3-phosphate Dehydrogenase Research Articles

Analyte stability in whole blood using experimental and datamining approaches

The analytical stability of laboratory tests relies mostly on internal and external quality control procedures. Summarized patient data has in several studies been shown to be a good supplement for monitoring analytical stability. In our present investigation, we evaluate a datamining method for retrospective evaluation and assessment of analyte stability in whole blood. Results from the laboratory information system were used as the basis for the datamining approach. Blood tests were requested by the general practitioners and drawing of the blood sample was either at the general practitioner's or at the hospital outpatient clinics. We were able to split data into groups based on sample collection place and time to analysis. The datamining approach was compared to experiments where samples were incubated at a single temperature as well as an experiment where the temperatures were changed during incubation. To demonstrate the method, we selected three laboratory tests considered representative: potassium, phosphate, and lactate dehydrogenase. The datamining approach showed results similar to the reference experiment. Furthermore, our results show that the analytes phosphate and potassium were not stable after short storage at a lower temperature.

Attenuation of carbohydrate metabolism and lipid profile by methanolic extract of Euphorbia helioscopia and improvement of beta cell function in a type 2 diabetic rat model

BackgroundTraditional plant-based remedies prescribed to treat diabetes have shown promise in research-based setting. Current research was conducted to examine the antidiabetic and antioxidant effects of methanolic extract of a folk herbal plant Euphorbia helioscopia in a rat model of type 2 diabetes.MethodsDiabetes was induced in male Wistar rats by administering 5% sucrose in drinking water and cafeteria diet for 8 weeks with subsequent nicotinamide and streptozotocin administration. Diabetic rats were then distributed into four individual groups (n = 8); Positive control (PC; no treatment), standard control (SC; Metformin @ 10 mg/kg bw), treatment 1 (EH1, E. helioscopia methanolic extract @200 mg/kg bw) and treatment 2 (EH2, E. helioscopia methanolic extract @400 mg/kg bw). After 21 days of treatments, the rats were decapitated for blood collection. Serum was evaluated for antidiabetic potential, antioxidant and lipid profile, thyroid hormone, amylin, leptin, and carbohydrate metabolic enzymes. Data were analyzed statistically by one-way analysis of variance (ANOVA).ResultsSerum levels of glucagon, glucose and C-peptide were significantly (P ≤ 0.05) decreased in EH1 (1915.33 ± 98.26a pg/ml, 122.59 ± 2.99a mg/dl, 277.59 ± 28.41a pg/ml respectively) and EH2 (1575.28 ± 56.46a pg/ml, 106.04 ± 5.21a mg/dl, 395.06 ± 42.55a pg/ml respectively) as compared to the PC (3135.78 ± 189.46bpg/ml, 191.24 ± 17.75bmg/dl, 671.70 ± 109.75b pg/ml respectively) group. A similar trend was observed in serum insulin levels in EH1 and EH2 groups. The plant’s methanolic extract effectively reduced the total oxidant status (TOS) and MDA levels in the diabetic rats and increased the total antioxidant capacity (TAC) along with an increased level of SOD, Catalase, Paraoxonase, and arylesterase. The plant extract also induced antihyperlipidemic activity and recovered the thyroid hormones, amylin, and leptin levels to normal. The activity of different carbohydrate metabolic enzymes like Pyruvate Kinase, Glucose 6 phosphate dehydrogenase, phosphofructokinase, and glucokinase has also been restored by the extract treatment.ConclusionCurrent study indicates the antioxidant and antidiabetic potential of E. helioscopia methanolic extract in normalizing the lipid profile, thyroid hormones, amylin, leptin, and carbohydrate metabolism in type 2 diabetic rat model.

Snakin-2 interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenase 1 to inhibit sprout growth in potato tubers.

The potato tuber is the main nutrient supply and reproductive organ; however, tuber sprouting can reduce its commercial value. Snakin-2 (StSN2) was first reported as an antimicrobial peptide that positively regulates potato disease resistance. Our recent study suggested StSN2 overexpression inhibited sprout growth, while the sprouting process was accelerated in StSN2 RNAi lines. Cytoplasmic glyceraldehyde-3- phosphate dehydrogenase 1 (StGAPC1) was identified as a candidate protein that interacts with StSN2 by coimmunoprecipitation/mass spectrometry (CoIP/MS) experiments. Here, we report that the expression levels of StSN2 and StGAPC1 decreased during sprouting compared with dormancy. Coexpression of StSN2 and StGAPC1 in bud eyes and apical buds was verified by immunofluorescence analysis of paraffin sections. In addition, interaction of StSN2 and StGAPC1 was confirmed by yeast two-hybrid, coimmunoprecipitation and split luciferase complementation assays. Overexpression of StGAPC1 depressed sprout growth, which is similar to the function of StSN2, and StSN2- and StGAPC1-overexpressing lines showed decreased glucose, fructose and galactose content. The interaction of StSN2 and StGAPC1 enhanced StGAPC1 activity and decreased its oxidative modification to inhibit sprout growth. Our results suggest that StSN2 plays a regulatory role in tuber sprout growth through interaction with StGAPC1.

Engineering of Promoters for Gene Expression in Pichia pastoris.

The availability of exceptionally strong and tightly regulated promoters is a key feature of Komagataella phaffii (syn. Pichia pastoris), a widely applied yeast expression system for heterologous protein production. Most commonly, the methanol-inducible promoter of the alcohol oxidase 1 gene (PAOX1) and the constitutive promoter of the glyceraldehyde 3 phosphate dehydrogenase gene (PGAP) have been used. Recently, also promising novel constitutive (PGCW14), regulated (PGTH1, PCAT1), and bidirectional promoters (histone promoters and synthetic hybrid variants) have been reported.As natural promoters showed so far limited tunability of expression levels and regulatory profiles, various promoter engineering efforts have been undertaken for P. pastoris . PAOX1, PDAS2, PGAP, and PGCW14 have been engineered by systematic deletion studies or random mutagenesis of upstream regulatory sequences. New engineering strategies have focused on PAOX1 core promoter modifications by random or rational approaches and transcriptional regulatory circuits to render PAOX1 independent of methanol induction. These promoter engineering efforts in P. pastoris have resulted in improved, sequence-diversified synthetic promoter variants allowing coordinated fine-tuning of gene expression for a multitude of biotechnological applications.

Role of Glucose 6 Phosphate Dehydrogenase (G6PD) in Diabetic Cataract

Introduction: Diabetes mellitus and its complications are spreading with increased rate in Asian population especially in Pakistan. Uncontrolled diabetes can lead to different micro vascular complications. Cataract is one of the complications of diabetes which may lead to lens degenerative changes and visual impairment. G6PD plays a vital role in preventive measurements from cataract development in normal population.
 Objectives: This Study was designed to estimate G6PD levels in diabetic without ocular manifestations & diabetic cataract population.
 Methodology: This cross sectional comparative study was done at the Department of Biochemistry LUMHS Jamshoro in collaboration with the Diabetic clinic, Institute of Ophthalmology & Diagnostic Research Laboratory LUMHS Jamshoro. 100 diagnosed subjects of diabetes were selected by Non-Probability type of sample technique with consent of subjects and they were divided in to two groups Group A as control 50 diabetic subjects with out ocular manifestation while Group B as case study group contain 50 subjects of diabetes with cataract. The fasting blood glucose level was estimated by Hexokinase Method while G6PD level was measured by kit method on SD Biosensor while HbA1c(% ) was estimated by TTAB methodology. The data was statistically analyzed by SPSS 21.
 Results: The mean level of G6PD in Group A was 15.63±2.45 u/Hb while in group B it was 9.01±3.11 u/G HB. This result finally concluded that there was significantly (<0.05) decline of G6PD diabetic cataract as compared with diabetic without cataract.
 Conclusion: This study concluded that there was significant decline in G6PD level in diabetic cataract. It is also concluded that the estimation of G6PD level in diabetic population will be beneficial to take early preventive measurements against diabetic vascular complications.

Diversity and function of microbial communities in the sand sheath of Agropyron cristatum by metagenomic analysis

The roots of most gramineous plants are surrounded by a variety of microorganisms; however, few studies have focused on the rhizosheath of psammophytes. Therefore, in this study, we used Illumina HiSeq high-throughput sequencing technology to analyse the composition and functional diversity of microbial communities in the rhizosheath of sand-grown Agropyron cristatum (L.) Gaertn. We found that the number of species and functions of microbial communities gradually decreased from the rhizosheath to the bulk soil. Thus, the microbial composition of the rhizosheath was richer and more diverse, and the abundance of bacteria, including Sphingosinicella, Rhizorhabdus, Friedmanniella, Geodermatophilus, Blastococcus, and Oscillatoria, was higher, and the abundance of fungi, such as Mycothermus, was higher. The abundance of CO2 fixation-related genes (acsA, Pcc, and cbbL) in the carbon cycle; NO3–, NO2–, NH2OH, and N2 transformation genes (nrtP, nirS, hao, and nifK) in the nitrogen cycle; soxB/A/C, Sat, and dsrB genes in the sulphur cycle; and 1-phosphate mannitol dehydrogenase (MtlD) gene and polyketide synthase gene (pks) were higher in the rhizosheath than in the bulk soil, as well as genes related to phosphorus uptake in the phosphorus cycle. Our findings showed that the rhizosheath may host the predominant microbial species related to the formation of a rhizosheath.

Mutations in the Hemoglobin Binding Domain of Band 3 Perturb Cellular Metabolism and Alter the Sickling of SS Erythrocytes

▪Under oxygenated conditions, 4 glycolytic enzymes that perform the terminal steps of glycolysis (phospho-fructoKinase [PFK], lactate dehydrogenase [LDH], aldolase [ALD] and glygeraldehyde 3 phosphate dehydrogenase [GAPDH]) bind to the cytoplasmic domain of band 3. Under deoxy conditions deoxy hemoglobin (Hb) is bound to band 3 and PFK, LDH, ALD and GAPDH are displaced (Campanella et al. PNAS 102, 2005; Blood 112, 2008). We generated transgenic mice in which the sequence encoding the first 35 amino acids of the wild type human band 3 cytoplasmic domain replaced the endogenous mouse band 3 sequences in the Slc4a1 gene, a mutant line in which human amino acids 12-21 were deleted removing the deoxy Hb binding site (-Hb) and a third line in which amino acids 1-11 were deleted creating a high affinity binding site for deoxyHb (++Hb). Erythrocytes from the mutant lines were insensitive to Oxygen concentration resulting in changes in oxygen dependent deformability and other physical properties compared to the wild type line (Chu et al. Blood 128, 2016, Zheng et al. JBC 294, 2019, Zhou et al. Sci. Adv. 5, 2019).We crossed our humanized band 3 mouse strains to the Townes Sickle Cell Disease (SCD) mouse model, maintaining both the human βA and βS alleles to generate human AA, AS and SS mice homozygous for each of the human band 3 cytoplasmic domain sequences. Using an assay in which SS red cells in phosphate buffer are deoxygenated to 6% oxygen over time (Dunkelberger et al., J. Phys. Chem. B 122, 2018), we observed that -Hb band 3/SS mice showed an accelerated rate of sickle cell formation and a higher percent of sickled cells than wild type band 3/SS mice (p<0.01). Conversely, ++Hb band 3/SS mice showed an inhibition of both the rate of sickling and the precent of sickled cells compared to wild type band 3/SS mice (p<0.05).We hypothesized that the inability of the glycolytic enzymes to reversibly bind to band 3 in the mutant mice were responsible for the differences in sickling. To test this hypothesis, we analyzed a panel of 28 cellular metabolites in 12 mice (6 female, 6 male) of each genotype: wild type band 3/AA, -AS and -SS, -Hb band 3/AA, -AS, -SS and ++Hb/AA, -AS, -SS. The metabolites were quantified by LC-MS/MS using an API 4500 triple quadrupole mass spectrometer (AB Sciex), with chromatographic resolution enabled on a polymeric amino column (apHera by Supelco) under alkaline mobile phase conditions (pH ~9.3). Stable isotope dilution and 8pt calibration curves allowed the absolute quantification of each metabolite.Consistent with the constitutive binding of the terminal glycolytic enzymes to band 3 in -Hb erythrocytes, glycolysis was inhibited after the phosphoenol pyruvate step, as evidenced by significant accumulation of the intermediates at top of the glycolysis pathway, including fructose 1,6 biphosphate (FBP; p<0.01), dihydroxyacetone phosphate/ glyceraldehyde-3-phosphate (G3P; p<0.01), and 3-phosphoglycerate/2-phosphoglycerate (PG; p<0.01). In the ++Hb mutant where the terminal glycolytic enzymes are constitutively displaced from band 3, significantly lower levels of FBP, G3P and PG were observed (p<0.01). The levels of these metabolites in wild type band 3/SS erythrocytes were intermediate between the two mutant strains.We hypothesized that the accumulation of FBP, G3P and PG contributed to the increased rate of sickling in the -Hb band 3/SS mice. To test this, we incubated wild type band 3/SS cells with either FBP or PG. Both intermediates increased the rate of sickle cell formation and percentage of sickled cells in a dose dependent fashion with no alteration in any RBC indices including MCV and osmotic fragility. We next hypothesized that reduction of the levels of glycolytic intermediates would have an antisickling effect. To test this, we incubated wild type band 3/SS cells with 2,3 diphosphoglycerol (DPG), which is a potent inhibitor of glycolysis. We found that DPG treatment led to a dose dependent decrease in the rate of sickle cell formation and percentage of sickled cells, again with no alteration in any RBC indices including MCV and osmotic fragility.We conclude that the accumulation of glycolytic intermediates leads to increased sickle cell formation. We propose that reduction in the levels of glycolytic intermediates either by accelerating the terminal stages of glycolysis or by redirection to the pentose phosphate pathway may offer a means to treat SCD. DisclosuresNo relevant conflicts of interest to declare.

Hemolytic anemia in a 2-years old caused by Glucose 6 Phosphate Dehydrogenase Deficiency (G6PD): a case report

Background: Glucose-6-Phosphate-Dehydrogenase (G6PD) deficiency is an enzyme abnormality related to sex chromosomes (x-linked), which is inherited. The activity or stability of the G6PD enzyme decreases, thus causing the breakdown of red blood cells when an individual is exposed to exogenous substances that can cause oxidative damage. This case study aims to describe a rare case of hemolytic anemia caused by glucose 6 phosphate dehydrogenase deficiency in a two-year-old boy. Case Presentation: A two-year-old boy presented with agitated, pale, yellowish eyes and body and projectile vomiting after consuming fava beans. He had a prior history of packed red cell transfusion. Physical examination found the conjunctivae were pale with the icteric sclera. Laboratory examination revealed leukocytosis, moderate normochromic normocytic anemia, increased reticulocytes count, and elevated total and unconjugated bilirubin. Peripheral blood smear examination found microcytes, spherocytes, fragmentocytes, and burr cells. Coomb test was negative with decreased G6PD result. Conclusion: G6PD deficient patients have no symptoms until exposed to oxidizing drugs, infections or after fava beans ingestion which may cause hemolytic anemia and jaundice. Several methods of screening and diagnosis of G6PD can be performed to avoid morbidity and mortality.

Soil biological properties as influenced by phospho-enriched compost and fertility levels in maize (Zea mays)

The present investigation was carried out to evaluate the impacts of phospho-enriched compost and fertility levels on soil biological properties in Typic Haplustepts soil of Sub-humid Southern Plain and Aravalli Hills Region in Rajasthan. The experiment was undertaken during kharif 2018 and 2019 at Instructional Farm (Agronomy), Rajasthan College of Agriculture, Udaipur (Rajasthan). The treatments comprised four levels of phospho-enriched compost (PEC), i.e. control, PEC @ 2.0, 4.0 and 6.0 t/ha and four levels of fertility (kg/ha) i.e. control, 50% RDF + foliar spray of Zn @ 0.5%, 75% RDF + foliar spray of Zn @ 0.5% and 100% RDF + foliar spray of Zn @ 0.5%, respectively. The experiment was laid out in a factorial randomized block design with three replications. Significant improvement in different soil biological properties, i.e. alkaline phosphate activity, dehydrogenase activity and soil microbial biomass carbon was observed with increasing levels of phospho-enriched compost and fertility up to 6 t/ ha and 100% RDF + foliar spray of Zn @ 0.5%, respectively after harvest of the maize (Zea mays L.) crop during both the years as well as in pooled analysis. In case of fertility levels, however, the microbial populations in soil after harvest of crop increase was significant up to 75% RDF + foliar spray of Zn @ 0.5% which was found statistically at par with 100% RDF + foliar spray of Zn @ 0.5%. The application of phospho-enriched compost @ 6 t/ha and 100% RDF + foliar spray of Zn @ 0.5% along with the recommended dose of fertilizer results in significantly higher biological properties of soil under Typic Haplustepts.

Analytical and Clinical Performance Evaluation of ARK Fentanyl II Assay on Beckman Coulter AU System

Abstract Introduction/Objective Drugs of abuse, specifically opioids, have been in recent years an important focus in both medicine and media due to the ‘opioid epidemic’ and large numbers of overdose deaths. Synthetic opioids such as fentanyl have contributed to this epidemic. The CDC reports that of roughly 50,000 opioid overdose deaths in 2019, nearly 73% were due to synthetic opioids. The objective of this study is to assess the analytical and clinical performance of the Fentanyl II Assay by ARK Diagnostics, Inc. Methods/Case Report Fentanyl was qualitatively determined by the ARK Fentanyl II Assay, a homogenous enzyme immunoassay based on competition between drug in the specimen and drug labeled with recombinant glucose-6- phosphate dehydrogenase (rG6PDH) for antibody binding sites. The presence of drug in urine increases enzyme activity, which converts nicotinamide adenine dinucleotide (NAD) to NADH in the presence of glucose-6-phosphate, resulting in an absorbance change measured by spectrophotometry. The evaluation was performed following CLSI guidelines. The analytical performance was evaluated for accuracy and precision. Results (if a Case Study enter NA) To evaluate the accuracy, twelve positive and eight negative specimens were tested by the ARK immunoassay performed on two Beckman Coulter instruments (AU480 and AU680) and by LC- MS/MS. The results from both instruments showed 100% agreement with the results from LC-MS/MS. On instrument AU480, the within-run CVs were 18.0% at the level of 22.510 ng/mL and 0.8% at the level of 552.628 ng/mL. The between-run CVs were 8.8% at the level of 68.928 ng/mL and 3.7% at the level of 158.947 ng/mL. On instrument AU680, the within-run CVs were 273.4% at the level of 1.052 ng/mL and 0.8% at the level of 523.788 ng/mL. The between-run CVs were 7.8% at the level of 53.779 ng/mL and 3.1% at the level of 145.263 ng/mL. In May 2021, 2,075 fentanyl assays were run at UAB. Of those, 476 returned a positive result with a positive rate of 22.9%, and 1,599 were negative with a negative rate of 77.1%. One positive result was confirmed by LC-MS/MS as negative for fentanyl but positive for norfentanyl, a metabolite of fentanyl. The clinical specificity was 99.9% and sensitivity was 100%. Conclusion In Conclusion, the ARK fentanyl Assay II on Beckman Coulter AU system has good accuracy, sensitivity and specificity. The precision at medium and high levels are good. However, the precision at low level needs to be improved.

Effect of Copper sulphate pollution and its antidote penicillamine on serum and brain tissues markers of albino rats

seventy albino rats were divided into seven equal groups each containing 10 rats (G1:control received distilled water) ; (G2: 0.1 LD50 of CuSO4) ; (G3: 0.2 LD50 of CuSO4) ; (G4: 0.4 LD50 of CuSO4) ; (G5: 0.1 LD50 of CuSO4 +100mg/kg/day of penicillamine) ; (G6: LD50 of CuSO4.+100 mg/kg/day of penicillamine) and (G7: LD50 of CuSO4+ 100 mg /kg/day of penicillamine) for 30 days and at the end of the experiment all rats were sacrificed and blood samples and brain tissues were collected for biochemical assaying of Fasting Blood Glucose (FBG), Serum Cu level, serum tyrosinase activity, oxidative stress marker Malondialdehyde (MDA) and Total Antioxidant Activity (TAC). also, DNA determination of relative gene expression of cerebral Adenosine monophosphate-activated Protein Kinase (AmpK), Protein Kinase (AKT), Phosphatidylinositol-3-Kinase (PI3K), Cytochrome c oxidase (Cyto co ), and Glucose -6- Phosphate Dehydrogenase (G6PD). Results: The result showed that administration of copper sulphate with different levels induced a significant increase in fasting blood glucose level, lipid peroxidation marker MDA, serum copper level, and serum tyrosinase activity, and a significant decrease in TAC. Moreover, copper sulphate administration elicited a significant downregulation ( AmpK, AKT, PI3K, Cytochrome c oxidase, G6PD).it could be approved that penicillamine could abolish the negative impact of copper sulphate on neural tissues and serum enzymes. Conclusion: D-penicillamine can reduce neurotoxicity and oxidative stress caused by copper pollution.

Risk Factors for Serum Bilirubin Rebound After Stopping Phototherapy in Neonatal Hyperbilirubinemia

Objective: To identify the factors affecting rebound increase in bilirubin levels after stopping phototherapy in neonatal hyperbilirubinemia. Setting: Tertiary-level neonatal unit. Patients: This was a hospital-based cross-sectional observational study. The study was conducted in neonatal division of rural tertiary care hospital. All neonates who were admitted for hyperbilirubinemia treatment and fulfilled the inclusion criteria were included in the study. Inclusion Criteria: All neonates 1.5 kg and above and treated in newborn intensive care unit for hyperbilirubinemia. Exclusion Criteria: (a) Critically ill patients requiring respiratory support any time after delivery, (b) neonates presenting with life-threatening surgical conditions, (c) patient with congenital malformation, (d) conjugated bilirubin elevation, (e) patients with birth asphyxia. Statistical Analysis: All the data were entered into a Microsoft Excel sheet. Appropriate tests of significance were applied. The various variables were sorted and presented as a number or percentage. The categorical variables used in the analysis were evaluated using the chi-square test (Yates’s correction and Fischer exact test were employed where applicable). The continuous data were analyzed by “t” test. The P value of less than 0.05 was taken to be level of statistical significance. Results: One hundred and fifty patients of neonatal hyperbilirubinemia were included in this study. In this study, 81 newborns (54.0%) were male and 78 (52%) of them were born by normal vaginal delivery. One hundred and nine babies (72.6%) were term babies. Fifty babies (33.3%) were low birth weight. Out of 150 neonates, 18(12%) had ABO incompatibility, 13(8.7%) Rh incompatibility, 23(15.7%) neonatal sepsis, and 4 (2.6%) had polycythemia. Out of 150 patients, 15(10%) babies had rebound hyperbilirubinemia at 24 h of life requiring phototherapy prematurity (particularly &lt;34 weeks) ( P value: .03), low birth weight (particularly &lt;2.0 kg) ( P value:.009), onset of jaundice requiring phototherapy before 72 h of life ( P value .04), blood group (both Rh and ABO) incompatibility ( P values: .001 and .002), neonatal sepsis ( P value: .004) were significantly associated with rebound hyperbilirubinemia. Glucose 6 phosphate dehydrogenase (G6PD) deficiency, polycythemia, maturity, mode of feeding, and gender did not show any statistical significant relationship with rebound hyperbilirubinemia. Conclusions: Rebound level of bilirubin need not be checked in all babies. Babies with risk factors like born preterm, low birth weight, having sepsis, requirement of phototherapy before 72 h of life, Rh and ABO group incompatibility, only need to be checked for serum bilirubin rebound. We recommend more studies with larger sample size to evaluate all these factors including polycythemia and G6PD deficiency.

Myostatin suppresses adipogenic differentiation and lipid accumulation by activating crosstalk between ERK1/2 and PKA signaling pathways in porcine subcutaneous preadipocytes.

The current study was undertaken to determine the effect of myostatin (MSTN) on lipid accumulation in porcine subcutaneous preadipocytes (PSPAs) and to further explore the potential molecular mechanisms. PSPAs isolated from Meishan weaned piglets were added with various concentrations of MSTN recombinant protein during the entire period of adipogenic differentiation process. Results showed that MSTN treatment significantly reduced the lipid accumulation, intracellular triglyceride (TG) content, glucose consumption, and glycerol phosphate dehydrogenase activity, while increased glycerol and free fatty acid release. Consistent with above results, the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway was obviously activated and thus key adipogenic transcription factors peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer-binding protein-alpha (C/EBP-α), and their downstream enzymes fatty acid synthase and acetyl-CoA carboxylase were all inhibited. However, chemical inhibition of ERK1/2 signaling pathway by PD98059 markedly reversed the decreased TG content by increasing PPAR-γ expression. In addition, MSTN activated the cyclic AMP/protein kinase A (cAMP/PKA) pathway and stimulated lipolysis by reducing the expression of antilipolytic gene perilipin, thus elevated key lipolytic enzymes adipose triglyceride lipase and hormone-sensitive lipase (HSL) expression and enzyme activity. On the contrary, pretreatment with PKA inhibitor H89 significantly reversed TG accumulation by increasing PPAR-γ expression and thus inhibiting ERK1/2, perilipin, and HSL phosphorylation, supporting the crosstalk between PKA and ERK1/2 pathways in both the anti-adipogenic and pro-lipolytic effects. In summary, our results suggested that MSTN suppressed adipogenesis and stimulated lipolysis, which was mainly mediated by activating crosstalk of ERK1/2 and PKA signaling pathways, and consequently decreased lipid accumulation in PSPAs, our findings may provide novel insights for further exploring MSTN as a potent inhibitor of porcine subcutaneous lipid accumulation.

Acute Intravascular Hemolysis in the Critical Phase of Severe Dengue

Background: Dengue and severe dengue have various hemorrhagic manifestations ranging from mild presentations such as petechiae, bleeding from injection sites, ecchymosis, and gum bleeding to severe presentations like major mucosal bleeding such as hematemesis, melena, menorrhagia, and concealed internal bleeding. Common causes of anemia in dengue are due to blood loss (secondary to thrombocytopenia and/or coagulopathy), transient suppression of the bone marrow, and rarely, hemolytic anemia. Clinical Description: We describe a 5-year-old boy who presented to us in the critical phase of severe dengue with features of capillary leakage and cola-colored urine. Salient investigation reports were anemia, thrombocytopenia, normal reticulocyte count, absence of coagulopathy, indirect hyperbilirubinemia, deranged transaminase, highly elevated lactate dehydrogenase, and low haptoglobin levels, and hemoglobinuria, suggestive of acute intravascular hemolysis (AIVH). Autoimmune and microangiopathic hemolytic anemia, malaria, ingestion of dyes, snake bite, Glucose 6 phosphate dehydrogenase deficiency, and incompatible blood transfusions were excluded. Thus, the final diagnosis was severe dengue with hemolytic anemia and AIVH. Management: Oxygen delivery by the Heated Humidified High-Flow Nasal Cannula, strict input/output charting, appropriate fluid therapy, and diuretic infusion were the mainstays of management in an intensive setting. The aim was to maintain ventilation, perfusion, balance hydration, achieve optimal urine output and prevent fluid overload. Conclusion: Clinicians should consider Dengue as a possible cause for AIVH in children with severe dengue and cola-colored urine.

Chebulagic acid attenuates HFD/streptozotocin induced impaired glucose metabolism and insulin resistance via up regulations of PPAR γ and GLUT 4 in type 2 diabetic rats

The aim of the present study was to evaluate the effects of chebulagic acid on enzymes involved in carbohydrate metabolism in high-fat diet/streptozotocin-induced diabetic rats. Diabetes was induced in albino male Wistar rats by feeding them with a high-fat diet comprising of 84.3% standard laboratory rat chow, 5% lard, 10% yolk powder, 0.2% cholesterol, and 5% bile salt for 2 weeks. After 2 weeks, the animals were kept in an overnight fast and injected with a low dose of streptozotocin (35 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.5). At the end of the experimental periods, the blood glucose, plasma insulin, hemoglobin, glycated hemoglobin levels, homeostatic model of insulin resistance, glycogen content, glycogen synthase, glycogen phosphorylase were measured in high-fat diet and streptozotocin-induced diabetic rats whereas the glucose metabolic enzymes such as hexokinase, glucose 6 phosphate dehydrogenase, glucose 6 phosphatase, fructose 1,6 bisphosphatase and protein expression of peroxisome proliferator-activated receptor–γ (PPAR-γ) and Glucose transporter 4 (GLUT4) were analyzed. Administration of chebulagic acid to HFD/STZ -induced diabetic rats for 30 days significantly decreased the levels of plasma glucose, homeostatic model assessment of insulin resistance, glycogen phosphorylase and glycosylated hemoglobin and increased the levels of insulin, glycogen content and glycogen synthase. On the other hand, the altered activity of carbohydrate metabolic enzymes, PPAR-γ and GLUT4 were brought back to near-normal levels on treatment with chebulagic acid. The effect produced by chebulagic acid on various parameters was comparable to that of metformin.

Identification and Evaluation of Reference Genes for Quantitative PCR Normalization in Alligator Weed Flea Beetle (Coleoptera: Chrysomelidae).

Stably expressed reference genes are critical internal standards for the quantification of gene transcription levels using quantitative real-time PCR. Housekeeping genes are commonly used as reference genes but their expressions were variable depending on experimental conditions in many insect species studied. Here we report the identification and evaluation of 10 housekeeping genes in alligator weed flea beetle, Agasicles hygrophila Selman & Vogt (Coleoptera: Chrysomelidae), a biocontrol agent of alligator weed. The 10 housekeeping genes are: beta-actin (Actin), ribosomal protein L13A (PRL13a), succinate dehydrogenase complex subunit A (SDHA), ribosomal protein S20 (RPS20), ribosomal protein S13 (RPS13), glyceraldehyde phosphate dehydrogenase (GAPDH), TATA-box-binding protein (TBP), ribosomal protein L32 (RPL32), tubulin alpha-1 chain (TUBULIN), and elongation factor-1 alpha (ELF). Five programs, geNorm, NormFinder, BestKeeper, ΔCt method, and RefFinder, were used to evaluate the expression stability of the 10 genes among various A. hygrophila body parts and with different nutrient types (starvation, diet types). The expression stability analysis showed that RPS32 and RPL13a were reliable reference genes for the study of gene transcription in different body parts; Actin and RPL13a were optimal reference genes for different nutrient types. The selections of reference genes were validated using a CarE gene (GeneBank No: KX353552). The results of this study provide useful bases for studies of gene expression in various aspects relating to A. hygrophila.

Enzymatic and Genetic Study in Blood Among Some Children with β – Thalassaemia in Mosul City, Iraq

The present study aimed to demonstrate the extent to which the activity of a number of enzymes and genetic variation of β-globin genes were affected in the blood of 65 children patient with β - thalassemia major of both sexes, their ages ranged between ( 2 – 15 ), who registered in the Thalassemia Center at Ibn Al-Atheer Teaching Hospital for Children in the city of Mosul / Iraq and who are continuing treatment, after they were diagnosed by specialist doctors, as well as 30 healthy children of both sexes with same ages of the patients and it was considered as a control group.&#x0D; The results showed a significant increase (P≤0.05) in the activity of Alanine transaminase (ALT), Aspartate transaminase (AST), Alkaline phosphatase (ALP), Glucose 6- phosphate dehydrogenase (G6PD) and Adenosine deaminase (ADA) in the serum of treated children patients with β - Thalassaemia major by 73% , 53%, 8%, 9% and 54% respectively, compared with the healthy children group (control group). Also the results showed a significant increase in the activity of G6PD and ADA in the hemolysis of RBC of the same children patients by 7% and 43% compared with control group.&#x0D; When determining the genetic variation of the β-globin gene the result which depends on PCR technique did not show any genetic variation in the size of PCR band ,while the result of the sequencing showed variation in the nucleotides and included converted the nucleotide (A) to (C) in position (250) , change nucleotide (T) to (C) in position (426), replacement (C) nucleotide to (A) in position (623), change (G) nucleotide to (A) in position (630) and replacement (T) to (A) in the position (724), also the result detection three Transversion mutation and two transition mutation in β-globin gene in babies with β-thalassemia

Rhubarb granule promotes diethylnitrosamine-induced liver tumorigenesis by activating the oxidative branch of pentose phosphate pathway via G6PD in rats

Ethnopharmacological relevanceRhubarb is a natural herbal medicine widely used clinically with numerous pharmacological activities including anti-cancer. Specifically, several studies reported that free anthraquinones from Rhubarb suppressed the proliferation of hepatoma cells. Nonetheless, recent studies revealed that Rhubarb caused hepatotoxicity in vivo, confirming its “two-way” effect on the liver. Therefore, the efficacy and safety of Rhubarb in the in vivo treatment of liver cancer should be further elucidated.Aim of the study: This study investigated the presence of hepatoprotection or hepatotoxicity of Rhubarb in diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Material and methodsA total of 112 male Sprague-Dawley rats weighing 190–250 g were enrolled. The rats were induced hepatocarcinogenesis using diethylnitrosamine (0.002 g/rat) until 17 weeks. Starting at week 11, Rhubarb granules (4 g/kg and 8 g/kg) were intragastrically administered daily for 7 weeks. All rats were euthanized at week 20 and the livers were analyzed via non-targeted metabolomics analysis. We established hepatic glucose 6 phosphate (6PG) levels and glucose 6 phosphate dehydrogenase (G6PD) activities to assess the pentose phosphate pathway (PPP). And the liver injuries of rats were analyzed via histological changes, hepatic function, as well as hepatic protein levels of alpha-fetoprotein (AFP), pyruvate kinase isozyme type M2 (PKM2), and proliferating cell nuclear antigen (PCNA). Furthermore, polydatin (0.1 g/kg/d) as a specific inhibitor of G6PD was used to treat rats. Notably, their histological changes, hepatic function, hepatic 6PG levels, hepatic G6PD activities, PCNA levels, and PKM2 levels were recorded. ResultsNon-targeted metabolomics revealed that Rhubarb regulated the PPP in the liver of Rhubarb-DEN-treated rats. Besides, Rhubarb activated the oxidative branch of the PPP by activating G6PD (a rate-limiting enzyme in the oxidative PPP) in the liver of Rhubarb-DEN-treated rats. Meanwhile, Rhubarb promoted DEN-induced hepatocarcinogenesis. Moreover, polydatin attenuated the promoting effect of Rhubarb on DEN-induced hepatocarcinogenesis. ConclusionsRhubarb promoted DEN-induced hepatocarcinogenesis by activating the PPP, indicating that the efficacy and safety of Rhubarb in the treatment of liver cancer deserve to be deliberated.

Effects of different fertilizers on some soil enzymes activity and chlorophyll contents of two cotton (G. hirsutum L.) varieties grown in a saline and non-saline soil

Cotton plant has two physiological development periods which are vegetative and generative periods. The activities of urease, dehydrogenase, glucosidase and alkaline phosphatase enzymes in the soil continue during the growth of plants. In this study, three different fertilizer sources (diammonium phosphate (DAP), vermicompost and manure) and two cotton varieties (Candia and Lima) were used in non-saline and saline soil conditions. The experimental layout was randomized plots with 3 replications. Total chlorophyll content of Candia and Lima cotton (Gossypium hirsutum L.) cultivars and urease, dehydrogenase, glucosidase and alkaline phosphate enzyme activities of the soil were determined. The chlorophyll content of cotton plants was significantly (p < 0.01) different in fertilizer treatments applied in non-saline and saline soils. The lowest chlorophyll value (17.83) in non-saline soil was obtained in the control, while the highest chlorophyll value (32.64) was recorded in the DAP fertilizer treatments. The lowest chlorophyll value (20.33) in saline soil was obtained from DAP fertilizer, while the highest chlorophyll value (24.11) was obtained in the vermicompost application. The fertilizer treatment significantly affected the urease activity in non-saline (p < 0.01) and saline (p < 0.05) soils. The highest mean urease enzyme activity in non-saline (102.74 µg N g−1 dry soil h−1) and saline (120.23 µg N g−1 dry soil h−1) soils was obtained in the DAP treatments. The highest mean dehydrogenase enzyme activity in non-saline (35.99 µg TPF g−1 dry soil 24 h−1) and saline (48.63 µg TPF g−1 dry soil 24 h−1) soils was obtained in cattle manure application. The difference between fertilizer applications was significant at p < 0.01 level in both soils.

Beneficial impact of Nesfatin-1 on reproductive dysfunction induced by nicotine in male rats: Possible modulation of autophagy and pyroptosis signaling pathways.

This study was conducted to explore the beneficial impact of nesfatin-1 on reproductive dysfunction induced by nicotine (NT) in male rats with possible modulation of autophagy and pyroptosis signaling pathways. This research was performed on 40 Wistar male rats. They were distributed into four groups: control, normal+nesfatin-1, NT, and NT+nesfatin-1. At the end of the experimental period, the serum was separated for assay of testosterone, FSH and LH. Also, sperm parameters were determined. Histopathological examination of testicular tissue and immunohistochemical analysis was done for mammalian target of rapamycin, AMP-activated protein kinase, and mitogen-activated protein kinases including phosphorylated extracellular signal regulated kinase and phosphorylated cJun N-terminal kinase. Relative gene expression was determined for testicular nucleotide oligomerization domain (NOD)-like receptors proteins and Caspase-1, and autophagy markers including microtubule-associated protein 1 light chain 3 alpha and Beclin-1. Also, the following testicular parameters were assayed: 3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, malondialdehyde, superoxide dismutase activity, catalase, glucose-6 phosphate dehydrogenase, reactive oxygen species, caspase-3 activity, IL-1β, IL-18, mitochondrial transmembrane potential and Complex-I activity. The results revealed that the normal+nesfatin-1 group showed insignificant changes as compared to the control group. Meanwhile, the NT group exhibited prominent reproductive dysfunction in male rats. On the other hand, in the NT+nesfatin-1 group nesfatin-1 notably attenuated this reproductive dysfunction as evidenced by improvement of hormonal assay, sperm parameters, histopathological picture, immunohistochemical evaluation and real time relative gene expressions. In conclusion: Nesfatin-1 alleviated the impairment of male reproductive functions induced by NT via enhancement of autophagy pathways, suppression of pyroptosis, apoptosis, mitochondrial dysfunction and ROS production. Thus nesfatin-1 may offer a novel protective or therapeutic access for treating male infertility.