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There is increasing evidence of the clinical importance of microparticles (MPs) and their role in blood transfusion-related side effects and the transmission of pathogens. The study aims to examine the red blood cell-derived MPs in blood bags during storage under standardized blood bank conditions. The samples were tested at various times to demonstrate the presence of RBC-derived MPs by flow cytometry. The quantitative assay was carried out in stored erythrocyte concentrate on days 0, 25 and 35 and their number from day 0 to 25 and 35 and the number of day 25 to day 35 were compared. The MPs were counted after being concentrated in a supernatant (labeled with the respective antibodies CD47, CD235a and Annexin V) obtained by a specific centrifugation procedure. The analysis showed that the number of Annexin V positive MPs increased between day 0 and day 35 (~ 0.001) and CD47 expression on MPs at day 25 and day 35 decreased compared to day 0 (~ 0.001). In addition, CD235a expression had shown minimal insignificant changes with an upward trend (> 0.05) during the storage period. It is concluded that monitoring the release of MPs from RBC units during storage is a sensitive approach to identifying MPs for transfusion drugs and, more broadly, for cell-based therapies. Key words: Red blood cells, phosphatidylserine, cell-derived microparticle.

Chitosan, obtained as a result of the deacetylation of chitin, one of the most important naturally occurring polymers, has antimicrobial properties against fungi, and bacteria. It is also useful in other fields, including: food, biomedicine, biotechnology, agriculture, and the pharmaceutical industries. A literature survey shows that its antimicrobial activity depends upon several factors such as: the pH, temperature, molecular weight, ability to chelate metals, degree of deacetylation, source of chitosan, and the type of microorganism involved. This review will focus on the in vitro and in vivo antimicrobial properties of chitosan and its derivatives, along with a discussion on its mechanism of action during the treatment of infectious animal diseases, as well as its importance in food safety. We conclude with a summary of the challenges associated with the uses of chitosan and its derivatives.

Emerging evidence indicates that many microRNAs (miRNAs) are indispensable regulators of osteoblast differentiation and bone formation. However, the role of miRNAs in mechanotransduction of osteoblasts remains to be elucidated. This study aimed to identify a mechanosensitive miRNA that regulates Activin A receptor type I (ACVR1)-induced osteogenic differentiation. After 4 weeks of hindlimb unloading (HLU) suspension of 6-month-old male C57BL/6J mice, femurs and tibias were harvested to extract total bone RNAs. Elevated levels of miR-208a-3p correlated with a lower degree of bone formation in whole-bone samples of HLU mice. However, in vitro overexpression of miR-208a-3p inhibited osteoblast differentiation, whereas silencing of miR-208a-3p by antagomiR-208a-3p promoted expression of osteoblast activity, bone formation marker genes, and matrix mineralization under mechanical unloading condition. Bioinformatics analysis and a luciferase assay revealed that ACVR1 is a target gene of miR-208a-3p that negatively regulates osteoblast differentiation under mechanical unloading environment. Further, this study also demonstrates that in vivo pre-treatment with antagomiR-208a-3p led to an increase in bone formation and trabecular microarchitecture and partly rescued the bone loss caused by mechanical unloading. Collectively, these results suggest that in vivo, inhibition of miRNA-208a-3p by antagomiR-208a-3p may be a potential therapeutic strategy for ameliorating bone loss.

Chronic oxidative stress fuels pathogenesis of a large set of diseases. Oxidative stress is the cause and consequence of numerous diseases including type 1 diabetes mellitus (T1DM), in which there is selective destruction of insulin producing pancreatic β-cells. Studies have documented that hyperglycemia produces profound stress. In vivo production of numerous reactive oxygen, nitrogen, chlorine species and lipid/sugar oxidation products in T1DM patients may be the result of persistent hyperglycemia. Post-translational modifications by reactive species may create new antigenic epitopes and play a role in the development of autoimmune response. In this paper our main focus was to establish the effect of existing hyperglycemia induced oxido-nitrosative stress in T1DM patients on the integrity of human serum albumin. Raised nitric oxide, carbonyl, RBC hemolysis, lowered ferric reducing antioxidant power (FRAP), thiol and deformed RBC in T1DM are all highly suggestive of persistent oxido-nitrosative stress. Hyperglycemia induced generation of advanced glycation end products (AGEs) was established by LCMS. Chronic oxido-nitrosative stress can modify HSA in T1DM patients, producing immunologically active albumin. Therefore, it is speculated that the aberrant HSA may play a role in the initiation/progression of T1DM.

Systemic lupus erythematosus (SLE) is an autoimmune disorder where the role of inflammatory processes in the etiopathogenesis is well documented. Despite extensive research, the trigger for initiation of the disease has not been identified. Peroxynitrite, a strong nitrating/oxidizing agent has been reported in SLE and other autoimmune diseases. In this study, human serum albumin (HSA) was exposed to peroxynitrite for 30min at 37°C. The structure of HSA was grossly perturbed when examined by various physico-chemical techniques. Peroxynitrite mediated nitration of HSA was confirmed by LCMS/MS. Furthermore, increase in hydrodynamic radius of peroxynitrite-modified-HSA suggests the attachment of nitro group(s). Aggregation in peroxynitrite-modified-HSA was evident in a TEM scan. Nitration, oxidation, cross linking, aggregation etc conferred immunogenicity on peroxynitrite-modified-HSA. High titre antibodies were elicited in rabbits immunized with peroxynitrite-modified-HSA. Induced antibodies were highly specific for peroxynitrite-modified-HSA but showed considerable binding with other nitrated molecules. Direct binding/inhibition ELISA carried out with autoantibodies in SLE sera showed preferential binding with peroxynitrite-modified-HSA. Anti-nDNA positive IgG from SLE sera showed preference for peroxynitrite-modified-HSA when subjected to immunoassay (direct binding and inhibition) and mobility shift assay. Our results reinforce the role of augmented inflammation in SLE progression.

In addition to being sources of carbohydrates, protein, and fiber, vegetables, and fruits also represent important sources of micro-nutrients, including antioxidants, and anti-mutagens in the form of carotenoids, and polyphenols, such as flavonoids. We describe the enhanced extraction of these bioactive compounds from radish (Raphanus sativus L.) by use of an enzymic pre-treatment to break up starch, and the complex mix of polysaccharides, cellulose, hemicellulose, and pectin, which constitute the plant cell wall. The enzymic cocktail included cellulases, pectinases, amylases, glucanases, and hemicellulases. Response surface methodology (RSM) was utilized to maximally explore the yield by estimating extraction conditions, enzyme concentration, incubation time, temperature, pH, and extraction solvents. Extracted antioxidant activity was assayed in terms of total phenolics content (TPC), free radical scavenging (DPPH and ABTS tests), inhibition of linolic peroxidation assay, Reducing potential , and Trolox equivalent antioxidant capacity (TEAC). Two different bacterial mutant strains (TA-98, TA-100, S. typhimurium) were used by applying Ames bacterial test. It was concluded that highest desirability was achieved at 3.53% (w/w) of enzyme formulation, at pH of 6.1, incubation time 66.08 min and 46.08 °C temperature produced 19.2 g/100 g FW of crude extract which contains 45.9 mg GAE/g FW total phenolics contents, 0.480 mg/mL Reducing Power, 85.9% LA inhibition and 271.7 mmole/g FW of TEAC in fresh radish root. We found that radish extract was an excellent source of antioxidants that might be further investigated for commercial exploitation.

The purpose of this study was to examine the possible mechanism underlying the protective effect of tetramethylpyrazine (TMP) against disuse-induced muscle atrophy. Sprague−Dawley rats were randomly assigned to receive 14 days of hindlimb unloading (HLU, a model of disuse atrophy) or cage controls. The rats were given TMP (60 mg/kg body mass) or vehicle (water) by gavage. Compared with vehicle treatment, TMP significantly attenuated the loss of gastrocnemius muscle mass (−33.56%, P < 0.01), the decrease of cross-sectional area of slow fiber (−10.99%, P < 0.05) and fast fiber (−15.78%, P < 0.01) during HLU. Although TMP failed to further improve recovery of muscle function or fatigability compared with vehicle treatment, it can suppress the higher level of lactate (−22.71%, P < 0.01) induced by HLU. Besides, TMP could effectually reduce the increased protein expression of muscle RING-finger protein 1 induced by HLU (−14.52%, P < 0.01). Furthermore, TMP can ameliorate the calcium overload (−54.39%, P < 0.05), the increase of malondialdehyde content (−19.82%, P < 0.05), the decrease of superoxide dismutase activity (21.34%, P < 0.05), and myonuclear apoptosis (−78.22%, P < 0.01) induced by HLU. Moreover, TMP significantly reduced HLU-induced increase of Bax to B-cell lymphoma 2 (−36.36%, P < 0.01) and cytochrome c release (−36.16%, P < 0.05). In conclusion, TMP attenuated HLU-induced gastrocnemius muscle atrophy through suppression of Ca2+/reactive oxygen species increase and consequent proteolysis and apoptosis. Therefore, TMP might exhibit therapeutic effect against oxidative stress, cytosolic calcium overload, and mitochondrial damage in disuse-induced muscle atrophy.

Systemic lupus erythematosus (SLE) is an inflammatory, autoimmune disorder of unknown etiology. The inflammatory stress in SLE patients may modify macromolecules and produce structural/functional abnormalities. The present study is aimed at examining the consequences of stresses on the structure of albumin in SLE patients. Albumin was isolated from the sera of SLE/healthy subjects. Multiple physicochemical techniques were used to elucidate, structure of albumin. Advanced glycation end products in SLE patients' albumin were identified by the AGE specific fluorescence. Quenching of tryptophan, tyrosine fluorescence and surface protein hydrophobicity was observed in SLE patients' albumin. Protein-bound carbonyls were elevated while free thiol, lysine, arginine, and alpha helicity was found to be decreased in SLE albumin. Furthermore, changes in the secondary structure of SLE albumin were observed as shift in the position of amide I/II bands. Functionality of SLE albumin was also compromised as its cobalt-binding ability was substantially declined. Adduction of moieties was detected by dynamic light scattering (DLS) and confirmed by matrix assisted laser desorption/ionization. DLS, thioflavin T and transmission electron microscopy results confirmed aggregates in SLE patients' albumin. This study may be helpful in understanding the role of modified albumin in the cofounding pathologies associated with SLE.

IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV-visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS-PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions.

We examined ultrastructure protective phenomena and mechanisms of slow and fast muscles in hibernating Daurian ground squirrels (Spermophilus dauricus). Some degenerative changes such as slightly decreased sarcomere length and vacuolization occurred in hibernation, but periaxonal capsular borders in intrafusal fibers remained distinct and the arrangement of extrafusal fibers and Z-lines unscathed. In soleus samples, the number of glycogenosomes more than tripled during hibernation. The expression of phosphorylated glycogen synthase remained unaltered while that of glycogen phosphorylase decreased during hibernation. The number of extensor digitorum longus glycogenosomes decreased and the expression of phosphorylated glycogen synthase decreased, while glycogen phosphorylase expression remained unaltered. The nuclei number remained unchanged. Kinesin and desmin, preventors of nuclear loss and damage, were maintained or just slightly reduced in hibernation. The single-fiber mitochondrial concentration and sub-sarcolemmal mitochondrial number increased in both muscle types. The expression of vimentin, which anchors mitochondria and maintains Z-line integrity, was increased during and after hibernation. Also, dynamin-related protein 1, mitochondrial fission factor, and adenosine triphosphate synthase were elevated in both muscle types. These findings confirm a remarkable ultrastructure preservation and show an unexpected increase in mitochondrial capacity in hibernating squirrels.

Nonenzymatic glycation of amino groups of DNA bases by reducing sugars can generate advanced glycation end products (AGEs). Cellular formation of AGEs under normal physiology is continuously scanned and removed by efficient system in the cells. However, excess formation and accumulation of AGEs may be cause or consequence of some human diseases. Mammalian DNA incubated with d-glucose for 28 days at 37°C showed structural changes in DNA as confirmed by UV, fluorescence, CD, melting temperature, S1 nuclease sensitivity and gel electrophoresis. Formation of DNA-AGE was confirmed by HPLC and LC-MS. Enzyme immunoassay and electrophoretic mobility shift assay of autoantibodies in type 2 diabetes patients' sera with disease duration of 5-15 years exhibited significantly high binding with DNA-AGE as compared to patients with 1-5 years of disease duration. Autoantibodies against aberrant DNA-AGE may be important in the assessment of initiation/progression of secondary complications in type 2 diabetes mellitus patients.

Skeletal unloading results in decreased bone formation and bone mass. During long-term space flight, the decreased bone mass is impossible to fully recover. Therefore, it is necessary to develop the effective countermeasures to prevent spaceflight-induced bone loss. Hindlimb Unloading (HLU) simulates effects of weightlessness and is utilized extensively to examine the response of musculoskeletal systems to certain aspects of space flight. The purpose of this study is to investigate the effects of a 4-week HLU in rats and subsequent reloading on the bone mineral density (BMD) and mechanical properties of load-bearing bones. After HLU for 4 weeks, the rats were then subjected to reloading for 1 week, 2 weeks and 3 weeks, and then the BMD of the femur, tibia and lumbar spine in rats were assessed by dual energy X-ray absorptiometry (DXA) every week. The mechanical properties of the femur were determined by three-point bending test. Dry bone and bone ash of femur were obtained through Oven-Drying method and were weighed respectively. Serum alkaline phosphatase (ALP) and serum calcium were examined through ELISA and Atomic Absorption Spectrometry. The results showed that 4 weeks of HLU significantly decreased body weight of rats and reloading for 1 week, 2 weeks or 3 weeks did not recover the weight loss induced by HLU. However, after 2 weeks of reloading, BMD of femur and tibia of HLU rats partly recovered (+10.4%, +2.3%). After 3 weeks of reloading, the reduction of BMD, energy absorption, bone mass and mechanical properties of bone induced by HLU recovered to some extent. The changes in serum ALP and serum calcium induced by HLU were also recovered after reloading. Our results indicate that a short period of reloading could not completely recover bone after a period of unloading, thus some interventions such as mechanical vibration or pharmaceuticals are necessary to help bone recovery.

Structural rearrangements and condensations of proteins under hyperglycemic stress have been implicated in various pathological disorders. This study aims to probe the role of methylglyoxal (MG) modified human immunoglobulin G (MG-IgG) in immuno-pathology of type 2 diabetes mellitus (T2DM). MG was found to perturb the structural integrity of IgG, affect its aromatic micro-environment and cause the generation of advanced glycation end products (AGEs) and aggregate adducts. It liberated the hydrophobic pockets of the protein, reduced its β pleated sheet structure and affected its tertiary conformation. Transition from β sheet to α helix and random coil was also observed in IgG upon modification by MG. It acted with strong oxidative potential and caused oligomerisation and disordered or amorphous type aggregation in the modified protein. Modified IgG had a cytotoxic and genotoxic impact. The MG modified IgG presented novel antigenic determinants that lead to an aggressive immune response. The antibodies had high affinity towards the immunogen. Auto-antibodies derived from T2DM patients exhibited strong affinity towards the modified IgG in comparison to the unmodified protein. Specificity of serum antibodies from T2DM patients was further confirmed by competitive-inhibition ELISA. The potential role of MG-IgG in the immunopathogenesis of T2DM has been discussed.

Imidacloprid (IC) is a systemic insecticide related to the tobacco toxin nicotine. IC is a toxic substance frequently used into combat insects, rodents and plants pests and other creatures that can pose problems for agriculture. We, therefore, planned this study to assess risk factors, biochemical and histological alterations associated with hepatotoxicity and nephrotoxicity. Forty-eight adult male albino mice were divided into four groups of 12 animals each. All the animals were given standard synthetic pellet diet. One group served as control, and the other three were served as experimental groups. Decrease in the body weight of the high dose group was observed at 15 mg/kg/day, and no mortality occurred during the treatment period. High dose of imidacloprid caused a significant elevation of serum clinical chemistry parameters, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvate kinase (SGPT), alkaline phosphatase (ALP) and total bilirubin (TBIL). Histology of liver and kidney indicates hepatotoxicity and nephrotoxicity at a high dose of imidacloprid. Based on the morphological, biochemical and histopathological analysis, it is evident that imidacloprid induced toxicological effects at 15 mg/kg/day to mice. The results of the present study demonstrate that IC had significant effects on body weight, liver functions and kidney (p < 0.05) at a dose of 15 mg/kg body weight. IC treatment 5 and 10 mg/kg/day may be considered as no observed adverse effect level (NOAEL) for mice. It was concluded that IC can cause hepatotoxicity and nephrotoxicity at a dose much lower than the LD50 (131 mg/kg body weight) in mice.

In this study, human serum albumin (HSA), the most abundant protein of blood plasma, was modified with varying concentrations of peroxynitrite. The peroxynitrite-induced changes in HSA was monitored by spectroscopy, SDS-PAGE, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal denaturation studies, and matrix-assisted laser desorption/inonization-time of flight mass spectrometry (MALDI-TOF MS). Aggregate formation was studied by thioflavin T binding and scanning electron microscopy (SEM). The results indicated formation of 3-nitrotyrosine, 6-nitrotryptophan, dityrosine, and carbonyls in modified samples and showed retarded mobility in SDS-polyacrylamide gel. Reduction in α-helicity and surface protein hydrophobicity confirmed the secondary and tertiary structure alterations in peroxynitrite-modified-HSA. Also, attachment of nitro group and increase in melting temperature was observed in modified sample. Furthermore, significant enhancement in the fluorescence intensity of ThT upon binding with peroxynitrite-modified-HSA and images under scanning electron microscope are suggestive of protein aggregation. It is, therefore, speculated that HSA modified by endogenously formed peroxynitrite might act as a trigger for nitration/aggregation and suggested the role of peroxynitrite-modified-HSA in SLE.

Objective: To study the role of peroxynitrite-modified human serum albumin (nitroxidized-albumin) in rheumatoid arthritis. Methods: Human serum albumin was exposed to peroxynitrite and changes in albumin structure were monitored by UV-visible, fluorescence and circular dichroism spectroscopy, thioflavin T, Congo red binding and attenuated total reflection-Fourier transformed infrared spectroscopy (ATR-FTIR). Antioxidant properties of nitroxidized-albumin were evaluated by free radical induced RBC hemolysis test. Markers of protein oxidation like carbonyl, thiol, dityrosine and RBC hemolysis were evaluated in RA patients’ sera. Binding of autoantibodies in RA sera (n=50) with nitroxidized-albumin was studied by direct binding, inhibition ELISA and electrophoretic mobility shift gel assay. Results: The nitroxidized-albumin indicated the generation of nitrotyrosine, nitrotryptophan, carbonyl, dityrosine and reduction in tyrosine and tryptophan fluorescence and α-helicity. Fluorescence emission intensities of thioflavin T and Congo red got augmented upon binding with nitroxidized-albumin. Furthermore, secondary and tertiary structures of nitroxidized-albumin were altered as evident by ATR- FTIR, far and near-UV CD. Autoantibodies in RA sera (or IgG purified from sera) showed enhanced binding with nitroxidized-albumin as determined by direct binding and inhibition ELISA. Protein carbonyls, dityrosine and RBC hemolysis were significantly high, but thiol was significantly low in RA sera compared to age- and sex- matched control. Conclusion: Endogenously available peroxynitrite can nitrate and oxidize albumin, leading to protein nitration/ oxidation and subsequent formation of crosslinks, aggregates and immunogenic nitroxidized-albumin. Therefore, nitroxidized-albumin may be a potential trigger for autoantibodies in RA patients.

Nonenzymatic glycosylation of proteins finally generates advanced glycation end products (AGEs). The Schiff's base and Amadori adduct are stages of early glycation. AGE-modified IgG may undergo conformational alterations and the final entity of the process may be involved in the pathogenesis of Rheumatoid Arthritis (RA). In this study, glycation of human IgG was carried out with varying concentrations of glucose. Effect of incubation period on glycation of IgG has also been studied. Amadori adduct was detected by nitroblue tetrazolium (NBT) dye. The glucose mediated structural alterations in IgG were studied by UV, fluorescence, CD, FT-IR, DLS and DSC spectroscopy, and SDS-PAGE. Glycation-induced aggregation in AGE-IgG was reported in the form of binding of thioflavin T and congo red. Furthermore, AGE-modified IgG exhibited hyperchromicity, decrease of tryptophan fluorescence accompanied by increase in AGE specific fluorescence, loss of β-sheet, appearance of new peak in FT-IR, increase in hydrodynamic size and melting temperature. SDS-PAGE results showed decrease in the band intensity of glycosylated-IgG compared to native IgG. Glycation-induced modifications and aggregation of IgG might be important in the pathogenesis of RA.

Using the closed-can technique, radon exhalation rate measurements have been carried out for shale and coal samples collected from various mines located in the Chakwal and Makarwal areas of Pakistan. For the two areas, the measured average values of the exhalation rates from shale are 1.45±0.13 and 0.67±0.25 Bq m −2 h −1 and for coal are 1.0±0.03 and 0.65±0.32 Bq m −2 h −1 , respectively. These values are much lower than the measured exhalation rates from alum-shale-based Nordic concrete which has values in the 50–200 Bq m −2 h −1 range. The lower values of the measured exhalation rates from the shale and coal deposits in the Chakwal and Makarwal areas are indicative of their lower uranium contents and mine workers in these areas do not face any abnormal health hazard due to radon since the exhalation rates have been found to be on the low side.

Berberis lycium Royle (family: Berberidaceae), a native to Pakistan, India and whole region to Himalyas is widely used like food and in folk medicine. A wide range of medicinally and nutritionally important phytochemical constituents have been isolated from plant such as alkaloids, cardioactive glycosides, saponins, tannins, anthocyanins, vitamins, carbohydrates, proteins, lipids, fiber content, β carotein, cellulose, phytic acid and phytate phosphorous. Plant possesses minerals such as Sodium, Calcium, Sulphur, Iron, Zinc, Copper, Lead, Manganese, Potassium and Phosphorus, which contribute to broad variety of biological processes and are valuable in the treatment of various disorders. Traditionally, the plant has been used against diarrhea, intestinal colic, piles, jaundice, internal wounds, rheumatism, diabetes, ophthalmia, gingivitis, throat pain, backache, scabies, bone fractures, sun blindness, pustules, manorrhagia, fever and as diuretic, expectorant and diaphoretic. B. lycium is known to possess antidiabetic, antihyperlipidemic, hepatoprotective, antibacterial, antifungal, anticoccidial, pesticidal, antimutagenic and wound healing properties, supporting its traditional uses. In this review, a comprehensive account of phytochemical constituents and pharmacological activities is presented along with traditional uses in a view of many recent findings and its potential for future research. To what extent, the findings about pharmacological activities are of potential clinical relevance and are unclear due to lack of clinical data. Key words: Antidiabetic, antihyperlipidemic, antimicrobial, antimutagenic, Berberis lycium,hepatoprotective.