Insights into in vitro spermatogenesis in mammals: Past,present, future

Considering the self‐renewal and differentiation ability of pluripotent stem cells, some studies have pointed out the possibility of stem cell‐derived sperm production. Most studies that test this hypothesis have been conducted on rodents, with some promising results; however, studies on humans are progressing slowly, and have encountered technical and ethical hurdles. Established methods to differentiate stem cells—including embryoid bodies, co‐culturing, and various feeder cells—may provide a niche that is similar to in vivo conditions and resolve epigenetic abnormalities, but a gonadal‐like three‐dimensional structure is still required to produce germ cells with the correct imprinting. In the last few years, sperm‐like cells with fertilizing capacity were produced from mouse embryonic stem cells, and the resulting embryos from these cells yielded live offspring. Future research should move towards the use of adult stem cells, however, owing to the unavailability of embryonic cells in adults. More intensive research and techniques are required since in vitro spermatogenesis provides hope to individuals without mature sperm who cannot be treated, and may be a useful system to study the precise mechanism of spermatogenesis. In this review, we describe recent studies of in vitro spermatogenesis mechanisms and related techniques in mammals. We also discuss the possible cell surface markers and culture conditions that might improve in vitro spermatogenesis.     

Critical scaffolding regions of the tumor suppressor Axin1 are natively unfolded

The Wnt pathway tumor-suppressor protein Axin coordinates the formation of a critical multiprotein destruction complex that serves to downregulate β-catenin protein levels, thereby preventing target gene activation. Given the lack of structural information on some of the major functional parts of Axin, it remains unresolved how the recruitment and positioning of Wnt pathway kinases, such as glycogen synthase kinase 3β, are coordinated to bring about β-catenin phosphorylation. Using various biochemical and biophysical methods, we demonstrate here that the central region of Axin that is implicated in binding glycogen synthase kinase 3β and β-catenin is natively unfolded. Our results support a model in which the unfolded nature of these critical scaffolding regions in Axin facilitates dynamic interactions with a kinase and its substrate, which in turn act upon each other.     

Serum Arsenic and Lipid Peroxidation Levels in Patients with Multiple Sclerosis

Oxidative stress plays a crucial role in the pathogenesis of multiple sclerosis (MS). Previous studies have shown that oxidative stress is one of the main underlying mechanisms of arsenic-induced cellular damage. The aim of this study was to assess the serum levels of arsenic and its relationship with lipid peroxidation in MS patients from Tabriz, as the third polluted city of Iran. The study population included 38 MS female patients and 38 age-matched healthy controls. Serum malondialdehyde (MDA) and arsenic levels were measured using thiobarbituric acid reactive substances (TBARS) assay and electrothermal atomic absorption spectrometry, respectively. The results showed that the arsenic (P?<?0.01) and MDA (P?=?0.03) levels were significantly higher in patients with MS than those in control. Moreover, serum levels of arsenic and MDA were positively correlated in MS patients. The elevated levels of serum arsenic might explain the increased oxidative stress in MS patients. We suggest that high arsenic levels in serum may lead to MS development, and therefore, exposure to this metal should be limited.     

Phylogenetic analysis of Aphanius from the endorheic Kor River Basin in the Zagros Mountains,South‐western Iran (Teleostei: Cyprinodontiformes: Cyprinodontidae)

Morphologically similar populations of Aphanius that are currently considered as A. sophiae inhabit the endorheic Kor River Basin in the Zagros Mountains. Using genetic analysis based on mtDNA (cytochrome b), combined with examination of morphology (morphometry, meristics, otoliths), we discovered that what is thought to be A. sophiae is actually two distinct species, one of which is described as A. shirini sp. n. The males of the new species can be distinguished from those of all other Iranian inland Aphanius species by having only 7–10 clearly defined white flank bars, which is the lowest number of flank bars among the Iranian inland Aphanius species. Both males and females differ from all other Iranian inland Aphanius species by having a significantly longer caudal peduncle and a smaller dorsal fin depth. Based on the PhyML and Bayesian likelihood trees, A. shirini is sister to A. vladykovi from the Karoun Basin in the Zagros Mountains. Our results indicate that an ancient exorheic Kor River Basin existed in the Late Miocene and Pliocene. The close phylogenetic relationship between A. shirini and A. vladykovi suggests that the pre‐Pliocene drainage in the ancient Kor River Basin was directed to the north‐west (to the Karoun Basin), and not to the south‐east as in the present‐day Kor Basin. Both A. shirini and A. vladykovi represent the highest altitude records for Aphanius. We conclude that the splits of A. shirini and A. vladykovi can be linked to tectonic events in the Middle to Late Miocene, which created the highest altitudes (>3000 m) in the Zagros Mountains, and led to isolation of populations. The present‐day endorheic Kor Basin is known to have formed in the Late Pleistocene or Early Holocene, and the ‘young’ age of A. sophiae is clearly related to this history. Our results contribute to elucidate the link between geological history and the present‐day species diversity in the tectonically still active Zagros Mountains of Iran.     

The effects of pulsed magnetic field exposure on the permeability of leukemia cancer cells

Purpose: The newer methods of cancer treatment require new idea of drug delivery in cancer cells. Due to numerous researches electromagnetic field affect on cell function and cell membrane for possible therapeutic and drug delivery. In this article, we determined in vitro uptake of fluorescent dyes into the attached K562 cells due to time-varying magnetic field exposure. Method and material: The K562 cells were exposed to magnetic pulses via Magstim stimulator and double 70?mm coil. The strength and duration of pulses in all experiments were the same and three different frequencies of 0.25, 1 and 10?Hz pulses for 56, 112 and 28 numbers of pulses were applied (nine experimental groups) and uptake of Ly and PI was measured in each group. Result: Our results show that magnetic field can efficiently increase permeability. Among the treatment groups, the system gives the optimal permeabilization when cells are exposed to a train of 28 pulses with 1?Hz frequency.     

The biochemical value of urinary metalloproteinases 3 and 9 in diagnosis and prognosis of bladder cancer in Egypt

Background

Matrix metalloproteinases (MMPs) have long been associated with cancer-cell invasion and metastasis. Few studies are available that describe this association with bladder cancer either related or unrelated to schistosoma infection.Evaluating the urinary levels of MMP3 and MMP9 as diagnostic and prognostic biomarkers in different stages of schistosomal and non schistosomal bladder cancer was the aim of the present study.Urine samples were collected from 70 patients with schistosomal and non schistosomal bladder cancer at early and advanced stages and also from12 healthy volunteers as controls. Urinary levels of MMP-3 and MMP-9 was measured by ELISA technique. Sensitivity and specificity of both markers were determined.

Results

Urinary levels of both MMP-3 and MMP-9 were significantly elevated in all bladder cancer patients compared with controls. MMP-3 started to elevate in early stages of schistosomal bladder cancer ( 0.173 ng/ml) and non-schistosomal bladder cancer patients (0.308 ng/ml) compared to control (0.016 ng/ml) and remained elevated in advanced stages (0.166, 0.235 ng/ml) of both types of bladder cancer patients. In contrast, MMP-9 showed a significant elevation in advanced stages only of both schistosomal and non schistosomal bladder cancer patients (10.33, 21.22 ng/ml) compared to control (0.409 ng/ml) and this elevation of both markers was much higher in non schistosomal bladder cancer. Both Metalloproteinases were specific for the diagnosis of the disease but MMP-3 was more sensitive and this sensitivity was evident in the early stage (84.85% for MMP3, 27.28% for MMP9).

Conclusions

MMP3 may be the recommended urinary metalloproteinases as early diagnostic biomarker in the early stages of both types of bladder cancer although both MMP9 and MMP3 can be used in the diagnosis of advanced stages. Further studies are required on large number of urine samples to confirm these results.     

RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.     

Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53''s response to DNA damage.     

The Effect of Pulsed Magnetic Field on the Molecular Uptake and Medium Conductivity of Leukemia Cell

The cell membrane acts as a barrier that hinders free entrance of most hydrophilic molecules into the cell. Owing to the numerous applications, the introduction of non-permeate molecules into biologic cells has drawn considerable attention in the past years. The aim of our study was to investigate the effect of time-varying magnetic field on transmembrane molecular transport by measuring bleomycin cytotoxicity and conductivity modifying in K562 cells. The cells were exposed to magnetic pulses of 2.2 T strength peak and about 250-μs duration via Magstim stimulator and double 70-mm coil. Three different frequencies of 0.25, 1, and 10 Hz pulses for 56,112, and 28 numbers of pulses, respectively, were applied (nine experimental groups) and uptake and conductivity was measured in each group. Our results show that time-varying magnetic field increase transmembrane molecular transport and media conductivity; this enhancement is greater for 28 pulses with 1 Hz frequency. The observed uptake enhancement due to magnetic exposure is considerable.     

Effect of pH on growth and biochemical responses of <Emphasis Type="Italic">Dunaliella bardawil</Emphasis> and <Emphasis Type="Italic">Chlorella ellipsoidea</Emphasis>

The goal of the present investigation was to study the effect of pH on growth and biochemical responses of Dunaliella bardawil and Chlorella ellipsoidea when exposed to different pH values. The two tested microalgae could grow in a wide range of pH (4–9 for D. bardawil and 4–10 for C. ellipsoidea). The dry weight gain and the biochemical components of D. bardawil were greatly enhanced at pH 7.5. In contrast, dry weight and carbohydrate content of C. ellipsoidea attained their maximum values at the alkaline pH. On the other hand, the protein content of C. ellipsoidea recorded its highest value at pH 4, while the pigment content of the same alga was highest at pH 4, 6, and 7.5 and decreased at alkaline pH. Both pH 6 and pH 9 stimulated the accumulation of β-carotene, vitamin E and vitamin C in D. bardawil, with the highest values of the three compounds recorded at pH 9. In the case of C. ellipsoidea, β-carotene content increased at pH 6 and pH 10 as compared with the control, but the amount of β-carotene was much higher at pH 6 than at pH 10. Vitamin E content was higher in C. ellipsoidea cells at pH 10 than at pH 6. Both pH 6 and pH 10 caused a significant decline in vitamin C content of C. ellipsoidea.