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Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the "activated" sugars used for biosynthesis of a wide range of glycoconjugates and serve as substrates themselves for the synthesis of other nucleotide sugars. NS analysis is complicated by the presence of multiple stereoisomers without diagnostic transition ions, therefore requiring separation by liquid chromatography. In this paper, we explored weak anion-exchange/reversed-phase chromatography on a hybrid column for the separation of 17 nucleotide sugars that can occur in humans. A robust and reproducible method was established with intra- and inter-day coefficients of variation below 10% and a linear range spanning three orders of magnitude. Application to patient fibroblasts with genetic defects in mannose-1-phosphate guanylyltransferase beta, CDP-l-ribitol pyrophosphorylase A, and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase showed abnormal levels of guanosine-5′-diphosphate-α-d-mannose (GDP-Man), cytidine-5′-diphosphate-l-ribitol (CDP-ribitol), and cytidine-5′-monophosphate-N-acetyl-β-d-neuraminic acid (CMP-Neu5Ac), respectively, in consonance with expectations based on the diagnosis. In conclusion, a novel, semi-quantitative method was established for the analysis of nucleotide sugars that can be applied to diagnose several genetic glycosylation disorders in fibroblasts and beyond. [ABSTRACT FROM AUTHOR]

Simple Summary: Ribosome biosynthesis has emerged as an excellent target for inhibition of cancer cell growth and proliferation. We used a high throughput screen to identify potent inhibitors of ribosome synthesis in malignant melanoma cells. Here, we show that the newly identified ribosome biogenesis inhibitor, RBI2, inhibits multiple cancer cell types by a mechanism of action that is distinct from previously described compounds. Using chromatin IP, isotopic labelling, and RNA sequencing, we conclude that treatment with RBI2 does not inhibit transcription initiation by RNA polymerase I, but appears to induce rapid polyadenylation and degradation of ribosomal RNA. This mechanism of action would be unique from previously described inhibitors of RNA polymerase I. Cancer cells are especially sensitive to perturbations in ribosome biogenesis as they rely on finely tuned protein homeostasis to facilitate their rapid growth and proliferation. While ribosome synthesis and cancer have a well-established relationship, ribosome biogenesis has only recently drawn interest as a cancer therapeutic target. In this study, we exploited the relationship between ribosome biogenesis and cancer cell proliferation by using a potent ribosome biogenesis inhibitor, RBI2 (Ribosome Biogenesis Inhibitor 2), to perturb cancer cell growth and viability. We demonstrate herein that RBI2 significantly decreases cell viability in malignant melanoma cells and breast cancer cell lines. Treatment with RBI2 dramatically and rapidly decreased ribosomal RNA (rRNA) synthesis, without affecting the occupancy of RNA polymerase I (Pol I) on the ribosomal DNA template. Next-generation RNA sequencing (RNA-seq) revealed that RBI2 and previously described ribosome biogenesis inhibitor CX-5461 induce distinct changes in the transcriptome. An investigation of the content of the pre-rRNAs through RT-qPCR revealed an increase in the polyadenylation of cellular rRNA after treatment with RBI2, constituting a known pathway by which rRNA degradation occurs. Northern blotting revealed that RBI2 does not appear to impair or alter rRNA processing. Collectively, these data suggest that RBI2 inhibits rRNA synthesis differently from other previously described ribosome biogenesis inhibitors, potentially acting through a novel pathway that upregulates the turnover of premature rRNAs. [ABSTRACT FROM AUTHOR]

Myotonic dystrophy type 1 (DM1), also known as Steinert's disease, is a chronic, progressive and disabling multisystemic disorder with a broad spectrum of severity that arises from an autosomal-dominant expansion of the Cytosine-Thymine-Guanine (CTG) triplet repeat in the 3′ untranslated region of the DMPK gene (19q13.3). In this study, we report the case of a family with several intergenerational expansions of the CTG repeat, with an additional case of a false suspicion of contraction phenomenon due to TP-PCR limitations. The meiotic instability of the (CTG)n repeats leads to genetic anticipation where increased size of DM1 mutation and a more severe phenotype have been reported in affected individuals across generations. Even if extremely rare, a decrease in the CTG repeat size during transmission from parents to child can also occur, most frequently during paternal transmissions. [ABSTRACT FROM AUTHOR]

Simple Summary: Two vascular endothelial growth factor (VEGF) receptors, FLT-1 and KDR, are expressed preferentially in proliferating endothelium. There is increasing evidence that soluble VEGF receptor domains that compete for VEGF binding may inhibit tumor angiogenesis, growth and metastatic spread. We tested whether soluble FLT-1 (sFLT-1) could inhibit breast cancer tumor growth in mice by blocking angiogenesis. A viral vector carrying the sflt-1 cDNA (Adsflt) was used to overexpress the receptor in MCF-7 cells, which produce VEGF. After six weeks, tumors were treated either with Adsflt or a negative control virus. After six months, average tumor volume in the Adsflt-treated group was decreased by 91% and vascular density was reduced 50%, relative to negative controls (p < 0.05), yet mice treated with Adsflt showed no delay in healing cutaneous wounds. These results provide evidence that viral sFLT-1 overexpression may be an effective anti-angiogenic therapy for cancer without the risk of systemic anti-angiogenic effects. Two vascular endothelial growth factor (VEGF) receptors, FLT-1 and KDR, are expressed preferentially in proliferating endothelium. There is increasing evidence that recombinant, soluble VEGF receptor domains interfering with VEGF signaling may inhibit in vivo neoangiogenesis, tumor growth and metastatic spread. We hypothesized that a soluble form of FLT-1 receptor (sFLT-1) could inhibit the growth of pre-established tumors via an anti-angiogenic mechanism. A replication-deficient adenovirus (Ad) vector carrying the sflt-1 cDNA (Adsflt) was used to overexpress the sFLT-1 receptor in a breast cancer animal model. MCF-7 cells, which produce VEGF, were used to establish solid tumors in the mammary fat pads of female nude mice. After six weeks, tumors were injected either with Adsflt or a negative control virus (AdCMV.βgal). After six months, average tumor volume in the Adsflt-infected group (33 ± 22 mm3) decreased by 91% relative to that of the negative control group (388 ± 94 mm3; p < 0.05). Moreover, 10 of 15 Adsflt-infected tumors exhibited complete regression. The vascular density of Adsflt-infected tumors was reduced by 50% relative to that of negative controls (p < 0.05), which is consistent with sFLT-1-mediated tumor regression through an anti-angiogenic mechanism. Moreover, cell necrosis and fibrosis associated with long-term regression of Adsflt–infected tumors were preceded by apoptosis of tumor vascular endothelial cells. Mice treated with Adsflt intratumorally showed no delay in the healing of cutaneous wounds, providing preliminary evidence that Ad-mediated sFLT-1 overexpression may be an effective anti-angiogenic therapy for cancer without the risk of systemic anti-angiogenic effects. [ABSTRACT FROM AUTHOR]

Objectives Anti‐TIF1-gamma autoantibodies can be detected with immunoprecipitation (IP), line blot (LB) and ELISA. We compared assay performance in patients with DM and the potential of these assays to detect anti-TIF1-gamma positive cancer-associated DM (CADM). Methods We included sera from 131 patients with DM followed at Karolinska University Hospital, Stockholm, Sweden and 82 healthy controls. Serum samples taken at DM diagnosis were tested for anti-TIF1-gamma autoantibodies with IP, two ELISAs (in-house and commercial) and LB. Cancer diagnosis and dates were obtained from the Swedish national cancer register. CADM was defined as a malignancy that developed within 3 years of DM diagnosis. Results Anti-TIF1-gamma autoantibodies were detected in 19/101 (18.8%), 15/113 (13.2%), 34/131 (26%) and 45/131 (34.4%) of the patients with IP, LB, in-house and commercial ELISA, respectively. The anti-TIF1-gamma results from the in-house ELISA were confirmed with IP in 93 of 101 (92%) cases, κ = 0.76, with a commercial ELISA in 110 of 131 (84%) cases, κ = 0.63, and with LB in 101 of 113 (89.3%) cases, κ = 0.67. Anti-TIF1-gamma results with IP were confirmed with LB in 85 of 92 (92.4%) cases, κ = 0.73. For detecting CADM, the anti-TIF1-gamma in-house ELISA had a sensitivity of 58% and specificity of 86%, the commercial ELISA had a sensitivity of 63% and specificity of 82%, IP had a sensitivity of 52% and specificity of 92%, LB had a sensitivity of 40% and specificity of 96%. Conclusion The two anti-TIF1-gamma ELISA assays had advantages both for autoantibody detection and to identify anti-TIF1-gamma-positive CADM. [ABSTRACT FROM AUTHOR]

• SPE techniques reduce matrix effect and improve Nt/Ns pretreatment and purification. • MPs and silica-fiber coating are remarkable sorbents for miniaturised SPE forms. • Chromatographic methods employing various detectors are used to detect Nt/Ns. • HILIC, along with tandem mass spectrometry, acquires prominence in Nt/Ns detection. Cordyceps genus is entomopathogenic mushrooms that have traditionally been used in ethnomedicine in Asian countries. Nucleosides (Ns), nucleotide(Nt), Nucleobases (Nb) and their analogues play a critically physiological role and have a great potential in drug development, such as pentostatin and cordycepin (COR). Due to their significance bioactivity, several Nt/Ns were used as markers for quality evaluation for medicinal Cordyceps, including adenosine, inosine, guanosine, uridine and COR. Among them, COR is the most considerable adenosine analogue, exhibiting significant therapeutic potential and has many intracellular targets. Nt/Ns contains polar compounds and the phosphate groups of Nt deprotonate and carry negative charges with a broad range of pH values. Recent years, various advanced methods of extraction and separation, and nanomaterials have been developed to extract, isolate and determine these molecules, such as ultrasound-assisted extraction (UAE), Supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) for the extraction, the solid phase extraction (SPE) methods (microextraction SPE (SPME), magnetic SPE (MSPE), and unique SPE materials based on the boronate affinity for the separation, and chromatography methods employing ultraviolet (UV), fluorescence, MS detection and electrospray ionization (ESI), along with matrix-assisted laser desorption/ ionization (MALDI) for the determination. COR derived from adenosine and its structure is very similar to that of 2′-deoxyadenosine (2′-dA) and adenosine, resulting in an incorrect identification, which will influence its therapeutic effects. Therefore, this review primarily focused on the characteristics of Nt/Ns, the advanced methods, strategies, nanomaterials for extracting and determining Nt/Ns (COR in particular) in Cordyceps spp, as well as the methods for distinguishing COR from its structure analogs. [ABSTRACT FROM AUTHOR]

A robust method using HPLC-UV was developed to improve the accuracy and repeatability of a quantitative method to detect 5 nucleotides (cytidine-monophosphate, uridine monophosphate, adenosine monophosphate, guanine monophosphate, and inosine monophosphate) in infant formulas. The results showed that efficient separation could not be achieved without strict conditions. The proposed method displayed a strong linear relationship (R2 > 0.9999) of single nucleotide in infant formula milk powder in the range of 10 to 1,000 mg/kg, a steady recovery (80.0% ∼110.0%) with relative standard deviation from 0.5% to 3.5%, under strict conditions of hydrophilic C 18 column with di-isopropyl at 62.5 ± 2.5°C (± standard deviation), 0.65 ± 0.1 mg/mL tetrabutylammonium bisulfate, and mobile phase of pH of 2.75 ± 0.02. By applying this method on a series of milk products in the Chinese market, we found a few of them exceeded the legal limits of nucleotides. [Display omitted] [ABSTRACT FROM AUTHOR]

Researchers increasingly wish to test hypotheses concerning the impact of environmental or disease exposures on telomere length (TL), and they use longitudinal study designs to do so. In population studies, TL is usually measured with a quantitative polymerase chain reaction (qPCR)-based method. This method has been validated by calculating its correlation with a gold standard method such as Southern blotting (SB) in cross-sectional data sets. However, in a cross-section, the range of true variation in TL is large, and measurement error is introduced only once. In a longitudinal study, the target variation of interest is small, and measurement error is introduced at both baseline and follow-up. In this paper, we present results from a small data set (n  = 20) in which leukocyte TL was measured twice 6.6 years apart by means of both qPCR and SB. The cross-sectional correlations between qPCR and SB were high at both baseline (r  = 0.90) and follow-up (r  = 0.85), yet their correlation for TL change was poor (r  = 0.48). Moreover, the qPCR data but not the SB data showed strong signatures of measurement error. Through simulation, we show that the statistical power gain from performing a longitudinal analysis is much greater for SB than for qPCR. We discuss implications for optimal study design and analysis. [ABSTRACT FROM AUTHOR]

The study focuses on engineering of recombinant Aspergillus niger to produce highly active xylanase. The xylanase G2 encoding gene originating from Aspergillus oryzae VTCC-F187 was cloned, amplified, and inserted into the pAN7.1GluA vector with specific primers possessing BamHI. The recombinant plasmid was introduced into Aspergillus niger VTCC-F017 by chemical methods. The recombinant strain was checked by polymerase chain reaction method and Southern blot. Next, the recombinant protein was expressed and purified by His-tag column. The molecular mass of the purified xylanase G2, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), was 21 kDa with a specific activity of 1025 IU/mg towards 0.5% (w/v) of birchwood xylan. The optimal temperature and pH were 55°C and pH 6.5, respectively. The enzyme was stable in a temperature ranges 25–40°C and a pH ranges 5–7. The presence of Tween 80 enhanced xylanase activity. Triton X-100, however, had no impact on the function of the enzyme. The xylanase activity was reduced by Tween 20, SDS, and organic solvents. The enzyme was completely inhibited by Hg2+ and partially by Zn2+, Fe2+, and Ag+, while it was slightly stimulated by K+ and EDTA. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is the most common type of dementia caused by irreversible neurodegeneration, with the onset mechanisms elusive. tRNA-derived RNA fragments (tRFs), a recently discovered family of small non-coding RNAs (sncRNAs), have been found to associate with many human diseases, including infectious, metabolic, and neurological diseases. However, whether tRFs play a role in human AD development is not known.Objective: This study aimed to explore whether tRFs are involved in human AD.Methods: Thirty-four postmortem human hippocampus samples were used. The expression of Drosha, Dicer, and angiogenin (ANG), three ribonucleases responsible for the biogenesis of sncRNAs, was determined by qRT-PCR and western blot. The tRFs in the hippocampus was detected by qRT-PCR or northern blot. We also used qRT-PCR to quantify NOP2/Sun RNA methyltransferase 2 (NSun2) and polyadenylation factor I subunit 1 (CLP1), two tRNA modification enzymes.Results: tRFs derived from a subset of tRNAs are significantly altered in the hippocampus of AD patients. The expression change of some tRFs showed age- and disease stage-dependent. ANG is significantly enhanced in AD, suggesting its role in inducing tRFs in AD. The expression of NSun2 in AD patients younger than 65 was significantly decreased. According to a previous report supporting NSun2-mediated tRNA methylation modification making tRNA less susceptible to ANG-mediated cleavage, our results suggested that the decrease in NSun2 may make tRNAs less methylated and subsequently enhanced tRF production from ANG-mediated tRNA cleavage.Conclusion: Our studies demonstrated for the first time the involvement of tRFs in human AD. [ABSTRACT FROM AUTHOR]

N-Linked glycosylation is a cellular process transferring sugars from glycosyl donors to proteins or lipids. Biopharmaceutical products widely produced by culturing mammalian cells such as Chinese hamster ovary (CHO) cells are typically glycosylated during biosynthesis. For some biologics, the N-linked glycan is a critical quality attribute of the drugs. Nucleotide sugars are the glycan donors and impact the intracellular glycosylation process. In current analytical methods, robust separation of nucleotide sugar isomers such as UDP glucose and UDP galactose remains a challenge because of their structural similarity. In this study, we developed a strategy to resolve the separation of major nucleotide sugars including challenging isomers based on the use of ion-pair reverse phase (IP-RP) chromatography. The strategy applies core-shell columns and connects multiple columns in tandem to increase separation power and ultimately enables high-resolution detection of nucleotide sugars from cell extracts. The key parameters in the IP-RP method, including temperature, mobile phase, and flow rates, have been systematically evaluated in this work and the theoretical mechanisms of the chromatographic behavior were proposed. [ABSTRACT FROM AUTHOR]

Temozolomide (TMZ) is a drug of choice in glioblastoma treatment. Its therapeutic applications expand also beyond high grade gliomas. However, a significant number of recurrences and resistance to the drug is observed. The key factor in each chemotherapy is to achieve the therapeutic doses of a drug at the pathologic site. Nonetheless, the rate of temozolomide penetration from blood to cerebrospinal fluid is only 20–30%, and even smaller into brain intestinum. That makes a challenge for the therapeutic regimens to obtain effective drug concentrations with minimal toxicity and minor side effects. The aim of our research was to explore a novel epigenetic mechanism of temozolomide action in therapeutic conditions. We analyzed the epigenetic effects of TMZ influence on different glioblastoma cell lines in therapeutically achieved TMZ concentrations through total changes of the level of 5-methylcytosine in DNA, the main epigenetic marker. That was done with classical approach of radioactive nucleotide post-labelling and separation on thin-layer chromatography. In the range of therapeutically achieved temozolomide concentrations we observed total DNA hypomethylation. The significant hypermethylating effect was visible after reaching TMZ concentrations of 10–50 μM (depending on the cell line). Longer exposure time promoted DNA hypomethylation. The demethylated state of the glioblastoma cell lines was overcome by repeated TMZ applications, where dose-dependent increase in DNA 5-methylcytosine contents was observed. Those effects were not seen in non-cancerous cell line. The increase of DNA methylation resulting in global gene silencing and consecutive down regulation of gene expression after TMZ treatment may explain better glioblastoma patients' survival. [ABSTRACT FROM AUTHOR]

The problem of inhibiting viral DNA ejection from bacteriophages by multivalent counterions, specifically Mg+2 counterions, is studied. Experimentally, it is known that MgSO4 salt has a strong and nonmonotonic effect on the amount of DNA ejected. There exists an optimal concentration at which the minimum amount of DNA is ejected from the virus. At lower or higher concentrations, more DNA is ejected from the capsid. We propose that this phenomenon is the result of DNA overcharging by Mg+2 multivalent counterions. As Mg+2 concentration increases from zero, the net charge of DNA changes from negative to positive. The optimal inhibition corresponds to the Mg+2 concentration where DNA is neutral. At lower/higher concentrations, DNA genome is charged. It prefers to be in solution to lower its electrostatic self-energy, which consequently leads to an increase in DNA ejection. By fitting our theory to available experimental data, the strength of DNA-DNA short range attraction energies, mediated by Mg+2, is found to be -0.004 kBT per nucleotide base. This and other fitted parameters agree well with known values from other experiments and computer simulations. The parameters are also in agreement qualitatively with values for tri- and tetravalent counterions. [ABSTRACT FROM AUTHOR]

Insulin resistance (IR) is a common pathophysiological feature of Type 2 diabetes. Although the mechanisms leading to IR are still elusive, evidence has shown that aerobic exercise can reverse this process. To investigate the effects of aerobic exercise on IR, the authors created an IR animal model by feeding C57BL/6 mice a high-fat diet for 8 wk. They then compared the effect of 6 wk of treadmill training (60 min/d) at 75% VO2max on mice in normal-diet (NE) and high-fat-diet (HE) groups with their sedentary control groups. Levels of skeletal-muscle AMPKα (AMP-activated protein kinase α), ACC (acetyl-CoA carboxylases), and CPT1 (carnitine palmitoyltransferase 1) mRNA and AMPKα, pAMPK-Thr172, ACC, pACC-Ser79, and CPT1 protein expressions were analyzed. In addition, fasting serum levels of insulin, triglyceride, and cholesterol were measured. The results demonstrate that 6 wk of exercise increased AMPKα mRNA expression by 11% and 25 % (p < .01) in the NE and HE groups, respectively, and AMPKα protein expression by 37.9% and 20.1% (p < .01) in NE and HE compared with their sedentary control. In addition, ACC mRNA and protein expressions declined, whereas CPT1 mRNA and protein expressions were elevated in both exercise groups compared with sedentary control groups. In addition, pAMPK-Thr172 and pACC-Ser79 expression increased significantly in the NE and HE groups compared with sedentary control groups. In conclusion, our results demonstrate that 6 wk of aerobic exercise can effectively ameliorate IR by increasing the expression of AMPKα and pAMPK-Thr172, thereby activating the key enzymes that facilitate lipid metabolism. [ABSTRACT FROM AUTHOR]

• Highly selective and efficient stationary phase providing separation of nucleotides and nucleobases. • New approach using HILIC proposed for determining the enzymatic activity of RihC. • The proposed approach is the most rapid known at the moment. Ribonucleoside hydrolase C (RihC, EC 3.2.2.1–3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases that catalyze the cleavage of both purine and pyrimidine ribonucleosides to nitrogenous bases and ribose. Its most efficient reaction is the cleavage of uridine with the highest reaction rate. The reaction cannot be detected by a simple spectrophotometric method because of the same absorption maximum for the substrate and reaction product or requires time- and labor-consuming sample preparation for ribose. Reversed-phase HPLC is currently used to register enzymatic activity, where the time of one chromatographic run takes about 10 min. Since a large number of analyses is required to measure the kinetics of an enzymatic reaction, the total time is significant. In this work, we obtained new recombinant RihC from Limosilactobacillus reuteri by gene cloning and expression in E.coli cells. We proposed a new approach for determining the enzymatic activity of the new RihC using hydrophilic interaction liquid chromatography (HILIC). The novel column was developed for this procedure providing the determination of uracil and uridine with high efficiency and retention times of 0.9 and 1.7 min, respectively. Kinetic parameters for RihC uridine cleavage were determined. The proposed approach provided significant rapidity for measurement of the enzyme kinetics being 5 times faster as compared to reversed-phase HPLC. [ABSTRACT FROM AUTHOR]

Telomere length is a heritable marker of cellular age that is associated with morbidity and mortality. Poor sleep behaviors, which are also associated with adverse health events, may be related to leukocyte telomere length (LTL). We studied a subpopulation of 3,145 postmenopausal women (1,796 European-American (EA) and 1,349 African-American (AA)) enrolled in the Women's Health Initiative in 1993–1998 with data on Southern blot-measured LTL and self-reported usual sleep duration and sleep disturbance. LTL-sleep associations were analyzed separately for duration and disturbance using weighted and confounder-adjusted linear regression models in the entire sample (AAs + EAs; adjusted for race/ethnicity) and in racial/ethnic strata, since LTL differs by ancestry. After adjustment for covariates, each additional daily hour of sleep beyond 5 hours, approximately, was associated with a 27-base-pair (95% confidence interval (CI): 6, 48) longer LTL in the entire sample. Associations between sleep duration and LTL were strongest among AAs (adjusted β = 37, 95% CI: 4, 70); a similar, nonsignificant association was observed for EAs (adjusted β = 20, 95% CI: −7, 48). Sleep disturbance was not associated with LTL in our study. Our models did not show departure from linearity (quadratic sleep terms: P ≥ 0.55). Our results suggest that longer sleep duration is associated with longer LTL in postmenopausal women. [ABSTRACT FROM AUTHOR]

Abstract The development of a high-performance liquid chromatography (HPLC)-based method, for guanosine monophosphate kinase activity assays, is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalyzed by Plasmodium vivax guanylate kinase (Pv GK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the Pv GK-catalyzed reaction to be associated with atypical kinetics, where substrate inhibition kinetics and non-Michaelis-Menten (sigmoidal) kinetics were found with respect to GMP and ATP, respectively. Additionally, the method was used in inhibition assays to screen twenty fragment-like compounds. The assays were robust and reproducible, with a signal window of 3.8 and a Z' factor of 0.6. For the best inhibitor, an IC 50 curve was generated. Graphical abstract Image 1 Highlights • Simple HPLC separation of nucleotides involved in the guanylate kinase reaction. • Direct and unambiguous determination of percent conversion of substrate into product. • Successful application to Plasmodium vivax guanylate kinase (Pv GK) activity studies. • Reaction catalyzed by Pv GK found to be associated with atypical kinetics. • Robust and reproducible inhibition assay for compound screening. [ABSTRACT FROM AUTHOR]

Buschke-Lowenstein tumors primarily are slow-growing giant condylomata accuminata of the anogenital region. They are locally destructive with a low rate of metastasis. Sexually transmitted oncogenic human papillomavirus type 6 and 11 are the greatest risk factors for Buschke-Lowenstein tumors or verrucous carcinomas, a type of squamous cell cancer. Grossly, the tumor appears as a large fungating, erythematous, cauliflower-like mass. Radical surgical excision of the tumor is the treatment of choice and close follow-up for recurrence is essential. The use of radiation or chemotherapy as adjunct treatments is controversial. This case report describes a patient with this rare condition. [ABSTRACT FROM AUTHOR]

Nucleotides and other phosphate-containing compounds are integral to enzymatic reactions such as those of the methylerythritol phosphate (MEP) pathway and glycolysis. Traditional chromatographic analysis of phosphates is often plagued by long run times and/or lack of MS compatibility. This study compares separation of five enzymatically-important nucleotides using ion-pair reversed phase (IP-RP), strong anion exchange (SAX), and hydrophilic interaction (HILIC) methods. These three methods were evaluated and compared based on separation parameters describing retention, resolution, efficiency, peak symmetry, selectivity, and inter- and intraday peak drift. Use of the FructoShell-N HILIC column led to separation of the five nucleotides isocratically with the shortest run time of all three methods tested. Additionally, the FructoShell HILIC method yielded a very low intraday variability and low peak asymmetry, issues that are often observed with HILIC separations on other stationary phases. To our knowledge, this column has not been applied to the separation of phosphates in biological samples and future work will focus on in vitro and in vivo analysis as well as broadening the applicability to other pathways. To this end, we have shown that the column will retain fructose bisphosphate, the substrate of the aldolase enzyme, under the same chromatographic conditions used for nucleotides. [ABSTRACT FROM AUTHOR]

Objectives mcr-1 -mediated colistin resistance in bacteria is concerning, as colistin is used in treating multidrug-resistant bacterial infections. And mcr-1 -producing bacteria have been identified in multiple sources. Up to 248 million people use public transportation daily in China, however; public transportation hasn't been studied as a potential source of community-based transmission of mcr-1. Herein we investigated mcr-1 -producing isolates from public transportation and explored the genomic characteristics of them. Methods Surface samples were collected from public transportation in Guangzhou, China, from October 2016 to April 2017. Polymerase chain reaction was performed to detect mcr-1 gene, plasmid replicon type and phylogenetic group. Minimum inhibitory concentrations (MICs) were determined by microdilution method. S1-nuclease digestion and pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were performed with mcr-1 -harboring plasmids. Whole-genome sequencing was performed with mcr-1 -producing isolates. Results Of the 737 samples with bacterial growth, 26 isolates were positive for mcr-1, including 23 Escherichia coli and 3 Klebsiella pneumoniae isolates. The E. coli isolates belonged to phylogroups A and B1. Most mcr-1 -producing isolates were resistant to ampicillin (25), cefotaxime (21), fosfomycin (16), and gentamicin (15). S1-PFGE, Southern blotting and replicon typing showed that mcr-1 was mainly located on ~33.3 kb to ~220 kb IncX4, IncI2 and IncHI2 plasmids in E. coli, while located on ~33.3 kb untyped plasmid in K. pneumoniae. Several sequence types (ST), including ST2253, ST101, ST10 complex and ST37, were revealed. Between 53 and 66 (mean = 61.8) resistance genes were identified among mcr-1 -producing isolates. Conclusions Public transportation may serve as a source of mcr-1 -producing bacteria. [ABSTRACT FROM AUTHOR]

The VicRK 2-component system of Streptococcus mutans regulates genes associated with cell wall biogenesis and biofilm formation. A putative RNase III-encoding gene ( rnc) is located downstream from the vicRKX operon. The goals of this study were to investigate the potential role of VicR in the regulation of adjacent downstream genes and evaluate transcription levels of vicR during planktonic and biofilm growth. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate whether vicRKX and adjacent downstream genes were cotranscribed. Binding of purified recombinant VicR protein to promoter regions of vicR, rnc, and syfA genes was confirmed by electrophoretic mobility shift assay and by chromatin immunoprecipitation analyses. VicR antisense (AS vicR) RNA was detected by Northern blotting and qRT-PCR assays. AS vicR overexpression mutants were constructed, and the biofilm biomass was determined by crystal violet microtiter assay. Adjacent downstream genes rnc, smc, syfA, smu.1511, and syfB were cotranscribed with vicRKX. The predicted promoter regions of vicR, rnc, and syfA genes were directly regulated by VicR. An AS vicR RNA transcript was detected upstream of the rnc gene. Expression of the AS vicR RNA transcript was elevated in planktonic cultures and repressed during biofilm growth. In addition, Western blot data showed that expression of the VicR protein decreased by 35% in planktonic as compared with biofilm cultures. Furthermore, we show that overexpression of AS vicR led to a reduction in biofilm formation. The downstream genes rnc, smc, syfA, smu.1511, and syfB are cotranscribed with vicRKX. VicR is autophosphorylated, and rnc and syfA are directly regulated by VicR. Expression of VicR protein correlated inversely with different levels of AS vicR RNA transcript and growth conditions. The biofilm biomass decreased in the AS vicR overexpression mutant. These data suggest a role for the AS vicR RNA transcript in posttranscriptional regulation of VicR protein production in S. mutans. [ABSTRACT FROM AUTHOR]

This paper is a personal review of the role developments in separation science over the last four decades have played in the diagnosis and understanding of purine and pyridine metabolism particularly in man. In 1967 the separation of nucleotides was used to demonstrate a new chromatography technique. This technique became known as HPLC and which continues to dominate the analysis of purines etc. The resolution and quantitation offered by even the earliest HPLC systems completely changed our understanding of matters such as nucleotide instability in cells and tissues, diagnosis of in born errors, etc. Capillary Electrophoresis also enabled high resolution as well as the quantitation of usual analytes such allantoin. Now LC-MS dominates the diagnostic field. This paper is based on the Anne Simmonds Lecture given by the author at PP17 in Gdansk in 2017. [ABSTRACT FROM AUTHOR]

Highlights • A distinctive miRNA signature between visceral and subcutaneous adipose tissues. • The novel role and function of miR-122 in adipose tissue. • The ratio of miR-122 between visceral and subcutaneous adipose tissues correlates with bariatric surgery outcome. Abstract The physiological roles and clinical impacts of the differences between visceral fat (VF) and subcutaneous fat (SF) are unclear. The present study aimed to compare the miRNA signatures between visceral fat (VF) and subcutaneous fat (SF) and study their influences on outcomes of bariatric surgery. To study the microRNA signatures of the VF and SF in obesity, we performed paired microRNA arrays of the adipose tissues from 20 bariatric surgery patients. The microRNA analysis identified miR-122 as the most significant signature between VF and SF. The tissue distribution, functions, and influences on adipogensis of miR-122 were analysed by Northern blotting, microRNA mimics and inhibitors, and whole-genome microarray analysis. The outcomes of body weight changes after bariatric surgery were analysed and correlated with the miR-122 abundances. Northern blotting confirmed that miR-122 was highly expressed in VF and SF. Bioinformatics analysis of the microarray revealed that proliferator activator receptor-γ (PPAR-γ) signalling was critically affected by miR-122. The modulation of PPAR-γ by miR-122 was confirmed in murine adipocytes and human adipose tissues. Furthermore, the differentiation of preadipocytes was significantly influenced by miR-122. In obese patients receiving bariatric surgery, the ratio of VF and SF miR-122 abundance correlated with 6-month and 1-year % excess body weight loss. Our findings indicate that miR-122 is highly expressed in adipose tissue. The abundance of miR-122 affects PPAR-γ signalling and adipocytes differentiation in vitro and human adipose tissues. Higher miR-122 in VF may be associated with greater body weight loss after bariatric surgery. [ABSTRACT FROM AUTHOR]

Summary: Tubular renal toxicity is a side‐effect of long‐term therapy with nucleos(t)ide analogue(s) (NA) in chronic hepatitis B (CHB). There are no established surrogate markers in plasma of early NA‐related toxicity. Neutrophil gelatinase‐associated lipocalin (NGAL) is a protein produced by tubular cells following renal damage. We aimed therefore to retrospectively compare conventional renal markers (estimated glomerular filtration rates (eGFR) and urinary protein/creatinine ratio uPCR) with a sensitive biomarker (NGAL) in CHB patients on long‐term NA therapy and assess the ability of new markers to predict NA‐related renal toxicity (new onset of nonalbumin proteinuria). A total of 192 naïve CHB patients (median age 41 years, 78% males, 25% HBeAg+, 35% cirrhosis) were NA treated for at least 5 years (median 8.34 years, range 5.54‐11.1 years). The eGFR and uPCR were compared at baseline and last clinical visit with serum NGAL concentrations measured by ELISA at same time‐points and assessed according to the presence/absence of nonalbumin proteinuria at last visit. While baseline and last visit eGFR were similar (median:78 vs 84 mL/min), serum NGAL concentrations increased during therapy (median:9.4 vs 16.4 ng/mL, P < .05). The proportion of patients with proteinuria (uPCR > 15) increased between baseline and last visit (4.6% vs 21.4%, P < .05), with 30 (16%) patients having de novo nonalbumin proteinuria at last visit. High baseline NGAL concentrations were exclusive to patients with de novo nonalbumin proteinuria (median:31.7 vs 7.8 ng/mL, P < .01) and baseline NGAL levels >25 mg/mL were predictive of nonalbumin proteinuria at last visit (AUROC = 0.813). In conclusion, serum NGAL can act as a surrogate marker of early renal injury (de novo nonalbumin proteinuria) in CHB on long‐term NA therapy. [ABSTRACT FROM AUTHOR]

Summary: Nucleot(s)ide analogues (NAs) reduce the risk of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB) patients. However, the risk of HCC is reportedly higher for NA‐treated patients than for patients in the inactive CHB phase. This study aimed to compare the long‐term outcomes of CHB patients with NA‐induced viral suppression and those of patients with inactive CHB. This retrospective study involved 1118 consecutive CHB patients whose HBV DNA level was continuously <2000 IU/mL during follow‐up with/without antiviral agents. The patients were classified into inactive CHB (n = 373) or NA groups (n = 745). The primary endpoint was overall survival. Secondary endpoints included development of HCC and other liver‐related events. The median duration of follow‐up was 41.0 (interquartile range = 26.5‐55.0) months. The difference in overall survival between the NA group vs. the inactive CHB group was not significant (hazard ratio [HR] = 0.78; 95% confidence interval [CI] = 0.33‐1.85; P = .57). The NA group showed a significantly higher risk of HCC (HR = 3.44; 95% CI = 1.82‐6.52; P < .01), but comparable risk for non‐HCC liver‐related events (HR = 1.02; 95% CI = 0.66‐1.59; P = .93), compared with the inactive CHB group. Among patients with cirrhosis, the NA group showed a significantly lower risk of death (HR = 0.31; 95% CI = 0.097‐0.998; P = .05) and non‐HCC liver‐related events (HR = 0.51; 95% CI = 0.31‐0.83; P < .01), but a slightly higher risk of HCC (HR = 2.39; 95% CI = 0.85‐6.75; P = .09), compared to the inactive CHB group. The overall survival of untreated patients with inactive CHB and of CHB patients achieving viral suppression with NA was comparable. However, NA treatment of cirrhotic patients was significantly associated with longer overall survival and lower risk of liver‐related events. [ABSTRACT FROM AUTHOR]

Summary: The nucleotide substitution G1896A on the precore (pc) region has been implicated in virological and serological responses during treatment in hepatitis B virus (HBV)‐infected patients. Whether this mutation affects the therapeutic course of HIV‐HBV co‐infected patients, especially from Western Africa, is unknown. In this prospective cohort study, 86 antiretroviral (ARV)‐naïve HIV‐HBV co‐infected patients from Côte d'Ivoire, initiating ARV‐treatment containing lamivudine (n = 53) or tenofovir (n = 33), had available baseline pc sequences. Association of the pcG1896A mutation with time to undetectable HBV‐DNA, hepatitis B "e" antigen (HBeAg) seroclearance (in HBeAg‐positive patients), and hepatitis B surface antigen (HBsAg) seroclearance was evaluated using Cox proportional hazards regression. At ARV‐initiation, median HBV‐DNA was 6.04 log10 copies/mL (IQR = 3.70‐7.93) with 97.7% harbouring HBV genotype E. Baseline pcG1896A mutation was identified in 51 (59.3%) patients, who were more commonly HBeAg‐negative (P < .001) and had basal core promotor A1762T/G1764A mutations (P < .001). Patients were followed for a median 36 months (IQR = 24‐36). Cumulative proportion of undetectable HBV‐DNA was significantly higher in patients with baseline mutation (pcG1896A = 86.6% vs no pcG1896A = 66.9%, P = .04), but not after adjusting for baseline HBV‐DNA levels and anti‐HBV agent (P = .2). No difference in cumulative proportion of HBeAg seroclearance was observed between mutation groups (pcG1896A = 57.1% vs no pcG1896A = 54.3%, P = .7). Significantly higher cumulative proportion of HBsAg seroclearance was observed in patients without this mutation (pcG1896A = 0% vs no pcG1896A = 36.9%, P < .001), even after adjusting for baseline HBsAg quantification and anti‐HBV agent (P < .001). In conclusion, lacking the pcG1896A mutation before ARV initiation appeared to increase HBsAg seroclearance rates during treatment. The therapeutic implications of this mutation need further exploration in this setting. [ABSTRACT FROM AUTHOR]

Although previous studies examining leukocyte telomere length (LTL) and all-cause mortality controlled for several confounders, the observed association could be biased due to unmeasured confounders, including familial factors. We aimed to examine the association of LTL with all-cause mortality in a Swedish twin sample while adjusting for familial factors and allowing for time-dependent effects. A total of 366 participants (174 twin pairs and 18 individuals) were recruited from the Swedish Twin Registry. LTL was assessed using the Southern blot method. All-cause mortality data were obtained through linkage with the Swedish Population Registry, updated through November 15, 2017. To control for familial factors within twin pairs, we applied a between-within shared frailty model based on generalized survival models. Overall, 115 (31.4%) participants were men and 251 (68.6%) were women. The average age of the study participants when blood was drawn was 79.1 years, and follow-up duration ranged from 10 days to 25.7 years (mean = 10.2 years). During the follow-up period, 341 (93.2%) participants died. Shorter LTL was associated with higher mortality rates when controlling for familial factors in the between-within shared frailty model. We found significant time-dependent effects of LTL on all-cause mortality, where the mortality rate ratios were attenuated with increasing age. [ABSTRACT FROM AUTHOR]

Stress-related changes in β-cell mRNA levels result from a balance between gene transcription and mRNA decay. The regulation of RNA decay pathways has not been investigated in pancreatic β-cells. We found that no-go and nonsense-mediated RNA decay pathway components (RDPCs) and exoribonuclease complexes were expressed in INS-1 cells and human islets. Pelo, Dcp2, Dis3L2, Upf2, and Smg1/5/6/7 were upregulated by inflammatory cytokines in INS-1 cells under conditions where central β-cell mRNAs were downregulated. These changes in RDPC mRNA or corresponding protein levels were largely confirmed in INS-1 cells and rat/human islets. Cytokine-induced upregulation of Pelo, Xrn1, Dis3L2, Upf2, and Smg1/6 was reduced by inducible nitric oxide synthase inhibition, as were endoplasmic reticulum (ER) stress, inhibition of Ins1/2 mRNA, and accumulated insulin secretion. Reactive oxygen species inhibition or iron chelation did not affect RDPC expression. Pelo or Xrn1 knockdown (KD) aggravated, whereas Smg6 KD ameliorated, cytokine-induced INS-1 cell death without affecting ER stress; both increased insulin biosynthesis and medium accumulation but not glucose-stimulated insulin secretion in cytokine-exposed INS-1 cells. In conclusion, RDPCs are regulated by inflammatory stress in β-cells. RDPC KD improved insulin biosynthesis, likely by preventing Ins1/2 mRNA clearance. Pelo/Xrn1 KD aggravated, but Smg6 KD ameliorated, cytokine-mediated β-cell death, possibly through prevention of proapoptotic and antiapoptotic mRNA degradation, respectively. [ABSTRACT FROM AUTHOR]

Aim: The telomere length of somatic cells is associated with systemic aging. The attrition of somatic telomere length is accelerated in pathological conditions, such as cardiovascular diseases. However, clinical parameters of cardiac function have not been well studied in this regard. The present study examined how cardiac function was affected by telomere length and the subtelomeric methylation of peripheral leukocytes. Methods: Telomere length was assessed by Southern blotting analysis of genomic DNA extracted from peripheral leukocytes. Subtelomeric methylation was assessed by comparison between the Southern blotting results with a restriction enzyme Msp I and those with Hpa II, a methylation‐sensitive isoschizomer of Msp I. Results: The following parameters were associated with telomere length and/or the subtelomeric methylation status in a sex‐associated manner: PR interval, the voltage of QRS complex, QRS interval, QT interval and T wave voltage in electrocardiogram; and ejection fraction, the diameter of the left ventricle at the end diastolic phase, aortic root diameter and inferior vena cava diameter in echocardiogram. Conclusions: Cardiac function correlates not only with telomere length, but also with the distribution of the telomere length and subtelomeric DNA methylation status. These imply that the loss of young cells, the accumulation of old cells and the acceleration of such changes in the cell population relate to phenotypes of cardiac aging with relative sex specificity. Furthermore, the PR interval showed a very close association with telomeric parameters in both sexes. Hence, PR is the most reliable candidate as an indicator of biological aging in both sexes. Geriatr Gerontol Int 2018; 18: 1415–1419. [ABSTRACT FROM AUTHOR]

Nickel is a major carcinogen that is implicated in tumor development through occupational and environmental exposure. Although the exact molecular mechanisms of carcinogenesis by low-level nickel remain unclear, inhibition of DNA repair is frequently considered to be a critical mechanism of carcinogenesis. Here, we investigated whether low concentrations of nickel would affect p53-mediated DNA repair, especially nucleotide excision repair. Our results showed that nickel inhibited the promoter binding activity of p53 on the downstream gene GADD45A, as a result of the disturbance of p53 oligomerization by nickel. In addition, we demonstrated that nickel exposure trigger the reduction of GADD45A-mediated DNA repair by impairing the physical interactions between GADD45A and proliferating cell nuclear antigen or xeroderma pigmentosum G. Notably, in the GADD45A-knockdown system, the levels of unrepaired DNA photoproducts were higher than wild-type cells, elucidating the importance of GADD45A in the nickel-associated inhibition of DNA repair. These results imply that inhibition of p53-mediated DNA repair can be considered a potential carcinogenic mechanism of nickel at low concentrations. [ABSTRACT FROM AUTHOR]

Background: Brown adipose tissue (BAT) contains both α- and β-adrenergic receptors. In the literature, the activity of α-adrenoreceptors is less documented, and their functions still remain puzzling. The present investigation has been undertaken to understand α-adrenoreceptors’ activity and their relation between uncoupling protein 1 (UCP1) mRNA expression and cyclic AMP (cAMP) generation in BAT. Methods: BAT precursor cells from young mice were grown in culture. Cells were exposed to norepinephrine (NE) and other agents. RNA was isolated after harvesting the cells, and northern blot was performed. Filters were exposed to film after hybridization with nick-translated complementary DNA probes, and results were evaluated by scanning. Amersham assay kit was used for cAMP measurement. Results: Treatment of prazosin and yohimbine separately with 1 μM of NE shows stimulation of UCP1 mRNA expression 106% and 154%, respectively, whereas with that of both drugs shows only 76%. cAMP generation occurs 282% with prazosin, 100% with yohimbine, and 382% with both drugs with 1 μM of NE, whereas it is 310%, 40%, and 358%, respectively, with 10 μM of NE. Conclusions: Stimulation of thermogenesis after treatment of prazosin and NE may be due to the inhibition of phosphodiesterase enzyme and with yohimbine and NE indicates the possibility of inhibition of the inhibitory effect of α2- and stimulation of α1-receptors. Increase of cAMP concentration with yohimbine and both drugs with NE are not correlated to UCP1 mRNA expression. This indicates that the relationship between cAMP elevation and stimulation of thermogenesis is not simple. This study clearly shows the interaction between β- and α-adrenoreceptor activities. [ABSTRACT FROM AUTHOR]

Research on gene expression (GE) provides insights into the physiology of a cell or group of cells at a given point in time. Studies of changes in GE can be used to identify patients at higher risk for various medical conditions, a higher symptom burden, and/or the adverse consequences associated with various treatments. The aims of this article are as follows: (1) to describe the different types of RNA transcripts, (2) to describe the processes involved in GE (i.e., RNA transcription, epigenetics, and posttranscriptional modifications), (3) to describe common sources of variation in GE, (4) to describe the most common methods used to measure GE, and (5) to discuss factors to consider when choosing tissue for a GE study. This article begins with an overview of the mechanisms involved in GE. Then, the factors that can influence the findings from GE experiments (e.g., tissue specificity, host age, host gender, and time of sample collection) are described and potential solutions are presented. This article concludes with a discussion of how the types of tissue used in GE studies can affect study findings. Given that the costs associated with the measurement of changes in GE are decreasing and the methods to analyze GE data are becoming easier to use, nurse scientists need to understand the basic principles that underlie any GE study. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the presence and molecular characteristics of oxazolidinone resistance genes cfr and optrA in staphylococci from household animals in rural China.Methods: Various samples were collected from household animals in 12 rural villages. Staphylococcal isolates showing florfenicol MICs ≥10 mg/L were identified and screened for the presence of cfr and/or optrA. PCR-positive isolates were characterized by antimicrobial susceptibility testing, S1 nuclease PFGE and Southern blotting. WGS data were analysed to identify the core-genome phylogenetic profile of each isolate as well as the genetic environment of cfr and/or optrA.Results: Nine optrA-positive (seven Staphylococcus sciuri and two Staphylococcus simulans) and 10 cfr-positive staphylococci were identified from eight and five villages, respectively. The gene optrA was chromosomally encoded in all nine isolates, whereas cfr was located on a plasmid in one S. sciuri and three Staphylococcus saprophyticus and in the chromosomal DNA of single Staphylococcus cohnii and Staphylococcus lentus isolates and two S. sciuri isolates. The remaining two cfr-carrying Staphylococcus haemolyticus isolates were indistinguishable by PFGE. Most optrA- or cfr-carrying staphylococci also harboured phenicol, tetracycline and/or macrolide-lincosamide-streptogramin B resistance genes. Genetic environment analysis showed that, for the first time, optrA was associated with transposon Tn6261, while cfr was adjacent to both a tnp (transposase) gene and a Tn558 transposon.Conclusions: The current study reveals for the first time the wide distribution of oxazolidinone resistance genes optrA and cfr in household animals in rural areas of China and is the first identification of optrA in S. simulans isolates. [ABSTRACT FROM AUTHOR]

Full recovery from column separation and matrix effects are the two factors that need to be considered for the high-precision analysis of stable Ca isotopes, but generally they are difficult to balance. In many cases, to get a pure Ca fraction, the interference of the matrix elements is reduced at the cost of discarding a fraction of Ca overlapping with other elements (e.g. Sr and K). However, quantitative evaluation using this approach is challenging but greatly needed. Our study investigates the influence of low Ca recovery on δ44/40Ca using thermal ionization mass spectrometry (TIMS) with the double spike technique. δ44/40Ca of IAPSO seawater, ML3B-G and BHVO-2 in different Ca subcuts (e.g. 0–20, 20–40, 40–60, 60–80 and 80–100%), display limited variation after iterative correction by 42Ca–43Ca double spike with the exponential law. Notably, δ44/40Ca of each Ca subcut with ∼20% recovery is consistent with that of the Ca cut with full recovery, within a margin of error. Our results indicate that 42Ca–43Ca double spike technique can simultaneously correct Ca isotopic fractionation, occurring during column separation, and TIMS determination, because both follow the exponential fractionation law well. Therefore, a “peak cut” procedure of column separation for Ca isotope measurement using the double spike technique on TIMS is proposed. Briefly, we can mix the double spike with the sample solution well before column separation, then collect the peak of the Ca cut and abandon both sides of the Ca eluate that may overlap with other elements. This procedure would eliminate matrix effects efficiently, especially for samples with low CaO contents which typically must be passed through the column twice (e.g. peridotite and dunite). [ABSTRACT FROM AUTHOR]

“ Crude extracts ” obtained from most natural sources cannot be used as is from a supercritical fluid (SCF) plant and have to be reprocessed to eliminate water, pigments and waxes so as to become “clean extracts” that are concentrated in active compounds for direct use as ingredients in food, cosmetics or nutraceuticals/pharmaceuticals. On the other hand, the solid or liquid raw material can be processed as the ingredient itself from which unwanted compounds are eliminated and/or in which a valuable solute is infused. Several on-line or off-line processes are proposed so as to obtain such “ready-for-use” ingredients by combination of supercritical fluid processes (extraction, fractionation, impregnation) and other techniques including vacuum and molecular distillation, centrifugation and dehydration, adsorption on specific adsorbents, cold filtration and winterisation. [ABSTRACT FROM AUTHOR]

In the screening of natural products for the development as cosmetic ingredients, the EtOAc-soluble fraction of Humulus japonicus showed tyrosinase inhibitory activity. HPLC-MS/MS coupled online tyrosinase assay of EtOAc-soluble fraction of H. japonicus characterized the twenty-eight constituents including two unknown ones and their tyrosinase inhibitory activity. Fractionation of H. japonicus using various chromatographic techniques yielded thirty-eight compounds. The chemical structures of isolated compounds were identified by spectroscopic analysis. As characterized by HPLC-MS/MS analysis, we isolated twenty-four predicted compounds and further identified two unknown ones, named humulusides A ( 1 ) and B ( 2 ). Additional ten compounds were also identified by purification. Tyrosinase inhibitory activity of isolated compounds were evaluated, which was closely correlated with the results from HPLC-MS/MS coupled online tyrosinase assay. Consistent with predicted data, two major compounds, trans - N -coumaroyltyramine ( 14 ) and cis - N -coumaroyltyramine ( 15 ) showed tyrosinase inhibition with IC 50 values of 40.6 and 36.4 μM. Taken together, H. japonicus is suggested as whitening ingredient in cosmetic products. In addition, HPLC-MS/MS coupled tyrosinase assay is powerful tool for predicting active compounds with short time and limited amounts, although identification of new compounds and verification of predicted data are also needs to be demonstrated by further experiment. [ABSTRACT FROM AUTHOR]

Background: Single nucleotide polymorphism (SNP) panels have been widely used to study genomic variations within and between populations. Methods of SNP discovery have been a matter of debate for their potential of introducing ascertainment bias, and genetic diversity results obtained from the SNP genotype data can be misleading. We used a total of 42 chicken populations where both individual genotyped array data and pool whole genome resequencing (WGS) data were available. We compared allele frequency distributions and genetic diversity measures (expected heterozygosity (He), fixation index (FST) values, genetic distances and principal components analysis (PCA)) between the two data types. With the array data, we applied different filtering options (SNPs polymorphic in samples of two Gallus gallus wild populations, linkage disequilibrium (LD) based pruning and minor allele frequency (MAF) filtering, and combinations thereof) to assess their potential to mitigate the ascertainment bias. Results: Rare SNPs were underrepresented in the array data. Array data consistently overestimated He compared to WGS data, however, with a similar ranking of the breeds, as demonstrated by Spearman's rank correlations ranging between 0.956 and 0.985. LD based pruning resulted in a reduced overestimation of He compared to the other filters and slightly improved the relationship with the WGS results. The raw array data and those with polymorphic SNPs in the wild samples underestimated pairwise FST values between breeds which had low FST (<0.15) in the WGS, and overestimated this parameter for high WGS FST (>0.15). LD based pruned data underestimated FST in a consistent manner. The genetic distance matrix from LD pruned data was more closely related to that of WGS than the other array versions. PCA was rather robust in all array versions, since the population structure on the PCA plot was generally well captured in comparison to the WGS data. Conclusions: Among the tested filtering strategies, LD based pruning was found to account for the effects of ascertainment bias in the relatively best way, producing results which are most comparable to those obtained from WGS data and therefore is recommended for practical use [ABSTRACT FROM AUTHOR]

Voluminous Mesozoic felsic volcanic rocks and granites in southeastern China provide a unique opportunity for studying the role of crustal magmatism in the evolution and modification of the crust in the eastern Cathaysia Block. The high-silica rhyolites of the upper volcanic sequence in eastern Zhejiang Province were investigated, focusing on their genesis and their relationship with contemporaneous granites. Rhyolites in the Tiantai, Yongkang and Liucheng basins were dated as late Early Cretaceous (from 111 Ma to 106 Ma in age). These rocks contain a large proportion of inherited zircons of ca. 130 Ma, corresponding to the age of the lower volcanic sequence in the area. However, the zircons of different ages have similar ranges of oxygen and Hf isotopes, implying similarities in the magmas from which they were generated. The rhyolites of the upper sequence also resemble those of the lower sequence in terms of their geochemistry. It is concluded that the former were derived by reworking of magma mush formed during the earlier magmatic episode via fractionation of feldspars and accessory minerals, e.g., zircon. Fractionation took place within the magma crystal mush by extraction of interstitial melts and accumulation of residual mineral phases, aided by the emplacement of contemporaneous basaltic magmas at the base of the crust. Overall, the geochemical features of the volcanic rocks in southeastern China indicate that episodic magmatism and reworking of crystal mush were essential mechanisms that drove the evolution of the igneous rocks and the hence crustal architecture in this area. [ABSTRACT FROM AUTHOR]

Weevils (Curculionoidea) comprise one of the most diverse groups of organisms on earth. There is hardly a vascular plant or plant part without its own species of weevil feeding on it and weevil species diversity is greater than the number of fishes, birds, reptiles, amphibians and mammals combined. Here, we employ ultraconserved elements (UCEs) designed for beetles and a novel partitioning strategy of loci to help resolve phylogenetic relationships within the radiation of Australasian smurf-weevils (Eupholini). Despite being emblematic of the New Guinea fauna, no previous phylogenetic studies have been conducted on the Eupholini. In addition to a comprehensive collection of fresh specimens, we supplement our taxon sampling with museum specimens, and this study is the first target enrichment phylogenomic dataset incorporating beetle specimens from museum collections. We use both concatenated and species tree analyses to examine the relationships and taxonomy of this group. For species tree analyses we present a novel partitioning strategy to better model the molecular evolutionary process in UCEs. We found that the current taxonomy is problematic, largely grouping species on the basis of similar color patterns. Finally, our results show that most loci required multiple partitions for nucleotide rate substitution, suggesting that single partitions may not be the optimal partitioning strategy to accommodate rate heterogeneity for UCE loci. [ABSTRACT FROM AUTHOR]

Background: Previous studies on physical activity and telomere length have relied largely upon self-reported physical activity data, and few studies have examined older adults. The association of objectively measured physical activity with leukocyte telomere length (LTL) is currently unknown.Methods: In this study, we examined cross-sectional associations between accelerometer-measured total, light, and moderate-to-vigorous physical activity (MVPA) and LTL, measured using Southern blot. The sample included 1,405 older (64-95 years old) white and African American women from the Women's Health Initiative. Multiple linear regression models adjusting for potential confounders were used to determine the association between accelerometer-measured physical activity and LTL.Results: Overall, the mean (standard deviation) of total, light, and moderate-to-vigorous activity was 5.5 (1.6), 4.7 (1.3), and 0.8 (0.5) h/d, respectively. Adjusting for accelerometer wear time, age, race/ethnicity, education, marital status, smoking, alcohol, body mass index, a history of chronic diseases, and hormone therapy use, LTL was 80 (95% confidence interval: 9, 150) base pairs longer among women with ≥2.5 compared with <2.5 h/wk of MVPA. Light activity was not significantly associated with LTL. For total activity, the most physically active women had significantly longer LTL than the least active women after adjustment for demographic and lifestyle characteristics; however, findings were not significant after further adjustment for health-related factors.Conclusions: Older women meeting current recommendations of ≥2.5 h/wk of MVPA, as assessed by accelerometer, had longer LTL. Additional studies using accelerometers in large, diverse cohorts of older women are needed to confirm and extend these findings. [ABSTRACT FROM AUTHOR]

MicroRNAs are novel biomolecules with a crucial function in normal cellular physiology and in pathophysiologic conditions, including cancer. Since the first report on the link between microRNAs and cancer was published in 2002, research has revealed the potential clinical implications of microRNAs. Oncology nurses play an important role in educating patients and their families about possible applications of microRNAs in oncology. [ABSTRACT FROM AUTHOR]

There has been a growing interest in visualization of metagenomic data. The present study focuses on the visualization of metagenomic data using inter-nucleotide distances profile. We first convert the fragment sequences into inter-nucleotide distances profiles. Then we analyze these profiles by principal component analysis. Finally the principal components are used to obtain the 2-D scattered plot according to their source of species. We name our method as inter-nucleotide distances profiles (INP) method. Our method is evaluated on three benchmark data sets used in previous published papers. Our results demonstrate that the INP method is good, alternative and efficient for visualization of metagenomic data. [ABSTRACT FROM AUTHOR]

Background Down syndrome ( DS) and Fragile X syndrome ( FXS) are the major genetic causes of intellectual disabilities. Here, we present a case of a 32-year-old woman with the diagnosis of both FXS and DS. She is the daughter of a 47-year-old pre-mutation woman who also has three sons with FXS. Methods Cytogenetic testing detected the presence of a complete trisomy 21. A combination of PCR and Southern blot analysis was utilized to document the presence of the FMR1 full mutation. Results The patient has physical characteristics and behavioural disturbances typical of both FXS and DS, which were confirmed by molecular testing. Her treatment plan included a trial of sertraline because of the severity of her shyness and lack of language. She had an excellent response to sertraline with improvement in shyness and social interactions, particularly with family members. Conclusions In this study, we report the case of a woman with both FXS and DS, which is the fifth case of FXS and DS in the world's literature. The patient is from Ricaurte, a small town in Colombia, South America, where there is the world's highest prevalence for FXS. [ABSTRACT FROM AUTHOR]

Objectives: To analyse the recombination events associated with conjugal mobilization of two multiresistance plasmids, pRUM17i48 and pLAG (formerly named pDO1-like), from Enterococcus faecium 17i48 to Enterococcus faecalis JH2-2.Methods: The plasmids from two E. faecalis transconjugants (JH-4T, tetracycline resistant, and JH-8E, erythromycin resistant) and from the E. faecium donor (also carrying a pHTβ-like conjugative plasmid, named pHTβ17i48) were investigated by several methods, including PCR mapping and sequencing, S1-PFGE followed by Southern blotting and hybridization, and WGS.Results: Two locations of repApHTβ were detected in both transconjugants, one on a ∼50 kb plasmid (as in the donor) and the other on plasmids of larger sizes. In JH-4T, WGS disclosed an 88.6 kb plasmid resulting from the recombination of pHTβ17i48 (∼50 kb) and a new plasmid, named pLAG (35.3 kb), carrying the tet(M), tet(L), lsa(E), lnu(B), spw and aadE resistance genes. In JH-8E, a 75 kb plasmid resulting from the recombination of pHTβ17i48 and pRUM17i48 was observed. In both cases, the cointegrates were apparently derived from replicative transposition of an IS1216 present in each of the multiresistance plasmids into pHTβ17i48. The cointegrates could resolve to yield the multiresistance plasmids and a pHTβ17i48 derivative carrying an IS1216 (unlike the pHTβ17i48 of the donor).Conclusions: Our results completed the characterization of the multiresistance plasmids carried by the E. faecium 17i48, confirming the role of pHT plasmids in the mobilization of non-conjugative antibiotic resistance elements among enterococci. Results also revealed that mobilization to E. faecalis was associated with the generation of cointegrate plasmids promoted by IS1216-mediated transposition. [ABSTRACT FROM AUTHOR]

Introduction: The FMR1 gene has four allelic variants according to the number of repeats of the CGG triplet. Premutation carriers with between 55 and 200 repeats are susceptible to developing pathologies such as tremor and ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI) syndrome. Case description: The patient was a 53-year-old female farmer with severe tremor in the upper limbs at rest that worsens with movement, tremor in the jaw and tongue, and generalized cerebral atrophy. She is a carrier of the FMR1 premutation diagnosed by PCR and Southern Blot, complying with the clinical and radiological criteria of FXTAS, and in addition, has a history of vagal symptoms suggestive of ovarian failure and menstrual cycle disorders that led to hysterectomy at age 33 and was subsequently diagnosed with FXPOI. Conclusion: An unusual case of FXTAS and FXPOI complying with clinical and radiological criteria is reported in a premutation carrier of the FMR1 gene. [ABSTRACT FROM AUTHOR]

Background: The current literature establishes the importance of gene functional category and expression in promoting or suppressing duplicate gene loss after whole genome doubling in plants, a process known as fractionation. Inspired by studies that have reported gene expression to be the dominating factor in preventing duplicate gene loss, we analyzed the relative effect of functional category and expression. Methods: We use multivariate methods to study data sets on gene retention, function and expression in rosids and asterids to estimate effects and assess their interaction. Results: Our results suggest that the effect on duplicate gene retention fractionation by functional category and expression are independent and have no statistical interaction. Conclusion: In plants, functional category is the more dominant factor in explaining duplicate gene loss. [ABSTRACT FROM AUTHOR]

A general and efficient method for the synthesis of pronucleotide (ProTide) 5'-phosphoramidate monoesters is reported. This method consists of a highly stereoselective 5'-phosphorylation mediated by dimethylaluminum chloride to afford the desired target ProTides in excellent yields without employing 3'-protection strategies. The application of this methodology to the synthesis of a number of pharmaceutically relevant compounds currently marketed or under investigation in clinical research is demonstrated. [ABSTRACT FROM AUTHOR]

Objective. The aim of this study was to identify a protein for urate transporter 1 (URAT1) regulation. Methods. The clinical dataset consisted of 492 case-control samples of Han Chinese (104 gout and 388 controls). Three alpha kinase 1 (ALPK1) and SLC22A12 loci associated with high gout risk and uric acid levels were genotyped. The overexpression of ALPK1 on URAT1 protein expression was evaluated in vivo in hALPKI transgenic mice. The in vitro protein levels of ALPK1 and URAT1 in ALPK1 small interfering RNA-transfected human kidney-2 cells with MSU crystal stimulation were examined. Results. ALPK1, which is a single nucleotide polymorphism (SNP) of rs11726117 (M861T; T), reduced the risk of gout via the SLC22A12 gene SNPs rs3825016 and rs475688, as compared with the subject of ALPK1 rs11726117 (C) allele {rs11726117 [CT + TT] vs rs3825016, odds ratio [OR] 0.39 [95% confidence interval (CI) 0.23, 0.67]; rs11726117 [CT + TT] vs rs475688, OR 0.39 [95% CI 0.23, 0.67]}. ALPK1-over- expressed mice demonstrated lower levels of URAT1 protein (P = 0.0045). Mouse endogenous ALPK1 proteins were detected in renal proximal tubule cells. MSU crystals inhibited URAT1 expressions through an upregulation of ALPK1 in human kidney-2 cells. Conclusion. Elevated ALPK1 expression decreased URAT1 expression. ALPK1 might prevent the impact of urate reuptake via SLC22A12 and appeared to be negatively associated with gout. ALPK1 is a potential repressor of URAT1 protein expression. [ABSTRACT FROM AUTHOR]

Ascorbate peroxidase (APX) acts indispensably in synthesizing L-ascorbate (AsA) which is pivotal to plant stress tolerance by detoxifying reactive oxygen species (ROS). Enhanced activity of APX has been shown to be a key step for genetic engineering of improving plant tolerance. However it needs a deeper understanding on the maintenance of cellular ROS homeostasis in response to stress. In this study, we identified and characterized an APX (CaAPX) gene from Camellia azalea. Quantitative real-time PCR (qRT-PCR) analysis showed that CaAPX was expressed in all tissues and peaked in immature green fruits; the expression levels were significantly upregulated upon cold and hot stresses. Transgenic plants displayed marked enhancements of tolerance under both cold and heat treatments, and plant growth was correlated with CaAPX expression levels. Furthermore, we monitored the activities of several ROS-scavenging enzymes including Cu/Zn-SOD, CAT, DHAR, and MDHAR, and we showed that stress tolerance was synchronized with elevated activities of ROS-scavenging. Moreover, gene expression analysis of ROS-scavenging enzymes revealed a role of CaAPX to orchestrate ROS signaling in response to temperature stresses. Overall, this study presents a comprehensive characterization of cellular response related to CaAPX expression and provides insights to breed crops with high temperature tolerances. [ABSTRACT FROM AUTHOR]

KIR2DP1 is an inactive member of the human lineage III KIR family, which includes all HLA-C-specific receptor genes. The lethal, and only, defect in KIR2DP1 is a nucleotide deletion in codon 88. Fixed in modern humans, the deletion is also in archaic human genomes. KIR2DP1 is polymorphic, with dimorphism at specificity-determining position 44. By repairing the deletion, we resurrected 11 alleles of KIR2DP1F, the functional antecedent of KIR2DP1. We demonstrate how K44-KIR2DP1F with lysine 44 recognized C1+HLA-C, whereas T44-KIR2DP1F recognized C2+HLA-C. Dimorphisms at 12 other KIR2DP1F residues modulate receptor avidity or signaling. KIR2DP1 and KIR2DL1 are neighbors in the centromeric KIR region and are in tight linkage disequilibrium. Like KIR2DL1, KIR2DP1 contributed to CenA and CenB KIR haplotype differences. Encoded on CenA, C1-specific K44-KIR2DP1F were stronger receptors than the attenuated C2-specific T44-KIR2DP1F encoded on CenB. The last common ancestor of humans and chimpanzees had diverse lineage III KIR that passed on to chimpanzees but not to humans. Early humans inherited activating KIR2DS4 and an inhibitory lineage III KIR, likely encoding a C1-specific receptor. The latter spawned the modern family of HLA-C receptors. KIR2DP1F has properties consistent with KIR2DP1F having been the founder gene. The first KIR2DP1F alleles encoded K44-C1 receptors; subsequently KIR2DP1F alleles encoding T44-C2 receptors evolved. The emergence of dedicated KIR2DL2/3 and KIR2DL1 genes encoding C1 and C2 receptors, respectively, could have led to obsolescence of KIR2DP1F. Alternatively, pathogen subversion caused its demise. Preservation of KIR2DP1F functional polymorphism was a side effect of fixation of the deletion in KIR2DP1F by micro gene conversion. [ABSTRACT FROM AUTHOR]