Melatonin ingestion after exhaustive late-evening exercise attenuate muscle damage,oxidative stress,and inflammation during intense short term effort in the following day in teenage athletes

ABSTRACT

The present study aimed to investigate whether nocturnal melatonin (MEL) ingestion has beneficial effects against exercise-induced oxidative stress and muscle damage in young athletes. Fourteen healthy-trained teenagers performed two-test sessions separated by at least, 1 week. During each session, participants completed the Running-Based Anaerobic Sprint Test (RAST) at 20:00 h. Then, they ingested a single 10-mg tablet of MEL or Placebo (PLA) in a double-blind randomized order at 22:00 h. The following morning (i.e., 07:30 h), participants performed the same test as the previous night. Blood samples were taken before and after exercise. MEL intake increased the peak power (P_peak) (p < .01), mean power (P_mean) (p < .001) and decreased the total time (TT) (p < .001) and the fatigue index (FI) (p < .05). Furthermore, MEL ingestion attenuated the hematologic parameters before and after exercise (White Blood Cells (WBC: p < .001 and p < .001, respectively); Neutrophiles (NE: p < .001 and p < .001, respectively); Lymphocytes (LY: p < .001 and p < .001, respectively)) and the ultra-sensitive C-reactive protein (us-CRP: p < .001 and p < .001; respectively) compared to PLA. Also, MEL reduced muscle and hepatic damage enzymes before and after exercise (creatine kinase (CK: p < .001 and p < .001; respectively), lactate dehydrogenase (LDH: p < .05 and p < .01; respectively), aspartate aminotransferase (ASAT: p < .01 and p < .001; respectively)), Malondialdehyde (MDA: p < .001 and p < .001; respectively) and Homocysteine (Hcy: p < .001 and p < .001; respectively)) from placebo. Plasma lactate [La] and glucose (GL) remained unchangeable during the two conditions. In summary, acute MEL ingestion after strenuous late-evening exercise attenuated transient leucocytosis and protected against lipid peroxidation and muscle damage induced by strenuous exercise the following morning in healthy male teenage athletes.     

Performances de la scintigraphie parathyroïdienne au 99mTc-Sestamibi dans l’hyperparathyroïdie secondaire (à propos de 20 cas)

Aim of the studyTo evaluate the performance of the ^99mTc-Sestamibi parathyroid scintigraphy and to compare it with the performance of cervical ultrasonography in patients with secondary hyperparathyroidism who are candidates for parathyroidectomy.Patients and methodsWe performed a retrospective study including 20 patients with severe secondary hyperparathyroidism who underwent parathyroid scintigraphy in the nuclear medicine department of Sfax, during the period between January 2009 and June 2012. Our two days protocol included dual-phase, MIBI/Tc subtraction and single photon emission photons (SPECT) techniques. We analyzed the results obtained from each technique alone, then from combinations thereof. For all patients, we have collected the surgical and histopathological data as well cervical ultrasound if available.ResultsThe subtraction technique was the best performing with a sensitivity of 47% and an accuracy of 55%. The combination of subtraction scintigraphy and SPECT has improved the sensitivity to 53%and accuracy to 57%. The combined lecture of ultrasound and scintigraphy has given the best performance with a sensitivity of 58%, a specificity of 83% and an accuracy of 66%.ConclusionParathyroid scintigraphy combining subtraction and SPECT showed better reliability. The coupling with ultrasound is essential to improve results. The poor performance of scintigraphy in secondary hyperparathyroidism implies that it should be required only to search for ectopic or supernumerary glands.     

Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris

In this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV‐G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV‐G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV‐G gene was not tremendously increased as compared with the non‐optimized one. In addition, similar levels of RABV‐G were obtained when α‐factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high‐level expression of RABV‐G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV‐G expression cassette indicating that folding was the limiting step for RABV‐G secretion. To circumvent this limitation, co‐overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV‐G reached 1230 ng ml^?1. Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.     

Associations of rs1883832 and rs4810485 polymorphisms of CD40 gene with myocardial infarction in the Tunisian population

Context: Cluster of differentiation 40 (CD40), and its ligand CD40L, are major co-stimulatory molecules whose interactions are important in both cellular and humoral immunity, and has been suggested to play a role in the pathogenesis of acute coronary syndrome.

Objective: The aim of this study was to examine the association of CD40 polymorphisms (-1?C>T (rs1883832) and 945G>T (rs4810485)) and myocardial infarction (MI), and to test the association of CD40 gene haplotypes with MI in Tunisians.

Materials and methods: Three hundred and fifty MI patients and 301 apparently healthy controls were included in the study. The polymorphisms of CD40 were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Results: There were significant differences in the genotype and allele frequencies of CD40 gene -1?C>T (rs1883832) polymorphism between cases and controls. Stratifying according to gender, the association between the TT genotype and MI was statistically significant in males, only. Haplotype analysis revealed that the C-T and T-G haplotypes were associated with an increased risk of MI (p?=?0.012 and p?<?0.001, respectively).

Conclusions: Our work showed a significant association between the -1?C>T (rs1883832) polymorphism of the CD40 gene and MI in the Tunisians.     

High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600mg/l while residual methanol level was maintained below 2g/l. Clarification of culture supernatant through a 0.1μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5×10(8)IU/mg. The optimised process has a great potential for large scale production of fully functional hIFNα2b.     

Use of Taguchi's methods as a basis to optimize hybridoma cell line growth and antibody production in a spinner flask

Taguchi’s methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or β 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the α subunit CD11b. Anti β 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi’s methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways. This revised version was published online in August 2006 with corrections to the Cover Date.     

The new medium MDSS2N, free of any animal protein supports cell growth and production of various viruses

The development of media free of serum and animal or human proteins is of utmost importance for increasing the safety of biologicals produced for therapy and vaccination. In order to reduce the risk of contamination, we have modified the serum free medium MDSS2, a very efficient serum free medium for the production of various biologicals including experimental vaccines using different cell lines (Merten et al., 1994), by replacing the animal derived products by plant extracts. The new serum and animal protein free medium (MDSS2N) can be efficiently used for biomass production of various cell lines. These cells grow equally well or better in this new serum-free medium than in the old formulation (MDSS2): • BHK-21/BRS cells, adapted to MDSS2N, showed an overall specific growth rate of 0.0197 h-1 (μ_max = 0.0510±0.0058 h-1), whereas those cultivated in MDSS2 grew with an average specific growth rate of 0.0179 h-1 (μ_max = 0.0305±0.0177 h-1). • Vero cells grew with an average specific growth rate of 0.0159 h-1 and 0.0153 h-1 in MDSS2 and MDSS2N, respectively. Very similar growth rates were obtained in microcarrier cultures in stirred tank reactors: the specific growth rates were 0.0161 h-1 and 0.0166 h-1 for MDSS2 and MDSS2N cultures, respectively. • For MDCK cells, when cultured on microcarriers in bioreactors, a higher average specific growth rate was observed in MDSS2N than in MDSS2; values of 0.0248 h-1 and 0.0168 h-1, respectively, were obtained. The capacity of MDSS2N to support the production of different viruses was equally evaluated and it could be established that for certain viruses there are no or insignificant differences between MDSS2N and MDSS2 (influenza and polio virus), whereas, the production of rabies virus is somewhat reduced in MDSS2N when compared to MDSS2. The use of MDSS2N for cell culture and the production of various viruses is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.