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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.

α-Glucosyl triazoles have rarely been tested as α-glucosidase inhibitors, partly due to inefficient synthesis of their precursor α-d-glucosylazide (αGA1). Glycosynthase enzymes, made by nucleophile mutations of retaining β-glucosidases, produce αGA1 in chemical rescue experiments. Thermoanaerobacterium xylanolyticus glucosyl hydrolase 116 β-glucosidase (TxGH116) E441G nucleophile mutant catalyzed synthesis of αGA1 from sodium azide and pNP-β-d-glucoside (pNPGlc) or cellobiose in aqueous medium at 45 °C. The pNPGlc and azide reaction product was purified by Sephadex LH-20 column chromatography to yield 280 mg of pure αGA1 (68% yield). αGA1 was successfully conjugated with alkynes attached to different functional groups, including aryl, ether, amine, amide, ester, alcohol, and flavone via copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reactions. These reactions afforded the 1,4-substituted 1,2,3-triazole-α-d-glucoside derivatives AGT2-14 without protection and deprotection. Several of these glucosyl triazoles exhibited strong inhibition of human lysosomal α-glucosidase, with IC50 values for AGT4 and AGT14 more than 60-fold lower than that of the commercial α-glucosidase inhibitor acarbose.

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.

Summary Background Juberg-Hayward syndrome (JHS; MIM 216100) is a rare autosomal recessive malformation syndrome, characterized by cleft lip/palate, microcephaly, ptosis, short stature, hypoplasia or aplasia of thumbs, and dislocation of radial head and fusion of humerus and radius leading to elbow restriction. Objective To report for the first time the molecular aetiology of JHS. Patient and methods Clinical and radiographic examination, whole exome sequencing, Sanger sequencing, mutant protein model construction, and in situ hybridization of Esco2 expression in mouse embryos were performed. Results Clinical findings of the patient consisted of repaired cleft lip/palate, microcephaly, ptosis, short stature, delayed bone age, hypoplastic fingers and thumbs, clinodactyly of the fifth fingers, and humeroradial synostosis leading to elbow restriction. Intelligence is normal. Whole exome sequencing of the whole family showed a novel homozygous base substitution c.1654C>T in ESCO2 of the proband. The sister was homozygous for the wildtype variant. Parents were heterozygous for the mutation. The mutation is predicted to cause premature stop codon p.Arg552Ter. Mutations in ESCO2, a gene involved in cohesin complex formation, are known to cause Roberts/SC phocomelia syndrome. Roberts/SC phocomelia syndrome and JHS share similar clinical findings, including autosomal recessive inheritance, short stature, cleft lip/palate, severe upper limb anomalies, and hypoplastic digits. Esco2 expression during the early development of lip, palate, eyelid, digits, upper limb, and lower limb and truncated protein model are consistent with the defect. Conclusions Our study showed that Roberts/SC phocomelia syndrome and JHS are allelic and distinct entities. This is the first report demonstrating that mutation in ESCO2 causes JHS, a cohesinopathy.

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.

Correction for ‘Spatiotemporal distribution and speciation of silver nanoparticles in the healing wound’ by Marco Roman et al., Analyst, 2020, 145, 6456–6469, DOI: 10.1039/D0AN00607F.

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.

Understanding, modeling and simulating the behavior of thermally and electrically conductive materials under simultaneous high electric current pulse and mechanical preload conditions has long been a topic of interest for various applications involving electromechanical systems. To this end, the present work describes a computational framework that enables the fully coupled electromagnetic and thermoelastic analysis of such systems. The partial differential equations (PDEs) representing the electrodynamic and thermodynamic conservation laws are utilized and encapsulated in a computational environment enabling their numerical solution. A specific contribution of the framework is that it is capable of solving the non-linear forms of the relevant PDEs that are formed due to the dependence of the material properties on state variables such as temperature. The proposed framework is applied for a specific high-current testing apparatus under construction in our laboratory. A high current pulse is conducted through a mechanically pretensioned specimen and generates Joule heating activating thermo-elastic strains in conjunction with Lorentz body forces influencing the associated dynamic thermo-structural response of specimens of interest. Application of the developed framework enables the generation of field predictions for the quantities of interest. Selective simulation results are presented to demonstrate the capabilities of the proposed framework followed by discussion and conclusions.

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.

We present the results obtained using an original open-source low-cost sensor (LCS) system developed to measure tropospheric O3 in a remote high altitude alpine site. We conducted our study at the Col Margherita Observatory (2543 m a.s.l.), a World Meteorological Organization Global Atmosphere Watch Regional Station (WIGOS Id: 0-380-0-MRG), located in the Italian Eastern Alps. The sensing system mounts three equivalent commercial low-cost sensors that have been calibrated using a laboratory standard (Thermo 49iPS), referenced to the Standard Reference Photometer #15 calibration scale by the WMO, before field deployment. Intra and inter-comparison between sensors and reference (Thermo 49c) have been conducted for six months from May to December 2018. The sensor’s dependence on the environmental meteorological variables has been considered and discussed. The evaluation of the analytical performances of this sensing system provides a limit of detection < 5 ppb, limit of quantitation < 17 ppb, linear dynamic range up to 250 ppb, intra-Pearson correlation coefficient (PCC) up to 0.96, inter-PCC > 0.8, bias > 3.5 ppb and ±8.5 at 95% of confidence. Thanks to the first implementation of an LCS System in an alpine site, we demonstrated how it is possible to obtain valuable data from a low-cost instrument in a remote harsh environment. This opens new perspectives for the adoption of a low-cost sensor network in atmospheric sciences. We further present our recent experience using LoRa to integrate the sensing system into a low-power wide-area network (LPWAN). We developed an end-node and a gateway, designing PCBs derived from the Arduino Mega, optimizing their power consumption and equipping them with batteries, a proper solar panel or wind turbine to ensure their autonomy while collecting environmental ozone and meteorological (T, RH, WS, WD) data. We drafted the communication software to send compressed data from end-nodes to gateways. The gateways are part of an openVPN with the main server located in Venice. The server also provides a postgreSQL database and a R-shiny web application for data visualization and manipulation. To enhance redundancy, the local data are also synchronized to a cloud database. In the next years, thanks to the Marie Skłodowska-Curie grant PIONEER, we will exploit our experiences to provide a comprehensive low-cost wireless sensor network to characterize transport of polluted air masses and provide long term climate data collection in support of the state-of-the-art instrumentation and established networks in remote alpine areas.BibliographyDallo F. et al.: Calibration and assessment of electrochemical low-cost sensors in remote alpine harsh environments, Atmospheric Measurement Techniques, amt-2020-483. In reviewAcknowledgements. This work was part of the Arctic Field Grant O3NET project, funded by the Research Council of Norway. The work received financial support by the National Project of Interest Next-Data (MIUR). The exploitation of the LoRa technology was performed with the ITIS "Max Planck" through the Remote Observatory SYstem (ROSY) "Alternanza scuola-lavoro" project. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 844526

Nearly all ice core archives from the Arctic and middle latitudes (such as the Alps), apart from some very high elevation sites in Greenland and the North Pacific, are strongly influenced by melting processes. The increases in the average Arctic temperature has enhanced surface snow melting even of higher elevation ice caps, especially on the Svalbard Archipelago. The increase of the frequency and altitude of winter “rain on snow” events as well as the increase of the length of the melting season have had a direct impact on the chemical composition of the seasonal and permanent snow layers due to different migration processes of water-soluble species, such as inorganic ions. This re-allocation along the snowpack of ionic species could significantly modify the original chemical signal present in the annual snow. This paper aims to give a picture of the evolution of the seasonal snow strata with a daily time resolution to better understand: a) the processes that can influence deposition b) the distribution of ions in annual snow c) the impact of the presence of liquid water on chemical re-distribution within the annual snow pack. Specifically, the chemical composition of the first 100 cm of seasonal snow on the Austre Broggerbreen Glacier (Spitsbergen, Svalbard Islands, Norway) was monitored daily from the 27th of March to the 31st of May 2015. The experimental period covered almost the entire Arctic spring until the melting season. This unique dataset gives us a daily picture of the snow pack composition, and helps us to understand the behaviour of cations (K+, Ca2+, Na+, Mg2+) and anions (Br−, I−, SO42−, NO3−, Cl−, MSA) in the Svalbard snow pack. We demonstrate that biologically related depositions occur only at the end of the snow season and that rain and melting events have different impacts on the snowpack chemistry.

In the field of air analysis, highlights within the period covered by this review included a wearable air sampler with an inbuilt GPS sensor which enabled autonomous sampling to be performed at specific sites once the device had been preprogrammed with the requisite location coordinates. A prototype high resolution laser absorption spectroscopic instrument was developed as a potential SI-traceable alternative to ID-CV-ICP-MS for determining mercury concentrations in air and its isotopic composition. Improvements in the capabilities of single-particle aerosol mass spectrometers were noted. New measurements using tools such as LDI-MS and PTR-MS are providing new insights into the composition of carbonaceous particles. A large amount of effort continues to be directed towards multi-element preconcentration methods for water analysis, using either liquid or solid phase extraction to boost the sensitivity of instrumentation. Species-specific preconcentration methods were developed for the determination of mercury or arsenic species and methodologies employed to isolate elements from matrices such as seawater before isotope-ratio determinations. The focus of elemental speciation has shifted towards elemental fractionation with the determination of nano-sized fractions. The advent of ICP-MS/MS instrumentation has made the analysis of non-metallic elements such as F or P possible with improved detection limits. An interesting development of the isotope dilution technique was the use of isotopically labelled cell gasses to investigate the possibility of carrying out isotope dilution analysis of monoisotopic elements such as As or Y. Much effort has been devoted to characterising new and existing matrix-matched reference materials for calibration and quality control in the determination of the elemental and isotopic composition of geological samples. Zircon geochronology is a very active area of research and there is now considerable interest in developing U-Pb dating methods for other accessory minerals and carbonates. The application of in situ techniques such as LA-ICP-MS, SIMS, and LIBS to obtain geochemical information at high spatial resolution is another continuing theme. The ongoing interest in the optimisation of methods for extraction of analytes from soil and plants included developments relating to nanoparticles, estimation of bioavailability and non-chromatographic speciation analysis. The relative maturity of the AAS, AES and AFS techniques means that there have been few reports of major advances in these fields. Many of the developments in ICP-MS have been driven by the increased availability of ICP-MS/MS instrumentation. The importance of sample preparation in LIBS has increasingly been recognised. Proximal analysis - in which chemometric approaches are applied to data obtained by atomic (and molecular) spectroscopy to infer physical or chemical properties of a sample e.g. the pH of a soil - are becoming more common, in particular in relation to field-portable techniques such as LIBS and pXRFS, with increased attention being paid to the opportunities arising from data fusion.

Nucleocidin 1 produced by