Citrate Buffer Research Articles

Abstract P3149: Effect Of SGLT2 Inhibitors On Atrial Fibrillation Susceptibility In Diabetic Mice

Background: Diabetes mellitus (DM) and heart failure (HF) are independent risk factors for atrial fibrillation (AF), the commonest clinical arrhythmia. A new class of drugs - sodium glucose cotransporter 2 inhibitors (SGLT2i) have beneficial effect in DM and HF and are now part of the guideline directed therapy for HF. However, conflicting evidence exists regarding their role and effect on cardiac arrhythmias including AF independent of their effect on HF and their exact mechanism(s) of action on the heart. Objective: Here we tested the effect of two commonly used SGLT2i on AF susceptibility in a type 2 DM (T2D) mouse model. Methods and Results: We used a T2D mouse model (high fat diet + low dose streptozocin) and a cohort of T2D mice were treated with SGLT2i - Empagliflozin (30mg/kg/d) or Dapagliflozin (5mg/kg/d) for 10 weeks in drinking water. We performed invasive electrophysiologic studies (EPS) to assess AF susceptibility by a rapid atrial burst pacing protocol in male mice aged 5 months at the time of EPS. T2D mice had increased AF susceptibility compared with non-diabetic littermate controls (normal chow diet + citrate buffer) [78% (14/18) vs. 28% (5/18), p < 0.01]. T2D mice treated with Empagliflozin were protected from increased AF susceptibility (22% (3/18) [p < 0.01]) while T2D mice treated with Dapagliflozin were not protected from increased AF susceptibility (58% (11/19) [p = 0.29]). Both Empagliflozin and Dapagliflozin treated T2D mice had normalization of their blood glucose levels to comparable levels with the non-diabetic control mice. T2D mice had mild LV dysfunction compared with non-diabetic controls (EF - 66% vs 81%, p < 0.01) and treatment with either Empagliflozin or Dapagliflozin attenuated the degree of LV dysfunction (EF - 71% in both groups). Conclusion: Taken together, our data support the concept that SGLT2i decrease AF susceptibility in hyperglycemia primed AF, in addition to attenuating LV dysfunction in T2D. However, the effect on AF was only observed with Empagliflozin but not Dapagliflozin treatment independent of their effect on hyperglycemia and LV dysfunction. Further investigations are necessary to determine if there is a class effect of SGLT2i on AF susceptibility and the specific mechanisms of action modulating this effect.

Development of Direct LDL Measurement by LDL Precipitation Combined with Sandwich ELISA Using In-House-Generated mAbs.

One of the major risk factors for the progression of cardiovascular diseases is a high blood level of low-density lipoprotein (LDL), so progression of the disease may be prevented by lowering the plasma LDL. Evaluation and monitoring of LDL concentrations are therefore necessary. Friedewald's equation is a calculation method that is currently used routinely to estimate plasma LDL concentrations in hospital laboratories. However, this method cannot be applied to samples with a high concentration of triglyceride (TG). To overcome this limitation, this study aimed to develop direct LDL measurements using in-house generated monoclonal antibodies against human LDL in combination with LDL precipitation using heparin-containing citrate buffer pH 5.04. The method was applied to measure the LDL concentration in 208 randomized samples from Suranaree University of Technology Hospital. The mean values obtained from the developed method and the hospital laboratory were 126.6 ± 43.1 mg/dL and 123.2 ± 42.3 mg/dL, respectively. Linear regression analysis showed a high correlation between these two methods (r = 0.8491, p < 0.0001). High concentrations of TG, total cholesterol, and HDL have no influence on the LDL values obtained by this method. In this study, we offer an alternative technique for the direct measurement of plasma LDL. Further development for more convenient and easy use can now be undertaken.

Stability of pesticide residues in citrus fruits during storage

Introduction. Storage of selected samples before transportation to the laboratory and immediately before analysis is an important component in the process of product quality control and evaluation.&#x0D; Purpose of the work was to evaluate the degradation of pesticide active substances in real samples of citrus fruit left for long periods of storage in deep freezing conditions.&#x0D; Materials and methods. High-performance liquid chromatography with triple quadrupole mass detector (HPLC-MS/MS) was used to identify and quantify pesticide active substances. According to the method of analysis MUK 4.1.3657–20, cryogenic grinding of samples was carried out in a cutter using dry ice. Sample preparation was carried out using the QuEChERS technology. To extract analytes from a homogenized sample acetonitrile was used in the presence of salts containing citrate buffer, followed by purification of the extract by SPE.&#x0D; Results. In the studied samples of citrus, the analytes showed stability. Thirty months after storage of samples in deep freezing conditions (temperature not higher than –20 °C), the identified levels of active ingredients of the pesticides imazalil, pyrimethanil and prochloraz did not change by more than 20% compared to the previously detected concentrations. Insignificant amounts of imidacloprid, thiabendazole and pyriproxyfen, as well as traces of azoxystrobin and trifloxystrobin, detected in the study of samples, were also found in samples after long-term storage. &#x0D; Limitation. The study is limited to the study of the stability of individual active substances found in real samples of citrus fruits intended for sale to the consumer.&#x0D; Conclusion. Proper storage of samples should ensure the preservation of the levels of active substances in the samples and exclude their change due to various processes (volatilization, enzymatic and hydrolytic decomposition, etc.), which requires the performance of preliminary experimental studies to assess their stability. The work showed the stability of residual quantities of imazalil, pyrimethanil, prochloraz, imidacloprid, thiabendazole, and pyriproxyfen in citrus fruit matrices under storage conditions in a freezer at not more than –20 °C for 30 months.

The Effect of Treatment With Aminoguanidine, an Advanced Glycation End Product Inhibitor, on Streptozotocin-Induced Diabetic Rats and Its Effects on Physiological and Renal Functions.

Diabetes is a multifactorial syndrome that affects the functioning of the renin-angiotensin system (RAS). The role of advanced glycation end products (AGEs) in diabetes is well known. In the present study, we hypothesized that the prevention of AGE accumulation or abrogation of AGE synthesis using an AGE inhibitor, aminoguanidine (AG), in streptozotocin (STZ)-induced diabetic animal modelswould affect the progression of diabetes and its related complications. We determined the effects of aminoguanidine (AG), an AGE inhibitor, in STZ-induced diabetic rats by determining various indices of RAS and renal functions. Additionally, we also investigated the effect of the drug, AG, on various hemodynamic and physiological functions in the body of the animals. Male Sprague Dawley rats weighing 200-250 g were assigned to four groups (n = 4-6): Vehicle, Vehicle+AG, STZ-induced, and STZ-induced+AG rats. Type 1 diabetes was induced by a single intraperitoneal (IP) injection of streptozotocin (55 mg/kg) dissolved in sodium citrate buffer. The control groups (Vehicle) were injected with buffer. The blood glucose levels were measured after 48 hours, and animals with blood glucose levels > 300 mg/dL were included in the study. Blood glucose levels in the vehicle rats were also determined to ensure non-diabetic conditions. After confirmation, AG was administrated at a dose of 1 g/L in drinking water for two weeks. Urine was collected to measure the glomerular filtration rate (GFR), and the immune reactivity for AT1 and AT2 proteins was analyzed by immunoblotting. Data were expressed as mean ± standard error of the mean(SEM), and a p-value < 0.05 was considered statistically significant. Diabetic rats had a significant drop in body weight, accompanied by increased food and water consumption. The diabetic rats exhibited significantly increased urine flow and GFR. These phenotypes were significantly or considerately reversed by AG treatment in the STZ+AG-treated diabetic rats. Aminoguanidineprevented the increase in blood sugar levels compared to STZ-induced diabetic rats alone (295.9 ± 50.69 versus 462.3 ± 18.6 mg/dL (p < 0.05)). However, it did not affect the glomerular filtration rate (GFR) and glomerular damage, as assessed by the renal histopathological studies. The STZ-induced diabetic rats had an increased sodium excretion (3.24 ± 0.40 mmol) and significantly increased expression of the AT2 receptor and that of the AT1 receptor, which was slightly reversed by the treatment with AG. Treatment with AG decreased sodium excretion (2.12 ± 0.63, as compared to the diabetic rats). These rats also had modestly decreased expression of the AT2 receptor (0.99 ± 0.07 versus 1.12 ± 0.08, as compared to the STZ-induced diabetic rats), while theAT1 receptors showed a slight increase in the STZ+AG-treated rats compared to the STZ-induced diabetic rats (1.1 ± 0.19 versus 1.08 ± 0.12). This study highlights the action of the drugAG in not exacerbating any damage in diabetic rats. Employing AG as a pharmacological intervention to prevent an increase in blood sugar adds a new dimension to controlling increased blood sugar and preventing diabetic complications. The employability and pharmacological intervention of the drug AG, in diabetes, therefore, need a renewed and further investigation.

Teneligliptin Co-Infusion Alleviates Morphine Tolerance by Inhibition of Spinal Microglial Cell Activation in Streptozotocin-Induced Diabetic Rats.

Morphine (MOR) is a commonly prescribed drug for the treatment of moderate to severe diabetic neuropathic pain (DNP). However, long-term MOR treatment is limited by morphine analgesic tolerance (MAT). The activation of microglial cells and the release of glia-derived proinflammatory cytokines are known to play an important role in the development of MAT. In this study, we aimed to investigate the effects of the dipeptidyl peptidase-4 inhibitor (DPP-4i) teneligliptin (TEN) on MOR-induced microglial cell activation and MAT in DNP rats. DNP was induced in four groups of male Wistar rats through a single intraperitoneal injection of streptozotocin (STZ) (50 mg/kg, freshly dissolved in 5 mmol/L citrate buffer, pH 4.5). Sham rats were administered with the vehicle. Seven days after STZ injection, all rats were implanted with an intrathecal (i.t) catheter connected to a mini-osmotic pump, divided into five groups, and infused with the following combinations: sham + saline (1 µL/h, i.t), DNP + saline (1 µL/h, i.t), DNP + MOR (15 µg/h, i.t), DNP + TEN (2 µg/h, i.t), and DNP + MOR (15 µg/h, i.t) + TEN (2 µg/h, i.t) for 7 days at a rate of 1 μL/h. The MAT was confirmed through the measurement of mechanical paw withdrawal threshold and tail-flick tests. The mRNA expression of neuroprotective proteins nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) in the dorsal horn was evaluated by quantitative PCR (qPCR). Microglial cell activation and mononucleate cell infiltration in the spinal cord dorsal horn were assessed by immunofluorescence assay (IFA) and Western blotting (WB). The results showed that co-infusion of TEN with MOR significantly attenuated MAT in DNP rats through the restoration of neuroprotective proteins Nrf2 and HO-1 and suppression of microglial cell activation in the dorsal horn. Though TEN at a dose of 2 μg has mild antinociceptive effects, it is highly effective in limiting MAT.

Electrochemical evaluation and voltammetric determination of some phenolic acids on the boron-doped diamond electrode

A simple, sensitive, and rapid method of determining phenolic acids, i.e. caffeic, ferulic, syringic, and vanillic using boron-doped diamond electrode (BDDE) by differential pulse voltammetry (DPV) was described. To the best of our knowledge, this is the first presentation of an electrochemical method to determine these compounds on BDDE. Under the conditions presented, the mentioned analytes undergo irreversible oxidation controlled by diffusion. The measurement conditions and composition of the supporting electrolyte were developed and optimized. Optimal measurement parameters ensure the usage of 0.02 mol L−1 ammonium citrate buffer pH 3.0 as a supporting electrolyte. The calibration graphs were linear with a correlation coefficient (r) >0.9959. The detection limits for the phenolic acids were within 0.01–0.03 mg L−1. Repeatability in all experiments, expressed by RSD%, was better than 5 %. The influence of interfering metal ions, such as Bi3+, Cd2+, Cu2+, Pb2+ and Tl+, was studied. The proposed method was successfully verified by measuring recoveries of phenolic acids in the model wine solution. The standard addition method was also applied in the determination of syringic acid in homemade and commercially available products: walnuts, walnut tincture, fresh and dried thyme. Effective application of the described analytical approach with the usage of BDDE not only enables the electrochemical detection of these four phenolic acids but also helps to reduce operational costs.

Investigating freezing-induced acidity changes in citrate buffers

Citrate buffers are commonly utilized in the field of biomolecule stabilization. We investigate their applicability in the frozen state within a range of initial pHs (2.5 to 8.0) and concentrations (0.02 to 0.60 M). Citrate buffer solutions subjected to various cooling and heating temperatures are examined in terms of the freezing-induced acidity changes, revealing that citrate buffers acidify upon cooling. The acidity is assessed with sulfonephthalein molecular probes frozen in the samples. Optical cryomicroscopy combined with differential scanning calorimetry was employed to investigate the causes of the observed acidity changes. The buffers partly crystallize and partly vitrify in the ice matrix; these processes influence the resulting pH and allow designing the optimal storage temperatures in the frozen state. The freezing-induced acidification apparently depends on the buffer concentration; at each pH, we suggest pertinent concentration, at which freezing causes minimal acidification.

The impact of liraglutide treatment on erectile function of the diabetic rats

Objectives: Glucagon like peptide-1 (GLP-1) is a hormone released from intestinal L-cells following nutrient consumption. It potentiates secretion of insulin from pancreatic beta-cells thus GLP-1 analogues are used for the treatment of type-2 diabetes mellitus( T2DM). This study aims to evaluate impact of GLP-1 receptor agonist liraglutide on erectile function of diabetic rats. Methods: Male Sprague-Dawley rats (n = 30, 13-weeks old, 240-335 gr) were fed with fatty diet for 2-weeks and divided into 3 groups (n = 10 each). The rats in the first group served as controls (Group C) whereas the rats in the remaining two groups were injected with streptozocin and became T2DM for forming diabetic group (Group D) and treatment group (Group DT). Rats in group D received citrate buffer injections whereas rats in the group DT received liraglutide injections (0.3 mg/kg/12h) subcutaneously. Erectile functions of all rats were evaluated with intracavernosal pressure (ICP)/mean arterial pressure (MAP) measurements. Moreover, plasma sex hormone levels (Testosterone, FSH, LH) were measured and histological assessment of midpenile tissue were performed (Collagen-Type-IV, rat epithelial antigen-1, nNOS). Results: Maximum ICP/MAP ratios were 0.790 ± 0.164, 0.263 ± 0.139 and 0.652 ± 0.131 in Group C, Group D and Group DT. Although mean ICP/MAP ratios were similar in Group C and Group DT (p = 0.076), mean ICP/MAP ratio was significantly lower in Group D (p &amp;lt; 0.001). Testosterone and FSH results were significantly lower in the Group D as well (p = 0.001). Histological analyses revealed that nNOS (p &amp;lt; 0.001), rat epithelial antigen-1 (p = 0.016) and muscle/collagen ratio (p = 0.015) were also lower in Group D, compared with the other groups. Conclusions: GLP-1 receptor agonist liraglutide demonstrated protective effects on the erectile tissues of the diabetic rats. Clinical trials are required to confirm if liraglutide treatment has similar beneficial effects on men who have T2DM.

Isolation, characterization and identification of cellulase (Endo-β-1,4-glucanase) producing bacteria from diverse locations

&#x0D; The objective of the study was to isolate cellulase producing bacteria from natural sources and identify the bacterial isolates that produce thermostable enzyme. Bacteria were isolated from samples collected from different locations like mango leaf litter, termite gut, composting yard (municipal solid waste, compost, and soil), soil from decaying paper waste, decaying wood, and saline coastal belt soil by enrichment in broth containing 1% cellulose and subsequent culturing on plates and the purified cultures stored in slants of the solidified agar medium of the same composition as enrichment medium. Primary screening of the isolates for cellulase (Endo β-1,4-glucanase) production was done by growing them on Carboxy Methyl Cellulose (CMC) agar medium and screening using indicator dye Congo red. Secondary screening of the positive isolates showing cellulolytic Index &gt; 4 was done by crude cellulase enzyme extraction and assay using 1% CMC in 1N citrate buffer (pH 5.0) at different pH values (4.8- 7.0) and temperatures (35- 70℃). Cellulase activity (EA) in U/mL was calculated based on the absorbance change per min. which depends on the amount of reducing sugar produced by the enzyme catalysed conversion of CMC to reducing sugar. Isolates were characterized morphologically and biochemically and six isolates (Gram -ve: T1, MSW, C1, S2 and Gram +ve: W2, P) having significantly higher EA were selected for DNA extraction. DNA was quantified and the DNA of four isolates (MSW, S2, W2, P) were selected for sequence analysis. The 16S rDNA sequencing was carried out through PCR amplification and Blast using NCBI blast similarity search tool resulted in the identification of MSW, S2, W2 as E.coli, Sulfitobacter pontiacus, Bacillus subtilis respectively and P as Bacillus cereus and Rhodococcus erythropolis strain e1. Isolates showed significantly higher cellulase activity (EA) at pH 6.2 and at varying temperatures. Bacterial isolates P and C2 showed significantly higher activity at 40℃, MSW at 60℃ and others at 50℃ (EA ranging from 0.0054 – 0.0604 U/mL). Isolates W2, P, S2 had EA ranging from 0.006 - 0.03 U/mL at 70℃. This study showed that the cellulase producers could be grown at different temperatures (40 - 70℃) on cellulose based substrate. In this study the substrate specificity indicates that the crude enzyme is an endo- β-1,4-glucanase that plays a prominent role in initiating and sustaining the hydrolytic process and randomly cleaves cellulose into glucose and olygomeric polysaccharides. Hence by selecting samples with cellulosic substance from different locations with extreme environmental conditions like high temperature, high salinity it is possible to isolate cellulolytic bacteria producing cellulase enzymes that are stable at higher processing temperatures and other harsh conditions which is of importance in industrial applications.&#x0D; Keywords: Endo-β-1,4-glucanase, Cellulolytic index, Cellulase activity, Thermostable

Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency.

The transfection potency of lipid nanoparticle (LNP) mRNA systems is critically dependent on the ionizable cationic lipid component. LNP mRNA systems composed of optimized ionizable lipids often display distinctive mRNA-rich "bleb" structures. Here, it is shown that such structures can also be induced for LNPs containing nominally less active ionizable lipids by formulating them in the presence of high concentrations of pH 4 buffers such as sodium citrate, leading to improved transfection potencies both in vitro and in vivo. Induction of bleb structure and improved potency is dependent on the type of pH 4 buffer employed, with LNP mRNA systems prepared using 300 mm sodium citrate buffer displaying maximum transfection. The improved transfection potencies of LNP mRNA systems displaying bleb structure can be attributed, at least in part, to enhanced integrity of the encapsulated mRNA. It is concluded that enhanced transfection can be achieved by optimizing formulation parameters to improve mRNA stability and that optimization of ionizable lipids to achieve enhanced potency may well lead to improvements in mRNA integrity through formation of the bleb structure rather than enhanced intracellular delivery.

Detection and Identification of an Unknown Impurity in Ephedrine HCl 5mg/mL Cyclic Olefin Syringes: Formulation Development.

An unknown impurity was detected in in-house prepared ephedrine hydrochloride (HCl) 5mg/mL prefilled sterilized syringes when applying a stability-indicating British Pharmacopoeia 2018 impurity method for ephedrine injection. Ultraviolet, chromatographic, mass spectral, and physicochemical methods were combined to identify the unknown impurity. The unknown impurity was identified as methcathinone, which is generated from ephedrine drug substance through an oxidation reaction. A formulation study, in which different process adjustments were tested, was carried out to reduce the amount of unknown impurity. Nitrogen gassing in combination with 0.05M citrate buffer addition proved to be the most potent process adjustment in reducing methcathinone formation in ephedrine HCl 5mg/mL prefilled sterilized syringes after 4months of storage in the dark at room temperature (20°C ± 5°C). More detailed research on the long-term stability of the reformulated ephedrine HCl drug product is currently underway, with promising results for up to 9months gathered already.

Cathepsin S inhibition in dendritic cells prevents Th17 cell differentiation in perivascular adipose tissues following vascular injury in diabetic rats.

In the context of diabetes mellitus (DM), the circulating cathepsin S (CTSS) level is significantly higher in the cardiovascular disease group. Therefore, this study was designed to investigate the role of CTSS in restenosis following carotid injury in diabetic rats. To induce DM, 60 mg/kg of streptozotocin (STZ) in citrate buffer was injected intraperitoneally into Sprague-Dawley rats. After successful modeling of DM, wire injury of the rat carotid artery was performed, followed by adenovirus transduction. Levels of blood glucose and Th17 cell surface antigens including ROR-γt, IL-17A, IL-17F, IL-22, and IL-23 in perivascular adipose tissues (PVAT) were evaluated. For in vitro analysis, human dendritic cells (DCs) were treated with 5.6-25 mM glucose for 24 h. The morphology of DCs was observed using an optical microscope. CD4+ T cells derived from human peripheral blood mononuclear cells were cocultured with DCs for 5 days. Levels of IL-6, CTSS, ROR-γt, IL-17A, IL-17F, IL-22 and IL-23 were measured. Flow cytometry was conducted to detect DC surface biomarkers (CD1a, CD83, and CD86) and Th17 cell differentiation. The collected DCs presented a treelike shape and were positive for CD1a, CD83, and CD86. Glucose impaired DC viability at the dose of 35 mM. Glucose treatment led to an increase in CTSS and IL-6 expression in DCs. Glucose-treated DCs promoted the differentiation of Th17 cells. CTSS depletion downregulated IL-6 expression and inhibited Th17 cell differentiation in vitro and in vivo. CTSS inhibition in DCs inhibits Th17 cell differentiation in PVAT tissues from diabetic rats following vascular injury.

1178-P: Interleukin-17 Signaling in Cerebrum of Offspring from Mother with Diabetes

A number of human cohort and animal studies suggest that offspring from mother with diabetes (OMD) has an increased risk of both short- and long-term complications, including various neurological issues including learning impairment and autism spectrum disorders. However, the underlying mechanism of the neurobehavioral issues is still not elucidated. The main objective of this study is to examine their mechanisms by examining gene expression in the cerebrum of OMD during the embryonic period. Six-week-old Crl: CD1 (ICR) females were injected intraperitoneally with 150 mg/kg of STZ for generating OMD. This OMD model showed learning impairment using accelerated rotarod as we previously reported at the 81st Scientific Sessions of the American Diabetes Association. Citrate buffer was used to generate the control group. After mating the females with healthy males, both maternal and fetal cerebrums were collected at embryonic day 17.5. RNA was extracted from the collected cerebrums using TRIzol, and RNA sequencing and quantitative PCR were performed. The number of differentially expressed genes common to three types of analysis (EdgeR, Deseq2, and TCC) was 40. Enrichment analysis of these 40 genes using the KEGG pathway identified a group of genes (Lipocalin-2 (Lcn-2), S100a8, and S100a9) related to the Interleukin 17 (IL-17) pathway. Quantitative PCR revealed mRNA expression of these genes was significantly higher in OMD. In order to investigate the distribution of Lcn-2, we next intraperitoneally administered biotinylated Lcn-2 to pregnant dam at gestational day 17.5 and collect maternal and fetal samples 30 minutes after the administration. Immunohistochemical staining showed that biotin expression was observed in the maternal cortex but not in the fetal one, suggesting that maternally derived Lcn-2 does not reach the fetal cortex. Altogether, our results demonstrate that IL-17 signaling in the fetal brain may be involved in learning impairments in OMD. Disclosure K.Sugai: None. M.Odawara: Speaker's Bureau; Eli Lilly Japan K.K., Novo Nordisk, Sanofi K.K. R.Suzuki: Consultant; Novo Nordisk A/S, Other Relationship; Sanofi K.K., Eli Lilly Japan K.K. J.Sasaki: None. K.Ishii: None. G.Li: None. M.Ito: None. M.Aierken: None. H.Suwanai: None. M.Torii: None. K.Hashimoto-torii: None.

1622-P: Complex Mechanisms Underlie the Antidiabetic Effect of Tempol in Experimentally Induced Diabetes Mellitus

Diabetes mellitus (DM) is a vastly growing worldwide health problem which imposes public health, social, as well as economic burden. Multifactorial and complicated pathophysiological pathways may underlie DM. Attention has been given for the search of polymodal therapeutic molecules. Herein, we tested the capacity of tempol, a known superoxide dismutase-mimetic in the treatment of experimentally induced DM. Thirty male Wistar rats weighing 150-200 g were equally divided into: 1- Control group (rats received a single intra-peritoneal injection of citrate buffer for 4 weeks), 2- Untreated diabetic group, and 3- Tempol treated diabetic group. At the end of the study blood samples were collected for the measurement of fasting blood glucose, fasting blood insulin, liver function, kidney function, oxidative stress markers, and inflammatory markers. Pancreas, skeletal muscles, and liver were removed for histopathological, immunohistochemical and RT-PCR studies. Treatment with tempol significantly improved glycemic status, insulin secretion, sensitivity and reserve. Tempol preserved both liver and kidney functions. Tempol also significantly enhanced energy status, and countered structural changes, inflammation, and apoptosis of pancreatic tissue. Tempol significantly upregulated pancreatic AMPK, and skeletal muscle and hepatic glucose transporters. Interestingly, tempol demonstrated an endogenous stem cells recruitment capacity. In conclusion, tempol exhibited a potential capacity to counter the complexity of the underlying mechanisms in DM. Disclosure Y.Naguib: None.

Investigation of the Influence of Wound-Treatment-Relevant Buffer Systems on the Colloidal and Optical Properties of Gold Nanoparticles.

Biocompatible gold nanoparticles (AuNPs) are used in wound healing due to their radical scavenging activity. They shorten wound healing time by, for example, improving re-epithelialization and promoting the formation of new connective tissue. Another approach that promotes wound healing through cell proliferation while inhibiting bacterial growth is an acidic microenvironment, which can be achieved with acid-forming buffers. Accordingly, a combination of these two approaches appears promising and is the focus of the present study. Here, 18 nm and 56 nm gold NP (Au) were prepared with Turkevich reduction synthesis using design-of-experiments methodology, and the influence of pH and ionic strength on their behaviour was investigated. The citrate buffer had a pronounced effect on the stability of AuNPs due to the more complex intermolecular interactions, which was also confirmed by the changes in optical properties. In contrast, AuNPs dispersed in lactate and phosphate buffer were stable at therapeutically relevant ionic strength, regardless of their size. Simulation of the local pH distribution near the particle surface also showed a steep pH gradient for particles smaller than 100 nm. This suggests that the healing potential is further enhanced by a more acidic environment at the particle surface, making this strategy a promising approach.

Peptide Hydrolysate of Telfairia occidentalis Hook f. Seed Protein Promotes Effective Glucose Homeostasis by Improving β-cell Dysfunction and Abating Carbohydrate Metabolic Disturbance in Diabetic Rats

Rising concerns with the use of synthetic antidiabetic drugs have promoted a shift towards the use of natural products. This study therefore investigated the antidiabetic activity of peptide hydrolysate of Telfairia occidentalis (T. occidentalis) seed protein (PHTOSP) in streptozotocin-induced diabetic rats. Thirty-six (36) experimental animals were randomly distributed into six groups (A–F) of six rats each (n = 6). Group A served as normal control while groups B, C, D, E, and F were treated with streptozotocin (STZ, 50 mg/kg body weight, b.w, i.p) dissolved in cold citrate buffer (0.1 M, pH 4.5) to induce type 2 diabetes. Groups C, D, and E were administered 50, 100, and 150 mg/kg b.w PHTOSP, respectively, while groups B and F were diabetic untreated and 5 mg/kg b.w glibenclamide-treated controls, respectively, in the experiment that lasted for 21 days. Subsequently, the analysis of biochemical parameters demonstrated a significant (p&lt;0.05) increase in serum insulin, hepatic glycogen, hexokinase, and glucose-6-phosphate dehydrogenase activities, accompanied by a significant (p&lt;0.05) reduction in fasting serum glucose, glucose-6-phosphatase, and fructose-1,6-bisphosphatase, along with an enhancement in relative body weight. Similarly, PHTOSP demonstrated a significant (p&lt;0.05) improvement on high-density (HDL) and low-density (LDL) lipoproteins, triglyceride (TG), total cholesterol (TC), and atherogenic index (AI) significantly (p&lt;0.05). In addition, histoarchitectural analysis revealed a reversal of congestion and proliferation of inflammatory cells in the pancreatic tissue following treatment with PHTOSP. Therefore, PHTOSP might possess potential antidiabetic properties such that it improves glycolytic pathway and promotes cell survival that are helpful in the management of diabetes mellitus (DM).

Tocotrienol-rich fraction reduces retinal inflammation and angiogenesis in rats with streptozotocin-induced diabetes

BackgroundDiabetic retinopathy (DR) is the second commonest microvascular complication of diabetes mellitus. It is characterized by chronic inflammation and angiogenesis. Palm oil-derived tocotrienol-rich fraction (TRF), a substance with anti-inflammatory and anti-angiogenic properties, may provide protection against DR development. Therefore, in this study, we investigated the effect of TRF on retinal vascular and morphological changes in diabetic rats. The effects of TRF on the retinal expression of inflammatory and angiogenic markers were also studied in the streptozotocin (STZ)-induced diabetic rats.MethodsMale Sprague Dawley rats weighing 200–250 g were grouped into normal rats (N) and diabetic rats. Diabetes was induced by intraperitoneal injection of streptozotocin (55 mg/kg body weight) whereas N similarly received citrate buffer. STZ-injected rats with blood glucose of more than 20 mmol/L were considered diabetic and were divided into vehicle-treated (DV) and TRF-treated (DT) groups. N and DV received vehicle, whereas DT received TRF (100 mg/kg body weight) via oral gavage once daily for 12 weeks. Fundus images were captured at week 0 (baseline), week 6 and week 12 post-STZ induction to estimate vascular diameters. At the end of experimental period, rats were euthanized, and retinal tissues were collected for morphometric analysis and measurement of NFκB, phospho-NFκB (Ser536), HIF-1α using immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Retinal inflammatory and angiogenic cytokines expression were measured by ELISA and real-time quantitative PCR.ResultsTRF preserved the retinal layer thickness (GCL, IPL, INL and OR; p < 0.05) and retinal venous diameter (p < 0.001). TRF also lowered the retinal NFκB activation (p < 0.05) as well as expressions of IL-1β, IL-6, TNF-α, IFN-γ, iNOS and MCP-1 (p < 0.05) compared to vehicle-treated diabetic rats. Moreover, TRF also reduced retinal expression of VEGF (p < 0.001), IGF-1 (p < 0.001) and HIF-1α (p < 0.05) compared to vehicle-treated rats with diabetes.ConclusionOral TRF provided protection against retinal inflammation and angiogenesis in rats with STZ-induced diabetes by suppressing the expression of the markers of retinal inflammation and angiogenesis.

Anti-Hyperglycemic and Hypoglycemic Activities of 80% Methanol Extract and Solvent Fractions of Ocimum lamiifolium Hochst Ex Benth. (Lamiaceae) Leaves in Mice.

Globally, the prevalence of diabetes mellitus is rising. Due to the scarcity, high cost, and many adverse effects of modern treatments, traditional medicine is commonly used in rural areas to treat a variety of illnesses, including diabetes mellitus. The aim of this study was to assess the antihyperglycemic and hypoglycemic effects of Ocimum lamiifolium Hochst ex Benth leaves. A crude methanol 80% extract's and its solvent fractions' effects on healthy, oral glucose-given, and STZ-induced diabetic mice were examined. Swiss albino mice of either sex were assigned into sixteen groups, each containing six mice, for the OGTT and hypoglycemia tests. Male mice were used in the study, and they were divided into groups for the negative control (citrate buffer for diabetic mice), the normal control (Tween 2%), the test groups, and a positive control (glibenclamide) for the antihyperglycemic test in STZ (200 mg/kg body weight)-induced diabetic mice. A crude 80% methanol extract of 200 mg/kg effectively lowered blood glucose levels (p <0.05) and none of the fractions extracts caused hypoglycemia shock in norma mice. The aqueous residue at 100, 200, and 400 mg/kg, the n-butanol fraction at 100 and 200 mg/kg, and the chloroform fraction at 200 mg/kg demonstrated higher glucose tolerance in orally glucose-loaded mice (p <0.05). The crude 400 mg/kg of an 80% methanol extract, 100 and 200 mg/kg of the n-butanol fraction, 200 and 400 mg/kg of the chloroform fraction, and 5 mg/kg of glibenclamide significantly reduced blood glucose levels in STZ-induced diabetic mice (p <0.05). The current research demonstrates that a crude 80% methanol extract of Ocimum lamiifolium Hochst ex Benth leaves, as well as its solvent fractions, significantly reduce blood sugar levels in mice that are healthy, loaded with glucose, and streptozotocin induced diabetic mice.

Investigation of excipients impact on polysorbate 80 degradation in biopharmaceutical formulation buffers

A study on the polysorbate 80 stability in various formulation buffers commonly used in biopharmaceuticals was performed, to investigate the excipients influence on polysorbate 80 degradation. Polysorbate 80 is a common excipient in biopharmaceutical products. However, its degradation will potentially impact the drug product quality, and may trigger protein aggregation and particles formation. Due to the heterogeneity of the polysorbates and the mutual effects with other formulation compositions, the study of polysorbate degradation is challenging. Herein, a real-time stability study was designed and performed. The polysorbate 80 degradation trend was monitored by fluorescence micelle-based assay (FMA), reversed-phase-ultra-performance liquid chromatography-evaporative light scattering detector (RP-UPLC-ELSD) assay, and LC-MS assay. These assays provide orthogonal results to reveal both the micelle-forming capability and the compositional changes of polysorbate 80 in different buffer systems. The degradation occurred after a period of storage under 25 °C in different trend, which indicates the excipients could impact the degradation kinetics. Upon comparison, the degradation is prone to happen in histidine buffer than in acetate, phosphate or citrate buffers. LC-MS confirms oxidation as an independent degradation pathway with detection of the oxidative aldehyde. Thus, it is necessary to pay more attention to the excipients selection and their potential impact on polysorbate 80 stability to achieve longer shelf life for the biopharmaceuticals. Besides, the protective roles of several additives were figured out, which could be applied as potential industrial solutions to the polysorbate 80 degradation issues.

Cardiovascular characterisation of a novel mouse model that combines hypertension and diabetes co-morbidities

Epidemiologic data suggest that the prevalence of hypertension in patients with diabetes mellitus is ∼1.5–2.0 times greater than in matched non-diabetic patients. This co-existent disease burden exacerbates cardiac and vascular injury, leading to structural and functional changes to the myocardium, impaired cardiac function and heart failure. Oxidative stress and persistent low-grade inflammation underlie both conditions, and are identified as major contributors to pathological cardiac remodelling. There is an urgent need for effective therapies that specifically target oxidative stress and inflammation to protect against cardiac remodelling. Animal models are a valuable tool for testing emerging therapeutics, however, there is a notable lack of appropriate animal models of co-morbid diabetes and hypertension. In this study, we describe a novel preclinical mouse model combining diabetes and hypertension to investigate cardiac and vascular pathology of co-morbid disease. Type 1 diabetes was induced in spontaneously hypertensive, 8-week old, male Schlager (BPH/2) mice via 5 consecutive, daily injections of streptozotocin (55 mg/kg in citrate buffer; i.p.). Non-diabetic mice received citrate buffer only. After 10 weeks of diabetes induction, cardiac function was assessed by echocardiography prior to post-mortem evaluation of cardiomyocyte hypertrophy, interstitial fibrosis and inflammation by histology, RT-PCR and flow cytometry. We focussed on the oxidative and inflammatory stress pathways that contribute to cardiovascular remodelling. In particular, we demonstrate that markers of inflammation (monocyte chemoattractant protein; MCP-1), oxidative stress (urinary 8-isoprostanes) and fibrosis (connective tissue growth factor; CTGF) are significantly increased, whilst diastolic dysfunction, as indicated by prolonged isovolumic relaxation time (IVRT), is elevated in this diabetic and hypertensive mouse model. In summary, this pre-clinical mouse model provides researchers with a tool to test therapeutic strategies unique to co-morbid diabetes and hypertension, thereby facilitating the emergence of novel therapeutics to combat the cardiovascular consequences of these debilitating co-morbidities.