osteogenesis imperfecta type VIII and dental anomalies in 4 siblings of a Karen tribe family. Materials and Methods Four patients and their unaffected parents were studied by clinical and radiographic examination. In situ hybridization of P3h1 during early murine tooth development, whole-exome sequencing, and Sanger direct sequencing were performed. Results A novel homozygous missense P3H1 mutation (NM_001243246.1; c.2141A>G; NP_001230175.1; p.Lys714Arg) was identified in all patients. Their unaffected parents were heterozygous for the mutation. The mutation is hypothesized to belong to isoform c of P3H1. Mutations in P3H1 are associated with autosomal recessive osteogenesis imperfecta type VIII. Hypodontia, a mesiodens, and single-rooted permanent second molars found in our patients have never been reported in patients with P3H1 mutations. Single-rooted second permanent molars or failure to form multiple roots implies effects of the P3H1 mutation on root development. Conclusions We report a novel P3H1 mutation as the underlying cause of osteogenesis imperfecta type VIII with dental anomalies. Our study suggests that isoform c of P3H1 is also a functional isoform of P3H1. We report, for the first time, to our knowledge, the association of P3H1 mutation and osteogenesis imperfecta type VIII with dental anomalies.

Parkinson's disease (PD) is a neurodegenerative disorder that affects movement, and its development is associated with environmental and genetic factors. Genetic variants in GBA and PARK2 are important risk factors implicated in the development of PD; however, their precise roles have yet to be elucidated. The present study aimed to identify and analyse proteins from the skin fibroblasts of patients with PD carrying heterozygous GBA and PARK2 variants, and from healthy controls. Liquid chromatography coupled with tandem mass spectrometry and label-free quantitative proteomics were performed to identify and compare differential protein expression levels. Moreover, protein-protein interaction networks were assessed using Search Tool for Retrieval of Interacting Genes analysis. Using these proteomic approaches, 122 and 119 differentially expressed proteins from skin fibroblasts of patients with PD carrying heterozygous GBA and PARK2 variants, respectively, were identified and compared. According to the results of protein-protein interaction and Gene Ontology analyses, 14 proteins involved in the negative regulation of macromolecules and mRNA metabolic processes, and protein targeting to the membrane exhibited the largest degree of differential expression in the fibroblasts of patients with PD with a GBA variant, whereas 20 proteins involved in the regulation of biological quality, NAD metabolic process and cytoskeletal organization exhibited the largest degree of differential expression in the fibroblasts of patients with PD with a PARK2 variant. Among these, the expression levels of annexin A2 and tubulin β chain, were most strongly upregulated in the fibroblasts of patients with GBA-PD and PARK2-PD, respectively. Other predominantly expressed proteins were confirmed by western blotting, and the results were consistent with those of the quantitative proteomic analysis. Collectively, the results of the present study demonstrated that the proteomic patterns of fibroblasts of patients with PD carrying heterozygous GBA and PARK2 variants are different and unique. Aberrant expression of the proteins affected by these variants may reflect physiological changes that also occur in neurons, resulting in PD development and progression.

Mandibular prognathism is characterized by a prognathic or prominent mandible. The objective of this study was to find the gene responsible for mandibular prognathism. Whole exome sequencing analysis of a Thai family (family 1) identified the ADAMTSL1 c.176C>A variant as the potential defect. We cross-checked our exome data of 215 people for rare variants in ADAMTSL1 and found that the c.670C>G variant was associated with mandibular prognathism in families 2 and 4. Mutation analysis of ADAMTSL1 in 79 unrelated patients revealed the c.670C>G variant was also found in family 3. We hypothesize that mutations in ADAMTSL1 cause failure to cleave aggrecan in the condylar cartilage, and that leads to overgrowth of the mandible. Adamtsl1 is strongly expressed in the condensed mesenchymal cells of the mouse condyle, but not at the cartilage of the long bones. This explains why the patients with ADAMTSL1 mutations had abnormal mandibles but normal long bones. This is the first report that mutations in ADAMTSL1 are responsible for the pathogenesis of mandibular prognathism.

Benzophenone synthase (BPS) catalyzes the production of 2,4,6-trihydroxybenzophenone via the condensation of benzoyl-CoA and three units of malonyl-CoA. The biosynthetic pathway proceeds with the formation of the prenylated xanthone α-mangostin from 2,4,6-trihydroxybenzophenone. Structural elucidation was performed to gain a better understanding of the structural basis of the function of Garcinia mangostana L. (mangosteen) BPS (GmBPS). The structure reveals the common core consisting of a five-layer αβαβα fold as found in other type III polyketide synthase enzymes. The three residues Met264, Tyr266 and Gly339 are proposed to have a significant impact on the substrate-binding specificity of the active site. Crystallographic and docking studies indicate why benzoyl-CoA is preferred over 4-coumaroyl-CoA as the substrate for GmBPS. Met264 and Tyr266 in GmBPS are properly oriented for accommodation of the 2,4,6-trihydroxybenzophenone product but not of naringenin. Gly339 offers a minimal steric hindrance to accommodate the extended substrate. Moreover, the structural arrangement of Thr133 provides the elongation activity and consequently facilitates extension of the polyketide chain. In addition to its impact on the substrate selectivity, Ala257 expands the horizontal cavity and might serve to facilitate the initiation/cyclization reaction. The detailed structure of GmBPS explains its catalytic function, facilitating further structure-based engineering to alter its substrate specificity and obtain the desired products.

Objective Juberg-Hayward syndrome (JHS; MIM 216100) is a rare autosomal recessive malformation syndrome, characterized by cleft lip/palate, microcephaly, ptosis, hypoplasia or aplasia of thumbs, short stature, dislocation of radial head, and fusion of humerus and radius leading to elbow restriction. A homozygous mutation in ESCO2 has recently been reported to cause Juberg-Hayward syndrome. Our objective was to investigate the molecular etiology of Juberg-Hayward syndrome in two affected Lisu tribe brothers. Materials and Methods Two patients, the unaffected parents, and two unaffected siblings were studied. Clinical and radiographic examination, whole exome sequencing, Sanger sequencing, Western blot analysis, and chromosome testing were performed. Results Two affected brothers had characteristic features of Juberg-Hayward syndrome, except for the absence of microcephaly. The elder brother had bilateral cleft lip and palate, short stature, humeroradial synostosis, and simple partial seizure with secondary generalization. The younger brother had unilateral cleft lip and palate, short stature, and dislocation of radial heads. The homozygous (c.1654C > T; p.Arg552Ter) mutation in ESCO2 was identified in both patients. The other unaffected members of the family were heterozygous for the mutation. The presence of humeroradial synostosis and radial head dislocation in the same family is consistent with both being in the same spectrum of forearm malformations. Chromosome testing of the affected patients showed premature centromere separation. Western blot analysis showed reduced amount of truncated protein. Conclusion Our findings confirm that a homozygous mutation in ESCO2 is the underlying cause of Juberg-Hayward syndrome. Microcephaly does not appear to be a consistent feature of the syndrome.

Transglycosidases are enzymes that have the potential to catalyze the synthesis of a wide range of high-value compounds starting from biomass-derived feedstocks. Improving their activity and broadening the substrate range are important goals to enable the widespread application of this family of biocatalysts. In this work, we engineered 20 mutants of the rice transglycosidase Os9BGlu31 and evaluated their catalysis in 462 reactions over 18 different substrates. This allowed us to identify mutants that expanded their substrate range and showed high activity, including W243L and W243N. We also developed double mutants that show very high activity on certain substrates and exceptional specificity towards hydrolysis, such as L241D/W243N. In order to guide a more general use of Os9BGlu31 variants as transglycosylation catalysts, we built cheminformatics models based on topological descriptors of the substrates. These models showed useful predictive potential on the external validation set and are allowing the identification of efficient catalytic routes to novel phytohormone and antibiotic glucoconjugates of interest.

The production of analytical amounts of azido sugars is used as a means of verifying catalytic acid/base mutations of retaining glycosidase, but application of this process to preparative synthesis has not been reported. The catalytic acid/base mutant of Thermoanaerobacterium xylanolyticus GH116 β-glucosidase, TxGH116D593A, catalyzed the gram scale production of 1-azido-β-D-glucose (1) from p-nitropheyl-β-D-glucopyranoside (pNPGlc) and azide via a transglucosylation reaction. Overnight reaction of the enzyme with pNPGlc and NaN3 in aqueous MES buffer (pH 5.5) at 55 °C produced 1 (3.27 g), which was isolated as a white foamy solid in 96% yield. This 1 was successfully utilized for the synthesis of fifteen 1,2,3-triazole-β-D-glucosyl derivatives (2–16) containing a variety of functional groups, via click chemistry.

Harboyan syndrome or corneal dystrophy and progressive deafness (MIM #217400) is characterized by congenital hereditary endothelial dystrophy (CHED) and progressive, sensorineural hearing loss. Mutations in SLC4A11 are responsible for this rare genetic syndrome. Eight patients from seven unrelated families affected with Harboyan Syndrome with mean follow-up of 12.0 ± 0.9 years were thoroughly investigated for the ocular, hearing, and kidney function abnormalities and the outcome of penetrating keratoplasty (PK). Mutation analysis of SLC4A11 was performed. All patients presented with bilateral cloudy corneas since birth. Sensorineural hearing loss was detected in all patients. Seven patients (11 eyes) underwent PK with the median age at surgery of 10.1 years (7.1-22.9). The overall corneal graft survival rate after primary PK was 72.7% (8/11 eyes). The mean graft survival time was 94.6 months (95% CI 83.1-126.0). All patients had unremarkable kidney function. The c.2264GA (p.Arg755Gln) mutation in SCL4A11 was detected in most patients (87.5%). All unrelated Karen tribe patients had p.Arg755Gln mutation, suggestive of founder effect. We found the allele frequency of this variant in the Karen population to be 0.01. The c.2263CT (p.Arg755Trp) mutation was found in one patient with mild phenotype and the novel truncating protein mutation c.2127delG (p.Gly710fsx*25) in SCL4A11 was identified in two Thai sisters. Visual outcome and graft survival after PK were satisfactory. Our study shows that all studied patients with SLC4A11 mutations had CHED and sensorineural hearing loss, and SLC4A11 mutations were not related to the onset and severity of hearing loss or outcome of keratoplasty.

Enzymes involved in β-glucan breakdown in plants include endoglucanases, exoglucanases and β-glucosidases. Glycoside hydrolase family 3 (GH3) exoglucanases from barley and maize and a few plant GH3 β-glucosidases have been characterized, but none from rice. A few of these enzymes have been expressed in recombinant yeast and plant systems, but bacterial expression of plant GH3 enzymes has not been successful. We expressed the rice GH3 exoglucanase OsExo2 in Escherichia coli as a thioredoxin fusion protein, while other active plant GH3 enzymes could not be produced in this system. The protein was purified over 2000-fold in three chromatographic steps. The enzyme hydrolyzed β-1,3- and β-1,4-linked oligosaccharides and polysaccharides, consistent with a role in cell wall remodeling. Of the oligosaccharides tested, it had highest catalytic efficiency toward laminaritriose, (apparent kcat/Km = 37.7 mM−1s−1). Among polysaccharides, OsExoII hydrolyzed barley mixed β-glucan and laminarin with similar efficiencies (apparent kcat/Km = 3.7 and 3.4 mL mg−1 s−1, respectively), but achieved its highest apparent kcat with lichenan (2.9 s−1). OsExoII was found to be stimulated by ethylene glycol, which increased the apparent kcat and decreased the Km and was transglycosylated. These results imply that E. coli expression may be successful for certain plant GH3 enzymes and OsExoII may be a useful enzyme for application to glycoside production.

Glycoside hydrolases (GH) bind tightly to the sugar moiety at the glycosidic bond being hydrolyzed to stabilize its transition state conformation. We endeavored to assess the importance of glucose-binding residues in GH family 116 (GH116) β-glucosidases, which include human β-glucosylceramidase 2 (GBA2), by mutagenesis followed by kinetic characterization, X-ray crystallography, and ONIOM calculations on Thermoanaerobacterium xylanolyticum TxGH116, the structural model for GH116 enzymes. Mutations of residues that bind at the glucose C3OH and C4OH caused 27–196-fold increases in KM for p-nitrophenyl-β-D-glucoside, and significant decreases in the kcat, up to 5000-fold. At the C6OH binding residues, mutations of E777 decreased the kcat/KM by over 60,000-fold, while R786 mutants increased both the KM (40-fold) and kcat (2–4-fold). The crystal structures of R786A and R786K suggested a larger entrance to the active site could facilitate their faster rates. ONIOM binding energy calculations identified D452, H507, E777, and R786, along with the catalytic residues E441 and D593, as strong electrostatic contributors to glucose binding with predicted interaction energies > 15 kcal mol−1, consistent with the effects of the D452, H507, E777 and R786 mutations on enzyme kinetics. The relative importance of GH116 active site residues in substrate binding and catalysis identified in this work improves the prospects for the design of inhibitors for GBA2 and the engineering of GH116 enzymes for hydrolytic and synthetic applications.

A promising means of rapid screening of extended-spectrum-β-lactamase (ESBL), AmpC β-lactamase, and co-production of ESBL and AmpC that combines resazurin chromogenic agar (RCA) with a combined disc method is here reported. Cefpodoxime (CPD) discs with and without clavulanic acid (CA), cloxacillin (CX) and CA+CX were evaluated against 86 molecularly confirmed β-lactamase-producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, nine co-producers of ESBL and AmpC and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also performed well in screening for co-existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is simple and inexpensive and provides results within 7 hr. It can be performed in any microbiological laboratory, in particular, in geographic regions in which ESBL, AmpC or co-β-lactamase-producing Enterobacteriaceae are endemic.

Hunter syndrome (or mucopolysaccharidosis type II, MPS II) is an X-linked recessive disorder induced by a deficiency of the iduronate 2-sulfatase (IDS) enzyme, resulting in the accumulation of glycosaminoglycan substrates, heparan sulfate and dermatan sulfate, in the lysosomes. The progressive accumulation of undegraded metabolites induces cell and tissue dysfunction, leading to multi-systemic pathology. The heterogeneity of clinical phenotypes, ranging from mild to severe forms, results from different mutations in the IDS gene. To date, >550 MPS II causal mutations have been reported in the IDS gene, of which ~10% are nonsense mutations that lead to premature protein termination. In the present study, the IDS mutation causing MPS II in an extended Thai family was identified using IDS enzyme assay and IDS gene exon sequencing. Three family members were enzymatically confirmed to have MPS II and to carry the novel IDS nonsense allele c.928C>T (p.Gln310*). The IDS mRNA levels were evaluated by reverse transcription-quantitative polymerase chain reaction, which demonstrated that all patients exhibited a reduction of IDS mRNA, suggesting its degradation by nonsense-mediated mRNA decay. Expression of wild type and mutant IDS in COS-7 cells revealed that the IDS p.Gln310* mutant lacked IDS activity, consistent with production of a nonfunctional, prematurely truncated protein. Taken together, these results indicate that the IDS c.928C>T (p.Gln310*) mutation is a severe disease-causing mutation for MPS II.