Late onset of type 2 diabetes is associated with mitochondrial tRNATrp A5514G and tRNASer(AGY) C12237T mutations

BackgroundMitochondrial dysfunctions caused by mitochondrial DNA (mtDNA) pathogenic mutations play putative roles in type 2 diabetes mellitus (T2DM) progression. But the underlying mechanism remains poorly understood.MethodsA large Chinese family with maternally inherited diabetes and deafness (MIDD) underwent clinical, genetic, and molecular assessment. PCR and sequence analysis are carried out to detect mtDNA variants in affected family members, in addition, phylogenetic conservation analysis, haplogroup classification, and pathogenicity scoring system are performed. Moreover, the GJB2, GJB3, GJB6, and TRMU genes mutations are screened by PCR‐Sanger sequencing.ResultsSix of 18 matrilineal subjects manifested different clinical phenotypes of diabetes. The average age at onset of diabetic patients is 52 years. Screening for the entire mitochondrial genomes suggests the co‐existence of two possibly pathogenic mutations: tRNA^Trp A5514G and tRNA^Ser(AGY) C12237T, which belongs to East Asia haplogroup G2a. By molecular level, m.A5514G mutation resides at acceptor stem of tRNA^Trp (position 3), which is critical for steady‐state level of tRNA^Trp. Conversely, m.C12237T mutation occurs in the variable region of tRNA^Ser(AGY) (position 31), which creates a novel base‐pairing (11A‐31T). Thus, the mitochondrial dysfunctions caused by tRNA^Trp A5514G and tRNA^Ser(AGY) C12237T mutations, may be associated with T2DM in this pedigree. But we do not find any functional mutations in those nuclear genes.ConclusionOur findings suggest that m.A5514G and m.C12337T mutations are associated with T2DM, screening for mt‐tRNA mutations is useful for molecular diagnosis and prevention of mitochondrial diabetes.     

tRNAIni CAT inhibits proliferation and promotes apoptosis of laryngeal squamous cell carcinoma cells

BackgroundLaryngeal squamous cell carcinoma (LSCC) brings a heavy blow to the patient''s voice. Transfer RNA (tRNA) is a common RNA, the roles of tRNAs in LSCC are largely unknown.MethodsThe tRNA expression profile in LSCC tissues and adjacent normal tissues was measured by a tRNA qRT‐PCR array. The expression level of tRNA^Ini _CAT in LSCC tissues and plasmas was detected by qRT‐PCR. The receiver operating characteristic (ROC) curve was established. tRNA^Ini _CAT was upregulated by a lentivirus vector in the LSCC cell line. Moreover, tRNA^Ini _CAT was upregulated in LSCC xenograft nude mouse model and the xenografts were used for pathological analysis and transmission electron microscope (TEM) observation.ResultsThe top 10 upregulated tRNAs were tRNA^Lys _CTT‐1, tRNA^Leu _TAA, tRNA^Phe _GAA, tRNA^Leu _CAG, tRNA^Tyr _ATA, tRNA^Met _CAT, tRNA^Tyr _GTA‐1, tRNA^Thr _CGT, tRNA^Tyr _GTA‐2, tRNA^Ala _AGC; and the top 10 downregulated tRNAs were tRNA^Ini _CAT, mt‐tRNA^Glu _TTC, tRNA^Val _CAC‐3, mt‐tRNA^Trp _TCA, mt‐tRNA^Tyr _GTA, mt‐tRNA^Lys _TTT, mt‐tRNA^Thr _TGT, mt‐tRNA^Asp _GTC, mt‐tRNA^Asn _GTT, mt‐tRNA^Pro _TGG. tRNA^Ini _CAT was downregulated in LSCC tissues and plasma. The area under the ROC curve (AUC) in LSCC tissues and the plasma of patients with LSCC was 0.717 and 0.808, respectively. tRNA^Ini _CAT inhibited LSCC cell proliferation and promoted apoptosis. The in vivo results showed that tRNA^Ini _CAT inhibited the growth of the xenografts and promoted apoptosis.ConclusionsThis is the first study to provide tRNA expression profiles for LSCC tissues. tRNA^Ini _CAT may be used as a new biomarker for the early diagnosis of LSCC. tRNA^Ini _CAT inhibits cell proliferation and promotes apoptosis in vitro and in vivo.     

Maternally transmitted nonsyndromic hearing impairment may be associated with mitochondrial tRNAAla 5601C>T and tRNALeu(CUN) 12311T>C mutations

BackgroundSequence alternations in mitochondrial genomes, especially in genes encoding mitochondrial tRNA (mt‐tRNA), were the important contributors to nonsyndromic hearing loss (NSHL); however, the molecular mechanisms remained largely undetermined.MethodsA maternally transmitted Chinese pedigree with NSHL underwent clinical, genetic, and biochemical assessment. PCR and direct sequence analyses were performed to detect mitochondrial DNA (mtDNA), GJB2, and SLC26A4 gene mutations from matrilineal relatives of this family. Mitochondrial functions including mitochondrial membrane potential (MMP), ATP, and ROS were evaluated in polymononuclear leukocytes (PMNs) derived from three deaf patients and three controls from this pedigree.ResultsFour of nine matrilineal relatives developed hearing loss at the variable age of onset. Two putative pathogenic mutations, m.5601C>T in tRNA^Ala and m.12311T>C in tRNA^Leu(CUN), were identified via PCR‐Sanger sequencing, as well as 34 variants that belonged to mtDNA haplogroup G2b2. Intriguingly, m.5601C>T mutation resided at very conserved nucleotide in the TψC loop of tRNA^Ala (position 59), while the T‐to‐C substitution at position 12311 located at position 48 in the variable stem of tRNA^Leu(CUN) and was believed to alter the aminoacylation and the steady‐state level of tRNA. Biochemical analysis revealed the impairment of mitochondrial functions including the significant reductions of ATP and MMP, whereas markedly increased ROS levels were found in PMNs derived from NSHL patients with m.5601C>T and m.12311T>C mutations. However, we did not detect any mutations in GJB2 and SLC26A4 genes.ConclusionOur data indicated that mt‐tRNA^Ala m.5601C>T and tRNA^Leu(CUN) 12311T>C mutations were associated with NSHL.     

Mutations in the selenocysteine insertion sequence-binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans

Selenium, a trace element that is fundamental to human health, is incorporated into some proteins as selenocysteine (Sec), generating a family of selenoproteins. Sec incorporation is mediated by a multiprotein complex that includes Sec insertion sequence-binding protein 2 (SECISBP2; also known as SBP2). Here, we describe subjects with compound heterozygous defects in the SECISBP2 gene. These individuals have reduced synthesis of most of the 25 known human selenoproteins, resulting in a complex phenotype. Azoospermia, with failure of the latter stages of spermatogenesis, was associated with a lack of testis-enriched selenoproteins. An axial muscular dystrophy was also present, with features similar to myopathies caused by mutations in selenoprotein N (SEPN1). Cutaneous deficiencies of antioxidant selenoenzymes, increased cellular ROS, and susceptibility to ultraviolet radiation-induced oxidative damage may mediate the observed photosensitivity. Reduced levels of selenoproteins in peripheral blood cells were associated with impaired T lymphocyte proliferation, abnormal mononuclear cell cytokine secretion, and telomere shortening. Paradoxically, raised ROS in affected subjects was associated with enhanced systemic and cellular insulin sensitivity, similar to findings in mice lacking the antioxidant selenoenzyme glutathione peroxidase 1 (GPx1). Thus, mutation of SECISBP2 is associated with a multisystem disorder with defective biosynthesis of many selenoproteins, highlighting their role in diverse biological processes.     

Selenoproteins in bladder cancer

Selenoproteins with genetically encoded selenium (Se) are very important in response to oxidative stress, redox balance and regulation of various metabolic and developmental processes. Although increased circulating Se has been associated with 33% risk reduction of bladder cancer, there are little data on selenoprotein expression at the protein and genetic level from both human and animal studies. Data from the Mammalian Gene Collection (MGC) Project clearly showed that highest mRNA expression in human urinary epithelium for TRXR1 (thioredoxin reductase 1), GPX1 (glutathione peroxidase 1), SEP15 (15 kDa selenoprotein), SELT (selenoprotein T) and SEPW1 (selenoprotein W1). Although bladder tumor has been characterized by increased Se, GPX and TRXR activity, circulating Se and GPX was interestingly decreased in these cancer patients. As such, selenoprotein expression in urinary epithelium may be involved in bladder cancer (development, progression and recurrence) and may play a significant role in chemotherapeutic intervention. Despite these findings, the role of selenoproteins in bladder cancer has rarely been investigated and the significance of selenoproteins in normal and malignant uroepithelium remains poorly understood.     

Clinical and molecular genetic analysis of a Chinese family with hereditary elliptocytosis caused by a novel mutation in the EPB41 gene

BackgroundHereditary elliptocytosis (HE) is a heterogeneous red blood cell membrane disorder characterized by the presence of elliptocytes on a peripheral blood smear. Clinical manifestations of HE vary widely from asymptomatic carriers to patients with severe transfusion‐dependent anemia. Most patients are asymptomatic or have mild anemia, which hinders diagnosis. The proband in this case had mild anemia and jaundice over a period of 4 years, the etiology of which was unclear. Hence, he was admitted to our hospital for further diagnosis.MethodsPeripheral blood smears and routine blood tests were performed and biochemical parameters of the proband, and his family members were determined. To confirm the diagnosis, gene mutations were screened in the proband using next‐generation sequencing (NGS) and verified by Sanger sequencing in other family members.ResultsA novel mutation (c.1294delA, p.Ser432 fs) in exon 15 of the EPB41 gene was detected in the proband and his family members. This mutation results in a frameshift and a premature stop codon at position 455, encoding a truncated protein. The variant was likely pathogenic according to the criteria of the American College of Medical Genetics and Genomics. SWISS‐MODEL protein structure prediction indicated partial loss of the spectrin and actin binding and C‐terminal domains.ConclusionA heterozygous mutation 1294delA in exon 15 of the EPB41 gene was identified using NGS and Sanger sequencing in members of a Chinese family. This identification expands the spectrum of EPB41 mutations and contributes to the genetic diagnosis of families with HE.     

遗传性凝血因子X缺陷症三例及其分子发病机制研究

目的 对3个遗传性FX缺陷症家系进行临床表型和基因型研究,并探讨其分子发病机制.方法 对3例先证者进行止凝血筛查,检测APTT和PT;利用凝固法和ELISA检测FX∶C和FX∶Ag.采用交叉纠正试验排除血浆中FX抑制物的存在,采用凝血酶生成试验检测3例先证者及健康对照者血浆中凝血酶的生成.采用蛋白印迹半定量方法检测血浆中FX抗原浓度和相对分子质量大小.对FX基因采用直接测序进行基因诊断.构建突变型表达质粒,瞬时转染293T细胞,分别测定转染细胞裂解液及培养上清液中FX∶Ag,测定上清液中FX∶C,实验重复3次.结果 先证者1的APTT和PT均明显延长,分别为113.4 s和62.3 s,交叉纠正试验被纠正,血浆中几乎没有凝血酶的生成.先证者2的APTT和PT分别为56.5 s和28.7 s,交叉纠正试验也被纠正,血浆中凝血酶生成为1 101.5 nmol·min.先证者3的APTT和PT分别为117.3 s和44.3 s,交叉纠正试验被纠正,凝血酶生成为782.5 nmol·min.先证者1的FX∶C和FX∶Ag分别为1.4%和3.6%,基因诊断结果显示,FX基因存在纯合突变Ser425→Pro,该突变体外表达显示能够在细胞中正常合成,但分泌障碍;先证者2的FX∶C和FX∶Ag分别为2.2%和5.5%,FX基因存在双杂合突变Ala-29→Pro和Phe324→Leu,Ala-29→Pro突变导致FX表达量在细胞裂解液和培养上清液中均明显减少,分别为野生型质粒的(41.32±5.21)%和(6.30±1.84)%,而Phe324→Leu突变在细胞中几乎不影响FX的合成;先证者3的FX∶C和FX∶Ag分别为2.2%和35%,FX基因存在双杂合突变Ala235→Thr和Arg347→Cys,这2种突变使FX在细胞裂解液中与野生型蛋白表达量相似,而细胞上清液中较野生型蛋白明显减低,分别为野生型的(23.03±1.92)%和(42.51±2.07)%.结论 本研究发现了5种新的FX基因突变;其中Ser425Pro、Phe324Leu、Ala235 Thr和Arg347Cys突变不影响FX突变蛋白的合成,而Ala-29Pro突变则导致FX突变蛋白合成减少,伴分泌障碍.     

遗传性蛋白S缺陷症一个新的基因突变

目的 研究一个遗传性蛋白S（PS）缺陷家系的遗传表型及基因型特征。方法 PS活性用凝固法测定,PS抗原用ELISA方法测定。用聚合酶链反应扩增5代家系34个成员中9个PS活性及抗原减低的PS1－15号外显子片段,用单链构象多态性（SSCP）分析cDNA变性后的差异,用测序方法检测突变点。结果 该家系5代9名成员游离PS抗原在10％－45％（正常值为55％－128％）。PS活性在13％－37％（正常值为70％－130％）,较正常参考范围明确降低,二者呈平行下降,但总PS抗原均在正常范围。在这些成员中均检测到10号外显子第163位核苷酸G→T突变,使AGT→ATT,在mRNA转录时,转录为终止密码子,阻断蛋白质的合成。结论 本家系的Ⅲ型PS缺陷症基因分析证明,先证者为杂合子型。在PS10号外显子上第163位核苷酸发生G→突变,在蛋白质合成过程中丝氨酸被终止密码子替代,为目前文献中尚未报道的一个新的基因突变点。     

Synthesis of new Pro-PYE ligands as co-catalysts toward Pd-catalyzed Heck–Mizoroki cross coupling reactions

The present research work describes the synthesis of five new ligands containing pyridinium amine, [H₂L¹][OTf]₂–[H₂L⁵][I]₂ from two new precursors, [P³_Et][I] and [P²_Me][CF₃SO₃]. The structure elucidations of the compounds were confirmed by multinuclear NMR (¹H, ¹³C), FT-IR and by single crystal XRD techniques. Theoretical DFT studies were carried out to get better insight into the electronic levels and structural features of all the molecules. These synthesized new Pro-PYE ligands [H₂L¹][OTf]₂–[H₂L⁵][I]₂ were found to be significantly active as co-catalysts for Pd(CH₃CO₂)₂ toward Heck–Mizoroki coupling reactions with wide substrate scope in the order of [H₂L¹][OTf]₂ ≫ [H₂L²][OTf]₂ > [H₂L³][OTf]₂ > [H₂L⁴][OTf]₂ > [H₂L⁵][I]_2.

The present research work describes the synthesis of five new ligands containing pyridinium amine, [H₂L¹][OTf]₂–[H₂L⁵][I]₂ from two new precursors, [P³_Et][I] and [P²_Me][CF₃SO₃].     

HDL metabolic activities in a boy with lipoprotein lipase deficiency and his family

BACKGROUND: Lipoprotein lipase (LPL) deficiency is a rare autosomal recessively inherited disease characterized by elevated triglyceride, low total cholesterol and quantitative and qualitative alterations of high-density lipoprotein (HDL). The aim of the present study was to explore HDL metabolic activities in a patient with LPL deficiency and in his family (n = 11). MATERIALS AND METHODS: Subjects were divided into four groups: proband (Ser447Stop/Arg170Leu carrier), Ser447Stop carriers, Arg170Leu carriers and silent mutation/wild-type carriers (controls). Cholesterol efflux from Fu5AH cells, lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), paraoxonase 1 (PON1) and platelet-activating factor acetylhydrolase (PAF-AH) activities were evaluated. RESULTS: Comparison between the proband and the control group revealed that the boy had significantly reduced cholesterol efflux (P < 0.001), conserved LCAT activity (P > 0.05) and increased CETP activity (P < 0.001). As regards antioxidant enzymes, while PON1 activity was higher in the proband than in the controls (P < 0.0001), PAF-AH activity was reduced (P < 0.05). The other groups did not show relevant differences in comparison with controls. CONCLUSIONS: The presence of one mutation was not enough to introduce important modifications in HDL functions. Markedly reduced HDL levels can keep certain normal enzymatic activities, which probably tend to counteract the deleterious effects of LPL deficiency.     

Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli

Amdinocillin (mecillinam) is a β-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 × 10⁻⁸ to 2 × 10⁻⁵ per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. The cysB gene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates of E. coli.     

The Rate of Synthesis of Glycosaminoglycans and Collagen by Fibroblasts Cultured from Adult Human Liver Biopsies

Adult human liver biopsies were cultured from normal, alcoholic hepatitis, chronic active hepatitis, fibrosis plus alcoholic hepatitis (active cirrhosis), inactive cirrhosis, and drug hepatitis. The synthesis of collagen was estimated in cultures from 58 livers by measuring the conversion of [¹⁴C]proline to the [¹⁴C]hydroxyproline of collagen; that of glycosaminoglycans in cultures from 57 livers by the incorporation of [³H]acetate and ³⁵SO₄ into glycosaminoglycans (GAG). The synthesis of procollagen was increased only in cultures from alcoholic hepatitis, both in the pulse medium (P < 0.05) and in the chase medium (P < 0.02). The synthesis of insoluble collagen was increased in cultures from chronic (active) hepatitis (P < 0.01), fibrosis plus alcoholic hepatitis (active cirrhosis) (P < 0.001), and inactive cirrhosis (P < 0.05). Essentially all radioactive GAG was soluble in culture media. The predominant GAG were chondroitin-4 or -6-SO₄. The synthesis of GAG was increased only in cultures from fibrosis plus alcoholic hepatitis (active cirrhosis) both in the pulse medium (P < 0.01) and chase medium (P < 0.001).     

The application of amino acid ionic liquids as additives in the ultrasound-assisted extraction of plant material

The aim of the present study was to determine the antioxidant activity of the aqueous extracts from Lycopodium clavatum, Cetraria islandica and Dipsacus fullonum obtained using aqueous solutions of ionic liquids by the ultrasound-assisted extraction (IL-UAE) method. Triethanolammonium salts [TEAH]⁺[AA]⁻ of four amino acids of different hydrophobicity – isoleucine – Ile, methionine – Met, threonine – Thr and arginine – Arg, were chosen as ionic liquids, because they are based on natural, bio-renewable raw materials, such as amino acids and contain a pharmaceutically and cosmetically acceptable counterion of triethanolamine. Triethanolammonium salts were synthesized, identified by spectroscopic methods (NMR and FT-IR) and characterized by thermal methods (DSC and TGA). The 2.5% w/v aqueous solutions of triethanolammonium amino acid salts were used as the solvents in combination with ultrasound assisted extraction (UAE). The estimation of antioxidant properties was carried out using the DPPH, FRAP and CUPRAC assays. Total polyphenol content was measured using the reagent Folin–Ciocalteu. The results showed that the use of [TEAH]⁺[Thr]⁻ or [TEAH]⁺[Met]⁻ aqueous solutions increased the antioxidant activity of extracts in comparison to that achieved for extracts with pure water. The use of [TEAH]⁺[Thr]⁻ as an additive for ultrasound-assisted extraction was characterized by obtaining plant extracts with the highest antioxidant potential, even 2.4-fold. The use of the AAIL-UAE method allowed obtaining higher amounts of polyphenols compared to pure water extracts, even 5.5-fold. The used method allowed the extraction of thermosensitive natural compounds, shortened the extraction time and lowered energy consumption.

The antioxidant activity of the aqueous extracts from Lycopodium clavatum, Cetraria islandica and Dipsacus fullonum obtained by ionic liquids and ultrasound-assisted extraction (IL-UAE) method was determined.     

Synthesis and evaluation of 3′-[18F]fluorothymidine-5′-squaryl as a bioisostere of 3′-[18F]fluorothymidine-5′-monophosphate

The squaryl moiety has emerged as an important phosphate bioisostere with reportedly greater cell permeability. It has been used in the synthesis of several therapeutic drug molecules including nucleoside and nucleotide analogues but is yet to be evaluated in the context of positron emission tomography (PET) imaging. We have designed, synthesised and evaluated 3′-[¹⁸F]fluorothymidine-5′-squaryl ([¹⁸F]SqFLT) as a bioisostere to 3′-[¹⁸F]fluorothymidine-5′-monophosphate ([¹⁸F]FLTMP) for imaging thymidylate kinase (TMPK) activity. The overall radiochemical yield (RCY) was 6.7 ± 2.5% and radiochemical purity (RCP) was >90%. Biological evaluation in vitro showed low tracer uptake (<0.3% ID mg⁻¹) but significantly discriminated between wildtype HCT116 and CRISPR/Cas9 generated TMPK knockdown HCT116^shTMPK−. Evaluation of [¹⁸F]SqFLT in HCT116 and HCT116^shTMPK− xenograft mouse models showed statistically significant differences in tumour uptake, but lacked an effective tissue retention mechanism, making the radiotracer in its current form unsuitable for PET imaging of proliferation.

[¹⁸F]SqFLT was developed to bypass thymidine kinase 1 (TK1) and evaluated for PET imaging of DNA synthesis.     

Colistin-Resistant Acinetobacter baumannii Clinical Strains with Deficient Biofilm Formation

In two pairs of clinical colistin-susceptible/colistin-resistant (Cst^s/Cst^r) Acinetobacter baumannii strains, the Cst^r strains showed significantly decreased biofilm formation in static and dynamic assays (P < 0.001) and lower relative fitness (P < 0.05) compared with those of the Cst^s counterparts. The whole-genome sequencing comparison of strain pairs identified a mutation converting a stop codon to lysine (*241K) in LpsB (involved in lipopolysaccharide [LPS] synthesis) in one Cst^r strain and a frameshift mutation in CarO and the loss of a 47,969-bp element containing multiple genes associated with biofilm production in the other.     

Modulation of Gene Expression by Human Cytosolic tRNase ZL through 5′-Half-tRNA

A long form (tRNase Z^L) of tRNA 3′ processing endoribonuclease (tRNase Z, or 3′ tRNase) can cleave any target RNA at any desired site under the direction of artificial small guide RNA (sgRNA) that mimics a 5′-half portion of tRNA. Based on this enzymatic property, a gene silencing technology has been developed, in which a specific mRNA level can be downregulated by introducing into cells a synthetic 5′-half-tRNA that is designed to form a pre-tRNA-like complex with a part of the mRNA. Recently 5′-half-tRNA fragments have been reported to exist stably in various types of cells, although little is know about their physiological roles. We were curious to know if endogenous 5′-half-tRNA works as sgRNA for tRNase Z^L in the cells. Here we show that human cytosolic tRNase Z^L modulates gene expression through 5′-half-tRNA. We found that 5′-half-tRNA^Glu, which co-immunoprecipitates with tRNase Z^L, exists predominantly in the cytoplasm, functions as sgRNA in vitro, and downregulates the level of a luciferase mRNA containing its target sequence in human kidney 293 cells. We also demonstrated that the PPM1F mRNA is one of the genuine targets of tRNase Z^L guided by 5′-half-tRNA^Glu. Furthermore, the DNA microarray data suggested that tRNase Z^L is likely to be involved in the p53 signaling pathway and apoptosis.     

Correction: Novel azobenzene-based amphiphilic copolymers: synthesis,self-assembly behavior and multiple-stimuli-responsive properties

Correction for ‘Novel azobenzene-based amphiphilic copolymers: synthesis, self-assembly behavior and multiple-stimuli-responsive properties’ by Yiting Xu et al., RSC Adv., 2018, 8, 16103–16113.

The final entry for Table 1 relating to Sample C-4 in the published paper showed an incorrect entry for the column [POSSMA] : [AZOMA] : [DMAEMA] : [CDB] : [AIBN]; this should read 12 : 50 : 100 : 0 : 0.20.The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Mechanism of Bilirubin Diglucuronide Formation in Intact Rats: BILIRUBIN DIGLUCURONIDE FORMATION IN VIVO

Although it is well established that bilirubin monoglucuronide is formed in the liver from bilirubin by a microsomal bilirubin uridine diphosphate (UDP)-glucuronosyltransferase, the subcellular site of conversion of monoglucuronide to diglucuronide and the molecular mechanism involved in diglucuronide synthesis have not been identified. Based on in vitro studies, it has been proposed that two fundamentally different enzyme systems may be involved in diglucuronide synthesis in rat liver: (a) a microsomal UDP-glucuronosyltransferase system requiring UDP-glucuronic acid as sugar donor or (b) a transglucuronidation mechanism that involves transfer of a glucuronosyl residue from one monoglucuronide molecule to another, catalyzed by a liver plasma membrane enzyme. To clarify the mechanism by which bilirubin monoglucuronide is converted in vivo to diglucuronide, three different experimental approaches were used. First, normal rats were injected with either equal amounts of bilirubin-IIIα [¹⁴C]monoglucuronide and unlabeled bilirubin-XIIIα monoglucuronide, or bilirubin-XIIIα [¹⁴C]monoglucuronide and unlabeled bilirubin-IIIα monoglucuronide. Analysis of radiolabeled diglucuronide excreted in bile showed that [¹⁴C]glucuronosyl residues were not transferred between monoglucuronide molecules. Second, in normal rats infused intravenously with dual-labeled [³H]bilirubin [¹⁴C]monoglucuronide, no transfer or exchange of the [¹⁴C]glucuronosyl group between injected and endogenously produced bilirubin monoglucuronide could be detected in the excreted bilirubin diglucuronide. Third, in homozygous Gunn rats, injected ¹⁴C-labeled or unlabeled bilirubin mono- or diglucuronides were excreted in bile unchanged (except that diglucuronide was hydrolyzed to a minor degree). This indicates that Gunn rats, which lack bilirubin UDP-glucuronosyltransferase activity, are unable to convert injected monoglucuronide to diglucuronide. Collectively, these findings establish that a transglucuronidation mechanism is not operational in vivo and support the concept that bilirubin diglucuronide is formed by a microsomal UDP-glucuronosyltransferase system.     

Protein S gene mutation c.946C > T (p.R316C) contributed to ischemic stroke in a man with von Willebrand disease type 3 caused by two novel VWF gene mutations,c.2328delT (p.A778Lfs* 23) and c.6521G > T (p.C2174F)

The risk factors for a family with VWD presenting with an ischemic stroke (IS) were explored. FVIII activity (FVIII:C), VWF antigen (VWF:Ag), and protein S activity were measured. Next generation sequencing (NGS) was performed targeting F8, F9, VWF, PROC, and PROS1. Sanger sequencing validation was performed on family members. The proband and his sister both had low FVIII:C (1 IU/dL) and VWF:Ag (3 IU/dL) levels, confirming the diagnosis of type 3 VWD. His father had nearly normal levels of FVIII:C (58 IU/dL) and VWF:Ag (57 IU/dL). His daughter had type 1 VWD with decreased FVIII:C (46 IU/dL) and VWF:Ag (19 IU/dL). NGS identified a heterozygous VWF c.2328delT (p.A778Lfs*23) frame shift mutation only in the proband and his sister. Another VWF missense mutation, c.6521G > T (p.C2174F), was found heterozygous in all members studied. A PROS1 mutation, c.946C > T (p.R316C), previously reported to relate to ischemic stroke, was found heterozygous in the patient, his father, and his daughter. Only the proband and daughter have a slightly decreased plasma protein S level. This may be the first case with type 3 VWD with severe VWF/FVIII deficiency presented with ischemic stroke contributed to by a protein S defect.     

Jejunal perfusion of simple and conjugated folates in tropical sprue

Absorption of labeled simple 3′,5′,9′-³H pteroylmonoglutamate, ([³H]PG-1) and conjugated pteroyl-μ[¹⁴C]glutamyl-γ-hexaglutamate, ([¹⁴C]PG-7) folates was assessed in six patients with tropical sprue, before and after 6 mo of treatment, utilizing jejunal perfusion and urinary recovery techniques. Degradation products of [¹⁴C]PG-7 which were produced during perfusion were identified by DEAE-cellulose column chromatography. Jejunal mucosal activities of folate conjugase, lactase, sucrase, and maltase were measured in every patient. Malabsorption of both [³H]PG-1 and [¹⁴C]PG-7 was found in every untreated patient, with significant improvement after therapy. The urinary excretion of ³H and ¹⁴C paralleled the luminal disappearance of both isotopes. The chromatographic patterns of intraluminal degradation products of [¹⁴C]PG-7 obtained during perfusion did not differ from those previously found in normal subjects and were similar in studies performed before and after treatment. The activity of folate conjugase was increased in the mucosa of the untreated patients when compared to the post-treatment levels while the activities of mucosal lactase, sucrase, and maltase were originally low and increased significantly after therapy. These observations suggest that folate conjugase originates at a different mucosal locus than the brush border disaccharidases, and are consistent with previous evidence that folate conjugase is an intracellular enzyme. The present studies have demonstrated unequivocal malabsorption of both simple and conjugated folates in tropical sprue. In tropical sprue, folate malabsorption is the reflection of impaired folate transport and not of impaired hydrolysis.