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This study aimed to evaluate the influence of different power-time ultrasound regimes of pasteurization on the physical, chemical, organoleptic properties, and lipid quality indices of goat curds characterized by a low cholesterol level. Cholesterol was eliminated by a percentage of 92.1 % by treating the raw goat milk with beta-cyclodextrin in the proportion of 0.6 %. Afterward, the goat milk was subjected to the following ultrasound regimes: 320 W for 1 (PA1), 3 (PA3), and 6 min (PA6) and 881 W for 1 (PP1), 3 (PP3), and 6 min (PP6) and then used for the curds production. Due to the ultrasound treatment, the milk suffered a concentration phenomenon, the most accentuated being registered for the PP6 sample. Considering the sensory properties, the most appreciated curd was the one obtained by the PP6 regime which recorded the highest scores for color and taste. Regarding the microbiological aspects, the ability of ultrasounds to inactivate microorganisms is observed and the most accentuated phenomenon is reported in the PP6 case. Thus, in comparison with the control sample, the total number of germs is reduced by a proportion of 91.85 %, the β-glucuronidase-positive Escherichia coli decreased by 93.15 %, while the coagulase-positive staphylococci were completely inactivated for the PP6 curd. The curds obtained for the PA6 and PP6 regimes registered the highest dry matter values as a cause of an accentuated syneresis process. The acidity values were higher for the curds obtained for PA1, PA3, and PA6 regimens due to more pronounced lactose hydrolysis and lower in the cases of PP3 and PP6 regimens compared to the control cheese. Twenty-five saturated, monounsaturated, and polyunsaturated fatty acids were identified in the curd samples and a rise in the unsaturated fatty acids proportion as the intensity of the applied ultrasound regime increased was observed. Also, AI, TI, and H/H lipid quality indices recorded better values as the power and time of the ultrasound action increased., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Endothelial hyper-permeability with plasma leakage and thrombocytopenia are predominant features of severe dengue virus infection. It is well established that heparanase, the endothelial glycocalyx degrading enzyme, plays a major role in various diseases with vascular leakage. It is yet to be elucidated whether heparanase activity plays a major role in dengue-associated plasma leakage. Moreover, the major source of heparanase secretion and activation in dengue remains elusive. Since a relatively high amount of heparanase is stored in platelets, we postulate that heparanase released by activated platelets contributes to the increased plasma heparanase activity during dengue virus infection., Methods: Heparanase activity (plasma and urine), and heparan sulfate and syndecan-1 (plasma levels) were measured in dengue patients with thrombocytopenia in acute phase (n=30), during course of disease (n=10) and in convalescent phase (n=25). Associations with clinical parameters and plasma leakage markers were explored. Platelets from healthy donors were stimulated with dengue non-structural protein-1, DENV2 virus and thrombin to evaluate heparanase release and activity ex vivo ., Results: Heparanase activity was elevated in acute dengue and normalized during convalescence. Similarly, glycocalyx components, such as heparan sulfate and syndecan-1, were increased in acute dengue and restored during convalescence. Increased heparanase activity correlated with the endothelial dysfunction markers heparan sulfate and syndecan-1, as well as clinical markers of plasma leakage such as ascites, hematocrit concentration and gall-bladder wall thickening. Notably, platelet number inversely correlated with heparanase activity. Ex vivo incubation of platelets with thrombin and live DENV2 virus, but not dengue virus-2-derived non-structural protein 1 induced heparanase release from platelets., Conclusion: Taken together, our findings suggest that the increase of heparanase activity in dengue patients is associated with endothelial glycocalyx degradation and plasma leakage. Furthermore, thrombin or DENV2 activated platelets may be considered as a potential source of heparanase., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Buijsers, Garishah, Riswari, van Ast, Pramudo, Tunjungputri, Overheul, van Rij, van der Ven, Alisjahbana, Gasem, de Mast and van der Vlag.)

Abstract: This study aimed to assess the associations of serum soluble klotho and fibroblast growth factor 23 (FGF-23) with the occurrence of carotid artery calcification. Peritoneal dialysis patients treated from June 2018 to June 2019 were retrospectively analyzed. They were divided into the carotid artery calcification and non-carotid artery calcification groups according to color Doppler ultrasound findings. Basic indicators in both groups were compared, and the influencing factors of carotid artery calcification were analyzed by logistic regression. Among the 73 continuous ambulatory peritoneal dialysis (CAPD) patients enrolled, 40 (54.8%) had carotid artery calcification. Significant differences were found in age (68.85 ± 7.45 vs 46.62 ± 5.51 years), dialysis time (8.15 ± 1.42 vs 6.02 ± 1.14 months), klotho amounts (325.56 ± 41.15 vs 436.65 ± 45.58 pg/mL) and FGF-23 levels (114.45 ± 15.56 vs 70.15 ± 12.23 pg/mL) between the carotid artery calcification and non-carotid artery calcification groups (all P < .001). The above factors were associated with carotid artery calcification occurrence in univariate analysis. Multivariate analysis showed that elevated age (odds ratio [OR] = 1.55, 95% confidence interval [CI] 1.13-1.74; P = .025) and FGF-23 (OR = 2.16, 95% CI 2.01-2.44; P = .042), and lower klotho (OR = 0.66, 95% CI 0.47-0.85; P = .036) were independent risk factors for carotid artery calcification in CAPD. Serum FGF-23 and age are risk factors for carotid artery calcification in patients with CAPD, whereas klotho is a protective factor., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Paenibacillus sp. 32352 is a soil-dwelling bacterium capable of producing an enzyme, Pn3Pase that degrades the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (Pn3P). Recent reports on Pn3Pase have demonstrated its initial characterization and potential for protection against highly virulent S. pneumoniae serotype 3 infections. Initial experiments revealed this enzyme functions as an exo-β1,4-glucuronidase cleaving the β(1,4) linkage between glucuronic acid and glucose. However, the catalytic mechanism of this enzyme is still unknown. Here, we report the detailed biochemical analysis of Pn3Pase. Pn3Pase shows no significant sequence similarity to known glycoside hydrolase (GH) families, thus this novel enzyme establishes a new carbohydrate-active enzyme (CAZy) GH family. Site-directed mutagenesis studies revealed two catalytic residues along with truncation mutants defining essential domains for function. Pn3Pase and its mutants were screened for activity, substrate binding and kinetics. Additionally, nuclear magnetic resonance spectroscopy analysis revealed that Pn3Pase acts through a retaining mechanism. This study exhibits Pn3Pase activity at the structural and mechanistic level to establish the new CAZy GH family GH169 belonging to the large GH-A clan. This study will also serve toward generating Pn3Pase derivatives with optimal activity and pharmacokinetics aiding in the use of Pn3Pase as a novel therapeutic approach against type 3 S. pneumoniae infections., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The fabrication of covalently cross-linked high-surface-area biopolymeric nanogel fibers by nanopore extrusion is reported for the first time. The biopolymer pullulan was functionalized with tert -butyl acetoacetate via a transesterification reaction to synthesize the water-soluble ketone-rich precursor pullulan acetoacetate (PUAA). PUAA and carbonic dihydrazide (CDH) as cross-linker were extruded through anodic aluminum oxide (AAO) nanoporous membranes, which possessed an average pore diameter of 61 ± 2 nm. By changing the concentration of PUAA, the flow rate, and extrusion time, the step polymerization cross-linking reaction was controlled so that the polymer can be extruded gradually during cross-linking through the membrane, avoiding the formation of macroscopic bulk hydrogels and rupture of the AAO membrane. Fibers with diameters on the order of 250 nm were obtained. This approach was also expanded to functionalized PUAA derivatives together with the fluorogenic substrate 4-methylumbelliferyl-β-d-glucuronide MUGlcU in (PUAA-MUGlcU), which exhibited a mean equilibrium swelling ratio of 5.7 and 9.0 in Milli-Q water and in phosphate-buffered saline, respectively. β-Glucuronidase was sensitively detected via fluorescence of 4-methylumbelliferone, which was liberated in the enzymatic hydrolysis reaction of PUAA-MUGlcU. Compared to hydrogel slabs, the rate of the hydrolysis was >20% higher in the nanogel fibers, facilitating the rapid detection of β-glucuronidase-producing Escherichia coli ( E. coli Mach1-T1). Nanopore extruded nanogel fibers are therefore considered a viable approach to enhance the functionality of hydrogels in surface-dominated processes.

There is a growing demand for rapid and sensitive detection approaches for pathogenic bacteria that can be applied by non-specialists in non-laboratory field settings. Here, the detection of the typical E. coli enzyme β-glucuronidase using a chitosan-based sensing hydrogel-coated paper sensor and the detailed analysis of the reaction kinetics, as detected by a smartphone camera, is reported. The chromogenic reporter unit affords an intense blue color in a two-step reaction, which was analyzed using a modified Michaelis-Menten approach. This generalizable approach can be used to determine the limit of detection and comprises an invaluable tool to characterize the performance of lab-in-a-phone type approaches. For the particular system analyzed, the ratio of reaction rate and equilibrium constants of the enzyme-substrate complex are 0.3 and 0.9 pM -1 h -1 for β-glucuronidase in phosphate buffered saline and lysogeny broth, respectively. The minimal degree of substrate conversion for detection of the indigo pigment formed during the reaction is 0.15, while the minimal time required for detection in this particular system is ~2 h at an enzyme concentration of 100 nM. Therefore, this approach is applicable for quantitative lab-in-a-phone based point of care detection systems that are based on enzymatic substrate conversion via bacterial enzymes.

The prevalence of chronic kidney disease (CKD) is increasing worldwide, and the mortality rate continues to be unacceptably high. The biomarkers currently used in clinical practice are considered relevant when there is already significant renal impairment compromising the early use of potentially successful therapeutic interventions. More sensitive and specific biomarkers to detect CKD earlier on and improve patients' prognoses are an important unmet medical need. The aim of this review is to summarize the recent literature on new promising early CKD biomarkers of renal function, tubular lesions, endothelial dysfunction and inflammation, and on the auspicious findings from metabolomic studies in this field. Most of the studied biomarkers require further validation in large studies and in a broad range of populations in order to be implemented into routine CKD management. A panel of biomarkers, including earlier biomarkers of renal damage, seems to be a reasonable approach to be applied in clinical practice to allow earlier diagnosis and better disease characterization based on the underlying etiologic process.

Currently, no treatment exists to improve semen quality in most infertile men. Here, we demonstrate systemic and direct effects of Fibroblast growth factor 23 (FGF23) and Klotho, which normally regulate vitamin D and mineral homeostasis, on testicular function. Direct effects are plausible because KLOTHO is expressed in both germ cells and spermatozoa and forms with FGFR1 a specific receptor for the bone-derived hormone FGF23. Treatment with FGF23 increased testicular weight in wild-type mice, while mice with global loss of either FGF23 or Klotho had low testicular weight, reduced sperm count, and sperm motility. Mice with germ cell-specific Klotho (gcKL) deficiency neither had a change in sperm count nor sperm motility. However, a tendency toward fewer pregnancies was detected, and significantly fewer Klotho heterozygous pups originated from gcKL knockdown mice than would be expected by mendelian inheritance. Moreover, gcKL mice had a molecular phenotype with higher testicular expression of Slc34a2 and Trpv5 than wild-type littermates, which suggests a regulatory role for testicular phosphate and calcium homeostasis. KLOTHO and FGFR1 were also expressed in human germ cells and spermatozoa, and FGF23 treatment augmented the calcium response to progesterone in human spermatozoa. Moreover, cross-sectional data revealed that infertile men with the highest serum Klotho levels had significantly higher serum Inhibin B and total sperm count than men with the lowest serum Klotho concentrations. In conclusion, this translational study suggests that FGF23 and Klotho influence gonadal function and testicular mineral ion homeostasis both directly and indirectly through systemic changes in vitamin D and mineral homeostasis., (© 2020 Federation of American Societies for Experimental Biology.)

Background: We sought to identify biomarkers that indicate low turnover on bone histomorphometry in chronic kidney disease (CKD) patients, and subsequently determined whether this panel identified differential risk for fractures in community-dwelling older adults., Methods: Among CKD patients who underwent iliac crest bone biopsies and histomorphometry, we evaluated candidate biomarkers to differentiate low turnover from other bone disease. We applied this biomarker panel to 641 participants in the Health Aging and Body Composition Study (Health ABC) study with estimated glomerular filtration rate (eGFR) less than 60 mL/min/1.73 m2 who were followed for fracture. Cox proportional hazards models evaluated the association of bone mineral density (BMD) with fracture risk and determined whether biomarker-defined low bone turnover modified fracture risk at any level of BMD., Results: In 39 CKD patients age 64 ± 13 years, 85% female, with mean eGFR 37 ± 14 mL/min/1.73 m2 who underwent bone biopsy, lower fibroblast growth factor (FGF)-23, higher ɑ-Klotho, and lower parathyroid hormone (PTH) indicated low bone turnover in accordance with bone histomorphometry parameters (individual area under the curve = 0.62, 0.73, and 0.55 respectively; sensitivity = 22%, specificity = 100%). In Health ABC, 641 participants with CKD were age 75 ± 3 years , 49% female, with mean eGFR 48 ± 10 mL/min/1.73 m2. For every SD lower hip BMD at baseline, there was an 8-fold higher fracture risk in individuals with biomarker-defined low turnover (hazard ratio 8.10 [95% CI, 3.40-19.30]) vs a 2-fold higher risk in the remaining individuals (hazard ratio 2.28 [95% CI, 1.69-3.08]) (Pinteraction = .082)., Conclusions: In CKD patients who underwent bone biopsy, lower FGF-23, higher ɑ-Klotho, and lower PTH together had high specificity for identifying low bone turnover. When applied to older individuals with CKD, BMD was more strongly associated with fracture risk in those with biomarker-defined low turnover., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mounting evidence indicates that there exists an association between heparanase (HPSE) and several physiological and pathological mechanisms in humans. However, the dynamics of the mechanisms involved in the regulation of HPSE expression in pancreatic cancer (PC) remain unclear. The aim of the present study was to assess the levels of HPSE in PC tissues and cell lines by western blotting and reverse transcription‑quantitative PCR (RT‑qPCR) analysis. Wound healing and Transwell assays were conducted to examine the effects of HPSE on migration and invasion in sh‑NC and sh‑HPSE PC cell lines. In addition, tumor growth was assessed in a mouse xenograft model in vivo. The expression levels of epithelial‑to‑mesenchymal transition (EMT)‑related biomarkers and the involvement of the Wnt/β‑catenin pathway were assessed by analyzing the results of western blot and RT‑qPCR assays. The results indicated that the expression of HPSE was substantially higher in PC tissues and cell lines, whereas experimental knockdown of HPSE suppressed the rates of migration and invasion of PC cells. Western blotting was used to assess the expression of EMT biomarkers and determine the function of HPSE in EMT. Furthermore, our results indicated that downregulation of HPSE expression decreased the expression of Wnt/β‑catenin associated proteins. In conclusion, HPSE appears to be a good candidate as a molecular target for the treatment of PC based on the finding of the present study.

Phosphaturic mesenchymal tumors (PMT) are tumors that cause hypophosphatemia/osteomalacia chiefly by secreting FGF23. We have identified FN1-FGFR1/FGF1 fusion genes in nearly half of PMT, suggesting a central role of FGFR1 pathways in the pathogenesis of PMT. Tumorigenic drivers are unknown for tumors where previous study detected neither fusion, including many in bone, where FISH failed because of tissue decalcification. To identify alternative fusions in PMT without known fusions, as well as to validate the positive FISH results and characterize the fusion junctions, 34 PMT were studied, including 12 with known FN1-FGFR1 fusion by FISH (Group A), 2 with FN1-FGF1 (B), 12 with neither fusion (C), and 8 with previous acid-based decalcification and hence unknown fusion status (D). In total, 23 archival samples were subjected to anchored multiplex PCR-based RNA-sequencing (AMP-seq) with primers targeting FN1, genes encoding the FGF/FGFR families, and KL (α-Klotho); five Group C cases were also studied with whole-transcriptomic and exome-captured RNA sequencing, respectively. The AMP-seq results were consistent with previous FISH and/or transcriptomic sequencing data, except in one old Group A sample. One case had a novel FGFR1 exon 9 breakpoint, confirmed by genomic DNA sequencing. One Group D bone tumor was found to harbor FN1-FGF1. All 3 RNA-sequencing platforms failed to identify convincing fusion genes in Group C (N = 10), which instead expressed significantly higher levels of either KL or KLB. This result was further confirmed with KL and KLB RNA CISH semi-quantification (RNAscope). Our results demonstrated the utility of AMP-seq, which was compromised by decalcification and prolonged archiving. Of potential importance, fusion-negative PMT frequently overexpressed α-Klotho (or instead β-Klotho less commonly), whose role as an obligatory co-receptor for FGF23-FGFR1 binding suggests its aberrant expression in osteocytes/osteoblasts might result in an FGF23-FGFR1 autocrine loop that in turn drives the overexpression of FGF23 and tumorigenesis through activated FGFR pathways.

Experimental and clinical studies aimed at investigating the mechanism(s) underlying vascular complications of diabetes indicate that a great number of molecules are involved in the pathogenesis of these complications. Most of these molecules are inflammatory mediators or markers generated by immune or adipose tissue. Some of them, i.e. resistin and sortilin, have been shown to be involved in the cross talk between adipocytes and inflammatory cells. This interaction is an attractive area of research, particularly in type 2 diabetes and obesity. Other proteins, such as adiponectin and visfatin, appear to be more promising as possible vascular markers. In addition, some molecules involved in calcium/phosphorus metabolism, such as klotho and FGF23, have an involvement in the pathogenesis of diabetic vasculopathy, which appears to be dependent on the degree of vascular impairment. Inflammatory markers are a promising tool for treatment decisions while measuring plasma levels of adipokines, sortilin, Klotho and FGF23 in adequately sized longitudinal studies is expected to allow a more precise characterization of diabetic vascular disease and the optimal use of personalized treatment strategies.

Introduction: Introduction: resistant starch (RS) is not completely digested in the human intestine but is fermented in the colon; intestinal pH decreases as short-chain fatty acids are produced. This is beneficial for health, and for preventing and treating rectal colon cancer. Pyrodextrinization and enzymatic hydrolysis are modifications to native starch (NS) that may increase the amount of RS. Objective: the objective of this project was to evaluate the effects of M. cavendish AAA native and both chemically and enzymatically modified starches on tumor markers in rats. Methods: modifications (chemical and enzymatic) were made to M. cavendish AAA NS, and were evaluated in rats with 1,2-DMH. Male Sprague Dawley rats (25) were used, divided into five experimental groups: PC, NC, NS, PI, and ERM. During 4 weeks they received the experimental diet assigned to each group. The PC, NS, PI and ERM groups received 2 weekly s.c. (subcutaneous) injections of 1,2-DMH (40 mg/kg) (third and fourth week). In feces, pH, β-glucuronidase enzyme, and short-chain fatty acids were evaluated, and a histopathological study was performed of the intestine to detect microscopic lesions. Results: the activity of β-glucuronidase decreased (p < 0.05) for NS, PI and ERM vs. PC. The highest proportion of butyric acid was observed in the NS (p < 0.05) vs. NC group. Sixty percent of enteritides were severe in grade in the PC group, and 40 % in the experimental groups. Conclusions: native starch granules resisted pyrodextrinization, but treatment with α-amylase broke the structure of the pyrodextrin granule. According to the treatments given to the rats, as the amount of RS present in the diet increases (NS), the neoplastic cells do not advance beyond the basement membrane, suggesting a possible cell-protective or anticancer effect.

Objective: This mini-review aims to discuss research works about heparanase published in 2016, 2017, 2018 and 2019 and provide a direction for therapy methods targeting heparanase., Patients and Methods: The relevant data were searched by using keywords "heparanase" "function", "diseases" and "inhibitors" in "PubMed", "Web of Science" and "China Knowledge Resource Integrated databases (CNKI)", and a hand-search was done to acquire peer-reviewed articles and reports about heparanase., Results: Except for tumor progression, pathological processes including procoagulant activities, preeclamptic placentas, inflammation and so on are all verified to be associated with heparanase activity. Also, these newly-found functions are closely related to certain cellular activities, including epithelial to Mesenchymal Transition (EMT)., Conclusion: It could be concluded that heparanase would be a potential and valuable therapy target., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Intrauterine growth restriction is often accompanied by placental vascular disease, of which histologic maternal vascular malperfusion is prominent. Maternal vascular malperfusion is characterized by accelerated villous maturation consistent with placental aging. Alpha klotho is an anti-aging protein produced by the placenta. We hypothesize that cord blood alpha klotho varies with maternal vascular malperfusion and small for gestational age infants through dysregulated angiogenesis., Methods: Nested case-control study of 54 preterm infants (N = 22 small for gestational age infants, 32 appropriate for gestational age infants, mean gestational age = 33.7 ± 2.7 weeks) and validation sample (N = 39) from a longitudinal birth cohort at Prentice Women's Hospital, Chicago, IL. Cord blood alpha klotho was measured via enzyme-linked immunoassay; concentrations were linked to multiplex data of cord blood angiogenic growth factors., Results: Median cord blood alpha klotho was decreased in small for gestational age infants (1200 [859, 2083] pg/mL) versus controls (3193 [1703, 3963] pg/mL; p < 0.01) and with severe maternal vascular malperfusion (1170 [760, 2645] pg/mL; P < 0.01), consistent with validation sample. Alpha klotho was decreased with maternal vascular malperfusion sublesions signifying accelerated villous maturation, including increased syncytial knots (1230 [805, 3606] pg/mL; p < 0.05) and distal villous hypoplasia (1170 [770, 3390] pg/mL; p < 0.05). Among 15 angiogenic markers, alpha klotho correlated directly with angiopoietin-2 (beta-coefficient = 2.6, p = 0.01)., Conclusions: Cord blood alpha klotho is decreased with small for gestational infants and maternal vascular malperfusion sublesions of accelerated placental villous maturation, and correlated with angiopoietin-2. Alpha klotho may play a role in vascular-mediated accelerated placental aging leading to intrauterine growth restriction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Central activation of fibroblast growth factor (FGF) receptors regulates peripheral glucose homeostasis and reduces food intake in preclinical models of obesity and diabetes. The current work was undertaken to advance our understanding of the receptor expression, as sites of ligand action by FGF19, FGF21, and FGF1 in the mammalian brain remains unresolved. Recent advances in automated RNAscope in situ hybridization and droplet digital PCR (ddPCR) technology allowed us to interrogate central FGFR/beta klotho (Klb) system at the cellular level in the mouse, with relevant comparisons to nonhuman primate and human brain. FGFR1-3 gene expression was broadly distributed throughout the CNS in Mus musculus, with FGFR1 exhibiting the greatest heterogeneity. FGFR4 expression localized only in the medial habenula and subcommissural organ of mice. Likewise, Klb mRNA was restricted to the suprachiasmatic nucleus (SCh) and select midbrain and hindbrain nuclei. ddPCR in the rodent hypothalamus confirmed that, although expression levels are indeed low for Klb, there is nonetheless a bonafide subpopulation of Klb+ cells in the hypothalamus. In NHP and human midbrain and hindbrain, Klb + cells are quite rare, as is expression of FGFR4. Collectively, these data provide the most robust central map of the FGFR/Klb system to date and highlight central regions that may be of critical importance to assess central ligand effects with pharmacological dosing, such as the putative interactions between the endocrine FGFs and FGFR1/Klb, or FGF19 with FGFR4., (© 2019 Wiley Periodicals, Inc.)

Aims: Fibroblast growth factor 23 (FGF-23) is known to be elevated in patients with congestive heart failure (CHF). As FGF-23 is expressed in the bone but can also be expressed in the myocardium, the origin of serum FGF-23 in CHF remains unclear. It is also unclear if FGF-23 expressed in the bone is associated with outcome in CHF. The aim of the present study was to investigate FGF-23 levels measured in bone marrow plasma (FGF-23-BM) and in peripheral blood (FGF-23-P) in CHF patients to gain further insights into the heart-bone axis of FGF-23 expression. We also investigated possible associations between FGF-23-BM as well as FGF-23-P and outcome in CHF patients., Methods and Results: We determined FGF-23-P and FGF-23-BM levels in 203 CHF patients (85% male, mean age 61.3 years) with a left ventricular ejection fraction (LVEF) ≤45% and compared them with those of 48 healthy controls (48% male, mean age 39.2 years). We investigated the association between FGF-23-BM and FGF-23-P with all-cause mortality in CHF patients, 32 events, median follow-up 1673 days, interquartile range [923, 1828]. FGF-23-P (median 60.3 vs. 22.0 RU/mL, P < 0.001) and FGF-23-BM (median 130.7 vs. 93.1 RU/mL, P < 0.001) levels were higher in CHF patients compared with healthy controls. FGF-23-BM levels were significantly higher than FGF-23-P levels in both CHF patients and in healthy controls (P < 0.001). FGF-23-P and FGF-23-BM correlated significantly with LVEF (r = -0.37 and r = -0.33, respectively), N terminal pro brain natriuretic peptide levels (r = 0.57 and r = 0.6, respectively), New York Heart Association status (r = 0.28 and r = 0.25, respectively), and estimated glomerular filtration rate (r = -0.43 and r = -0.41, respectively) (P for all <0.001) and were independently associated with all-cause mortality in CHF patients after adjustment for LVEF, estimated glomerular filtration rate, New York Heart Association status, and N terminal pro brain natriuretic peptide, hazard ratio 2.71 [confidence interval: 1.18-6.20], P = 0.018, and hazard ratio 2.80 [confidence interval: 1.19-6.57], P = 0.018, respectively., Conclusions: In CHF patients, FGF-23 is elevated in bone marrow plasma and is independently associated with heart failure severity and all-cause mortality. The failing heart seems to interact via FGF-23 within a heart-bone axis., (© 2019 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Understanding the fate of fecal pollution in the landscape is required for microbial risk analysis. The aim of this study was to assess the patterns and dynamics of beta-d-glucuronidase (GLUC), which has been suggested as a surrogate for fecal pollution monitoring, in a stream draining an agricultural headwater catchment. Automated enzymatic on-site measurements of stream water and sediments were made over two years (2014-2016) to quantify the sources and pathways of GLUC in a stream. The event water fraction of streamflow was estimated by stable isotopes. Samples from field sediments on a hillslope, streambed sediment and stream water were analyzed for GLUC and with a standard E. coli assay. The results showed ten times higher GLUC and E. coli concentrations during the summer than during the winter for all compartments (field and streambed sediments and stream water). The E. coli concentrations in the streambed sediment were approximately 100 times those of the field sediments. Of the total GLUC load in the study period, 39% were transported during hydrological events (increased streamflow due to rainfall or snowmelt); of these, 44% were transported when the stream contained no recent rainwater. The results suggested that a large proportion of the GLUC and E. coli in the stream water stemmed from resuspended streambed sediments. Moreover, the results strongly indicated the existence of remnant populations of GLUC-active organisms in the catchment., (Copyright © 2019. Published by Elsevier B.V.)

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

This study used automated enzymatic activity measurements conducted from a mobile research vessel to detect the spatial variability of beta‑d‑glucuronidase (GLUC) activity in large freshwater bodies. The ship-borne observations provided the first high-resolution spatial data of GLUC activity in large water bodies as rapid indication of fecal pollution and were used to identify associations with hydrological conditions and land use. The utility of this novel approach for water quality screening was evaluated by surveys of the Columbia River, the Mississippi River and the Yahara Lakes, covering up to a 500 km river course and 50 km 2 lake area. The ship-borne measurements of GLUC activity correlated with standard E. coli analyses (R 2  = 0.71) and revealed the effects of (1) precipitation events and urban run-off on GLUC activity in surface waters, (2) localized point inlets of potential fecal pollution and (3) increasing GLUC signals along gradients of urbanization. We propose that this ship-borne water quality screening to be integrated into future water inventory programs as an initial or complementary tool (besides established fecal indicator parameters), due to its ability to provide near real-time spatial information on potential fecal contamination of large surface water resources and therefore being helpful to greatly reduce potential human health risks., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

β-Glucuronidase (GLU) as a vital factor in enterohepatic circulation and drug-inducing enteropathy has been given more and more attention in recent years. In this study, an off-on near-infrared (NIR) fluorescent probe (DDAO-glu) for selectively and sensitively sensing GLU was developed on the basis of its substrate preference. DDAO-glu can rapidly and selectively respond to bacterial GLU under physiological conditions for detecting the real-time intestinal GLU bioactivity of complex biological systems such as human feces in clinic. Meantime, DDAO-glu has been successfully applied for visualization of endogenous GLU in bacterial biofilm, thallus, and even in vivo. Using this NIR probe, we successfully visualized the real distribution of intestinal GLU in the enterohepatic circulation. Furthermore, a high-throughput screening method was successfully established by our probe, and a potent natural inhibitor of GLU was identified as (-)-epicatechin-3-gallate (ECG) for effectively preventing NSAIDs-inducing enteropathy in vivo. DDAO-glu could serve as a powerful tool for exploring real physical functions of intestinal GLU in enterohepatic circulation, under physiological and pathological contexts, and developing the novel inhibitors of GLU to therapy acute drug-inducing enteropathy in clinic.

Lack of an efficient transformation system has hampered the molecular breeding of Agaricus bisporus. Here, we describe a highly efficient Agrobacterium-mediated transformation system for A. bisporus using foxtail millet (Setaria italica L. Beauv) grains as cultivation and infection media. Mycelium-millet complexes were prepared for co-culture and treated with ultrasonication for 10 s to improve infection efficiency. After a 72-h culture period, the newly grown mycelium-surrounded millet grain was transferred to selection medium supplemented with 200 μg/mL cefotaxime and 15 μg/mL hygromycin B (hyg) to screen positive transformants. Putative transformants were analyzed for the presence of the hyg gene by polymerase chain reaction and Southern blotting. Expression of eGFP in A. bisporus transformants was detected by fluorescence imaging, and the β-glucuronidase (GUS) protein was detected by histochemical staining. Our protocol resulted in an average 53.85% transformation frequency, and over 85% of the transformants tested remained mitotically stable, even after five successive rounds of subculturing. A feasible method for A. bisporus mushroom transformation using foxtail millet as an innovative culture medium was developed, which will benefit future functional genetic studies of this mushroom., (Copyright © 2018. Published by Elsevier B.V.)

Mobilization of transposable elements (TEs) in plants has been recognized as a driving force of evolution and adaptation, in particular by providing genes with regulatory modules that impact their transcription. In this study, we employed an ATCOPIA93 long-terminal repeat (LTR) promoter- GUS fusion to show that this retrotransposon behaves like an immune-responsive gene during pathogen defense in Arabidopsis We also showed that the endogenous ATCOPIA93 copy " EVD ", which is activated in the presence of bacterial stress, is negatively regulated by both DNA methylation and polycomb-mediated silencing, a mode of repression typically found at protein-coding and microRNA genes. Interestingly, an ATCOPIA93 -derived soloLTR is located upstream of the disease resistance gene RPP4 and is devoid of DNA methylation and H3K27m3 marks. Through loss-of-function experiments, we demonstrate that this soloLTR is required for the proper expression of RPP4 during plant defense, thus linking the responsiveness of ATCOPIA93 to biotic stress and the co-option of its LTR for plant immunity., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

This study was designed to evaluate the influence of phosphorus (P) restriction on the deleterious effects of high fat diets on mineral metabolism. Twenty-four rats were allotted to 3 groups (n = 8 each) that were fed different diets for 7 months. Rats in group 1 were fed normal fat-normal P (0.6%) diet (NF-NP), rats in group 2 were fed high fat- normal P diet (HF-NP) and rats in group 3 were fed high fat-low P (0.2%) diet (HF-LP). Blood, urine and tissues were collected at the end of the experiments. When compared with the control group (NF-NP), rats fed HF diets showed increases in body weight, and in plasma concentrations of triglycerides and leptin, and decreased plasma calcitriol concentrations. In rats fed HF-NP plasma fibroblast growth factor 23 (FGF23) was higher (279.6±39.4 pg/ml vs 160.6±25.0 pg/ml, p = 0.018) and renal klotho (ratio klotho/GAPDH) was lower (0.75±0.06 vs 1.06±0.08, p<0.01) than in rats fed NF-NP. Phosphorus restriction did not normalize plasma FGF23 or renal klotho; in fact, rats fed HF-LP, that only ingested an average of 22.9 mg/day of P, had higher FGF23 (214.7±32.4 pg/ml) concentrations than rats fed NF-NP (160.6±25.0 pg/ml), that ingested and average of 74.4 mg/day of P over a 7 month period. In conclusion, our results demonstrate that severe P restriction over a prolonged period of time (7 months) does not normalize the increase in circulating FGF23 induced by HF diets. These data indicate that the deleterious effects of high fat diet on the FGF23/klotho axis are not eliminated by reduced P intake., Competing Interests: The authors have declared that no competing interests exist.

Background: Increased vascular permeability is a pathophysiological hallmark of sepsis and results in increased transcapillary leakage of plasma fluid, hypovolemia, and interstitial edema formation. 6% hydroxyethyl starch (HES 130/0.4) is commonly used to treat hypovolemia to maintain adequate organ perfusion and oxygen delivery. The present study was designed to investigate the effects of 6% HES 130/0.4 on glycocalyx integrity and vascular permeability in lipopolysaccharide (LPS)-induced pulmonary inflammation and systemic inflammation in mice., Methods: 6% HES 130/0.4 or a balanced electrolyte solution (20 ml/kg) was administered intravenously 1 h after cecal ligation and puncture (CLP) or LPS inhalation. Sham-treated animals receiving 6% HES 130/0.4 or the electrolyte solution served as controls. The thickness of the endovascular glycocalyx was visualized by intravital microscopy in lung (LPS inhalation model) or cremaster muscle (CLP model). Syndecan-1, hyaluronic acid, and heparanase levels were measured in blood samples. Vascular permeability in the lungs, liver, kidney, and brain was measured by Evans blue extravasation., Results: Both CLP induction and LPS inhalation resulted in increased vascular permeability in the lung, liver, kidney, and brain. 6% HES 130/0.4 infusion led to significantly reduced plasma levels of syndecan-1, heparanase, and hyaluronic acid, which was accompanied by a preservation of the glycocalyx thickness in postcapillary venules of the cremaster (0.78 ± 0.09 μm vs. 1.39 ± 0.10 μm) and lung capillaries (0.81 ± 0.09 μm vs. 1.49 ± 0.12 μm)., Conclusions: These data suggest that 6% HES 130/0.4 exerts protective effects on glycocalyx integrity and attenuates the increase of vascular permeability during systemic inflammation.

Klotho deficiency is relevant to renal fibrosis and podocyte injury in vivo and in vitro. We examined whether histological findings of renal biopsy specimens were associated with the levels of soluble klotho in humans. We investigated renal biopsy specimens of 67 patients and detailed microscopic findings were reviewed. Soluble serum/urinary klotho and urinary angiotensinogen were assessed by enzyme-linked immunosorbent assays, and tissue klotho expression was assessed by immunohistochemical staining. The median age of the study participants was 35.6 years. High serum klotho levels (≥14 pg/mL) were associated with decreased odds ratios (ORs) of interstitial fibrosis (OR = 0.019, P = 0.003) and segmental sclerosis (OR = 0.190, P = 0.022) in multivariable logistic regression analysis. Patients with a lower urinary klotho-to-creatinine ratio (UKCR) were significantly more likely to have diffuse foot process effacement (OR = 0.450, P = 0.010). The area under the receiver-operating characteristic curve (AUC) of serum klotho for predicting interstitial fibrosis was 0.920 (95% CI, 0.844-0.996), and the best cut-off value of serum klotho was 138.1 pg/mL. The AUC of UKCR for predicting diffuse foot process effacement was 0.754 (95% CI, 0.636-0.872), and the best cut-off value of UKCR was 96.7 pg/mgCr. Urinary angiotensinogen-to-creatinine ratio was not associated with serum klotho, UKCR, or any pathological finding. Our data suggested that soluble serum and urinary klotho levels represent a potential biomarker to predict renal fibrosis and podocyte injury in humans.

Background/aims: Klotho is a multifunctional protein expressed predominantly in kidney tubular epithelium. Here, we investigated the protective effects of Klotho on necroptosis in renal ischemic-reperfusion injury (IRI) and the role of oxidative stress in this process., Methods: Mice were subjected to bilateral renal pedicle clamping. Mouse renal tubular epithelial (TCMK-1) cells were exposed to hypoxia/reoxygenation (H/R) or H2O2. Kidney samples from acute kidney injury (AKI) patients and controls were examined by immunofluorescence. Klotho protein and N-acetyl-L-cysteine (NAC) were used to define their roles in mediating necroptosis. Necroptosis was assessed by TUNEL staining, immunoblotting, and real-time PCR. Oxidative stress was studied via ELISA, immunoblotting, colorimetric, and thiobarbituric acid reactive substances assays., Results: Renal IRI induced Klotho deficiency in the serum and kidney, but an increase in the urine. The levels of the necroptotic markers receptor-interacting protein kinase (RIP) 1, RIP3, IL-1β, and TUNEL-positive cells increased after IRI; all increases were ameliorated by Klotho. In TCMK-1 cells, Klotho and NAC attenuated the elevation in RIP1, RIP3, and LDH release induced by H/R or H2O2. Moreover, Klotho decreased the levels of oxidative stress biomarkers and elevated superoxide dismutase 2 expression in both in vivo and in vitro experiments. Studies in human samples further confirmed the Klotho deficiency and increased formation of RIP3 puncta in AKI kidneys., Conclusion: Klotho protects tubular epithelial cells from IRI and its anti-necroptotic role may be associated with oxidative stress inhibition., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The heparan sulfate endoglycosidase heparanase (HPSE) is involved in tumor growth, chronic inflammation and fibrosis. Since a role for HPSE in chronic liver disease has not been demonstrated to date, the current study was aimed at investigating the involvement of HPSE in the pathogenesis of chronic liver injury. Herein, we revealed that HPSE expression increased in mouse livers after carbon tetrachloride (CCl 4 )-mediated chronic induction of fibrosis, but with a trend to decline during progression of the disease. In mouse fibrotic liver tissues HPSE immunostaining was restricted in necro-inflammatory areas, co-localizing with F4/80 macrophage marker and TNF-α. TNF-α treatment induced HPSE expression as well as HPSE secretion in U937 macrophages. Moreover, macrophage-secreted HPSE regulated the expression of α-SMA and fibronectin in hepatic stellate LX-2 cells. Finally, HPSE activity increased in the plasma of patients with liver fibrosis but it inversely correlated with liver stiffness. Our results suggest the involvement of HPSE in early phases of reaction to liver damage and inflammatory macrophages as an important source of HPSE. HPSE seems to play a key role in the macrophage-mediated activation of hepatic stellate cells (HSCs), thus suggesting that HPSE targeting could be a new therapeutic option in the treatment of liver fibrosis.

Metabolized forms of benzodiazepines (benzos) can cause issues with mass spectrometry identification. Benzodiazepines undergo a process called glucuronidation during metabolism that attaches a glucuronic acid for increased solubility. Often in clinical testing an enzymatic hydrolysis step is implemented to increase the sensitivity of benzodiazepines by hydrolyzing β-D-glucuronic acid from benzodiazepine-glucuronide conjugates in urine samples using the β-Glucuronidase enzyme. In this study resorufin β-D-glucuronide, a substrate of the β-Glucuronidase enzyme, was added to patient samples to determine if proper hydrolysis had occurred. The presence of resorufin as an Internal Hydrolysis Indicator (IHI) shows the activity and efficiency of the enzyme in each patient sample. Synthetic/patient urine samples were obtained and mixed with hydrolysis buffer containing resorufin β-D-glucuronide. The β-Glucuronidase enzyme was used to hydrolyze the benzodiazepine analytes as well as resorufin β-D-glucuronide. The enzymatic hydrolysis addition increased the positivity rate of benzodiazepines by 42.5%. The β-Glucuronidase substrate resorufin (IHI) displayed variability in area counts between patient samples. Comparative studies with internal standards and resorufin (IHI) showed no correlation between recovery and analyte variability. Hydrolysis reactions greatly improved the sensitivity of benzodiazepines by liquid chromatography time-of-flight mass spectrometry analysis. The large variation in resorufin (IHI) area counts amongst patient samples indicates possible variability in enzymatic hydrolysis activity. The enzymatic hydrolysis step is a part of the extraction procedure and should be controlled for in each patient sample., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Klotho is a recently discovered anti‑aging gene, which has been reported as a tumor suppressor in numerous human malignancies; however, the role of Klotho in human ovarian cancer remains to be elucidated. The aim of the present study was to detect the expression of Klotho and evaluate its association with the progression of human ovarian cancer. A clinical follow‑up study of 120 patients with ovarian cancer and 78 normal controls was conducted. The expression levels of Klotho were determined by western blotting and immunohistochemistry. The results demonstrated that high Klotho expression levels were detected in all normal controls, whereas the positive rate of Klotho was 61.6% in the ovarian cancer group, which was significantly decreased compared with in the control group (P<0.01). Furthermore, reduced Klotho expression was significantly correlated with decreased survival rates in patients with ovarian cancer (P=0.025). Subsequently, Klotho levels were detected in seven human ovarian cancer cell lines by western blotting. The results demonstrated that the highest levels of Klotho were detected in CaOV3 cells, medium levels of Klotho were detected in CaOV4 and SKOV‑3 cells, and almost no Klotho was detected in the other four cell lines: OVCA 432, OVCAR‑5, OVCAR‑8 and A2780 cells. The association between Klotho levels and cell proliferation was determined by MTT assay, and the results indicated that higher levels of Klotho inhibited the proliferation of A2780 and OVCAR‑5 cells, whereas reduced Klotho expression promoted cell growth of CaOV3 and SKOV‑3 cells. In addition, the plasma levels of inflammatory cytokines in tumor‑bearing mice and normal control mice were detected by enzyme‑linked immunosorbent assay, and plasma interleukin (IL)‑6 and IL‑1β levels were elevated in all tumor‑bearing mice. Notably, the mRNA expression levels of IL‑6 were significantly higher in the liver, ovaries and kidneys of Klotho‑/‑ mice compared with in wild type mice (P<0.01), thus indicating that aberrant Klotho expression may contribute to systemic inflammation in Klotho‑/‑ mice. Finally, the in vivo antitumor role of aberrant Klotho expression was determined in a nude mice model. A2780 cells were transfected with pCMV6‑Klotho and the stably transfected cells were screened; the mice were injected with the stably transfected cells. The results indicated that tumor volume and tumor weight were significantly decreased in the pCMV6‑Klotho group compared with in the pCMV6 vector group (P<0.01). These findings suggest that overexpression of Klotho may suppress tumor growth in animal models. In conclusion, Klotho was demonstrated to act as a potent tumor suppressor in human ovarian cancer cells. Reduced Klotho expression was detected in the specimens of patients with ovarian cancer, and overexpression of Klotho significantly inhibited cell proliferation of human ovarian cancer cells. Therefore, Klotho may be considered a useful key target for the molecular therapy of human ovarian cancer.

In many karst regions in developing countries, the populations often suffer from poor microbial water quality and are frequently exposed to bacterial pathogens. The high variability of water quality requires rapid assays, but the conventional cultivation-based analysis of fecal indicator bacteria, such as Escherichia coli (E. coli), is very time-consuming. In this respect, the measurement of the enzymatic activity of E. coli could prove to be a valuable tool for water quality monitoring. A mobile automated prototype device was used for the investigation of β-d-glucuronidase (GLUC) activity at a remote karst spring, connected to a sinking surface stream, in Northern Vietnam. To assess the relationship between GLUC activity, discharge dynamics and contamination patterns, multiple hydrological, hydrochemical, physicochemical and microbiological parameters, including discharge, turbidity, particle-size distributions, and E. coli, were measured with high temporal resolution during ten days of on-site monitoring. A complex contamination pattern due to anthropogenic and agricultural activities led to high E. coli concentrations (270 to >24,200 MPN/100ml) and a GLUC activity between 3.1 and 102.2 mMFU/100ml. A strong daily fluctuation pattern of GLUC activity and particle concentrations within small size classes (<10μm) could be observed, as demonstrated by autocorrelations. A Spearman's rank correlation analysis resulted in correlation coefficients of rs=0.56 for E. coli and GLUC activity and rs=0.54 for GLUC activity and the concentration of 2-3μm particles. On an event scale, correlations were found to be higher (rs=0.69 and 0.87, respectively). GLUC activity and E. coli displayed a general contamination pattern, but with significant differences in detail, which may be explained by interferences of e. g. viable but non-culturable cells. Although further evaluations are recommended, GLUC activity is a promising, complementary parameter for on-site and near real-time water quality monitoring., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A suitable promoter greatly enhances the efficiency of target gene expression of plant molecular breeding and farming; however, only very few promoters are available for economically important non-graminaceous ornamental monocots. In this study, an 868-bp upstream region of defense-related LsGRP1 of Lilium, named P LsGRP1 , was cloned by genome walking and proven to exhibit promoter activity in Nicotiana benthamiana and Lilium 'Stargazer' as assayed by agroinfiltration-based β-glucuronidase (GUS) expression system. Many putative biotic stress-, abiotic stress- and physiological regulation-related cis-acting elements were found in P LsGRP1 . Serial deletion analysis of P LsGRP1 performed in Nicotiana tabacum var. Wisconsin 38 accompanied with types of treatments indicated that 868-bp P LsGRP1 was highly induced upon pathogen challenges and cold stress while the 131-bp 3'-end region of P LsGRP1 could be dramatically induced by many kinds of abiotic stresses, biotic stresses and phytohormone treatments. Besides, transient GUS expression in a fern, gymnosperms, monocots and dicots revealed good promotor activity of P LsGRP1 in many phylogenetically distinct plant species. Thus, pathogen-inducible P LsGRP1 and its 131-bp 3'-end region are presumed potential as tools for plant molecular breeding and farming., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Context and Objective:: Acute kidney injury (AKI) is still a headache for clinicians and scientists as a possible reason for increased death among intensive care unit (ICU) patients after invasive cardiac surgery. Furthermore, the diagnostic process for AKI using conventional biomarkers is not sufficient to ensure early warning of this condition because of the morbid influence of non-renal factors that definitively delay the time for the prognosis. These imposed limitations have led to significant amounts of research targeted towards identifying novel biomarkers for AKI with a sustained degree of sensitivity and specificity. Here, we reviewed previous studies conducted on the Klotho, CYR61 and YKL-40 biomarkers in relation to AKI., Design and Setting:: Review of the literature conducted in the Institute of Clinical Chemistry & Biochemistry, Ljubljana University Medical Center, Slovenia., Methods:: The literature was searched in PubMed and the Cochrane Library. From the database of this specialty, we selected 17 references that matched our context for detailed analysis and further investigation., Results:: The studies reviewed showed notable differences in their results relating to the diagnostic impact of Klotho, CYR61 and YKL-40 on early prediction of AKI., Conclusions:: The results regarding the Klotho, CYR61 and YKL-40 biomarkers showed markedly equivocal performance in the previous studies and did not fulfill the expectations that these factors would form valid possible biomarkers for AKI.

Early detection and diagnosis have great practical significances for the effective prevention and treatment of cancer. In this study, we developed a novel, facile and ultra-sensitive fluorescence assay for the determination of tumor invasive biomarker β-glucuronidase (GLU) based on the inner-filter effect (IFE). The nitrogen-doped carbon quantum dots (N-CQDs) with green photoluminescence were employed as the fluorophore in IFE, and 4-nitrophenyl-β-D-glucuronide (PNPG) was used to act as GLU substrate, and GLU catalytic product (p-nitrophenol (PNP)) was capable of acting as the robust absorber in IFE to turn off the fluorescence of N-CQDs due to the complementary overlap between the absorption of PNP and the excitation of N-CQDs. Thus, signal of GLU activity could be recorded by the fluorescence intensity of N-CQDs. Unlike other fluorescence sensing mechanism such as fluorescence resonance energy transfer (FRET) or photoinduced electron transfer (PET), IFE has no requirement for electron or energy transfer process or any chemical modification of fluorophore, which makes our assay more flexible and simple. The proposed method exhibited a good linear relationship from 1UL(-1) to 60UL(-1) (R(2)=0.9967) with a low detection limit of 0.3UL(-1). This method was also successfully applied to the analysis of serum samples and the inhibitor screening from natural product. The developed sensor platform was proven to be reliable, facile, sensitive, and selective, making it promising as a candidate for GLU activity detection in clinic tumor diagnose and anti-tumor drug screening., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tumor metastatic lymph node mapping has been widely used to predict the metastatic spread of primary tumor and guide the lymph node dissection in clinical practice. In this research, a new near-infrared (NIR)-emitting low molecular weight heparin (LMWHEP)-modified Cy7-loaded nanoliposome (LMWHEP-NLips/Cy7) was developed and had the particle size of about 80 nm and the fluorescence intensity of about 2300, which is optimal for metastatic lymph node uptake and imaging. The NIR-emitting nanoliposomes were designed by LMWHEP coating on the surface of Cy7-loaded nanoliposome (NLips/Cy7) according to electrostatic attraction. The LMWHEP-NLips/Cy7 with negligible cytotoxicity for Hela and RAW264.7 cells and was found to be fluorescent stability compared with the Cy7-free dye at room temperature. The BALB/c nude mice bearing tumor lymphatic metastasis was established at eighth week post-injection by subcutaneously injecting Hela cells suspension. Heparanase (HPA) expression concentrations quantitatively measured by ELISA kit respectively were 237.42U/mL, 214.82U/mL and 128.45U/mL in the extracellular Hela cells, metastatic popliteal and iliac lymph node. LMWHEP-NLips/Cy7 successfully increased fluorescence signal in the metastatic lymph node compared with normal lymph node and achieve in vivo and ex vivo high fluorescence signal within 10 min and retention time up to 4 h post-injection. Maximum mean fluorescence intensity of the LMWHEP-NLips/Cy7 group was significantly more than NLips/Cy7 group (increase 2-fold in the metastatic popliteal lymph node and 4.8-fold in the metastatic iliac lymph node, p < 0.05). The experimental results demonstrating LMWHEP-NLips/Cy7 have the potential utility as specific, biosafe and stable near-infrared imaging contrast agents for HPA-expression tumor metastatic lymph node mapping. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Chronic kidney failure involves abnormalities of mineral metabolism, skeletal and of cardiovascular system (so called CKD - MBD) that have a major impact on the survival of renal patient. Increasingly complex pathophysiological mechanisms have been discovered in recent years with evidence of new molecules involved in the development of CKD - MBD. Besides the classical PTH / Vitamin D axis, the most recent discovery of a new FGF23 / Klotho axis has expanded knowledge on the mechanisms of mineral homeostasis but also on the more complex mechanisms of cellular aging, vascular calcification and cardiac remodeling. The importance of bone as an endocrine organ has become even more evident following the discovery of molecules such as Sclerostin (involved in the regulation of osteoblastic proliferation and differentiation) and Sibling (a family of proteins that regulate both local and systemic mineral metabolism). The ability to characterize as biomarkers of CKD - MBD for these new molecules depends on their eventual ability to express a specific pathophysiological processes, identify patients at risk, highlight the response to a therapeutic treatment and to be easily identifiable and quantifiable on biological fluids. As of today, it seems that we can recognize FGF23 as a biomarker of CKD-MBD, while the remaining molecules as still waiting for a more definite settlement.

Introduction: α-klotho is an anti-aging protein, potentially important in preeclampsia (PE). Produced by kidney, brain and placenta, and by mRNA splicing is both a full-length membrane-bound and a truncated soluble protein in the circulation. The membrane-bound protein is an obligate co-receptor for fibroblast growth factor 23 (FGF23) and its action on receptor (FGFR), but ADAM proteinases also cause its shedding. The aims of this study were to investigate levels of maternal plasma, placental, and fetal membrane α-Klotho and their association with placental accelerated villous maturation (AVM) in PE. In addition, placental and membrane levels of ADAM17 and FGFR were measured in the same patients., Methods: Maternal blood, placenta and fetal membranes from 61 women (31 with PE and 30 controls) between 32 and 40 weeks gestation were collected. Plasma α-klotho was measured by ELISA, and quantitative immunohistochemistry used for α-klotho, ADAM17 and FGFR1 in tissues. Placental AVM was histologically assessed., Results: Maternal plasma levels of α-Klotho were higher in PE compared to controls (p = 0.01) and patients with the highest levels had significantly less AVM (p = 0.03). α-Klotho, ADAM17, and FGFR were all present in syncytiotrophoblast and cytotrophoblast of membranes. Between 32 and 40 weeks gestation, all placental levels decreased in controls respectively (p = 0.04, p = 0.004, p = 0.05), but not in PE. Fetal membrane levels were unchanged., Discussion: Maternal plasma α-Klotho was increased in PE and its levels associated with reduced placental AVM. Changes in placental α-Klotho, ADAM17, and FGFR suggest their involvement in the pathophysiology of PE., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Plants transport photoassimilates from source organs to sink tissues through the phloem translocation pathway. In the transport phloem, sugars that escape from the sieve tubes are released into the apoplasmic space between the sieve element/companion cell complex (SE/CC) and phloem parenchyma cells (PPCs) during the process of long-distance transport. The competition for sugar acquisition between SE/CC and adjoining PPCs is mediated by plasma membrane translocators. YFP-tagged AtSWEET4 protein is localized in the plasma membrane, and PromoterAtSWEET4-GUS analysis showed that AtSWEET4 is expressed in the stele of roots and veins of leaves and flowers. Overexpression of AtSWEET4 in Arabidopsis increases plant size and accumulates more glucose and fructose. By contrast, knock-down of AtSWEET4 by RNA-interference leads to small plant size, reduction in glucose and fructose contents, chlorosis in the leaf vein network, and reduction in chlorophyll content in leaves. Yeast assays demonstrated that AtSWEET4 is able to complement both fructose and glucose transport deficiency. Transgenic plants of AtSWEET4 overexpression exhibit higher freezing tolerance and support more growth of bacterium Pseudomonas syringae pv. phaseolicola NPS3121. We conclude that AtSWEET4 plays an important role in mediating sugar transport in axial tissues during plant growth and development.

Background: Trans-metatarsal operation to diabetic foot necrosis is a common procedure although only half of the patients do not need a second amputation due to surgery wound ischemia. No current tools are available for early prediction of surgery success and the clinical decision for a second operation may take weeks. Heparanase protein is involved in inflammation, angiogenesis and coagulation activation. The aim of the study was to evaluate heparanase level and procoagulant activity as an early predictor for success or failure of diabetic foot trans-metatarsal surgery., Methods: The study group included 40 patients with diabetic foot necrosis requiring trans-metatarsal surgical intervention. Eighteen patients designated as necrotic group, developed post-surgery necrosis at the surgery wound within the first month, requiring a second more proximal amputation. Skin biopsies from the proximal surgery edge were stained for heparanase, tissue factor (TF), TF pathway inhibitor (TFPI) and by hematoxylin and eosin. Plasma samples were drawn pre-surgery and at 1h, 1week and 1month post-surgery. Samples were tested for heparanase levels by ELISA and TF+heparanase activity, TF activity and heparanase procoagulant activity., Results: Skin biopsy staining did not predict subsequent necrosis. In the non-necrotic group a significant rise in TF+heparanase activity, heparanase activity and heparanase levels were observed 1h and 1week post-surgery. The most significant increase was in heparanase procoagulant activity at the time point of 1h post-surgery (P<0.0001). Pre-surgery TF activity was significantly lower in the non-necrotic group compared to the necrotic group (P<0.05)., Conclusions: Measuring heparanase procoagulant activity pre-surgery and 1h post-surgery could potentially serve as an early tool to predict the procedure success. The present results broaden our understanding regarding early involvement of heparanase in the wound healing process., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Aerobic vaginitis (AV) is a recently proposed term for genital tract infection in women. The diagnosis of AV is mainly based on descriptive diagnostic criteria proposed by Donders and co-workers. The objective of this study is to report AV prevalence in southwest China using an objective assay kit based on preformed enzymes and also to determine its characteristics., Materials and Methods: A total of 1948 outpatients were enrolled and tested by a commercial diagnostic kit to investigate the AV prevalence and characteristics in southwestern China. The study mainly examined the vaginal ecosystem, age distribution, Lactobacillus amount, and changes in pH. Differences within groups were analyzed by Wilcoxon two-sample test., Results: The AV detection rate is 15.40%. The AV patients were usually seen in the sexually active age group of 20-30 years, followed by those in the age group of 30-40 years. The vaginal ecosystems of all the patients studied were absolutely abnormal, and diagnosed to have a combined infection [aerobic vaginitis (AV) + bacterial vaginitis (BV) 61.33%; 184/300]. Aerobic bacteria, especially Staphylococcus aureus and Escherichia coli, were predominantly found in the vaginal samples of these women., Conclusion: AV is a common type of genital infection in southwestern China and is characterized by sexually active age and combined infection predominated by the AV and BV type., (Copyright © 2016. Published by Elsevier B.V.)

Aims: Klotho is a trans-membrane protein that serves as a tumour suppressor in a wide array of malignancies. Recent data suggest it as an epigenetically silenced tumour suppressor in cervical cancer. Yet, the expression pattern of klotho in cervical cancer has not been determined. We aimed to study the expression of klotho in squamous cell carcinomas (SQCC) and adenocarcinoma (ADC) of the cervix., Methods: Klotho expression was analysed by immunohistochemistry in 44 SQCC samples, 38 ADC samples and the adjacent normal tissue. For each sample, percentage of positive stained cells, staining intensity and a combined staining score were recorded. Staining was validated by measuring klotho mRNA levels, using quantitative RT-PCR, in 18 of the samples., Results: Klotho expression was high in all endocervical and exocervical normal tissues adjacent to tumour. No expression of klotho was noted in 7 out of 38 (18.4%) ADC samples and in 2 out of 44 (4.5%) SQCC samples. Staining intensity, number of positively stained cells and combined intensity score were all lower in tumours compared with normal adjacent tissues in ADC and SQCC. Klotho mRNA levels highly correlated with immunohistochemical (IHC) staining (p=0.008)., Conclusions: We found reduced klotho expression in cervical carcinoma, especially in ADC, compared with normal adjacent tissue. Our results support the role of klotho as a potential tumour suppressor in cervical cancer. Further studies are required in order to establish the therapeutic role of klotho in cervical carcinoma and identify patients who may benefit from it., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Peel Plate™ EC is a low-profile plastic, 47 mm culture dish with an adhesive top that contains a dried medium with Gram-negative selective agents and with enzyme substrate indicators for β-galactosidase (coliform) and β-glucuronidase (Escherichia coli). The method provides a conventional quantitative coliform (red) and E. coli (blue/purple/black) count with simple rehydration and incubation for 24 ± 2 h at 35 ± 1°C, while providing a total coliform result, sum of E. coli, and coliform without color differential in dairy products at 32 ± 1°C for 24 ± 2 h. Dairy matrixes claimed and supported with total coliform data are whole milk, skim milk, chocolate milk (2% fat), heavy cream (35% fat), pasteurized whole goat milk, ultra-high-temperature pasteurized milk, powdered milk, lactose-reduced milk, strawberry milk, shredded cheddar cheese, raw cow milk, raw goat milk, raw sheep milk, sour cream, condensed milk, eggnog, vanilla ice cream, condensed whey, yogurt, and cottage cheese. Matrixes claimed for E. coli and total coliform detection are raw ground beef, mixed cellulose 0.45 μm filtered bottled water, environmental sponge of stainless steel, raw ground turkey, dry dog food, liquid whole pasteurized eggs, milk chocolate, leafy green (mixed greens) rinse/flume water, irrigation water, poultry carcass rinse, and large animal carcass sponge. The method has been independently evaluated for total coliform in whole milk, skim milk, chocolate milk, and heavy cream. The method was also independently evaluated for E. coli and coliform in ground beef, filtered bottled water, and sponge rinse from stainless steel surfaces. In inclusivity and exclusivity studies, the method detected 57 of 58 different strains of coliform and E. coli at 32 ± 1°C and 35 ± 1°C in and excluded 31 of 32 different noncoliform strains consisting of Gram-negative and Gram-positive bacteria. In the matrix study, each matrix was assessed separately at each contamination level in comparison to an appropriate reference method. Colony counts were determined for each level and then log10 transformed. The transformed data were evaluated for repeatability, log-mean comparison between methods with 95% confidence interval, and r(2). A 95% confidence interval range of -0.5 to 0.5 on the mean difference was used as the acceptance criterion to establish significant statistical difference between methods. The evaluations demonstrate that the Peel Plate EC method provides no statistical differences across most of the matrixes. The coliform r(2) values were greater than 0.9 except in the case of skim milk (r(2) = 0.77 and 0.69), sheep milk (0.84), and chocolate (0.81). In the case of skim milk, the three highest concentrations were significantly biased low compared with the reference method, whereas in the case of chocolate, the highest concentration was significantly biased high. The E. coli r(2) values were greater than 0.9 except in the case of hog rinse (0.89), flume water (0.82), and chocolate (0.77). The lower values were generally from only a 1 log difference between highest and lowest concentrations except in the case of chocolate, in which the highest concentration was biased high compared with the reference method. Within-method repeatability of Peel Plate EC was similar to the reference method, with relative SDs generally less than 5% when log10 means were ≥1.5. QC data support that the Peel Plate EC is stable for 1 year when refrigerated. Incubation temperature ranges, 30-36°C, and times, 22-26 and 48 h for yogurt, were not significantly different in paired t-test comparison. The method is selective without the need for confirmation, although confirmation of coliform and E. coli was performed as part of the validation work.

A sensitive field-portable fluorimeter with incubating capability and triplicate sample chambers was designed and built. The system was optimised for the on-site analysis of E. coli in recreational waters using fluorescent based enzyme assays. The target analyte was β-D-Glucuronidase (GUS) which hydrolyses a synthetic substrate 6-Chloro-4-Methyl-Umbelliferyl-β-D-Glucuronide (6-CMUG) to release the fluorescent molecule 6-Chloro-4-Methyl-Umbelliferyl (6-CMU). The system was calibrated with 6-CMU standards. A LOD of 5 nM and a resolution of less than 1 nM was determined while enzyme kinetic tests showed detection of activities below 1 pmol min(-1) mL(-1) of sample. A field portable sample preparation, enzyme extraction protocol and continuous assay were applied with the system to analyse freshwater and marine samples. Results from a one day field trial are shown which demonstrated the ability of the system to deliver results on-site within a 75 min period., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Upregulation of heparanase has been reported in an increasing number of human cancer tissues. However, the level of salivary heparanase and its clinical significance in patients with salivary gland tumors remain unclear., Methods: Salivary heparanase levels in patients with salivary gland tumors were detected using enzyme-linked immunosorbent assays (ELISAs) and the clinical significance was evaluated by analyzing the correlations among salivary heparanase levels, clinicopathological parameters, and clinical outcomes., Results: The levels of salivary heparanase were significantly higher in patients with malignant salivary gland tumors than in benign tumors and normal controls (P<0.0001). High salivary heparanase levels were positively correlated with increased lymph node metastasis (P = 0.0235) and poorer tumor node metastasis stage (TNM) (P = 0.0183). Survival analyses revealed that high salivary heparanase levels were associated with worse overall survival (P = 0.0023) and disease-free survival (DFS) (P = 0.0025)., Conclusions: The study shows that salivary heparanase levels, as detected by the ELISAs, can be used to diagnose and provide an accurate prognosis for malignant salivary gland tumors. Salivary heparanase level was an independent predictor in patients with malignant salivary gland tumors.

We developed a fluorescent β-d-glucuronidase activity (BGA)-based assay for detecting and quantifying Escherichia coli in samples to assess the biocide efficacy of hand antiseptics. The fluorescence level is proportional to the number of viable E. coli organisms present. We compared our assay results to those of the E. coli plate count method specified by the European standard for testing hygienic hand rub disinfectant products (EN1500). The plate count method requires excessive handling and materials and is not valid if the number of organisms per plate is too low or high for counting in many of the samples. We optimized the fluorescent assay based on the cleavage of 4-methylumbelliferyl-β-d-glucuronide by adding 4-nitrophenyl-β-d-glucuronide, a nonfluorogenic BGA substrate, to induce glucuronidase activity and reduce assay time. Furthermore, our method can be automated and eliminates the need for multiple dilutions. Fluorescence was temporally monitored, and the time required to reach a specific value of fluorescence was correlated with the initial number of viable E. coli organisms on the samples. There was a positive correlation (P < 0.05) with a high correlation coefficient (R(2) = 0.82) between the E. coli counts by plate count and fluorescence methods. Reported effects in fluorescent BGA were compared to the EN1500 plate count method with five hand disinfectants. We found our method more advantageous, because it was as sensitive as the EN1500 method, requires less time to complete, and is less expensive and less laborious than conventional plating techniques., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The carA and carB genes code the small and large subunits of carbamoyl-phosphate synthase (CPS) that responsible for arginine and pyrimidine production. The purpose of this work was to study the gene organization and expression pattern of carAB operon, and the biological functions of carA and carB genes in Xanthomonas citri subsp. citri., Methods: RT-PCR method was employed to identify the full length of carAB operon transcript in X. citri subsp. citri. The promoter of carAB operon was predicted and analyzed its activity by fusing a GUS reporter gene. The swimming motility was tested on 0.25% agar NY plates with 1% glucose. Biofilm was measured by cell adhesion to polyvinyl chloride 96-well plate., Results: The results indicated that carAB operon was composed of five gene members carA-orf-carB-greA-rpfE. A single promoter was predicted from the nucleotide sequence upstream of carAB operon, and its sensitivity to glutamic acid, uracil and arginine was confirmed by fusing a GUS reporter gene. Deletion mutagenesis of carB gene resulted in reduced abilities in swimming on soft solid media and in forming biofilm on polystyrene microtiter plates., Conclusions: From these results, we concluded that carAB operon was involved in multiple biological processes in X. citri subsp. citri.

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus (DCMV), and an endogenous plant pararetroviral sequence (DvEPRS, formerly known as DMV-D10) were reported from dahlia (Dahlia spp). Promoter elements from these dahlia-associated pararetroviruses were identified and characterized. The TATA box, the CAAT box, the transcription start site, the polyadenylation signal, and regulation factors, characteristic of caulimovirus promoters, were present in each of these promoter regions. Each of the promoter regions was separately cloned into a binary vector containing β-glucuronidase (GUS) reporter gene and delivered into Agrobacterium tumefaciens by electroporation followed by agroinfiltration into Nicotiana benthamiana. The activity of the 35S promoter homologs was determined by transient expression of the GUS gene both in qualitative and quantitative assays. The length of the promoter regions in DMV, DCMV, and DvEPRS corresponded to 438, 439, and 259 bp, respectively. Quantitative GUS assays showed that the promoters from DMV and DCMV resulted in higher levels of gene expression compared to that of DvEPRS in N. benthamiana leaf tissue. Significant differences were observed among the three promoters (p < 0.001). Qualitative GUS assays were consistent with quantitative GUS results. This study provides important information on new promoters for prospect applications as novel promoters for their potential use in foreign gene expression in plants.

Background/aims: The potential physiologic roles of Klotho in acute kidney injury (AKI) have recently been demonstrated in animal models. However, to date, there have been no human studies investigating the expression of renal Klotho in AKI., Methods: We retrospectively collected biopsy specimens and clinical data of AKI patients between January 2001 and December 2012. Klotho expression was determined by immunohistochemical staining, and the clinical-pathological correlation was examined., Results: Among the 34 patients diagnosed with acute tubular necrosis or acute tubulointerstitial nephritis, 21 patients without chronic histological lesions were included. The mean age was 37.3 ± 18.5 years and the mean peak creatinine level was 8.2 ± 5.5 mg/dL. In total, 10 patients (47.6%) received temporary renal replacement therapy (RRT); however, 17 patients (81%) showed functional recovery with creatinine levels of < 1.3 mg/dL after 1 month. The intensity of Klotho expression was scored as a percentage of Klotho-positive area. The renal Klotho score showed a significant negative correlation with the initial or peak creatinine level. When the patients were divided into three groups according to the Klotho score (low, middle, high), the low group had a significantly higher peak creatinine level and a more frequent requirement for RRT. However, the Klotho score was not a significant predictor of renal recovery., Conclusions: The results demonstrated that renal Klotho expression in humans decreased significantly according to the severity of AKI, regardless of the etiology, and that low expression was associated with a poor short-term outcome.

Klotho plays an important role in the pathogenesis of cardiovascular disease in chronic kidney disease (CKD). Klotho is highly expressed in the kidney and parathyroid glands, but its presence in the vasculature is debated. Renal Klotho is decreased in CKD, but the effect of uremia on Klotho in other tissues is not defined. The effect of vitamin D receptor activator therapy in CKD on the expression of Klotho in various tissues is also in debate. In uremic rats (surgical 5/6th nephrectomy model), we compared 3 months of treatment with and without paricalcitol on Klotho immunostaining in the kidney, parathyroid glands, and aorta. With uremia, Klotho was unchanged in the parathyroid, significantly decreased in the kidney (66%) and the intimal-medial area of the aorta (69%), and significantly increased in the adventitial area of the aorta (67%) compared with controls. Paricalcitol prevented the decrease of Klotho in the kidney, increased expression in the parathyroid (31%), had no effect in the aortic media, but blunted the increase of Klotho in the aortic adventitia. We propose that fibroblasts are responsible for the expression of Klotho in the adventitia. In hyperplastic human parathyroid tissue from uremic patients, Klotho was higher in oxyphil compared with chief cells. Thus, under our conditions of moderate CKD and mild-to-moderate hyperphosphatemia in rats, the differential expression of Klotho and its regulation by paricalcitol in uremia is tissue-dependent.

Klotho is an evolutionarily highly conserved protein related to longevity. Increasing evidence of a vascular protecting effect of the Klotho protein has emerged and might be important for future treatments of uremic vascular calcification. It is still disputed whether Klotho is locally expressed in the vasculature or whether its vascular effects arise uniquely from its presence in the circulation.