Assessment of Biochemical Bone Turnover Markers in Polish Healthy Children and Adolescents.

BACKGROUND: Assessing bone turnover in paediatric populations is crucial for understanding the physiological changes occurring during skeletal development and identifying potential abnormalities. The objective of this study was to assess osteocalcin (OC), bone alkaline phosphatase (BALP), and C-terminal telopeptide of type I collagen (CTX-I) levels reflecting bone formation and resorption for age and sex in Polish healthy children and adolescents. MATERIALS AND METHODS: A total of 355 healthy normal-weight children and adolescents (46.5% girls) aged 1-18 years old were recruited. Total body less head (TBLH) and spine L1-L4 were used in children to assess bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA). Bone marker concentrations were determined by immunoenzymatic methods. RESULTS: Bone marker levels in girls and boys started with higher values in the first year of life and subsequently decreased until reaching a nadir during the prepubertal period. The pubertal peak values of bone markers were reached at 11-13 years old in boys and at 9-11 years old in girls. After puberty, the adolescents showed a gradual decline in bone marker concentrations to the values observed in adults. We found positive correlations between OC level and TBLH-BMD (r = 0.329, p = 0.002), TBLH-BMD Z-score (r = 0.245, p = 0.023), and L1-L4 BMD (r = 0.280, p = 0.009) in the prepubertal group. CONCLUSIONS: We showed serum levels of bone turnover markers-BALP, OC, and CTX-I-in relation to age and sex in healthy Polish children and adolescents. The age intervals of these markers for girls and boys aged 1-18 years old may be clinically useful in the assessment of bone metabolism in individuals with skeletal disorders.

Assessment of clinical chemistry and hematological parameters in female Sprague-Dawley rats following a 7-day oral exposure to three different species of Echinacea.

Echinacea has grown in popularity due to its broad therapeutic benefits. Despite its popularity, comprehensive safety evaluations for three medicinal species are limited. In this study, female Sprague-Dawley rats received oral doses (0, 25, 50, 100, 200 mg/kg/d) of 75% (v/v) ethanol extract from the aerial parts of 9 Echinacea samples of three species - Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida - over a 7-day period. Blood and serum samples, collected twenty-four hours post the final dose, were analyzed for hematology and clinical chemistry parameters. The results revealed varied effects across the tested samples, with many parameters showing no discernible impacts at administered doses. Subtle alterations were observed in parameters such as relative liver weight, alkaline phosphatase (ALP), and platelet count. Parameters like relative spleen weight, alanine transaminase (ALT), glucose, urea, hematocrit, hemoglobin, and RBC count exhibited effects in only one out of the nine samples tested. These findings emphasize the heterogeneity in the effects of Echinacea. While the results suggest that Echinacea samples might be considered relatively safe, potential clinical implications warrant caution and underscore the importance of extended testing. A comprehensive toxicity profile assessment remains paramount to conclusively ascertain the safety of three Echinacea species.

Assessment of the Enzymatic Dephosphorylation Kinetics in the Assemblies of a Phosphopentapeptide that Forms Intranuclear Nanoribbons.

Although the formation of peptide assemblies catalyzed by alkaline phosphatase (ALP) has received increasing attention in inhibiting cancer cells, the detailed enzyme kinetics of the dephosphorylation of the corresponding phosphopeptide assemblies have yet to be determined. We recently discovered that assemblies from a phosphopentapeptide can form intracellular nanoribbons that kill induced pluripotent stem cells or osteosarcoma cells, but the kinetics of enzymatic dephosphorylation remain unknown. Thus, we chose to examine the enzyme kinetics of the dephosphorylation of the phosphopentapeptide [NBD-LLLLpY (1)] from concentrations below to above its critical micelle concentration (CMC). Our results show that the phosphopeptide exhibits a CMC of 75 µM in phosphate saline buffer, and the apparent Vmax and Km values of alkaline phosphatase catalyzed dephosphorylation are approximately 0.24 µM/s and 5.67 mM, respectively. Despite dephosphorylation remaining incomplete at 60 min in all the concentrations tested, dephosphorylation of the phosphopeptide at concentrations of 200 µM or above mainly results in nanoribbons, dephosphorylation at concentrations of CMC largely produces nanofibers, and dephosphorylation below the CMC largely generates nanoparticles. Moreover, the formation of nanoribbons correlates with the intranuclear accumulation of the pentapeptide. By providing the first examination of the enzymatic kinetics of phosphopeptide assemblies, this work further supports the notion that the assemblies of phosphopentapeptides can act as a new functional entity for controlling cell fates.

Assessment of degradation mechanism of imidacloprid residues in grape rhizosphere soil by UHPLC-Orbitrap™-MS and its residual impact on soil enzyme activity.

Imidacloprid (IM) is a systemic insecticide persistent in the environment and possesses a negative impact on the non-targeted ecosystem. The objective of the present study was to evaluate the dissipation and degradation mechanism of IM residues in grape rhizosphere soil and to investigate its residual effect on soil enzyme activity at different IM spiking levels. The half-life of IM residue in soil was 27, 36, and 43.5 days at a spiking level of 1, 10, and 50 mg kg-1, respectively following a bi-phasic first + first-order dissipation kinetics. UHPLC-Orbitrap™-MS analysis by targeted metabolomics approach revealed that IM metabolites such as IM-amine analogue, guanidine (reduction), 5-hydroxy IM (hydroxylation), IM-Urea (oxidation), reduced NO analogue of IM (oxidation), and olefin of guanidine IM (dehydrogenation) were identified and proposed the degradation mechanism in grape rhizosphere soil. Toxicity of IM residues on five extracellular enzymes, viz., dehydrogenase, acid phosphatase, alkaline phosphatase, ß-glucosidase, and urease revealed that activity of dehydrogenase, acid phosphatase, and alkaline phosphatase remained unaffected at 60th day of sampling. The ß-glucosidase and urease were negatively affected throughout the incubation period indicating the influence of IM residues on carbon and nitrogen mineralization in soil. Thus, long-term exposure of IM to grape rhizosphere through soil drenching could affect soil enzyme activity which has a negative effect on the soil nutrient cycle and soil microbiome.

Integrating Fluorescence and Magnetic Resonance Imaging in Biocompatible Scaffold for Real-Time Bone Repair Monitoring and Assessment.

In situ monitoring of bone tissue regeneration progression is critical for the development of bone tissue engineering scaffold. However, engineered scaffolds that can stimulate osteogenic progress and allow for non-invasive monitoring of in vivo bone regeneration simultaneously are rarely reported. Based on a hard-and-soft integration strategy, a multifunctional scaffold composed of 3D printed microfilaments and a hydrogel network containing simvastatin (SV), indocyanine green-loaded superamphiphiles, and aminated ultrasmall superparamagnetic iron oxide nanoparticles (USPIO-NH2 ) is fabricated. Both in vitro and in vivo results demonstrate that the as-prepared scaffold significantly promotes osteogenesis through controlled SV release. The biocomposite scaffold exhibits alkaline phosphatase-responsive near-infrared II fluorescence imaging. Meanwhile, USPIO-NH2 within the co-crosslinked nanocomposite network enables the visualization of scaffold degradation by magnetic resonance imaging. Therefore, the biocomposite scaffold enables or facilitates non-invasive in situ monitoring of neo-bone formation and scaffold degradation processes following osteogenic stimulation, offering a promising strategy to develop theranostic scaffolds for tissue engineering.

[Clinical value of arterial stiffness assessment on risk prediction of vascular stiffness in the octogenarian elderly].

Objective: This study aimed to analyze clinical factors related to arterial stiffening and establish a risk prediction nomogram of arterial stiffening in the octogenarian(≥80 years). Methods: This study was a retrospective cross-sectional study, which enrolled the octogenarian elderly who underwent physical examination and secondary prevention intervention in the outpatient department of Chinese People's Liberation Army General Hospital from April 2022 to August 2022. Clinical data including demographics, biochemical indicators and medical history were collected. Brachial-ankle pulse wave velocity (baPWV) was detected during the clinical visit. Participants were divided into the control group (baPWV≤1 800 cm/s) and vascular sclerosis group (baPWV>1 800 cm/s). The risk factors of arterial stiffness were analyzed by univariate and logistic regression analysis, and the nomogram model was constructed by R programming language. The predictive effect of the nomogram model was evaluated by the receiver operating characteristic curve (ROC). Results: The median age of the 525 participants was 87.0 (82.0, 92.0) years, 504 (96.0%) were male, 82 in the control group, 443 in the vascular sclerosis group. The baPWV, age, systolic blood pressure, mean arterial pressure and diastolic blood pressure were significantly lower in the control group than those in the vascular sclerosis group (all P<0.05). Logistic regression analysis showed that high-density lipoprotein cholesterol, alanine aminotransferase and amylase were protective factors, and alkaline phosphatase and creatinine were risk factors of arterial stiffening (all P<0.05). The combined nomogram model scores including age, mean arterial pressure and the above five laboratory indicators indicated that mean arterial pressure and serum creatinine levels were strongly correlated with vascular sclerosis. The ROC curve suggested that the nomogram model had good prediction ability. Conclusions: Age, mean arterial pressure, high-density lipoprotein cholesterol, alanine aminotransferase, alkaline phosphatase, amylase and creatinine are independently determinants for increased vascular stiffness. The combined prediction model in this study can provide reference for individualized clinical risk prediction of vascular sclerosis in the octogenarian elderly.

The use of serum alkaline phosphatase as a choledocholithiasis marker to mitigate the cost of magnetic resonance cholangiography.

OBJECTIVE: To assess the predictive value of preoperative serum laboratory test results for identifying choledocholithiasis and reduce the use of cholangioresonance and its inherent costs. METHODS: Patients aged 21-69 years who underwent preoperative cholangioresonance examination at our institute were included. Patients with a history of fluctuating jaundice or biliary pancreatitis, bile duct dilatation on ultrasonography, and elevated levels of canalicular enzymes (alkaline phosphatase >100U/L and gamma-glutamyl transferase >50U/L) underwent cholangioresonance-guided surgery. Cases of choledocholithiasis confirmed by cholangioresonance were compared with those without choledocholithiasis. Serum laboratory data were evaluated and the diagnostic capabilities of these examinations were analyzed. RESULTS: A total of 104 patients were included. For detecting choledocholithiasis using alkaline phosphatase, the cut-off point was 78U/L, sensitivity was 97.6% (95%CI: 87.4-99.9), and specificity was 72.6% (95%CI: 59.8-83.1). In the binary logistic regression analysis, age (OR= 0.92; 95%CI: 0.86-0.98) and alkaline phosphatase level (OR= 1.02; 95%CI: 1.01-1.05) were selected for the final model. CONCLUSION: Serum alkaline phosphatase levels may aid preoperative diagnosis of asymptomatic choledocholithiasis. After a global clinical assessment of the patient, serum laboratory findings may contribute to a reduction in cholangioresonance-related heathcare costs.

Histochemical assessment of osteoclast-like giant cells in Rankl-/- mice.

OBJECTIVES: We examined mice with gene deletion of Receptor activator of nuclear factor-κB (Rank) ligand (Rankl) to histologically clarify whether they contained progenitor cells committed to osteoclastic differentiation up to the stage requiring RANK/RANKL signaling. METHODS: The tibiae and femora of ten-week-old male wild-type, c-fos-/-, and Rankl-/- mice were used for immunohistochemistry and transmission electron microscopy (TEM). RESULTS: In Rankl-/- mice, we observed osteoclast-like giant cells, albeit in low numbers, with single or two nuclei, engulfing the mineralized extracellular matrix. TEM revealed that these giant cells contained large numbers of mitochondria, vesicles/vacuoles, and clear zone-like structures but no ruffled borders. They often engulfed fragmented bony/cartilaginous components of the extracellular matrix that had been degraded. Additionally, osteoclast-like giant cells exhibited immunoreactivity for vacuolar H+-ATPase, galectin-3, and siglec-15 but not for tartrate-resistant acid phosphatase, cathepsin K, or MMP-9, all of which are classical hallmarks of osteoclasts. Furthermore, osteoclast-like giant cells were ephrinB2-positive as they were near EphB4-positive osteoblasts that are also positive for alkaline phosphatase and Runx2 in Rankl-/- mice. Unlike Rankl-/- mice, c-fos-/- mice lacking osteoclast progenitors and mature osteoclasts had no ephrinB2-positive osteoclast-like cells or alkaline phosphatase-positive/Runx2-reactive osteoblasts. This suggests that similar to authentic osteoclasts, osteoclast-like giant cells might have the potential to activate osteoblasts in Rankl-/- mice. CONCLUSIONS: It seems plausible that osteoclast-like giant cells may have acquired some osteoclastic traits and the ability to resorb mineralized matrices even when the absence of RANK/RANKL signaling halted the osteoclastic differentiation cascade.

Association of Liver Function Tests with Mortality in an Insurance Applicant Population.

OBJECTIVES: -Determine the relationship between liver function test (LFT) results (GGT, alkaline phosphatase, AST, ALT and albumin) and all-cause mortality in life insurance applicants. METHOD: -By use of the Social Security Master Death File, mortality was examined in 15,272,955 insurance applicants for whom blood samples were submitted to the Clinical Reference Laboratory. There were 268,593 deaths observed in this study population, after an average follow-up time of 10.9 years. Results were stratified by sex and by age less/greater than 60, creating 4 groups. Liver function test values were grouped using percentiles of their distribution within these age/ sex groups - so as to update the results generated in prior publications. Additional models were fit using different exclusions and percentile groups within single year age groups. Also, LFTs were treated as continuous variables and included in Cox models with age and smoking status. RESULTS: -Using the risk of the middle 50% of the population by distribution as a reference, relative mortality observed for GGT and alkaline phosphatase was linear with a steep slope from very low to high values. AST showed a J-shaped association with mortality. ALT showed a low-magnitude inverse correlation with mortality. Albumin demonstrated a higher-magnitude inverse correlation with mortality, especially at values below the median. The overall risk associated with LFTs was durable over at least 10 years of follow-up. CONCLUSION: -Liver function tests show a strong and durable correlation to mortality in a large group of insurance applicants. The durability over time suggests that even older values of LFTs found in medical records could be of use in mortality risk prediction.

Feasibility of Global Assessment of Bone Metastases in Prostate Cancer with 18F-Sodium Fluoride-PET/Computed Tomography.

18F-sodium fluoride (NaF) PET/computed tomography (CT) allows detection of bone metastases in patients with prostate cancer (PCa). The aim of this study was to test the feasibility of assessing global metastatic bone disease in patients with PCa by using a threshold-based PET segmentation technique. This retrospective analysis was performed in 32 patients with PCa with known bone metastases who underwent NaF-PET/CT imaging. An adaptive contrast-oriented thresholding technique was used to segment NaF avid lesions. The mean metabolic volumetric product (MVPmean), partial volume-corrected MVPmean (cMVPmean), and metabolically active volume (MAV) were calculated. Lesional values were summed within each patient to obtain the global PET disease burden. Pearson correlation analysis was used to assess the associations between global NaF-PET/CT metrics and clinical biomarkers of metastatic disease activity. Global MVPmean, cMVPmean, and MAV were significantly correlated with alkaline phosphatase (ALP) levels (p < 0.05). No correlation was observed between global NaF-PET/CT measures and prostate-specific antigen (PSA) levels. Global assessment is a feasible method to quantify metastatic bone disease activity in patients with PCa. Convergent validity was supported by demonstrating a significant correlation between NaF-PET/CT parameters and blood ALP levels.

The Evaluation of Clinical Status of Endoscopic Retrograde Cholangiography for the Placement of Metal and Plastic Stents in Cholangiocarcinoma Therapy.

Objective: Cholangiocarcinoma is a common malignant tumor that occurs in the bile duct system, which can be treated by using the endoscopic retrograde cholangiography (ERCP). This study was aimed at exploring the therapeutic effect of ERCP with metal stent and plastic stent for cholangiocarcinoma. Methods: The clinical data of 71 patients with cholangiocarcinoma treated by ERCP in our hospital from June 2020 to October 2021 were retrospectively analyzed. According to different stent types, the patients were divided into plastic stent group (n = 43) and metal stent group (n = 28). Patients in the plastic stent group and metal stent group were received with plastic stent and metal stent, respectively. The indexes of liver function (serum alkaline phosphatase (ALT), direct bilirubin (DBIL), glutamic oxaloacetic transaminase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL)), postoperative complications, success rate of stent implantation, and survival time of patients in the two groups were determined. Logistic multivariate regression analysis was used to analyze the prognostic factors of postoperative cholangiocarcinoma. Results: The liver function indexes of the two groups were significantly improved after treatment with the stent, in which the ameliorative effect in the metal stent group was better than that in the plastic stent group (P < 0.05). The incidence of postoperative complications in the plastic stent group and the metal stent group was 53.49% and 14.29%, respectively, and the success rate of stent placement was 60.47% and 96.43%, respectively. The incidence of complications in the metal stent group was lower than that in the plastic stent group, and the success rate of stent placement was higher than that in the plastic stent group (P < 0.05). The median survival time of patients in the plastic stent group and the metal stent group was 8.15 and 11.83 months, respectively. The survival time of patients in the metal stent group was longer than that of the plastic stent group. The median survival time of patients with types I, II, III, and IV was 12.73, 11.54, 10.57, and 9.36 months, respectively. The survival time of patients with stage I was significantly higher than that of patients with types II, III, and IV. There was an inverse relationship between the disease type and the survival time of patients. Logistic multivariate regression analysis showed that tumor diameter ≥ 5 cm, portal vein invasion, lymph node metastasis, and classification of hilar cholangiocarcinoma were the risk factors (P < 0.05) and metal stent type was the protective factor (P < 0.05). Conclusion: In the clinical treatment of patients with cholangiocarcinoma, the placement of metal stent and plastic stent under ERCP plays an important role. The placement of the metal stent under ERCP has a higher success rate and better prognosis and can prolong the survival time of patients to a greater extent, but the price of the metal stent is relatively expensive. For patients with an expected survival period of more than 4-6 months, the metal stent should be considered; otherwise, the plastic stent can be used to maintain cost-effectiveness. Therefore, it is necessary to comprehensively analyze the patient's economic affordability, expected survival time, stent drainage time, and personal needs and then select an appropriate treatment method.

Semiquantitative assessment of 99mTc-MIBI uptake in parathyroids of secondary hyperparathyroidism patients with chronic renal failure.

Objective: To explore the valuably influential factors and improve the diagnostic accuracy and efficiency of 99mTc-methoxyisobutylisonitrile (MIBI) uptake in parathyroids of secondary hyperparathyroidism (SHPT) patients with chronic renal failure (CRF). Methods: The correlation analysis was performed between clinical indices related to CRF and 99mTc-MIBI uptake intensity TBR (the gray value mean ratio between the parathyroid target and the bilateral neck background, semiquantitatively calculated with ImageJ software). All clinical indices and TBRs were compared by a three- or two-level grouping method of MIBI uptake, which was visually qualitatively assessed. The three-level grouping method comprised slight, medium, and high groups with little, faint, and distinct MIBI concentration in parathyroids, respectively. The two-level grouping method comprised insignificant and significant groups with TBR greater than or less than 0.49-0.71, respectively. Results: MIBI uptake was significantly positively related to patient age, CRF course, hemodialysis vintage, serum parathyroid hormone (PTH), and alkaline phosphatase (AKP) but was significantly negatively related to serum uric acid (UA). MIBI washout was significantly positively related to patient age but was significantly negatively related to serum phosphorus (P) and calcium (Ca) × P. Oral administration of calcitriol and calcium could significantly reduce the MIBI uptake. MIBI uptake tendency might alter. Such seven indices, namely the MIBI uptake, CRF course, hemodialysis vintage, serum AKP, calcium, cysteine proteinase inhibitor C, and PTH, were comparable between the slight and medium groups but were significantly different between the slight and high groups or between the medium and high groups. The above seven indices plus blood urea nitrogen/creatinine were all significantly different between the insignificant and significant groups. All above significances were with P < 0.05. Conclusions: Patient age, CRF course, hemodialysis vintage, serum PTH, AKP, UA, phosphorus, Ca × P, oral administration of calcitriol and calcium, and parathyroids themselves can significantly influence MIBI uptake in parathyroids of SHPT patients with CRF. The two-level grouping method of MIBI intensity should be adopted to qualitatively diagnose the MIBI uptake.

Assessment of hazardous impact of nickel oxide nanoparticles on biochemical and histological parameters of gills and liver tissues of Heteropneustes fossilis.

BACKGROUND: The aim of the present study was to assess the hazardous impact of nickel oxide nanoparticles (NiO NPs) on gills and liver of Heteropneustes fossilis. METHODS: Fishes were treated with four concentrations of NiO NPs for a period of 14 days. Nickel accumulation, lipid peroxidation, antioxidant enzymes activities (superoxide dismutase, catalase, glutathione s transferase & glutathione reductase), liver enzymes activities (aspartate amino transferase, alanine transaminase, & alkaline phosphatase), Na+/K+ ATPase activity, FTIR, metallothionein content, ethoxyresorufin-o-deethylase activity, immunohistochemistry, histology and scanning electron microscopy were analyzed in both gills and liver tissues. RESULTS: Results revealed increased accumulation of nickel in both the tissues of exposed fishes. Lipid peroxidation and activities of different antioxidant enzymes increased (except superoxide dismutase) in both the tissues after exposure. Fluctuations in liver enzymes activities and variation in the activity of Na+/K+ ATPase were also observed. FTIR data revealed shift in peaks position in both the tissues. Level of metallothionein and its expression as well as activity of ethoxyresorufin-o-deethylase and expression of CYP1A also increased in both the target tissues of treated fishes. Furthermore, histological investigation and scanning electron microscopy showed structural damages in gills as well as liver tissues of exposed fishes. CONCLUSION: Our results suggest that NiO NPs cause deteriorating effects on the gill and liver tissues of fish, therefore effluents containing these nanoparticles should be treated before their release into water bodies.

Assessment of spent hemp biomass as a potential ingredient in ruminant diet: nutritional quality and effect on performance, meat and carcass quality, and hematological parameters in finishing lambs.

Spent hemp biomass (SHB), a byproduct of cannabinoid extraction from the production of industrial hemp has not been approved by FDA-CVM since its effects on animal health, performance, and product quality are unknown. Our objective was to investigate the effects of feeding two levels of SHB and a 4-wk withdrawal period on performance, carcass characteristic, meat quality, and hematological parameters in finishing lambs. A total of 35 weaned, Polypay male lambs kept in single pens were randomly assigned to five feeding treatments (n = 7) and fed diets containing either no SHB (CON) or SHB at 10% (LH1) or 20% (HH1) for 4 wk with 4 wk of clearing period from SHB, or SHB at 10% (LH2) or 20% (HH2) for 8 wk. Chemical analysis revealed SHB to have a nutritive quality similar to alfalfa with no mycotoxin, terpenes, or organic residuals as a result of the extraction process. Feed intake of lambs was negatively affected by 20% SHB in period 1 but not in period 2 where feed intake was the greatest in HH1 and LH2. In contrast, none of the performance data, including liveweight gains, were different across the groups and periods. In period 1, blood glucose, cholesterol, calcium, paraoxonase, and tocopherol were decreased by the level of SHB fed, while bilirubin and alkaline phosphatase (ALP) were increased. In period 2, the concentration in blood of urea, magnesium, bilirubin, ALP, and ferric reducing ability of the plasma (FRAP) were higher in LH2 and HH2 as compared with CON, while ß-hydroxybutyrate was lower in HH2. Blood parameters related to liver health, kidney function, immune status, and inflammation were unaffected by feeding SHB. Most carcass and meat quality parameters did not differ across feeding groups either. Except carcass purge loss and meat cook loss were larger in lambs that were fed 20% SHB. Although lower feed intake of lambs that were fed 20% SHB initially in period 1 suggested SHB was not palatable to the lambs, increased feed intake at a lower level of inclusion at 10% in period 2 may point to a positive long-term effect of feeding SHB.

The use of hemp by-products in livestock diets holds promise for reducing feed costs and achieving greater resource-use efficiency through integration of livestock production and rapidly growing hemp farming. Spent hemp biomass (SHB), the byproduct of the extraction process of cannabidiol from hemp can potentially be included in the ruminant diets due to its desirable nutritional properties. However, the potential accumulation of tetrahydrocannabinol­a psychotropic compound in animal tissues and its effect on animal health, production, and product quality are still unknown. Therefore, we conducted an indoor feeding study to investigate the effects of varying levels of SHB and a withdrawal period on feed intake, performance, health, and meat quality of lambs at Oregon State University. Our findings indicated that SHB can be included in lamb diets without causing any major detrimental effects on performance, meat quality, or health of the lambs.

Preclinical Toxicological Assessment of A Novel siRNA, SLN360, Targeting Elevated Lipoprotein (a) in Cardiovascular Disease.

SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated small interfering RNA (siRNA) with a promising profile for addressing lipoprotein (a)-related cardiovascular risk. Here, we describe the findings from key preclinical safety studies. In vitro, SLN360 specifically reduced LPA expression in primary human hepatocytes with no relevant off-target effects. In rats, 10 mg/kg subcutaneous SLN360 was distributed specifically to the liver and kidney (peak 126 or 246 mg/g tissue at 6 h, respectively), with <1% of peak liver levels observed in all other tested organs. In vitro, no genotoxicity and no effect on human Ether-a-go-go Related Gene currents or proinflammatory cytokine production was observed, whereas in vivo, no SLN360-specific antibodies were detected in rabbit serum. In rat and nonhuman primate 29-day toxicology studies, SLN360 was well tolerated at all doses. In both species, known GalNAc-conjugated siRNA-induced microscopic changes were observed in the kidney and liver, with small increases in alanine aminotransferase and alkaline phosphatase observed in the high dose rats. Findings were in line with previously described siRNA-GalNAc platform-related effects and all observations were reversible and considered nonadverse. In cynomolgus monkeys, liver LPA messenger RNA and serum lipoprotein (a) were significantly reduced at day 30 and after an 8-week recovery period. No dose-related changes in safety assessment endpoints were noted. No SLN360-induced cytokine production, complement activation, or micronucleus formation was observed in vivo. The toxicological profile of SLN360 presented here is restricted to known GalNAc siRNA effects and no other toxicity associated with SLN360 has been noted. The preclinical profile of SLN360 confirmed suitability for entry into clinical studies.

Phosphate-triggered ratiometric fluoroimmunoassay based on nanobody-alkaline phosphatase fusion for sensitive detection of 1-naphthol for the exposure assessment of pesticide carbaryl.

The excessive use of carbaryl has resulted in the risk of its exposure. In this study, we isolated six nanobodies (Nbs) from a camelid phage display library against the biomarker of carbaryl, 1-naphthol (1-NAP). Owing to its characteristics of easy genetic modifications, we produced a nanobody-alkaline phosphatase (Nb-CC4-ALP) fusion protein with good stability. A dual-emission system based ratiometric fluoroimmunoassay (RFIA) for quick and highly sensitive determination of 1-NAP was developed. Silicon nanoparticles (SiNPs) was used as an internal reference and for aggregation-induced emission enhancement (AIEE) of gold nanoclusters (AuNCs), while AuNCs could be quenched by MnO2 via oxidation. In the presence of ALP, ascorbic acid phosphate (AAP) can be transformed into ascorbic acid (AA), the later can etch MnO2 to recover the fluorescence of the AuNCs. Based on optimal conditions, the proposed assay showed 220-fold sensitivity improvement in comparison with conventional monoclonal antibody-based ELISA. The recovery test of urine samples and the validation by standard HPLC-FLD demonstrated the proposed assay was an ideal tool for screening 1-NAP and provided technical support for the monitoring of carbaryl exposure.

Assessment of the Impacts of Centipeda minima (L.) on Cell Viability, and Osteogenic Differentiation of Mesenchymal Stem Cell Spheroids.

Background and Objectives: Centipeda minima (L.) is a well-known and traditional pharmaceutical that has been utilized to treat different conditions controlling rhinitis, soothe pain, and decrease swelling. We assessed the impacts of Centipeda minima (L.) extricates (CMTs) on the osteogenic differentiation of cell spheroids made of human-bone-marrow-derived mesenchymal stem cells. Materials and Methods: Mesenchymal stem cells (MSCs) in spheroid 3D culture were generated and propagated in the presence of CMTs ranging from 0 to 1 µg/mL. Cell morphology was measured on Days 1, 3, 5, and 7. The quantitative cellular viability was evaluated on Days 1, 3, 5, and 7. Alkaline phosphatase activity assays were designed to measure the osteogenic differentiation of mesenchymal stem cell spheroids on Day 7. Alizarin Red S staining was performed to investigate the mineralization of cell spheroids on Days 7 and 14. Real-time polymerase chain reactions were used to measure the expression levels of RUNX2 and COL1A1 on Day 14. Western blot techniques were performed to identify the protein expression of Runt-related transcription factor 2 and type I collagen. Results: The control group's mesenchymal stem cells displayed a spheroid shape. There was no noticeable change in morphology with the addition of CMTs at final concentrations of 0.001, 0.01, 0.1, and 1 µg/mL compared with the untreated (control) group. The application of CMTs did not induce a significant change in cell viability. The relative alkaline phosphatase activity values in the 0.001, 0.01, 0.1, and 1 µg/mL CMT groups were 114.4% ± 8.2%, 130.6% ± 25.3%, 87.8% ± 3.4%, and 92.1% ± 6.8%, respectively, considering a control of 100% (100.0% ± 17.9%). On Day 14, calcium deposits were clearly observed in each group. The relative values of Alizarin Red S staining in the 0.001, 0.01, 0.1, and 1 µg/mL CMT groups were 100.1% ± 8.9%, 105.9% ± 0.0%, 109.7% ± 19.1%, and 87.0% ± 40.9%, respectively, considering a control of 100% (100.0% ± 28.7%). The addition of CMT significantly increased RUNX2 expression in the 0.01 µg/mL group and COL1A1 in the 0.001 and 0.01 µg/mL groups. Normalization of protein expression showed that the addition of CMTs significantly increased type I collagen expression in the 0.001, 0.01, and 1 µg/mL groups. Conclusions: In conclusion, CMTs influence the osteogenic differentiation of bone-marrow-derived mesenchymal stem cells and the use of CMTs may positively influence the osteogenic differentiation of cell spheroids.

Prevalence and management of vitamin D deficiency in children with newly diagnosed coeliac disease: cohort study.

BACKGROUND: Coeliac disease (CD) causes deficiency of various micronutrients including vitamin D, and there are no specific guidelines for treatment. AIMS: To determine the prevalence of vitamin D deficiency in children newly diagnosed with CD and the role of oral high-dose vitamin D in its treatment. METHODS: Calcium intake, sun exposure and biochemical and radiological parameters related to vitamin D deficiency were compared between 60 children aged 0-18 years diagnosed with CD and 60 healthy age- and sex-matched controls. The cases with serum 25(OH)D (<20 ng/ml) were given oral vitamin D (60,000 IU/week) and calcium (500 mg/day) for 12 weeks, along with a gluten-free diet (GFD); they were re-evaluated within a week of completion. The primary outcome measure was the serum 25(OH)D level, and secondary measures included serum calcium, phosphorus, alkaline phosphatase, parathormone and clinical and/or radiological rickets. RESULTS: The prevalence of vitamin D deficiency (25(OH)D <20 ng/ml) was significantly greater in the cases (n=38, 63.3%) than in the controls (n=27, 45.0%). Upon treatment, all 38 cases with vitamin D deficiency showed a significant rise in 25(OH)D levels along with normalisation of other biochemical abnormalities. Two children had 25(OH)D levels >100 ng/ml with no other feature suggestive of vitamin D toxicity. CONCLUSIONS: Vitamin D deficiency is more prevalent in children with CD. Administration of oral high-dose vitamin D for 12 weeks along with a GFD leads to a robust response, indicating rapid mucosal recovery. The vitamin D dosage recommended for malabsorption states may be excessive in CD.Abbreviations: ALP: alkaline phosphatase; CaBP: calcium-binding proteins; CD: coeliac disease; GFD: gluten-free diet; PTH: parathormone; RU: reproducibility units; 25(OH)D: 25 hydroxy vitamin D.

Assessment of renal and hepato-protective potential of guava leaves in male Sprague dawley rats.

Present research project was an attempt to explore the functional/nutraceutical worth of guava leaves from two locally grown varieties (Ruby & Safeda). For the purpose, guava leaves extract was fed to experimental male Sprague Dawley rats to explore the nutraceutical potential of guava leaves against hepatotoxicity. Two studies were performed on two types of rats i.e. study I (normal rats), study II (hepatotoxic rats). In both studies, 250 mg/kg each of pink guava leaves extracts (T1) and white guava leaves extracts (T2) was added in the feed. Feed intake and body weights of the rats were recorded. At the end of the first and eighth week of study, the blood samples of the rats were analyzed to check the effect of guava leaves extracts on renal functioning (Alkaline Phosphatase, Alanine Transaminase and Aspartate Transaminase) as well as liver functioning parameters including urea and creatinine. In both studies, comparatively higher feed consumption was observed in the control group than the rest of the treatments. At the end of study I, the highest weight (207±9.21 g) was observed in T0 whereas, during study II, the maximum value (202±5.58 g) was found in T2 (rats consuming white guava leaves extract) that indicates its effectiveness against hepatotoxicity. Regarding renal functioning tests, pink guava leaves were more effective in decreasing urea and creatinine levels in rats as compared to the white guava leaves in both study plans. Likewise, in each of study trial, pink guava leaves were more effective in reducing AST, ALT and ALP than white guava leaves and control. From the present investigation, it is deduced that guava leaves were effective against hepatotoxicity.

Prevalence of low alkaline phosphatase activity in laboratory assessment: Is hypophosphatasia an underdiagnosed disease?

BACKGROUND: Tissue-nonspecific alkaline phosphatase (TNSALP) encoded by the ALPL gene is of particular importance for bone mineralization. Mutation in the ALPL gene can lead to persistent low ALP activity resulting in the rare disease Hypophosphatasia (HPP) that is characterized by disturbed bone and dental mineralization. While severe forms are extremely rare with an estimated prevalence of 1/100.000, recent studies suggest that moderate form caused by heterozygous mutations are much more frequent with an estimated prevalence of 1/508. The purpose of this study was to estimate the prevalence of low AP levels in the population based on laboratory measurements. METHODS: In this study, the prevalence of low AP activity and elevated pyridoxal-5-phosphate (PLP) levels was analyzed in 6.918.126 measurements from 2011 to 2016 at a single laboratory in northern Germany. Only laboratory values of subjects older than 18 years of age were included. Only the first measurement was included, all repeated values were excluded. RESULTS: In total, 8.46% of the measurements of a total of 6.918.126 values showed a value < 30 U/L. 0.59% of the subjects with an ALP activity below 30 U/L had an additional PLP measurement. Here, 6.09% showed elevated pyridoxal-5-phosphate (PLP) levels. This suggest that 0.52% (1:194) of subjects show laboratory signs of HPP. CONCLUSION: These data support the genetic estimation that the prevalence of moderate forms of HPP may be significantly higher than expected. Based on these data, we recommend automatically measurement of PLP in the case of low ALP activity and a notification to the ordering physician that HPP should be included in the differential diagnosis and further exploration is recommended.