The influence of unconventional ultrasonic pasteurization on the characteristics of curds obtained from goat milk with the low cholesterol content.

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 ß-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.

Associations of serum soluble klotho and fibroblast growth factor 23 with carotid artery calcification in patients undergoing continuous ambulatory peritoneal dialysis: A retrospective study.

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 ±â€Š7.45 vs 46.62 ±â€Š5.51 years), dialysis time (8.15 ±â€Š1.42 vs 6.02 ±â€Š1.14 months), klotho amounts (325.56 ±â€Š41.15 vs 436.65 ±â€Š45.58 pg/mL) and FGF-23 levels (114.45 ±â€Š15.56 vs 70.15 ±â€Š12.23 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.

Enzyme-Responsive Biopolymeric Nanogel Fibers by Extrusion: Engineering of High-Surface-Area Hydrogels and Application in Bacterial Enzyme Detection.

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-ß-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. ß-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 ß-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.

Quantitative E. coli Enzyme Detection in Reporter Hydrogel-Coated Paper Using a Smartphone Camera.

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 ß-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-1h-1 for ß-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.

Increased Plasma Heparanase Activity and Endothelial Glycocalyx Degradation in Dengue Patients Is Associated With Plasma Leakage.

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.

Characterization of the ß-glucuronidase Pn3Pase as the founding member of glycoside hydrolase family GH169.

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-ß1,4-glucuronidase cleaving the ß(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.

New Potential Biomarkers for Chronic Kidney Disease Management-A Review of the Literature.

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.

Heparanase potentiates the invasion and migration of pancreatic cancer cells via epithelial­to­mesenchymal transition through the Wnt/ß­catenin pathway.

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 transcription­quantitative PCR (RT­qPCR) analysis. Wound healing and Transwell assays were conducted to examine the effects of HPSE on migration and invasion in sh­NC and sh­HPSE PC cell lines. In addition, tumor growth was assessed in a mouse xenograft model in vivo. The expression levels of epithelial­to­mesenchymal transition (EMT)­related biomarkers and the involvement of the Wnt/ß­catenin pathway were assessed by analyzing the results of western blot and RT­qPCR 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/ß­catenin 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.

Influence of FGF23 and Klotho on male reproduction: Systemic vs direct effects.

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.

Biomarkers of Bone Turnover Identify Subsets of Chronic Kidney Disease Patients at Higher Risk for Fracture.

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.

Biomarkers of vascular disease in diabetes: the adipose-immune system cross talk.

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.

New Advances of Heparanase in Human Diseases.

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.

Frequent overexpression of klotho in fusion-negative phosphaturic mesenchymal tumors with tumorigenic implications.

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 ß-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.

[Anticancer effect of modified banana (Musa cavendish AAA) starch in rats with 1.2-dimethylhydrazine]. / Efecto anticancerígeno del almidón modificado de banano (Musa cavendish AAA) en ratas con 1,2-dimetilhidrazina.

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, ß-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 ß-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.

INTRODUCCIÓN: Introducción: el almidón resistente (AR) no se digiere completamente en el intestino humano sino que se fermenta en colon; disminuye el pH intestinal, ya que se producen ácidos grasos de cadena corta, interviniendo de manera benéfica en el tratamiento preventivo y curativo del cáncer de colon rectal. La pirodextrinización y la hidrólisis enzimática son modificaciones al almidón nativo (AN) que pueden incrementar la cantidad de AR. Objetivo: el objetivo de este proyecto fue evaluar los efectos del almidón nativo de M. cavendish AAA y de los almidones modificados química y enzimáticamente sobre diversos marcadores tumorales en ratas. Métodos: se realizaron modificaciones (química y enzimática) del AN del banano M. cavendish AAA y se evaluaron en ratas tratadas con 1,2-DMH. Se utilizaron 25 ratas Sprague Dawley machos divididas en cinco grupos experimentales: CP, CN, AN, PI y MER. Durante 4 semanas recibieron la dieta experimental asignada a cada grupo. Los grupos CP, AN, PI y MER recibieron 2 inyecciones s.c. (subcutáneas) semanales de 1,2-DMH (40 mg/kg) (semanas 3 y 4). En las heces se evaluaron el pH, la enzima ß-glucuronidasa y los ácidos grasos de cadena corta, y se realizó un estudio histopatológico del ciego y el colon para detectar lesiones microscópicas. Resultados: la actividad de ß-glucuronidasa disminuyó (p < 0,05) para los grupos AN, PI y MER en comparación con el CP. La mayor proporción de ácido butírico se observó en el AN (p < 0,05) frente al CN. El 60 % de las enteritis fueron de grado severo en el CP, mientras que en los grupos experimentales fueron de 40 %. Conclusiones: los gránulos de almidón nativo resistieron la pirodextrinización pero el tratamiento con α-amilasa rompió la estructura del gránulo de pirodextrina. De acuerdo a los tratamientos suministrados a las ratas, conforme mayor es la cantidad de AR presente en la dieta (AN), las células neoplásicas no avanzan más allá de la membrana basal, sugiriendo un posible efecto protector o anticancerígeno celular.

Cord blood alpha klotho is decreased in small for gestational age preterm infants with placental lesions of accelerated aging.

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 ±â€¯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.

Bone marrow and plasma FGF-23 in heart failure patients: novel insights into the heart-bone axis.

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.

A simplified and miniaturized glucometer-based assay for the detection of ß-glucosidase activity.

ß-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 ß-glucosidase activity. Single-factor experiments showed that optimum ß-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 ß-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 ß-dextranase, snailase, ß-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 ß-glucosidase activity. The easy-to-operate method was successfully used to detect a series of ß-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 ß-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

The central fibroblast growth factor receptor/beta klotho system: Comprehensive mapping in Mus musculus and comparisons to nonhuman primate and human samples using an automated in situ hybridization platform.

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.

Event-transport of beta-d-glucuronidase in an agricultural headwater stream: Assessment of seasonal patterns by on-line enzymatic activity measurements and environmental isotopes.

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.

Spatial patterns of enzymatic activity in large water bodies: Ship-borne measurements of beta-D-glucuronidase activity as a rapid indicator of microbial water quality.

This study used automated enzymatic activity measurements conducted from a mobile research vessel to detect the spatial variability of beta­d­glucuronidase (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 km2 lake area. The ship-borne measurements of GLUC activity correlated with standard E. coli analyses (R2 = 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.