Homogenate Protein Research Articles

К вопросу оценки качества сырья из каспийской атерины (Atherina boyeri caspia (Risso, 1810))

The purpose of this work was to establish the informative value of methods of structural analysis of biological fluids in determining the prospects of using atherina (Atherina boyeri caspia (Risso, 1810)) as a source of dietary protein in aquaculture. For the first time, solid-phase structures of atherina muscle homogenates were studied. It is known that the solid phase of biological fluids formed under conditions of wedge-shaped dehydration is a visualization of biochemical processes occurring in an organism or organ. The study of the morphological picture of a dried drop (facies) makes it possible to establish associative links between the features of the structuring of the studied liquid and its composition, as well as to determine the nature of the impact of environmental factors on a living system. The results of the study made it possible to establish the main type of homogenate facies and its morphological parameters, which indicate the consistency of biochemical processes and the presence of high-grade protein in the muscle tissue of the Caspian atherina, caught both in the spring and autumn seasons. The size and weight parameters of fish, the content of water-soluble protein in muscle tissue are characterized. For the first time, a comparative analysis of the level of water-soluble protein and solid-phase structures of muscle homogenates of Caspian atherina caught in different seasons in the coastal waters of Dagestan was carried out. Atherina is a food component for many fish living in the Caspian Sea. It was determined that in October, the content of muscle water-soluble protein in atherina tended to increase relative to March. During the feeding period, the accumulated part of the protein components turns into lipid components, which accumulate intensively, therefore, there are no significant seasonal differences in the content of water-soluble protein. It has been established that the biochemical processes occurring in muscle tissues are reflected in the solid-phase structures of biological fluids formed under conditions of wedge-shaped dehydration. The obtained results of the trend towards an increase in water-soluble muscle proteins in the body and an increase in the population in recent years indicate the importance of the use of Caspian atherina in the quality of live feed at fish farms.

In Vitro to In Vivo Scalars for Drug Clearance in Nonalcoholic Fatty Liver and Steatohepatitis.

In vitro-in vivo extrapolation (IVIVE) allows prediction of clinical outcomes across populations from in vitro data using specific scalars tailored to the biologic characteristics of each population. This study experimentally determined scalars for patients with varying degrees of nonalcoholic fatty liver disease (NAFLD), ranging from fatty liver to nonalcoholic steatohepatitis (NASH) and cirrhosis. Microsomal, S9, and cytosol fractions were extracted from 36 histologically normal and 66 NAFLD livers (27 nonalcoholic fatty liver [NAFL], 13 NASH, and 26 NASH with cirrhosis). Corrected microsomal protein per gram liver (MPPGL) progressively decreased with disease severity (26.8, 27.4, and 24.3 mg/g in NAFL, NASH, and NASH/cirrhosis, respectively, compared with 35.6 mg/g in normal livers; ANOVA, P < 0.001). Homogenate, S9, and cytosolic protein showed a consistent trend of decline in NASH/cirrhosis relative to normal control (post-hoc t test, P < 0.05). No differences across the groups were observed in homogenate, S9, cytosolic, and microsomal protein content in matched kidney samples. MPPGL-based scalars that combine protein content with liver size revealed that the reduction in MPPGL in NAFL and NASH was compensated by the reported increase in liver size (relative scalar ratios of 0.96 and 0.99, respectively), which was not the case with NASH/cirrhosis (ratio of 0.63), compared with healthy control. Physiologically based pharmacokinetics-informed global sensitivity analysis of the relative contribution of IVIVE scalars (hepatic CYP3A4 abundance, MPPGL, and liver size) to variability in exposure (area under the curve) to three CYP3A substrates (alprazolam, midazolam, and ibrutinib) revealed enzyme abundance as the most significant parameter, followed by MPPGL, whereas liver volume was the least impactful factor. SIGNIFICANCE STATEMENT: Nonalcoholic fatty liver disease-specific scalars necessary for extrapolation from in vitro systems to liver tissue are lacking. These are required in clearance prediction and dose selection in nonalcoholic fatty liver and steatohepatitis populations. Previously reported disease-driven changes have focused on cirrhosis, with no data on the initial stages of liver disease. The authors obtained experimental values for microsomal, cytosolic, and S9 fractions and assessed the relative impact of microsomal scalars on predicted exposure to substrate drugs using physiologically based pharmacokinetics.

Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%–80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.

Carcinogenic effect of human tumor-derived cell-free filtrates in nude mice.

Cancer remains a worldwide cause of morbidity and mortality. Investigational research efforts have included the administration of tumor-derived extracts to healthy animals. Having previously demonstrated that the administration of non-transmissible, human cancer-derived homogenates induced malignant tumors in mice, here, we examined the consequences of administering 50 or 100µg of protein of crude homogenates from mammary carcinoma, pancreatic adenocarcinoma, and melanoma samples in 6 inoculations per week during 2months. The concurrent control mice received homogenates of healthy donor-skin cosmetic surgery fragments. Mammary carcinoma homogenate administration did not provoke the deterioration or mortality of the animals. Multiple foci of lung adenocarcinomas with a broad expression of malignity histomarkers coexisting with small cell-like carcinomas were found. Disseminated cells, positive to classic epithelial markers, were detected in lymphoid nodes. The administration of pancreatic tumor and melanoma homogenates progressively deteriorated animal health. Pancreatic tumor induced poorly differentiated lung adenocarcinomas and pancreatic islet hyperplasia. Melanoma affected lungs with solid pseudopapillary adenocarcinomas. Giant atypical hepatocytes were also observed. The kidney exhibited dispersed foci of neoplastic cells within a desmoplastic matrix. Nuclear overlapping with hyperchromatic nuclei, mitotic figures, and prominent nuclear atypia was identified in epidermal cells. None of these changes were ever detected in the control mice. Furthermore, the incubation of zebrafish embryos with breast tumor homogenates induced the expression of c-Myc and HER-2 as tumor markers, contrasting to embryos exposed to healthy tissue-derived material. This study confirms and extends our hypothesis that tumor homogenates contain and may act as vectors for "malignancy drivers," which ultimately implement a carcinogenesis process in otherwise healthy mice.

Protective effects of vitamin B12 against cisplatin induced nephrotoxicity in adult mice

Cisplatin (CP) is one of the most widely used and highly effective cytotoxic agents in the treatment of various cancers, but its clinical use is associated with considerable nephrotoxicity. This characteristic of CP makes it harmful to the cancer patient's kidneys. In the present study, we report the effects of vitamin B12 on rat kidneys treated with cisplatin using single doses of both compounds. CP was administered to adult albino mice at a dose of 1 mg/kg of body weight for 10 days. CP caused oxidative stress by significantly (p∠0.01) suppressing the expressions of NRF-2 and HO-1, followed by the upregulation (p∠0.01) of inflammatory markers like NF-kB, TNF-α and IL-1β proteins in the kidney's homogenates. The co-administration of vitamin B12 at a dose of (500 mg/kg) significantly (p∠0.01) reduced the oxidative stress (activated NRF-2 and HO-1 proteins) mediated by inflammatory mediators (NF-kB, TNF-α, and IL-1β proteins) to decrease the intensity of the toxicity in the kidneys of the adult albino mice. The results suggest that vitamin B12 is an effective chemoprotective agent against nephrotoxicity induced by the antitumoral cisplatin in adult mice.

Neuroprotective properties of GABA and its derivatives in diabetic encephalopathy in old animals

The aim of the work was to evaluate the GABA neuroprotective properties and its structural analogues in old animals after seven months of hyperglycemia.Materials and methods. Diabetes mellitus was modeled in white outbred male rats (12 months old) by the administration of a streptozotocin (65 mg/kg) and nicotinamide (230 mg/kg) combination. After 6 months, the animals with a postprandial glycemia level between 11 and 18 mmol/l were selected for the study. After the groups had been formed, the animals were administrated with GABA and GABAergic compounds (Compositions МРВАand PPC), respectively, for 1 month, the control group animals were administrated with saline. After the treatment, an oral glucose tolerance test and a set of behavioral tests aimed at studying sensory-motor (Open Field, Adhesion test, Rotarod) and cognitive functions (New Object Recognition and Morris Water Maze), as well as the functional state evaluation of the endothelium were performed. Further on, sampling of blood and brain tissues for a biochemical and enzyme immunoassay (the level of glucagon-like peptide-1 (GLP-1) and TNF-α in serum and the level of Klotho protein, BDNF, Nrf2, NF-κB and malondialdehyd (MDA) in brain homogenates), as well as a morphological analysis of changes in CA1 and CA3 neurons of the hippocampus and somatosensory cortex, was carried out.Results. GABA and compositions with its derivatives had a pronounced neuroprotective effect in old animals with prolonged hyperglycemia. The hypoglycemic effect of the studied compositions was accompanied by an increase in the production of GLP-1. In the animals with DM, after 6 weeks of the test substances administration, higher rates of sensory-motor and cognitive functions and a less structural damage to the sensory-motor cortex and the brain hippocampus were recorded. These effects may be due to higher levels of the Klotho proteins, Nrf2 and BDNF, as well as lower levels of NF-κB, which may underlie the suppression of the oxidative stress, the reduction of MDA and inflammation (TNF-α).Conclusion. After 6 weeks of the administration, GABA and its compositions in old animals (19 months old) significantly improved sensory-motor and cognitive functions, reduced negative structural changes in the hippocampus and somatosensory cerebral cortex.

Evaluation of cerebrospinal fluid levels of synaptic vesicle protein, VAMP-2, across the sporadic Alzheimer’s disease continuum

BackgroundSynapse loss is an early event that precedes neuronal death and symptom onset and is considered the best neuropathological correlate of cognitive decline in Alzheimer’s disease (AD). Vesicle-associated membrane protein 2 (VAMP-2) has emerged as a promising biomarker of AD-related synapse degeneration in cerebrospinal fluid (CSF). The aim of this study was to explore the CSF profile of VAMP-2 across the AD continuum in relation to core AD biomarkers, other synaptic proteins, neurogranin (Ng) and synaptosomal-associated Protein-25 kDa (SNAP-25) and cognitive performance.MethodsWe developed a digital immunoassay on the Single Molecule Array platform to quantify VAMP-2 in CSF and used existing immunoassays to quantify Ng, SNAP-25 and core CSF AD biomarkers. The clinical study included 62 cognitively unimpaired AD biomarker-negative subjects and 152 participants across the AD continuum from the SPIN cohort (Sant Pau Initiative on Neurodegeneration). Cognitive measures of episodic, semantic, executive and visuospatial domains and global cognition were included. Statistical methods included χ2 tests, spearman correlation, and ANCOVA analyses.ResultsThe VAMP-2 assay had a good analytical performance (repeatability 8.9%, intermediate precision 10.3%). Assay antibodies detected native VAMP-2 protein in human brain homogenates. CSF concentrations of VAMP-2, neurogranin and SNAP-25 were lower in preclinical AD stage 1 compared to controls and higher at later AD stages compared to AD stage 1 and were associated with core AD biomarkers, particularly total tau (adj. r2 = 0.62 to 0.78, p < 0.001). All three synaptic proteins were associated with all cognitive domains in individuals on the AD continuum (adj. r2 = 0.04 to 0.19, p < 0.05).ConclusionsOur novel digital immunoassay accurately measures VAMP-2 changes in CSF, which reflect AD biomarkers and cognitive performance across multiple domains.

Circadian rhythm disruptions associated with opioid use disorder in synaptic proteomes of human dorsolateral prefrontal cortex and nucleus accumbens

Opioid craving and relapse vulnerability is associated with severe and persistent sleep and circadian rhythm disruptions. Understanding the neurobiological underpinnings of circadian rhythms and opioid use disorder (OUD) may prove valuable for developing new treatments for opioid addiction. Previous work indicated molecular rhythm disruptions in the human brain associated with OUD, highlighting synaptic alterations in the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc)—key brain regions involved in cognition and reward, and heavily implicated in the pathophysiology of OUD. To provide further insights into the synaptic alterations in OUD, we used mass-spectrometry based proteomics to deeply profile protein expression alterations in bulk tissue and synaptosome preparations from DLPFC and NAc of unaffected and OUD subjects. We identified 55 differentially expressed (DE) proteins in DLPFC homogenates, and 44 DE proteins in NAc homogenates, between unaffected and OUD subjects. In synaptosomes, we identified 161 and 56 DE proteins in DLPFC and NAc, respectively, of OUD subjects. By comparing homogenate and synaptosome protein expression, we identified proteins enriched specifically in synapses that were significantly altered in both DLPFC and NAc of OUD subjects. Across brain regions, synaptic protein alterations in OUD subjects were primarily identified in glutamate, GABA, and circadian rhythm signaling. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24-h cycle, we were able to map circadian-related changes associated with OUD in synaptic proteomes associated with vesicle-mediated transport and membrane trafficking in the NAc and platelet-derived growth factor receptor beta signaling in DLPFC. Collectively, our findings lend further support for molecular rhythm disruptions in synaptic signaling in the human brain as a key factor in opioid addiction.

Neuroprotective Potential of Vitamin B-12 Against Cadmium–Induced Neuroinflammation Mediated Memory Dysfunctions in Adult Albino Mice

Cadmium induces neurotoxicity in brain and results in increased enzymatic functions, accumulation of Amyloid beta plaque and Neuo Fibrillary Tangles (NFTs) causing neurodegenerative diseases. The purpose of the present work was to investigate the anti-inflammatory or anti-oxidative potential of vitaminB12 against Cd -induced memory impairment and mechanism of signaling pathway of vitamin-B12 protection in Cd-induced neuroinflammatory in mice model. Male albino mice of BALB/C trait of age 7 to 8 weeks and weighing about (30-32±3g) were used as model animal in the study. All mice were divided in three groups. Control Group (CG) treated with 0.9% saline (1mL/Kg), Cd Cl2 administered Group (CD) (1mg/kg) and Cd +Vitamin administered group (CV) B-12 (500µg/kg). CD group was treated with Cd Cl2 for three weeks on alternate days while CV group was treated with Vitamin B12 and toxin CdCl2 for the next two weeks intraperitonealy. Behavior tests like Morris Water Maze (MWM) and Y-Maze (YM) tests were conducted on the experimental mice to study the neuro protective potential of Vitamin-B12 against CdCl2 induced memory dysfunctions. After completing the above procedures, mice were sacrificed and the brains of each group were collected for protein quantification step and western blotting. The Immuno blots of Iba-1, NF-kB, TNF-α, IL-1β, BACE1 and Aβ proteins in the brain homogenates of CD and CV mice confirm the anti-neuroinflammatory potential of vitamin-B12 against Cadmium –induced Neuroinflammation. Escape latency of CV group mice receiving Methylcobalamine was less and showed better performance in MWM test. The results of Y-maze test also showed that the spontaneous percentage alternation of CD group mice was less than CV and CG group of mice. Immuno blot of p-Akt confirm the signaling pathways of vitamin-B12 protection in Cd-induced neuroinflammatory mediated memory-impairment in the model mice. Our findings suggest that Vitamin-B12 could be a potent candidate drug for treating cognitive dysfunctions due to its anti-inflammatory and anti-oxidative potential. Our study also shows that Vitamin-B12 activates p-Akt signaling pathway along with decreasing the NF-Κb, TNF-α and IL-1β to protect the brain of mice against CdCl2 neurotoxicity.

6-Aminoflavone Activates Nrf2 to Inhibit the Phospho-JNK/TNF-α Signaling Pathway to Reduce Amyloid Burden in an Aging Mouse Model.

In the current study, we examined the antioxidant activity and anti-amyloidogenic potential of 6-aminoflavone in an adult mice model of d-galactose-induced aging. Male albino eight-week-old mice were assigned into four groups: 1. the control group (saline-treated), 2. d-galactose-treated mice (100 mg/kg/day, intravenously) for eight weeks, 3. d-galactose-treated mice (100 mg/kg/day, intravenously for eight weeks) and 6-AF-treated mice (30 mg/kg/day, intravenously for the final four weeks), and 4. 6-AF-treated mice (30 mg/kg/day i.p. for four weeks). We conducted many assays for antioxidant enzymes, including lipid peroxidation, catalase, glutathione (GSH), peroxidase (POD), and sulfoxide dismutase (SOD) (LPO). Western blotting was used to assess protein expression while the Morris water maze (MWM) and Y-maze (YM) were used to study behavior. The findings show that 6-AF greatly improved neuronal synapse and memory impairment brought on by d-galactose and it significantly inhibited BACE1 to reduce the amyloidogenic pathway of A (both amyloid β production and aggregation) by upregulating Nrf2 proteins (validated through molecular docking studies) and suppressing phosphorylated JNK and TNF-α proteins in adult albino mice's brain homogenates. These findings suggest that 6-AF, through the Nrf2/p-JNK/TNF-α signaling pathway, can diminish the oxidative stress caused by d-galactose, as well as the amyloidogenic route of A formation and memory impairment.

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7days) and consumed drinking water with or without 1mM sodium nitrate. Compared with the contralateral control limb, 3days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P< 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3days decreased FSR in both SS (P=0.009) and IMF (P=0.031) mitochondria. Additionally, 3days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7days of immobilization. However, in contrast to 3days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

Melissa officinalis extract suppresses endoplasmic reticulum stress-induced apoptosis in the brain of hypothyroidism-induced rats exposed to γ-radiation

The purpose of this study was to demonstrate the neuroprotective effect of Melissa officinalis extract (MEE) against brain damage associated with hypothyroidism induced by propylthiouracil (PTU) and/or γ-radiation (IR) in rats. Hypothyroidism induction and/or exposure to IR resulted in a significant decrease in the serum levels of T3 and T4 associated with increased levels of lipid peroxidation end product, malondialdehyde (MDA), and nitrites (NO) in the brain tissue homogenate. Also, hypothyroidism and /or exposure to IR markedly enhance the endoplasmic reticulum stress by upregulating the gene expressions of the protein kinase RNA-like endoplasmic reticulum kinase (PERK), activated transcription factor 6 (ATF6), endoplasmic reticulum-associated degradation (ERAD), and CCAAT/enhancer-binding protein homologous protein (CHOP) in the brain tissue homogenate associated with a proapoptotic state which indicated by the overexpression of Bax, BCl2, and caspase-12 that culminates in brain damage. Meanwhile, the PTU and /or IR-exposed rats treated with MEE reduced oxidative stress and ERAD through ATF6. Also, the MEE treatment prevented the Bax and caspase-12 gene expression from increasing. This treatment in hypothyroid animals was associated with neuronal protection as indicated by the downregulation in the gene expressions of the microtubule-associated protein tau (MAPT) and amyloid precursor protein (APP) in the brain tissue. Furthermore, the administration of MEE ameliorates the histological structure of brain tissue. In conclusion, MEE might prevent hypothyroidism-induced brain damage associated with oxidative stress and endoplasmic reticulum stress.

Screening of Conditions for Manganese Peroxidase Production from White Rot Fungi (Pleurotus djamor)

The need for alternative and environmentally friendly methods for waste clean-up has led to the use of enzymes in bioremediation. In this study, white rot fungi were isolated from decaying plant parts using standard microbiological and biochemical techniques. The isolated fungi were identified and screened using standard substrates (2, 6-DMP) to determine their capability to produce manganese peroxidase. Pure manganese peroxidase was achieved after four distinct purification phases. Crude extract of the homogenate proteins obtained through optimized submerged fermentation system were precipitated using ammonium sulfate. Ammonium sulfate used at 60% concentration at pH 4.5 precipitated proteins with highest Manganese peroxidase activity (322 U/mg). The precipitated proteins were desalted through dialysis for twelve hours with buffer exchange at interval of six hours and activity of 343.91 U/mg was recorded afterwards. DEAE-cellulose was used for the ion exchange purification of the dialyzed protein, NaCl gradients of 0.3-0.6 M was found to be best in washing off bound protein from the exchange resin and activity of 434.18 U/mg was recorded from the pooled fraction tubes. Sephadex G-100 was used for separation of the proteins into molecular sizes and weights. 2.8 purification folds of the enzyme were achieved after ion exchange (DEAE-cellulose) and gel filtration (sephadex G-100) with percentage yield of 2.20%. Specific activity of the enzyme increased to 602.00% after gel filtration. The partial purified enzyme was further characterized for determination of optimal pH (4.5), temperature (40 OC) and kinetic properties Km of 3.4 mM and Vmax of 250µmol/min.

A Comparative Study on GC-MS Profiles and Effect of Ethanolic Extracts of Stem-Bark and Leaves of Daniellia oliveri on Cerebral Sodium Pump and Thiols Status in Fenton Reaction Treated Rat Organs In-vitro

Daniellia oliveri is one of the most extensively utilized medicinal plants in Nigeria and some West African countries for the treatment of various ailments. In the present study, the GC-MS profiles and effect of ethanolic extracts of stem bark and leaves of Daniellia oliveri on lipid peroxidation, Na+/K+-ATPase activity and thiols status in H2O2 and Fe2+ (fenton reaction) assaulted cerebral and hepatic tissue homogenates was investigated. The stem bark and leaves were extracted with absolute ethanol for 72hrs and concentrated in a rotary evaporator. Wistar rats were euthanized and the brains and livers were removed, homogenized, centrifuged and the supernatants were used for lipid peroxidation, Na+/K+-ATPase activity, and thiol assays in the presence of prooxidants (1mM H2O2 or 10μM Fe2+ or H2O2 and Fe2+).The results revealed that ethanol extract of both the leaf and stem-bark inhibited lipid peroxidation induced by H2O2, Fe2+ and combination of H2O2 and Fe2+ and this was evident in the reduction of lipid peroxidation adducts formation in rat liver and brain homogenates with the stem-bark possessing more efficacy. Furthermore, the results of Na+/K+-ATPase and thiol assays revealed that H2O2 and Fe2+ inhibited the activity of cerebral Na+/K+-ATPase, while H2O2/Fe2+ caused a marked reduction in the levels of both protein and non-protein thiols in the tissues homogenates. However, the ethanolic extract of Daniellia oliveri extirpated this anomaly by increasing the activity of cerebral Na+/K+-ATPase as well as the thiol level in the rat hepatic and cerebral tissues. Finally, GC-MS analysis was carried out on the ethanolic extracts of the stem bark and leaves of Daniellia oliveri and results revealed the presence of 21 compounds including 1,2-Benzisothiazol-3-amine, 1H-Indole-2-carboxylic acid, 2-Ethylacridine, Ethyl 2-(2-chloroacetamido)-3,3,3-trifluoro-2-(3-fluoroanilino) propionate and trans-1-Butyl-2-methylcyclopropane in the stem bark and 10 compounds including Methylthio-acetonitrile, 2-Methyl-Z,Z-3,13-octadecadienol, Desulphosinigrin and 4H-1,2,4-triazole-3,5-diamine in the leaves, justifying the observed biological activities of Daniellia oliveri and the higher potency of the stem bark may be attributed to the presence of more bioactive constituents found in it. This study therefore justifies the medicinal usage of leaves and stem-bark of Daniellia oliveri and suggests its consideration in the treatment and management of degeneration diseases with etiology associated with oxidative stress.

Investigation of the Protective Effects of Urtica dioica, Capsella bursa-pastoris and Inula racemosa on Acetaminophen-Induced Nephrotoxicity in Swiss Albino Male Mice

Acetaminophen (APAP) is the most commonly used nonprescription antipyretic-analgesic drug. This medication is thought to be safe at the suggested dosage (4 g/24 h), but its overdose (up to 2.5 g/kg) can cause severe injuries to the human body, including renal injury. APAP has various toxic effects on nephrons, as it leads to an excessive free radical generation that, in turn, results in a disturbance in the redox homeostasis of cells, causing oxidative stress. To replenish this oxidative stress, there is an ultimate urge for natural therapies that can retain the cellular homeostasis of nephrons by diminishing the overdose impression of acetaminophen. The principle objective of this work is to appraise nephrotoxicity due to APAP and its amelioration through the antioxidant properties of aqueous extracts of selected medicinal plants: Urtica dioica, Capsella bursa-pastoris, and Inula racemosa (UD, CBP, and IR, respectively). The pH stability of the nutraceuticals used was examined by determining the impact of pH 4, pH 7 and pH 9 on the DPPH radical scavenging activity of aqueous plant extracts. Gas chromatography-Mass spectroscopy (GC–MS) analytical technique was performed to determine the volatile organic phytochemical profiles of all three medicinal plants. Male Swiss albino mice were used for the present investigation. The animals were distributed into five groups of (n = 6), a total of 30 mice, for in vivo analysis. Group 1 served as the control group; group 2 received a single IP dose of APAP (600 mg/kg); group 3 received APAP pretreated with UD (300 mg/kg); group 4 received APAP pretreated with CBP (300 mg/kg); and group 5 received APAP pretreated with IR (300 mg/kg). Overdose of the APAP- induced a significant (p &lt; 0.05) alterations in the total protein concentration, weight and the nephrological architecture in renal tissue, as observed through biochemical assays and histopathological examinations. Due to nephrotoxicity, there was a substantial (p &lt; 0.05) drop in body weight and total protein contents in the APAP alone group when compared to the treatment groups. There was remarkable protection against APAP-induced alterations in the total protein of renal homogenate in the treatment groups. Histopathological analysis (H&amp;E staining) of the mice kidneys indicated severe deterioration in the APAP alone group, whereas the therapy groups showed considerable nephroprotection towards APAP-induced abnormalities. The biochemical findings and histopathological study of the kidneys revealed that the herbal extracts (UD, CBP, and IR) have a nephroprotective potential against APAP-induced nephropathy. The trend of efficacy was observed as UD &gt; CBP &gt; IR. However, extensive study is needed to determine the likely ameliorative mechanism of these nutraceuticals.

Implantation with SHED sheet induced with homogenate protein of spinal cord promotes functional recovery from spinal cord injury in rats.

Introduction: After spinal cord injury (SCI) occurs, the lesion is in a growth inhibitory microenvironment that severely hinders neural regeneration. In this microenvironment, inhibitory factors are predominant and factors that promote nerve regeneration are few. Improving neurotrophic factors in the microenvironment is the key to treating SCI. Methods: Based on cell sheet technology, we designed a bioactive material with a spinal cord-like structure -SHED sheet induced with homogenate protein of spinal cord (hp-SHED sheet). Hp-SHED sheet was implanted into the spinal cord lesion for treating SCI rats with SHED suspensions as a control to investigate the effects on nerve regeneration. Results: Hp-SHED sheet revealed a highly porous three-dimensional inner structure, which facilitates nerve cell attachment and migration. Hp-SHED sheet in vivo restored sensory and motor functions in SCI rats by promoting nerve regeneration, axonal remyelination, and inhibiting glial scarring. Discussion: Hp-SHED sheet maximally mimics the microenvironment of the natural spinal cord and facilitate cell survival and differentiation. Hp-SHED sheet could release more neurotrophins and the sustained action of neurotrophins improves the pathological microenvironment, which effectively promotes nerve regeneration, axonal extension, and inhibits glial scarring, thereby promoting the in situ centralis neuroplasticity. Hp-SHED sheet therapy is a promising strategy for effective treatment of SCI based on neurotrophins delivery.

Use of fungal and bacterial protease preparations to enhance extraction of lipid from fish roe: Effect on lipidomic profile of extracted oil.

The present study investigated the hydrolysis of protein in hoki roe homogenate using a HT (bacterial), a FP-II (fungal) protease preparations and Alcalase (bacterial) to enhance lipid yield extraction. The degree of hydrolysis was determined at various pH, temperature and time using casein and hoki roe. Total lipid extraction and lipidomic analysis was carried out following proteolysis of hoki roe homogenate. The degree of hydrolysis and SDS-PAGE revealed that the hydrolytic capability of Alcalase was better than HT and FPII. The total extracted lipid yield was better following hydrolysis with Alcalase (19.29%), compared to HT (18.29%) and FPII (18.33%). However, the total phospholipid (PL) and n-3 fatty acid yields were better from HT hydrolysed hoki roe homogenate (PL=30.7μmol/g; n-3=10.5%), compared to Alcalase (PL=22μmol/g; n-3=5.95%). Overall, this study indicates that HT protease preparation hydrolysis of fish roe homogenate can both enhance lipid extraction and retain lipid quality.

АКТУАЛЬНОСТЬ ПРИМЕНЕНИЯ ПАКЕТА ПРОГРАММНОЙ СРЕДЫ R-MALDIQUANT ДЛЯ ПРЕОБРАЗОВАНИЯ НЕОБРАБОТАННЫХ МАСС- СПЕКТРОВ ДИРОФИЛЯРИЙ

Diagnosis of human tissue helminthiasis remains a complex problem of practical medicine. Therefore, the search for alternative direct methods of their verification is an important area of laboratory diagnostics. Proteomic research methods are one of the most modern areas of laboratory practice. Updating the database of reference protein profiles of parasitic pathogens is one of the main priorities of mass spectrometry. To convert digital data of protein extract spectra, the R-MALDIquant software package for dirofilaria differentiation (Dirofilaria immitis, Dirofilaria repens) was used. For the study, two D. repens females were taken, removed from the subcutaneous fat of two patients (one from each patient), two D. immitis females obtained from the heart after autopsy of two fallen dogs. In the study, only the head ends were taken (where the stichocytes are concentrated), without reproductive organs (where the main excretory-secretory antigens of the parasite are located). The profiles of the mass spectra of the homogenate proteins were obtained using the standard Sepsityper Kit 50 and the “Formic Acid Extraction” for Microflex LT MALDI–TOF MS (Bruker Daltonics) with flexControl software (Bruker Daltonics). Visualization was performed using Flexanalysis 3.3 software (Bruker Daltonics). Preliminary processing of mass spectra was carried out using the R software environment in the MALDIquant package. The coincidence rate in the dendrogram sections for D. immitis individuals was 96-99 %, and for D. repens – 73-100 %, which is probably due to phenotypic differences in helminths isolated from different patients. The results obtained by us of processing two types of dirofilaria using the R-MALDIquant software environment are the first step in creating a library of reference protein profiles of nematodes of the genus Dirofilaria. Key words: Dirofilaria repens, Dirofilaria immitis, MALDI-TOF MS, R package, MALDIquant. erroneous identification of the causative agent of the disease D. latum, which did not affect the results of treatment

Identification of regenerating island-derived protein 3E in dogs.

Regenerating islet-derived protein (REG) 1A (aka pancreatic stone protein) and REG3A (aka pancreatitis-associated protein) are upregulated in humans with sepsis, pancreatitis, and gastrointestinal diseases, but little is known about this protein family in dogs. Our aim was to identify REG1 and REG3 family members in dogs. REG-family genes were computationally annotated in the canine genome and proteome, with verification of gene expression using publicly available RNA-seq data. The presence of the protein in canine pancreatic tissue and plasma was investigated with Western blot and immunohistochemistry, using anti-human REG1A and REG3A antibodies. Protein identity was confirmed with mass spectrometry. Two members of the REG3 subfamily were found in the canine genome, REG3E1 and REG3E2, both encoding for the same 176 AA protein, subsequently named REG3E. Anti-human REG3A antibodies demonstrated cross-reactivity with the canine REG3E protein in pancreas homogenates. In canine plasma, a protein band of approximately 17 kDa was apparent. Mass spectrometry confirmed this protein to be the product of the two annotated REG3E genes. Strong immunoreactivity to anti-human REG3A antibodies was found in sections of canine pancreas affected with acute pancreatitis, but it was weak in healthy pancreatic tissue. Recombinant canine REG3E protein underwent a selective trypsin digestion as described in other species. No evidence for the presence of a homolog of REG1A in dogs was found in any of the investigations. In conclusion, dogs express REG3E in the pancreas, whose role as biomarker merits further investigations. Homologs to human REG1A are not likely to exist in dogs.

Transplantation of human cord blood-derived multipotent stem cells (CB-SCs) enhances the recovery of Parkinson in rats

Earlier published research showed that cord blood-derived multipotent stem cells (CB-SCs) exhibited the intrinsic expression of specific transcription factors (e.g., En1, Nurr1 and Wnt1) and seems to be induced to form dopamine neurons in vitro. In this research, we further investigated the therapeutic potential of CB-SCs in 6-hydroxydopamine lesioned Parkinson's disease (PD) rats. The results of PCR analysis showed that CB-SCs could express transcription factors associated with pluripotentiality and dopaminergic differentiation (e.g., Klf4, c-Myc, Nanog, Sox2, Ngn2, and Nurr1). After being transplanted into the striatum and substantia nigra of PD rats, most of CB-SCs (>90%) developed a fate commitment to dopaminergic differentiation, expressed as the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT). The improvement effect of cell transplantation on dyskinesia in PD rats was better than that in sham control group. Moreover, higher levels of TH protein in brain homogenates further demonstrated that there were more surviving dopamine neurons in the brain of transplanted PD rats. Study concluds, CB SCS transplantation could promote the regeneration of dopamine neurons and behavioral recovery of PD rats.