Inhibition of Autophagy in Heat-Stressed Sperm of Adult Mice: A Possible Role of Catsper1, 2 Channel Proteins.

Objective: Various phenomena guarantee gamete maturation and formation at all stages of evolution, one of which is autophagy playing a critical role in the final morphology of gametes, particularly sperms. Autophagy is influenced by oxidative stress, disturbances of calcium homeostasis, and hyperthermia conditions. The current study aimed to assess the autophagy-related proteins along with the activity of sperm calcium channel (CatSper) proteins following the induction of heat stress (HS). Methods: The study sample includes two groups of adult mice: sham and HS groups. In the HS group, the right testis was transferred to the abdominal cavity for 120 hours and then returned to the scrotum where it remained for 7 days. After 7 days, the testis and epididymis were removed to conduct real-time, immunohistochemical studies, sperm parameter evaluation, and seminiferous tubule assessment. In this study, the expression and distribution of autophagy proteins were measured. Plus, CatSper1 and CatSper2 were evaluated as proteins of calcium channels. Results: The results of the present study demonstrated that the expression intensity of autophagy indices in seminiferous tubules decreased significantly after HS induction, which was associated with a decrease in the distribution of CatSper proteins in the sperms. HS led to morphological changes in sperm, reduced motility and viability of sperm, and decreased spermatogenesis indices. Conclusion: In this study, following heat stress, the decrease in CatSper protein distribution may lead to the structural disorder of CatSper channels, which could strongly affect autophagic activity. Also, disruption of spermatogenesis and sperm parameters may be the consequence of decreased autophagy activity.

Autophagy comparative after decompression of tunica albuginea in testicular torsion in mature and immature rat.

BACKGROUND: One of the types of catabolic processes in cells is autophagy, which maintains cell homeostasis. It is also known as a defense response to oxidative stress. This study aimed to track the autophagic activity of testicular tissue in mature and immature rats in 3 conditions: (1) physiologic, (2) under increased pressure in the tunica albuginea compartment following the induction of testicular torsion, and (3) decompression of this compartment by flap technique. METHODS: This study was performed on 72 Wistar rats divided into 3 mature and 3 immature groups, each of the groups including Sham group, testicular torsion induction group for 6 hours (Torsion Detorsion), and testicular torsion induction group for 6 hours, followed by the flap technique. RESULTS: In the physiologic state or Sham, autophagic indices (LC3, Beclin-1) are active, and more activity was observed in mature rats, compared with immature ones, which indicated the activity of this process during development and maturity. In the Torsion Detorsion group, significant expression of autophagy and apoptosis indices (caspase3) was observed along with the degradation of spermatogenesis and steroidogenesis. It seems that in this state, the activation of autophagy leads to programmed death and is in line with apoptosis. Finally, in the flap technique group, an increase in the expression of autophagy indices was accompanied by a reduction in the expression of apoptotic indices and improved spermatogenesis and steroidogenesis. CONCLUSION: Decompression of tunica albuginea by flap technique can activate of autophagy process to save tissue function and eliminate stress caused by testicular torsion.

Evaluation of Sertoli cell autophagy associated with laminin, fibronectin, and caspase-3 proteins' alteration, following testicular torsion rat.

Autophagy is a vital process that maintains cellular homeostasis by joining lysosomes and providing energy production substrates. In testicular tissue, Sertoli cells play functional roles in spermatogenesis and steroidogenesis. It is well known that autophagy physiologically occurs in the Sertoli cells. Under pathological conditions, such as testicular torsion, autophagy can be activated under high-stress stimuli. It is worth noting that Sertoli cells receive autophagy-induced signals through some extracellular matrix proteins, e.g. laminin and fibronectin. The present study aims to evaluate Sertoli cells' autophagy-associated extracellular matrix proteins' alteration following testicular torsion in rat model. The animals were divided into two groups as sham and testicular torsion/detorsion groups. In the testicular torsion/detorsion group, testicular torsion was maintained for 6 hr, followed by detorsion for 14 days. The obtained results revealed that testicular torsion-induced oxidative stress leads to increased autophagy in Sertoli cells as well as the whole testicular tissue. Moreover, extracellular matrix proteins including laminin and fibronectin act as autophagy-regulating proteins, in which their expression levels are reduced and increased respectively. In addition, the level of caspase-3, as an autophagy inhibitory protein, did not increase significantly in the cytoplasm of Sertoli cells as opposed to whole testicular tissue, indicating that autophagy is active after testicular torsion in these cells.

Improving sciatic nerve regeneration by using alginate/chitosan hydrogel containing berberine.

Peripheral nerve injuries are the common results of trauma that lead to pain and handicap in patients. Berberine due to its properties like antibiotic, immunostimulant, antitumor, antimotility, and positive effect on neurological disorders can be used to enhance peripheral nerve injuries. In this study, alginate/chitosan hydrogel containing different concentrations of berberine (0, 0.1, 1, 10% (w/v)) was created, evaluated, and applied as a scaffold for sciatic nerve regeneration. To prepare hydrogel, sodium alginate was dissolved in distilled water and cross-linked with CaCl2, and chitosan was dissolved in acetic acid and cross-linked with ß-glycerol phosphate. The structure, release, swelling, weight loss, cytocompatibility, and hemocompatibility of the prepared hydrogels were assessed. The sciatic nerve crush was created in rats and fabricated hydrogels were injected, and functional analysis was used to evaluate their effectiveness. The results of physical characterization of the hydrogel indicated that the initial average pore size was about 39 µm and about 70% of the main weight of hydrogels was lost after incubation for 21 days and hemocompatibility of hydrogels was also confirmed. The MTT assay showed the cytocompatiblity of hydrogels and also indicated that berberine has dose-dependence effect on cell proliferation. The in vivo results showed the positive effect of berberine especially the hydrogel contained 1% of berberine on regeneration of sciatic nerve. Based on this study, Alg/Chit hydrogel can be applied as a treatment to heal peripheral nerve injuries. Graphical abstract.

Apelin-13 Protects PC12 Cells Against Methamphetamine-Induced Oxidative Stress, Autophagy and Apoptosis.

Methamphetamine (METH) is a potent psychomotor stimulant that has a high potential for abuse in humans. In addition, it is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to METH causes psychosis and increases the risk of Parkinson's disease. Apelin-13 is a novel endogenous ligand which studies have shown that may have a neuroprotective effect. Therefore, we hypothesized that Apelin-13 might adequately prevent METH-induced neurotoxicity via the inhibition of apoptotic, autophagy, and ROS responses. In this study, PC12 cells were exposed to both METH (0.5, 1, 2, 3, 4, 6 mmol/L) and Apelin-13 (0.5, 1.0, 2.0, 4.0, 8.0 µmol/L) in vitro for 24 h to measure determined dose, and then downstream pathways were measured to investigate apoptosis, autophagy, and ROS responses. The results have indicated that Apelin-13 decreased the apoptotic response post-METH exposure in PC12 cells by increasing cell viability, reducing apoptotic rates. In addition, the study has revealed Apelin-13 decreased gene expression of Beclin-1 by Real-Time PCR and LC3-II by western blotting in METH-induced PC12 cells, which demonstrated autophagy is reduced. In addition, this study has shown that Apelin-13 reduces intracellular ROS of METH-induced PC12 cells. These results support Apelin-13 to be investigated as a potential drug for treatment of neurodegenerative diseases. It is suggested that Apelin-13 is beneficial in reducing oxidative stress, which may also play an important role in the regulation of METH-triggered apoptotic response. Hence, these data indicate that Apelin-13 could potentially alleviate METH-induced neurotoxicity via the reduction of oxidative damages, apoptotic, and autophagy cell death.

Nesfatin-1 protects PC12 cells against high glucose-induced cytotoxicity via inhibiting oxidative stress, autophagy and apoptosis.

OBJECTIVE: Diabetic neuropathy (DN) is the most common complication of diabetes mellitus. It is thought that neuronal cell death which is mainly due to reactive oxygen species (ROS) overproduction in the cells is responsible for most symptoms of this disorder. Nesfatin-1 has identified recently as a novel endogenous neuropeptide which recent studies have shown that it may have a protective effect. Therefore, we postulated that Nesfatin-1 might adequately prevent from high glucose-induced cell injury via inhibition of apoptotic, autophagy, and ROS responses. METHODS: In this study, PC12 cells were pretreated with different concentrations of Nesfatin-1 (1-100 ng/ml) and then co-treated with Nesfatin-1 and glucose (125 mM) for 48 h, and downstream pathways then were evaluated to investigate ROS, apoptosis, and autophagy. RESULTS: Results of this study showed that Nesfatin-1 can not only inhibit from intracellular ROS overproduction-induced by high glucose in PC12 cells (p < 0.0001) but also reduce the apoptotic cell death in PC12 cells following high glucose exposure by increasing cell viability and reducing apoptotic rates (p < 0.05). Furthermore, Nesfatin-1 decreased the LC3-II levels by western blotting (p < 0.0001), which showed a reduction in autophagy. CONCLUSION: These results support the idea that Nasfatin-1can protect PC12 cells against high glucose-induced cell injury by inhibition of apoptosis, autophagy and ROS production and can be considered as a potential drug for treatment of diabetic neuropathy.

Predictive and therapeutic biomarkers in chimeric antigen receptor T-cell therapy: A clinical perspective.

The adoptive transfer of genetically engineered T cells modified to express a chimeric antigen receptor (CAR) has shown remarkable activity and induces long-term remissions in patients with advanced hematologic malignancies. To date, little is known about predictive indicators of therapeutic efficacy or serious toxicity after CAR T-cell therapy in clinical practice. Biomarkers are not only potentially able to inform physicians and researchers of immunotherapy targets in particular but could also be used to monitor the effectiveness of treatments and to predict incidence of side effects in some circumstances. Identification of new biomarkers can therefore not only contribute to the development of new therapeutic and prognostic strategies for CAR T-cell therapy for cancer but also help to generate improved clinical practices for early recognition and minimization of adverse effects while preserving the antitumor activity of the CAR T cells. Herein, we will consider a variety of predictive and therapeutic biomarkers in CAR T-cell therapy and the state of current understanding of their clinical utility. The incorporation of biomarker studies in CAR T-cell clinical trials and practice will help to realize the potential clinical benefit of biomarker-guided therapy.

Exosomes and microRNAs: New potential therapeutic candidates in Alzheimer disease therapy.

Exosomes are biological nanocarriers which could be involved in a variety of basic physiological events. They exert their effects via targeting their cargos (i.e., DNAs, messenger RNAs, microRNAs [miRNAs], and proteins) to host cells, which led to change behaviors of recipient cells. One of the important aspects of exosomes is the roles of them in disease conditions. Increasing evidence indicated that exosomes are one of the main players in Alzheimer's disease (AD) pathogenesis. Hence, it seems that these nanocarriers could be used as diagnostic and therapeutic biomarkers in AD treatment. Another important player in AD pathogenesis is miRNA. MiRNAs are short noncoding RNAs which exert their effects as epigenetic regulators. These molecules involved in different stages of AD. Therefore, miRNAs could be used as prognostic, diagnostic, and therapeutic biomarkers in AD. Here, we summarized various roles of exosomes and application of them in AD pathogenesis. Moreover, we highlighted the utilization of miRNAs as a therapeutic option in AD therapy.

Gene-knocked out chimeric antigen receptor (CAR) T cells: Tuning up for the next generation cancer immunotherapy.

Recently clinical trials utilizing genetically engineered T cells expressing a chimeric antigen receptor (CAR) that is half monoclonal antibody and half T-cell receptor have demonstrated remarkable response in patients with advanced cancers like relapsed or refractory acute lymphoblastic leukemia (ALL) and lymphoma. Moreover, emerging chimeric genome editing tools such as zinc-finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas composed of sequence-specific DNA binding module(s) linked to a non-specific DNA cleavage domain have made possible to dramatically expand the ability to manipulate cells aim to treat and/or study a wide range of diseases including cancer. Here, we will discuss how joint application of these two chimeras will help us to manipulate CAR T cells aiming to enhance the efficacy of CAR T cell therapy in preclinical and clinical settings.

MicroRNAs and exosomes in depression: Potential diagnostic biomarkers.

Depression is known as one of important psychiatric disorders which could be associated with disability among various populations. Diagnostic and statistical manual of mental disorders, 4th edition (DSM-IV) and international statistical classification of diseases and related health problems (ICD-10) could be used as subjective diagnostic schemes for identification of mental disorders such as depression. Utilization of subjective diagnostic schemes are associated with limitations. Hence, it seems that employing of new diagnosis platforms is required. Multiple lines of evidence indicated that measurement of several biomarkers could be useful for detection patients with depression. Among of various types of biomarkers, microRNAs (miRNAs) have been emerged as powerful tools for diagnosis patients with depression. MiRNAs are small non-coding RNAs which act as epigenetic regulators. It has been showed that these molecules have critical roles in pathogenesis of many diseases such as depression. These molecules exert their effects via targeting a variety of cellular and molecular pathways involved in initiation and progression of depression. Hence, miRNAs could be used as diagnostic and therapeutic biomarkers in patients with depression. Besides miRNAs, exosomes as nano- carriers could have been emerged as diagnostic biomarkers in several diseases such as depression. These vesicles are able to carry several cargos such as DNAs, proteins, mRNAs, and miRNAs to recipient cells. Here, we summarized several miRNAs involved in pathogenesis and response to treatment of depression which could be used as diagnostic biomarkers. Moreover, we highlighted exosomes as new diagnostic biomarkers for patients with depression.

Breast cancer diagnosis: Imaging techniques and biochemical markers.

Breast cancer is a complex disease which is found as the second cause of cancer-associated death among women. Accumulating of evidence indicated that various factors (i.e., gentical and envirmental factors) could be associated with initiation and progression of breast cancer. Diagnosis of breast cancer patients in early stages is one of important aspects of breast cancer treatment. Among of various diagnosis platforms, imaging techniques are main diagnosis approaches which could provide valuable data on patients with breast cancer. It has been showed that various imaging techniques such as mammography, magnetic resonance imaging (MRI), positron-emission tomography (PET), Computed tomography (CT), and single-photon emission computed tomography (SPECT) could be used for diagnosis and monitoring patients with breast cancer in various stages. Beside, imaging techniques, utilization of biochemical biomarkers such as proteins, DNAs, mRNAs, and microRNAs could be employed as new diagnosis and therapeutic tools for patients with breast cancer. Here, we summarized various imaging techniques and biochemical biomarkers could be utilized as diagnosis of patients with breast cancer. Moreover, we highlighted microRNAs and exosomes as new diagnosis and therapeutic biomarkers for monitoring patients with breast cancer.

The siRNA-Mediated Down-Regulation of Vascular Endothelial Growth Factor Receptor1.

BACKGROUND: Angiogenesis is an important biological process involved in the proliferation of endothelial cells, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is considered as a prominent regulator of angiogenesis which exerts the aforementioned effect(s) through its respective receptors (VEGFR1 and VEGFR2). VEGF receptors are targeted as a therapeutic candidate for cancer growth inhibition. RNAi as a new and promising strategy has provided a useful means to specifically suppress gene expression in cancer cells. OBJECTIVES: The current study aimed to down-regulate expression of the VEGFR1 using siRNA. MATERIALS AND METHODS: This experimental study designed specific siRNAs against VEGFR1. Total RNA was extracted from human umbilical vain endothelial cell (HUVEC) and subsequently cDNA was synthetized. PCR was performed using specific primers to amplify the target gene. After double digestion and purification, the gene was cloned into pEFGP-N1 expression vector. Then, AGS cells were transfected with recombinant pEGFP-N1 using lipofectamin. The gene expression and down-regulation were evaluated by fluorescence scanning, reverse transcription PCR (RT-PCR) and Western blot techniques. RESULTS: Fluorescent scanning, RT-PCR (27.68%) and western blot analysis (31.06%) showed that the expression of VEGFR1 was suppressed effectively. CONCLUSIONS: The results of the current study showed that specifically designed siRNA can be considered as an appropriate strategy to suppress gene expression and might be a promising tool to prevent angiogenesis.

Utilization of Failure Mode and Effects Analysis (FMEA) Method in Increasing the Revenue of Emergency Department; a Prospective Cohort Study.

INTRODUCTION: The balance between revenue and cost of an organization/system is essential to maintain its survival and quality of services. Emergency departments (ED) are one of the most important parts of health care delivery system. Financial discipline of EDs, by increasing the efficiency and profitability, can directly affect the quality of care and subsequently patient satisfaction. Accordingly, the present study attempts to investigate failure mode and effects analysis (FMEA) method in identifying the problems leading to the loss of ED revenue and offer solutions to help fix these problems. METHODS: This prospective cohort study investigated the financial records of ED patients and evaluated the effective errors in reducing the revenue in ED of Imam Hossein hospital, Tehran, Iran, from October 2007 to November 2009. The whole department was divided into one main system and six subsystems, based on FMEA. The study was divided into two phases. In the first phase, the problems leading to the loss of revenue in each subsystem were identified and weighted into four groups using risk priority number (RPN), and the solutions for fixing them were planned. Then, in the second phase, discovered defects in the first phase were fixed according to their priority. Finally, the impact of each solution was compared before and after intervention using the repeated measure ANOVA test. RESULTS: 100 financial records of ED patients were evaluated during the first phase of the study. The average of ED revenue in the six months of the first phase was 73.1±3.65 thousand US dollars/month. 12 types of errors were detected in the predefined subsystems. ED revenue rose from 73.1 to 153.1, 207.06, 240, and 320 thousand US dollars/month after solving first, second, third, and fourth priority problems, respectively (337.75% increase in two years) (p<0.001). 111.0% increase in the ED revenue after solving of first priority problems revealed that they were extremely indispensable in decreasing the revenue (p<0.0001). CONCLUSION: The findings of the present study revealed that FMEA could be considered as an efficient model for increasing the revenue of emergency department. According to this model, not recording the services by the nursing unit, and lack of specific identifying code for the patients moving from ED to any other department, were the two first priority problems in decreasing our ED revenue.