Upstream or swim up processing technique: which one is more effective to select human sperm with high chromatin integrity

Background: sperm processing methods separate motile sperms with good morphology from dead and abnormal forms of sperms, immature germ cells, and non-sperm cells

Objective: the propose of this study was to compare the efficacy of upstream and swim-up processing techniques to separate sperms with the high quality especially in relation to sperm chromatin integrity

Materials and Methods: this experimental study used semen samples from 60 normozoospermic men. Specimens were divided into equal aliquots for processing by swim up [group A], and upstream [group B] methods and compare with control by raw semen [group C]. Sperm concentration, morphology, motility, DNA fragmentation and chromatin maturation were measured in these three groups

Results: the results revealed that sperm concentration in the swim up samples was significantly greater than upstream samples [p/= 0.4]. In addition, sperm DNA fragmentation and chromatin maturation were not significantly different between the three groups [p >/= 0.1]

Conclusion: according to results, apparently the upstream method had no significant efficiency to separate good quality sperms compare to swim up. Therefore, swim up seems to be a simple, inexpensive, reliable and widely available method with an efficient yield to separate motile sperm with good morphology and better chromatin integrity for insemination in the infertility clinics

Retinopathy of Prematurity-assist: Novel Software for Detecting Plus Disease

PURPOSE: To design software with a novel algorithm, which analyzes the tortuosity and vascular dilatation in fundal images of retinopathy of prematurity (ROP) patients with an acceptable accuracy for detecting plus disease. METHODS: Eighty-seven well-focused fundal images taken with RetCam were classified to three groups of plus, non-plus, and pre-plus by agreement between three ROP experts. Automated algorithms in this study were designed based on two methods: the curvature measure and distance transform for assessment of tortuosity and vascular dilatation, respectively as two major parameters of plus disease detection. RESULTS: Thirty-eight plus, 12 pre-plus, and 37 non-plus images, which were classified by three experts, were tested by an automated algorithm and software evaluated the correct grouping of images in comparison to expert voting with three different classifiers, k-nearest neighbor, support vector machine and multilayer perceptron network. The plus, pre-plus, and non-plus images were analyzed with 72.3%, 83.7%, and 84.4% accuracy, respectively. CONCLUSIONS: The new automated algorithm used in this pilot scheme for diagnosis and screening of patients with plus ROP has acceptable accuracy. With more improvements, it may become particularly useful, especially in centers without a skilled person in the ROP field.

Efficacy of intrauterine injection of granulocyte colony stimulating factor [G-CSF] on treatment of unexplained recurrent miscarriage: a pilot RCT study

Background: Endometrium undergoes several changes in structure and cellular composition during pregnancy. Granulocyte Colony-stimulating Factor [GCS-F] is an important cytokine with critical role in embryo implantation and pregnancy. The aim of the present study was to evaluate the impact of intrauterine injection of G-CSF in patients who suffer from unexplained recurrent miscarriage [RM]

Methods: In the present randomized clinical trial, a total of 68 patients were randomly allocated into two study groups including intrauterine G-CSF [n=23, 300mg] injection and control group [n=27, no G-CSF injection]. Eighteen out of 68 patients were excluded from the final analysis due to different reasons. All patients were in Ovulation Induction [I/O] cycle. In G-CSF group, intrauterine injection of G-CSF was done twice in the cycle. All enrolled patients were under 40 years old and had at least two unexplained pregnancy losses. Pregnancy was evaluated by titer of bhCG, presence of gestational sac [implantation] and fetal heart rate [clinical pregnancy] was assessed by vaginal ultrasonography. Student's T test and Mann-Whitney U were used for analysis. The p

Results: No significant differences were observed between the two study groups when the rates of chemical pregnancy [26.1%vs.29.6%, p=0.781], implantation [26.1%vs.22.2%, p=0.750], clinical pregnancy [17.4% vs.11.1%, p=0.689] and abortion [33%vs.37.5%, p=0.296] were compared

Conclusion: In our study, no significant difference was observed between the two study groups when the rates of chemical pregnancy, implantation, clinical pregnancy and abortion were compared

Reconstruction of mammalian oocytes by germinal vesicle transfer: a systematic review

Nuclear transfer procedures have been recently applied for clinical and research targets as a novel assisted reproductive technique and were used for increasing the oocyte activity during its growth and maturation. In this review, we summarized the nuclear transfer technique for germinal vesicle stage oocytes to reconstruct the maturation of them. Our study covered publications between 1966 and August 2017. In result utilized germinal vesicle transfer techniques, fusion, and fertilization survival rate on five different mammalian species are discussed, regarding their potential clinical application. It seems that with a study on this method, there is real hope for effective treatments of old oocytes or oocytes containing mitochondrial problems in the near future

Rational development of a polycistronic plasmid with a CpG-free bacterial backbone as a potential tool for direct reprogramming

Objective: Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector

Materials and Methods: In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells

Results: In the present study, we developed a nonviral episomal vector named pLENSO/Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming

Conclusion: According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future

Effect of rocker bar ankle foot orthosis on functional mobility in post-stroke hemiplegic patients: timed up and go and gait speed assessments

Objectives: Ankle Foot Orthoses [AFOs] are widely utilized to improve walking ability in hemiplegic patients. The present sludy aimed to evaluate the effect of Rocker bar Ankle Foot Orthosis [RAFO] on functional mobility in posl-stroke hemiplegic patients

Methods: Fifteen hemiplegic patients [men and women] who were at leasl 6-months posl-stroke and able to walk without an assisUve device for at lea

The patients were examined for three conditions: shoes only, with SAFO and with RAFO. Their functional mobility was evaluated through 10-meter walk test and Timed Up and Go [TUG] tesl In addition, paired t-tesl; was used to analyze the obtained data

Results: When the patients used RAFO, their gait speed significantly increased [P<0.05]. Moreover, the time of performing TUG tesl experienced a significant decrease using RAFO compared with utilizing shoe only [P<0.05]

Discussion: RAFO led to a significant improvement in functional mobility in hemiplegic patients posl stroke. This may be due to the positive effect of rocker modification on improving push off and transferring weight during the Glance phase of gait

Expression and Activity Evaluation of Reteplase in Escherichia coli TOP10

Reteplase is a part of tissue plasminogen activator [t-PA] used for the removal of thrombi in blood vessels. In the present study we express the Reteplase gene in Escherichia coli TOP10 and then its thrombolytic activity was measured. The recombinant plasmid pBADgIIIA was transformed into the competent Escherichia coli TOP10 and then transformed bacteria was seeded into bioreactor containing 1.5 L LB medium and induced by 0.02% L-Arabinoseat 37 degreeC, pH 7, and 180 rpm until OD 600 of 0.6 was reached. Samples were analyzed by SDS-PAGE and western blotting and the expression of Reteplase was examined. Finally the activity of this recombinant protein was evaluated using Chromogenic Activity Assay Kit. The presence of Reteplase in transformed Escherichia coli TOP10 was examined by western blotting which revealed that the target protein in form inclusion body was expressed as a unique band at 39 and the refolded Reteplase was 66 KDa. The amount of protein produced was 90.5 microg/mL and its activity was determined as 0.8 units. In this study, the expression of Reteplase in Escherichia coli TOP10 was scaled up under optimum condition. Furthermore we earned Reteplase with partially suitable thrombolytic activity

Inactivation of aprE gene in bacillus subtilis 168 by homologus recombination

One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis [B. Subtilis]. One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate [FITC]-labeled casein-substrate. The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase

Obstructive sleep apnea and postoperative complications in patients undergoing coronary artery bypass graft surgery: a need for preventive strategies

Obstructive sleep apnea [OSA] is very frequent and often unrecognized in surgical patients. OSA is associated with perioperative complications. We evaluated the effects of OSA on postoperative complications and hospital outcomes in patients undergoing coronary artery bypass graft [CABG] surgery. Candidates of elective CABG were evaluated by the Berlin questionnaire for OSA. After surgery, patients were assessed for postoperative complications, re-admission to the Intensive Care Unit [ICU], duration of intubation, re-intubation, days spent in the ICU and the hospital. We studied 61 patients who underwent CABG from which 25 [40.9%] patients had OSA. Patients with OSA had higher body mass index [29.5 +/- 3.9 vs. 26.0 +/- 3.7 kg/m2, P = 0.003] and higher frequency of hypertension [68.0% vs. 30.5%, P = 0.003], dyslipidemia [36.0% vs. 5.5%, P = 0.004], and pulmonary disease [16.0 vs. 2.7%, P = 0.08]. Regarding the surgical outcomes, OSA patients had longer intubation duration [0.75 +/- 0.60 vs. 0.41 +/- 0.56 days, P = 0.03]. Obstructive sleep apnea is frequent, but unrecognized among patients undergoing CABG. In these patients, OSA is associated with prolonged intubation duration. Preventing these problems may be possible by early diagnosis and management of OSA in cardiac surgery patients. Further studies with larger sample of patients and longer follow-ups are required in this regard

Molecular cloning and expression of rat micro -opioid receptor in Escherichia coli [BL21]

The micro [mu] opioid receptors, which mediate the effects of morphine, are widely distributed in brain. The purpose of this study was to design a simple expression system for rat micro -receptor in Escherichia coli [BL21]. In this laboratory study, rat micro -receptor cDNA was isolated from pcDNA3 vector using Xba1 and Hind3 restriction enzymes. pET-15b was digested by Nco1 restriction enzyme. micro -receptor cDNA and pET-15b formed a recombinant DNA that was transformed to Escherichia coli [BL21]. The insert presence was proved by Rsa1 restriction enzyme and the induction of its expression was performed using IPTG. Finally, the presence of desired insert was confirmed using RSA1, and the colonies that had correct orientation in gene containing plasmid were used for further studies. On the SDS-page gel electrophoresis, a 33 kDa band was observed when IPTG was used at 0.5 and 1 mM concentrations, that is equal to calculated molecular weight of rat micro -receptor. At the end of this project, the expression of rat micro -receptor by IPTG induction was successfully performed

Common proteomic technologies, applications, and their limitations

Proteomics refers to the analysis of expression, localization, functions, posttranslational modifications, and interactions of proteins expressed by a genome at a specific condition and at a specific time. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. In this review, we have focused on the proteomics methods: gel-based and gel-free techniques and discussed their applications and challenges in the field of proteomics

Cloning and expression of functional reteplase in escherichia coli TOP10

Production of tissue Plasminogen Activator protein [t-PA] in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli [E. coli] bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method. Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification. We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein

Preparation and in vitro characterization of chitosan nanoparticles containing Mesobuthus eupeus scorpion venom as an antigen delivery system

Hydrophilic nanoparticles have been widely investigated in recent years as delivery systems for therapeutic macromolecules such as antigens. In the present study Mesobuthus eupeus venom-loaded chitosan nanoparticles were prepared via ionic gelation of tripolyphosphate (TPP) and chitosan. The optimum encapsulation efficiency (91.1 percent) and loading capacity (76.3 percent) were obtained by a chitosan concentration of 2 mg/mL, chitosan-to-TPP mass ratio of 2 and M. eupeus venom concentration of 500 µg/mL. The average nanoparticle size at optimum conditions was determined by Zetasizer (Malvern Instruments, UK). The nanoparticle size was about 370 nm (polydispersity index: 0.429) while the zeta potential was positive. Transmission electron microscope (TEM) imaging showed a spherical, smooth and almost homogenous structure for nanoparticles. Fourier transform infrared (FTIR) spectroscopy confirmed tripolyphosphoric groups of TPP linked with ammonium groups of chitosan in the nanoparticles. The in vitro release of nanoparticles showed an initial burst release of approximately 60 percent in the first ten hours, followed by a slow and much reduced additional release for about 60 hours. It is suggested that the chitosan nanoparticles fabricated in our study may provide a suitable alternative to traditional adjuvant systems.(AU)

Optimization of the expression of genes encoding poly [3-hydroxyalkanoate] synthase from pseudomonas aeruginosa PTCC 1310 in escherichia coli

Over the years, the use of plastics has complicated the problem of disposal of solid wastes. One strategy to reduce plastic waste is the use of biodegradable plastics. A group of these plastics are polyhydroxyalkanoates [PHAs]. To date more than 250 different microorganisms are known to synthesize and accumulate PHA. Most Pseudomonas strains are able to accumulate mcl-PHA. In previous studies, the phaC1 and phaC2 genes were identified in Pseudomonas aeruginosa [P.aeruginosa] PTCC 1310 and were cloned. The aim of this study was to express these genes and optimize the conditions for their expression. The inserts obtained from vectors pTZPHAC1 and pTZPHAC2 were subcloned into pET15b expression vector. After transformation of competent Escherichia coli [E.coli] BL21 [DE3] cells with recombinant plasmids, expression was induced using IPTG. By changing expression conditions such as IPTG concentration, time and temperature of incubation with IPTG, the expression conditions for these enzymes were optimized, and the obtained results were compared using proper statistical analysis. The PHA synthase genes were induced with IPTG and the expressed 62 kDa protein was observed and purified. By changing expression conditions, 1 mM IPTG, 37°C and a 2 hr incubation provided the highest level of protein production in E.coli cells. These results suggest that induction condition of PhaC genes can influence expression of PHA synthase enzymes

Effectiveness of massage therapy on the mood of patients after open-heart surgery

Cardiovascular diseases have the highest death rates in human society. Coronary artery disease is among the most important of these diseases. No treatment of cardiovascular disease has as much impact on the quality of life of the patients as the heart surgery. The recovery from heart surgery is associated with symptoms of pain and psychological distress. In the early recovery period, the patients will face moderate symptoms of anxiety and depression. In this regard, various measures of nursing, as complementary therapy practices have been performed to help the patients for overcoming the physical and psychological needs. One of these methods, in recent years has been the use of complementary and alternative therapies, particularly massage therapy, after heart surgery. Thus, the aim of this study was to determine the effectiveness of massage therapy on the mood of patients after open-heart surgery in Isfahan Chamran Hospital during 2010-11. In this study 72 patients, who underwent coronary artery bypass surgery, were selected. They were randomly assigned to the two case and control groups. The patients of the case group [n = 36] received Swedish massage for 20 minutes in 4 sessions in 4 consecutive days, 3 to 6 days after the open-heart surgery. The patients in the control group received only the routine care. The mood questionnaire [POMS] which was used in this study has been completed the day before the start of the study and intervention and again after the last day of the intervention. SPSS software version 12 and descriptive and inferential statistical methods were used for data analysis. The comparison of study results showed that massage decreased the overall rating of the patients' mood after the surgery. The use of massage therapy as an effective nursing intervention can improve the patients' mood after openheart surgery. Due to the low cost and simplicity of this method, it can perhaps be used as a complement to drug therapy and postoperative interventions used in these patients.