Phosphatase Test Research Articles

Fluorescence of Intrinsic Milk Chromophores as a Novel Verification Method of UV-C Treatment of Milk.

The European Food Safety Authority (EFSA) has approved the use of a 1045 J/L UV-C dose as an adjunct to pasteurization to increase the shelf life and vitamin D3 content of milk. However, there are no verification methods analogous to the alkaline phosphatase test for pasteurized milk to ensure that the desired UV-C dose has been correctly applied. The aim is to develop a real-time in-line detector based on fluorescence spectroscopy. In this study, 22 different UV-C doses (ranging from 0 to 2000 J/L) were applied to milk to assess the impact of photooxidation on intrinsic photosensitive chromophores. Fluorescence spectroscopy (90°-angle) was employed as the method of analysis for monitoring the changes in the fluorescence spectra of chromophores in milk without sample pretreatment. Three important chromophore areas (CAs) were identified: CA 1 (riboflavin), CA 3 (vitamin A and dityrosine) and CA 4 (tryptophan), with statistically significant changes at around 1045 J/L and 1500 J/L. The findings of our preliminary study support our hypothesis that the fluorescence of intrinsic chromophores can be used as verification of the applied UV-C dose.

Difference in microbial community structure along a gradient of crater altitude: insights from the Nushan volcano.

The variation in the soil microbial community along the altitude gradient has been widely documented. However, the structure and function of the microbial communities distributed along the altitude gradient in the crater still need to be determined. We gathered soil specimens from different elevations within the Nushan volcano crater to bridge this knowledge gap. We investigated the microbial communities of bacteria and fungi in the soil. It is noteworthy that the microbial alpha diversity peaks in the middle of the crater. However, network analysis shows that bacterial (nodes 760 vs 613 vs 601) and fungal (nodes 328 vs 224 vs 400) communities are most stable at the bottom and top of the crater, respectively. Furthermore, the soil microbial network exhibited a decline, followed by an increase across varying altitudes. The core microorganisms displayed the highest correlation with pH and alkaline phosphatase (AP, as determined through redundancy analysis (RDA) and Mantel tests for correlation analysis. The fungal community has a higher number of core microorganisms, while the bacterial core microorganisms demonstrate greater susceptibility to environmental factors. In conclusion, we utilized Illumina sequencing techniques to assess the disparities in the structure and function of bacteria and fungi in the soil.IMPORTANCEThese findings serve as a foundation for future investigations on microbial communities present in volcanic soil.

Design and Development of Continuous Ohmic Heater for Milk and Its Performance Study

Present study aimed for the design and development of a continuous ohmic heating system for heating of milk. The holding capacity of the designed heater was 8.24 litre and had a 'tube in tube' type arrangement. The heat transfer area of the ohmic heater was increased by employing two stages, consisting of SS-316 pipes. Process was controlled by using an electrical panel equipped with all the required electrical components. The designed ohmic heater for milk was optimized for heating of milk temperature up to 73°C. Efficient heating of milk was heated at optimized conditions of 137.7 V (stage-1) and 72.2V(stage-2) with 1.5 l/min flowrate. The average heating rate for milk 4.6˚C/min. At optimized conditions, system performance co-efficient was found to be 86.95. The milk heated using ohmic heater showed no significant difference in the composition and sensory attributes when compared to the conventional heating method. Moreover, the alkaline phosphatase test was also parallel to the thermal heating system and milk was highly acceptable by the sensory panel and cost is considerably lower than the thermal system.

The Role of Graphene and BiodentineTM on Proliferation and Odontoblastic Differentiation of Pulp Stem Cells

Objective: The need for new biomaterials with biocompatibility, mechanical, and antimicrobial properties continues in regenerative endodontic clinical applications in dentistry. Biodentine™ is successfully used in vital pulp therapies and has regenerative effects. Additionally, graphene, which possesses good physicochemical, mechanical, and biological properties, promotes cellular biomineralization and osteogenic differentiation in dentistry, along with its neuroregenerative effect. This study aims to investigate the effects of graphene and Biodentine™ on cell proliferation, oxidative stress, and odontogenic differentiation in human dental pulp stem cells (hDPSCs). Methods: Cryopreserved hDPSCs purchased from American Type Culture Collection (ATCC) were used in our study (Cat No: PT-5025). hDPSCs were seeded into the E-plate wells and subsequently four different doses of graphene (12.5, 25, 50 and 100 µg/ml) and Biodentine™ (2, 4, 8 and 16 µg/ml) were added. Results of MTT, total antioxidant capacity (TAC), total oxidant status (TOS) and alkaline phosphatase (ALP) tests were obtained at the end of the 24th hr. 96 hr-real time cell index data were collected with xCELLigence® system. Resulting data were compared using one-way analysis of variance (ANOVA). Results: 12.5 µg/ml graphene and 2 µg/ml Biodentine™ were found to be the subgroups with the highest levels of cell proliferation and the lowest oxidative stress. Antioxidative effect was determined in all Biodentine™ doses but only in 12.5 µg/ml graphene. Odontogenic differentiation was observed in all doses of graphene and Biodentine™. Conclusion: 12.5 µg/ml graphene and 2 µg/ml Biodentine™ were observed to have positive impacts on the proliferation, oxidative stress and odontogenic differentiations of hDPSCs. Keywords: Cell proliferation. Graphene. Oxidative stress. Odontogenic differentiation. Tricalcium silicate

Incorporating mesoporous SiO2-HA particles into chitosan/hydroxyapatite scaffolds: A comprehensive evaluation of bioactivity and biocompatibility

In this work, composite scaffolds with various composition ratios of chitosan (CS), hydroxyapatite (HA), and mesoporous SiO2 particles co-synthesized with hydroxyapatite (SiO2-HA) were fabricated via the freeze-drying method for bone tissue engineering applications. Morphological studies showed that adding mesoporous particles resulted in a structure with a more uniformly porous geometry, subsequently leading to reduced biodegradation rates and water absorption in the scaffolds. The bioactivity results showed the introduction of mesoporous particles notably enhanced the coverage of the scaffold surface with apatite films. Moreover, biocompatibility assessments using sarcoma osteogenic cell line (SAOS-2) highlighted mesoporous particles' positive impact on cell adhesion and growth. The fluorescence images showed spindle-shaped cells with a greater number and normal cell nuclei for the scaffolds containing mesoporous SiO2-HA particles. The MTT cytotoxicity results indicated that the scaffolds containing mesoporous particles showed approximately 25 % higher cell survival more than single chitosan-based ones. What is more, the mesoporous-containing scaffolds occurred to have the best alkaline phosphatase test (ALP) activity among all scaffolds. It is important to add that CS/HA/mesoporous SiO2-HA scaffolds including SAOS-2 cells showed no sign of either early or late apoptosis. These findings affirm the potential of CS/HA/mesoporous SiO2-HA scaffolds as promising implants for bone tissue engineering.

Ginsenoside Rg1 modulates PI3K/AKT pathway for enhanced osteogenesis via GPER

BackgroundOsteoporosis is a systemic skeletal disorder characterized by decreased bone density and the degradation of bone tissue microarchitecture. Ginsenoside Rg1, derived from Panax ginseng, has been a part of traditional Chinese medicine in China for centuries, particularly for treating osteoporosis. However, there remains limited research on the osteogenic potential of Rg1 within the glucocorticoid-induced osteoporosis (GIOP) model and its specific mechanisms. PurposeThe primary objective of this study is to investigate the osteogenic potential of Rg1 within the GIOP model and explore the signaling pathways associated with its in vivo and in vitro effects. MethodsCell proliferation, differentiation and mineralization were evaluated by the Cell counting kit 8(CCK8) assay, alkaline phosphatase (ALP) test and Alizarin Red S staining, respectively. The qPCR technique was used to determine the relative expression of mRNA and the western blot was used to determine the relative expression of protein. In vivo experiments, spinal vertebrae staining in zebrafish larvae was accomplished by alizarin red S staining. ResultsZebrafish larvae's hatching, survival, malformation, and heart rate were unaffected by 50 μM of Rg1 in vivo, while the MEC3T3-E1 cell line's proliferation was unaffected by 50 μM of Rg1 in vitro. Meanwhile, Rg1 was shown to improve osteogenic differentiation or bone formation as well as the level of mRNA expression of osteogenic markers in vivo and in vitro. Treatment with Rg1 significantly increased the expression of G protein-coupled estrogen receptor (GPER) and pAKT. In addition, the GPER inhibitor G15 could significantly reduce the mRNA and protein expression levels of GPER and phosphorylated AKT, LY294002, a PI3K/AKT pathway inhibitor, markedly suppresses the expression of phosphorylated AKT, yet shows no significant impact on GPER expression. Both G15 and LY294002 can significantly blocked the Rg1-mediated enhancement of osteogenesis capacity in the GIOP model. In contrast, when both the agonists G1 of GPER and LY294002 were added, G1 increased the relative expression of mRNA and protein of GPER, but not the expression of osteogenic capacity and osteogenic markers. ConclusionsThis study investigates the mineralization effects and mechanisms of Ginsenoside Rg1 both in vitro and in vivo. For the first time, we propose that Rg1 might regulate osteogenesis by modulating AKT phosphorylation through mediating GPER expression within the PI3K/AKT pathway in the GIOP model. This discovery introduces novel targets and avenues for osteoporosis treatment.

Identification of antidiabetic inhibitors from Allophylus villosus and Mycetia sinensis by targeting α-glucosidase and PPAR-γ: In-vitro, in-vivo, and computational evidence

Diabetes mellitus (DM) is a metabolic disorder arising from insulin deficiency and defectiveness of the insulin receptor functioning on transcription factor where the body loses control to regulate glucose metabolism in β-cells, pancreatic and liver tissues to homeostat glucose level. Mainstream medicines used for DM are incapable of restoring normal glucose homeostasis and have side effects where medicinal plant-derived medicine administrations have been claimed to cure diabetes or at least alleviate the significant symptoms and progression of the disease by the traditional practitioners. This study focused on screening phytocompounds and their pharmacological effects on anti-hyperglycemia on Swiss Albino mice of n-hexane, ethyl acetate, and ethanol extract of both plants Mycetia sinensis and Allophylus villosus as well as the in-silico investigations. Qualitative screening of phytochemicals and total phenolic and flavonoid content estimation were performed significantly in vitro analysis. FTIR and GC–MS analysis précised the functional groups and phytochemical investigations where FTIR scanned 14, 23 & 17 peaks in n-hexane, ethyl acetate, and ethanol extracts of Mycetia sinensis whereas the n-hexane, ethyl acetate, and ethanol extracts of Allophylus villosus scanned 11 peaks, 18 peaks, and 29 peaks, respectively. In GC–MS, 24 chemicals were identified in Mycetia sinensis extracts, whereas 19 were identified in Allophylus villosus extracts. Moreover, both plants' ethyl acetate and ethanol fractioned extracts were reported significantly (p < 0.05) with concentrations of 250 mg and 500 mg on mice for oral glucose tolerance test, serum creatinine test and serum alkaline phosphatase test. In In silico study, a molecular docking study was done on these 43 phytocompounds identified from Mycetia sinensis and Allophylus villosus to identify their binding affinity to the target Alpha Glucosidase (AG) and Peroxisome proliferator-activated receptor gamma protein (PPARG). Therefore, ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis, quantum mechanics-based DFT (density-functional theory), and molecular dynamics simulation were done to assess the effectiveness of the selected phytocompounds. According to the results, phytocompounds such as 2,4-Dit-butyl phenyl 5-hydroxypentanoate and Diazo acetic acid (1S,2S,5R)-2-isopropyl-5-methylcyclohexyl obtained from Mycetia sinensis and Allophylus villosus extract possess excellent antidiabetic activities.

Antioxidant, hepatoprotective & nephroprotective potential of a novel synthetic compound 2′, 3′-dihydroxybenzylidene in paracetamol intoxicated rats

Paracetamol is a commonly used analgesic and antipyretic drug, but at a high dose level, it leads to deleterious side effects. The need to investigate new hepatoprotective drugs is driven by the lack of safety and efficiency of existing medications. A newly synthesized compound 2′, 3′-dihydroxybenzylidene (DHB) was evaluated in the present study for its antioxidant, hepatoprotective and nephroprotective potential compared to ascorbic acid in paracetamol intoxicated rats. DHB and ascorbic acid were evaluated against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) for assessment of the antioxidant potential. Male albino rats (n = 20) were categorized into 5 groups with 4 rats each and the test compounds were administered for 14 days consecutively. On day 15th, the rats were anesthetized, and blood was collected through heart puncture for the evaluation of hematological and serological parameters. Subsequently, the rats were dissected for the histopathology of liver and kidney. Alanine Transaminase (ALT), Alkaline Phosphatase (ALP), Serum Bilirubin (SBR), Cholesterol level and Renal Function Tests (RFTs) showed a substantial increase in the paracetamol treated group. Healing in liver and kidney tissues was observed in the DHB treated groups compared to paracetamol intoxicated group. The hemoglobin (HB), mean corpuscular hemoglobin (MCH), RBCs and mean corpuscular hemoglobin concentration (MCHC) were found significantly elevated while the total leukocytes count (TLC), platelets (PLT) and neutrophils significantly decreased in the DHB treated group compared to the paracetamol intoxicated group. It is concluded that DHB possesses antioxidant, hepatoprotective, nephroprotective, and anti-inflammatory potential against paracetamol induced hepatotoxicity and nephrotoxicity in rats.

A Novel Nanocomposite Scaffold Based on Polyurethane (PU) Containing Cobalt Nanoparticles (CoNPs) for Bone Tissue Engineering Applications.

Bone tissue engineering, as a relatively new approach, has focused on combining biodegradable scaffolds, cells, and biologically active molecules for the recovery of different damaged tissues, such as bone defects. Polyurethane (PU), as a synthetic polymer, benefits from a porous structure which impersonates bone's natural environment. However, PU lacks osteoinduction activities. Cobalt nanoparticles (CoNPs) stimulate angiogenesis and biomineralization, which greatly favors osteogenesis. Here, we designed a novel scaffold based on PU and combined it with CoNPs for bone regeneration applications. The composition and structure of PU-CoNPs nanocomposite were characterized using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC). MTT and AO data showed biocompatibility and enhanced viability and proliferation of fibroblasts on PU-CoNPs scaffold. Ascorbic acid-2-phosphate, β-glycerophosphate, and dexamethasone-induced osteogenesis for 14 days. The alkaline phosphatase test asserts the increased mineralization of hADSCs cultured on PUCoNPs compared to pure PU scaffold. Further, the results disclosed an elevated osteogenic differentiation at the level of genes and proteins using immunocytochemical analysis (ICC) and quantitative real-time PCR (qPCR). These findings provide an evidence that PU-CoNPs nanocomposite might be a promising candidate for bone repair applications.

Characterization of Pectobacterium Carotovoratum Subsp. Carotovorum causing Bacterial Soft Rot Disease of Turnip in Pakistan

This study aims to identify the Pectobacterium carotovorum subsp. carotovorum (Pcc) causing bacterial soft rot disease of turnip in Pakistan through conventional and molecular techniques. During the survey, four different bacterial isolates (VRSPCC01-04) were obtained from symptomatic turnip steckling (cv. purple top) at Vegetable Research Station Sahiwal, Punjab, Pakistan, and subjected for further analysis. All the isolates were characterized and identified based on morphological, biochemical, and molecular analysis (16S Ribosomal RNA). During morphological studies, the colony characters found deep cavities or pits formation in Crystal violet pectate medium (CVP), Creamy yellowish colony color was found on Yeast Extract Glucose calcium carbonate Medium (YGC), while creamy in color with the oval-shaped colony was observed on nutrient agar (NA) medium. In addition, the Pathogenicity test proved that all the tested isolates developed a typical water-soak symptom after 24 h and initiation of soft rot began the 48 h after inoculation on turnip roots. Results of biochemical tests showed that all isolates had a positive reaction for catalase, citrate, and an adverse reaction for Phosphatase and Indole test. The 16S rRNA sequences of isolates were 99-100% similar to the corresponding 16S rRNA sequence of the Pectobacterium carotovorum (PCC) reference isolates. Based on this, all identified bacterial isolates are first time reported as a causative agent of soft rot disease of turnip in Pakistan.

Impact of In-Process Crystallinity of Biodegradable Scaffolds Fabricated by Material Extrusion on the Micro- and Nanosurface Topography, Viability, Proliferation, and Differentiation of Human Mesenchymal Stromal Cells

Due to affordability, and the ability to parametrically control the vital processing parameters, material extrusion is a widely accepted technology in tissue engineering. Material extrusion offers sufficient control over pore size, geometry, and spatial distribution, and can also yield different levels of in-process crystallinity in the resulting matrix. In this study, an empirical model based on four process parameters-extruder temperature, extrusion speed, layer thickness, and build plate temperature-was used to control the level of in-process crystallinity of polylactic acid (PLA) scaffolds. Two sets of scaffolds were fabricated, with low- and high-crystallinity content, and subsequently seeded with human mesenchymal stromal cells (hMSC). The biochemical activity of hMSC cells was tested by examining the DNA content, lactate dehydrogenase (LDH) activity, and alkaline phosphatase (ALP) tests. The results of this 21-day in vitro experiment showed that high level crystallinity scaffolds performed significantly better in terms of cell response. Follow-up tests revealed that the two types of scaffolds were equivalent in terms of hydrophobicity, and module of elasticity. However, detailed examination of their micro- and nanosurface topographic features revealed that the higher crystallinity scaffolds featured pronounced nonuniformity and a larger number of summits per sampling area, which was the main contributor to a significantly better cell response.

Effect of Different Routes of Blood Collection on the Results of Liver and Kidney Function Test of Adult Wistar Rats

Assessment of blood parameters is often used to monitor animal health and to determine the success of dietary and pharmacological intervention in preclinical animal models. It is important to consider whether hematological and metabolic measurements might differ in relation to different blood sampling sites at the time of blood collection. This research is aimed at evaluating the effects of the different routes of blood collection on the results of kidney function tests and liver function tests of adult Wistar rats. A total of 15 rats weighing 110g-150g were separated into three groups of 5 rats each and then allowed to acclimatize for 2 weeks. Blood was collected from group A using ocular puncture and from groups B and C through cardiac puncture and neck laceration mode of blood collection respectively. Liver function tests to determine serum levels of liver enzymes - aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) and kidney function tests to determine the serum levels of urea and creatinine were carried out and the results recorded. Our results showed that the different routes of blood collection used have no effect on the serum level of AST, ALT, ALP, urea and creatinine. Hence, it is safe to say that the different routes of blood collection have no effect on the result of the kidney and liver function tests.

Effect of Azadirachta indica against Sodium Benzoate Induced Hepatorenal Toxicity in Wistar Rats- An Experimental Interventional Study

Introduction: Azadirachta indica (Neem) is one of the widely used plants which has various medicinal properties like antipyretic, antimicrobial, antitumour, anti-inflammatory, antiulcer and antidiabetic effects. Silymarin-a milk thistle derivative has its own clinical significance. Sodium Benzoate (SB) is a versatile food preservative used in packaged food and drink industries which is consumed by the people unknowingly above World Health Organisation (WHO) standards which causes potential cytotoxicity. Aim: To evaluate the effect of Azadirachta indica leaf extract and the combination effect of Azadirachta indica leaf extract (Neem) with Silymarin against SB induced hepatorenal toxicity in adult male albino wistar rats. Materials and Methods: This experimental interventional animal study was conducted at Central Animal House at Aarupadai Veedu Medical College and Hospital, Puducherry, India from July 2021 to August 2021 for a period of 14 days. Total 30 male wistar rats were randomised into five groups with six rats into each group. The groups were Group 1 Control received only distilled water, Group 2 SB 200 mg/kg bw (SB) alone, Group 3 SB+Silymarin 100 mg/kg bw, Group 4 SB+ Azadirachta indica aqueous extract 400 mg/kg bw, Group 5 SB+Silymarin 100 mg/ kg bw+Azadirachta indica aqueous extract 400 mg/kg bw for 14 days through oral gavage. Doses and duration were determined based on previous studies. Blood was drawn from a retroorbital puncture, animals were sacrificed by euthanasia, a part of Liver and Kidney samples were sent for histopathological examination-cell structure, integrity, inflammation. Biochemical parameters of liver function tests-Serum Alanine Transaminase (ALT), Serum Aspartate aminotransferase (AST), Serum Alkaline Phosphatase (ALP) and Kidney function tests: urea, creatinine and uric acid were measured in serum. Statistical analysis were done by one-way Analysis of Variance (ANOVA) followed by Dunnet’s post-hoc test used for intergroup comparison, p-value &lt;0.05 considered to be significant. Results: There was a significant increase in the activities of liver enzymes ALT, AST, ALP and kidney function (creatinine, uric acid) in the SB alone treated group when compared with the control group. Hepatorenal protection of Neem extract was shown by significant decrease in liver and renal parameters which was comparable to that of control and Silymarin standard drug. Combination of silymarin and neem showed significant protection in liver (ALT, AST, ALP) and kidney function (urea, creatinine, uric acid) when compared to neem alone treated group. All the results were substantiated by histopathological examination of liver and kidney tissues. Conclusion: This study suggests effect of Azadirachta indica leaf extract with Silymarin possess hepatorenoprotective effect against SB induced damage in rats. Combination effect of Azadirachta indica leaf extract with Silymarin significantly proved the hepatorenal protectivity when compared with Neem alone treated group.

An artificial bone filling material of poly l-lactic acid/collagen/nano-hydroxyapatite microspheres: Preparation and collagen regulation on the property

Artificial bone materials are in great need due to a lot of bone injuries. Herein, collagen/nano-hydroxyapatite (Col/nHA, C-H) composite nanospheres were obtained by in-situ mineralization, and poly L-lactic acid/collagen/nano-hydroxyapatite (PLLA/Col/nHA, P-C-H) was further prepared by high-speed shear emulsification method. The interfacial properties and structure between PLLA and nHA are regulated by the adhesive property of Col. The morphology, structure and properties of P-C-H microsphere were characterized in detail by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET) and simulated degradation of PBS in vitro. The results show that C-H is uniformly distributed in P-C-H microspheres, and a mesoporous material with a “pomegranate” structure and a particle size of 5–30 μm is self-assembled based on C-H multiple composite microspheres. It is beneficial to the sustained-release degradation of P-C-H and the retention/release of Ca2+. The 60-day PBS degradation shows that PLLA delays the degradation of nHA, making the degradation rate of P-C-H basically consist with the human bone healing cycle. The co-culture of P-C-H with MC3T3-E1 cells shows that P-C-H has high biocompatibility and no cytotoxicity. The cell viability is higher than 100 % in 72 h, indicating P-C-H has a proliferation effect on cell growth. Alkaline phosphatase and quantitative real-time PCR test show a positive promotion of P-C-H in cell proliferation and differentiation. The multi-layered P-C-H microspheres have an application potential in bone tissue engineering.

Osteogenic differentiation of pre-conditioned bone marrow mesenchymal stem cells with Nisin on modified poly-L-lactic-acid nanofibers.

IntroductionHuman bone marrow-derived mesenchymal stem cells (hBM-MSCs) are undifferentiated cells with self-renewing ability and multi-lineage differentiation beneficial for regenerative medicine. Nano scaffolds are novel materials employed in bone repair and regeneration. Nisin is a prebiotic that can increase stem cells’ lifespan and proliferation. This study attempted to provide a proper strategy for bone marrow mesenchymal stem cells differentiation into the Osteocytes on a Poly-L-lactic-acid (PLLA) scaffold after pretreating with Nisin.MethodsMSC osteogenic differentiation was evaluated by measuring Calcium, Alkaline phosphatase, and quantitative tests such as Real-Time PCR, Acridine Orange, Alizarin Red, Von Kossa, and others.ResultsThe result of the MTT test showed that the optimal dose of Nisin prebiotic for the MSCs’ preconditioning was 200 IU/mL on the 1st, 3rd, and 5th days of culture. Real-time PCR data indicated that the expression rate of ALP, Osteonectin, Osteocalcin, and Collagen I have increased in the presence of Nisin, while the RUNX-2 gene expression has decreased. Furthermore, the results of Alizarin Red and Von Kossa tests, as well as Scanning electron microscopy (SEM), revealed that the cell proliferation in the preconditioned samples with Nisin increased significantly.ConclusionsThe study concluded that the cell proliferation and differentiation increased in samples pretreated with Nisin on the PLLA Nano scaffolds.

Aneuploidy Rate and Stemness in Low-Level Mosaic Human Embryonic Stem Cells in the Presence/Absence of Bortezomib, Paclitaxel, and Lapatinib

Human embryonic stem cells (hESCs) are predisposed to aneuploidy through continual passages. Some reports indicate more sensitivity of aneuploid hESCs cells to anticancer drugs. The present study was designed to investigate the cytotoxicity of three anticancer drugs (including bortezomib, paclitaxel, and lapatinib) and their effect on aneuploidy rate in hESCs. To create a low-level mosaic cell line, normal hESCs (80%) and trisomic hESCs for chromosomes 12 and 17 (20%) were mixed. The effect of the 3 mentioned anticancer drugs on the chromosomal status was assessed by metaphase spread analysis after selection of the nontoxic conditions. Expression of pluripotency genes was analyzed, and an alkaline phosphatase test was performed to assess pluripotency preservation. Our data showed that treatment with bortezomib, paclitaxel, and lapatinib was nontoxic at 0.01, 0.01, and 0.2 μ<sc>M</sc> concentrations, respectively. Alkaline phosphatase and pluripotency gene expression analyses revealed maintenance of pluripotency following treatment with above-noted nontoxic concentrations. Aneuploid cells were dominant in treated and control groups with a minimum abundance of 70%, with no significant differences between groups. Drug treatments had no negative effect on pluripotency. Insensitivity of aneuploid cells in treatment groups could be related to the specific characteristics of each cell line in response to the drug and the proliferative superiority of cells with trisomies 12 and 17.

The Togo national proficiency test pilot programme for basic clinical chemistry tests.

BackgroundA national proficiency test (PT) programme is not currently implemented in most low-income countries. However, participation in such PT programmes assists improves test performance and result accuracy.ObjectiveThis study assessed how well 11 government hospital laboratories performed 18 basic clinical chemistry tests and identified areas needing improvement.MethodsA cross-sectional study was carried out by the Division of Laboratories of the Ministry of Health of Togo from 01 July 2016 to 31 December 2016. The test performance was evaluated using panels provided by One World Accuracy, Canada (Vancouver). The Clinical Laboratory Improvement Amendments criteria were used in evaluating the laboratories, and their success rates were compared with the World Health Organization Regional Office for Africa’s target of 80%.ResultsThe overall rate of acceptable results at the laboratories was over 80% for glucose, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase and triglycerides tests. The laboratories using fully automated spectrophotometers had an acceptable results rate of 89% (p = 0.001). The overall performance of the laboratories by cycles varied from 71% to 82%.ConclusionThis national PT programme identified the tests, which laboratories must improve their performance (urea, creatinine, uric acid, bilirubin, cholesterol, total protein, calcium, magnesium, phosphorus). It demonstrated the need for the use of routine appropriate internal quality control in all laboratories. The proficiency test programme should be extended to all clinical laboratories and target all biology disciplines.

Function alteration of engineered heart tissues carrying a shorter filamin C protein induced by CRISPR/Cas9

Dilated cardiomyopathy (DCM) is characterized by ventricular dilation and systolic dysfunction beginning in young adults and leading to heart failure with few treatment options. Loss-of-function mutations in the FLNC gene, encoding Filamin C, including splicing mutations, have been strongly associated with an arrhythmogenic DMC phenotype. FLNC-related cardiomyopathies are autosomal dominant disease and the mechanisms leading to the phenotype are not fully understood. In vitro models are needed to improve knowledge of pathogenic mechanisms. A control iPSc line (ICAN-403.3) was established from healthy donor fibroblasts and a mutated FLNC subclone (ICAN-FLNC-42.1) was derived using CRISPR/Cas9 technology. The iPSc cells were then differentiated into cardiomyocytes and beating pseudo-tissues in order to study the functional effects of the mutation. We generated two iPS clones, one derived from the other after mutation induced by non-homologous CRISPR/Cas9-dependent DNA repair. The clones were characterized for their pluripotency (immunoflurescence and qPCR of genes expressed in the pluripotent state, alkaline phosphatase test), their genomic integrity (exome, karyotype) and for their capacity for differentiation in the 3 layers (ScoreCard). More specifically, these cells can be differentiated into beating cardiomyocytes (> 85% of cells express troponin T) and exhibit sarcomeric striation by immunostaining of actinin-2. Sequencing shows that the mutation induced by CRISPR/Cas9 is a deletion of the 3′ terminal of exon 42 of the FLNC gene found in the homozygous state [Chr7(GRCh38):g.128854898_128854915del] and lead to the deletion of the consensus donor splice site of exon 42. Additionally, no off-target in cardiac expressed genes has been detected by exome sequencing. Analysis by RT-PCR and immunoblot shows that the mutation induces a exon 42 skipping and reduced expression of a protein of lower molecular weight. Cardiac tissues records for 3 weeks [engineered heart tissue (EHT) generated from the two iPS clones differentiated into cardiomyocytes] show, for the mutant, a lower beat frequency and a higher arrhythmia index. This model will make it possible to understand the pathophysiological mechanism of DCM due to FLNC mutations. These models are also useful for testing potential therapeutic pathways.

산성인산화효소 검사법 (Acid Phosphatase Test)에서 정액예비시험용 시약 (AP Reagent)이 DNA 분석에 미치는 영향

Generally, DNA of crime scene evidence is obtained from body fluids such as blood, semen and saliva. The detection and identification of body fluids are the most important step in forensic investigation. Especially, the determination of semen is very significant in case of sexual violence. Prostatic acid phosphatase test as the presumptive test for searching presence of semen is commonly used because of its convenience and cost/time-effectiveness. In this study, we tried to figure out the effect of AP reagent on DNA analysis according to the time dividing into two groups, control and experimental (AP reagent reaction). The degree of DNA condition was evaluated by the DNA concentration, degradation index and STR analysis. The result of degradation index of experimental group is higher than the control group. Also, in STR analysis, peak height and peak height ratio (PHR) of experimental group is lower than the control group increasing the effect of AP reagent on DNA analysis over time. Therefore, we confirmed that it is necessary to be cautious using the AP reagent on the whole of forensic evidence for high-quality DNA analysis.

Histology and radiography studies of effects of Lepidium sativum seeds on bone healing in male albino rats

Lepidium sativum (garden cress) is a folk medicine that exhibited several therapeutic applications including anti-asthma, antihypertension, and bone healing. This study aimed to investigate the effects of L. sativum seeds on healing of fractured bone in male albino rats (Rattus rattus albinus). Twenty male albino rats were randomly divided into four groups (each group contains 5 rats); Non-Broken Control (NBC), Broken Control Non-Treated (BCNT), Broken Treated with 1 g/kg of seed powder (BT1g), and Broken Treated with 5 g/kg of seeds powder (BT5g). Right tibial closed fractures were created in the rats using a three-point bending technique. Serum Ca2+ level and Alkaline phosphatase tests were examined in all groups. Digital radiography was applied for three times, at the beginning, after 3 weeks, and 6 weeks. Histopathological study was done for right limb tibia bones. Results showed that the highest level of ALP was in the BT5g group after 3 weeks. However, after 6 weeks ALP level was the lowest in the BT5g group followed by the BT1g group. Serum Ca2+ level was higher in BT5g group after 6 weeks as compared to same group after 3 weeks. The X-ray photography shows that in treatment groups a clear improvement in bone healing that was revealed by an obvious callus and to a large extent a blurred fracture line. We also observed that after 6 weeks, the healing and recovery of broken bones were almost completed in L. sativum treated groups, especially in the BT5g group. Histology data further confirm the distinctive features of bone healing after feeding of seeds powder. Overall, our data suggested that seed of L. sativum has potential to speed up the callus formation, fracture line repairing, and new bone regeneration. This study highlights the importance of folk herbs in healing of fractured bones.