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Objective: The ability to generate lung alveolar epithelial type II [ATII] cells from pluripotent stem cells [PSCs] enables the study of lung development, regenerative medicine, and modeling of lung diseases. The establishment of defined, scalable differentiation methods is a step toward this goal. This study intends to investigate the competency of small molecule induced mouse embryonic stem cell-derived definitive endoderm [mESC-DE] cells towards ATII cells
Materials and Methods: In this experimental study, we designed a two-step differentiation protocol. mESC line Royan B20 [RB20] was induced to differentiate into DE [6 days] and then into ATII cells [9 days] by using an adherent culture method. To induce differentiation, we treated the mESCs for 6 days in serum-free differentiation [SFD] media and induced them with 200 nM small molecule inducer of definitive endoderm 2 [IDE2]. For days 7-15 [9 days] of induction, we treated the resultant DE cells with new differentiation media comprised of 100 ng/ml fibroblast growth factor [FGF2] [group F], 0.5 micro g/ml hydrocortisone [group H], and A549 conditioned medium [A549 CM] [group CM] in SFD media. Seven different combinations of factors were tested to assess the efficiencies of these factors to promote differentiation. The expressions of DE- and ATII-specific markers were investigated during each differentiation step
Results: Although both F and H [alone and in combination] promoted differentiation through ATII-like cells, the highest percentage of surfactant protein C [SP-C] expressing cells [37%] were produced in DE-like cells treated by F+H+CM. Ultrastructural analyses also confirmed the presence of lamellar bodies [LB] in the ATII-like cells
Conclusion: These results suggest that hydrocortisone can be a promoting factor in alveolar fate differentiation of IDE2- induced mESC-DE cells. These cells have potential for drug screening and cell-replacement therapies
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@#Background: There is a meaningful necessity for a targeted therapy of essential tremor (ET), as medications have not been developed specifically for ET. For nearly a century, many drugs have been applied in the treatment of tremor but the drug treatment of ET remains still unknown. Some potential therapeutic factors such fingolimod (FTY720) can be effectively used to treat ET in animals. In the present research, the effect of FTY720, the immunomodulatory sphingosine 1-phosphate (S1P) analog, on degeneration of cerebellar and olivary neurons induced by harmaline in male rats was investigated. Methods: The animals were allotted into control dimethyl sulfoxide (DMSO), saline + harmaline [30 mg/kg, intraperitoneally, (i.p.)], harmaline + FTY720 (1 mg/kg, i.p, 1 h and 24 h before harmaline injection) groups (n = 10). The cerebellum and inferior olive nucleus (ION) were studied for neuronal degeneration using immunohistochemistry (IHC) and ultrastructural study by transmission electron microscopy (TEM) techniques. Results: Harmaline caused neuronal cell loss, caspase-3 mediated apoptosis, astrocytosis and ultrastructural changes in cerebellar Purkinje cells and inferior olive neurons. FTY720 exhibited neuroprotective effects on cerebellar Purkinje cells and inferior olivary neurons. Conclusion: These results suggest that FTY720 has potential efficacy for prevention of ET neurodegeneration and astrocytosis induced by harmaline in male rats.
ABSTRACT
Type 1 diabetes mellitus [T1DM] is a disease where destruction of the insulin producing pancreatic beta-cells leads to increased blood sugar levels. Both genetic and environmental factors play a part in the development of T1DM. Currently, numerous loci are specified to be the responsible genetic factors for T1DM; however, the mechanisms of only a few of these genes are known. Although several environmental factors are presumed responsible for progression of T1DM, to date, most of their mechanisms remain undiscovered. After several years of hyperglycemia, late onsets of macrovascular [e.g., cardiovascular] and microvascular [e.g., neurological, ophthalmological, and renal] complications may occur. This review and accompanying figures provides an overview of the etiological factors for T1DM, its pathogenesis at the cellular level, and attributed complications
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Objective: CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid
Materials and Methods: In this experimental study, single homology arm donor was applied along with a single guide RNA [sgRNA] specific to the homology region, and either Cas9 or its mutant nickase variant [Cas9n]. Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells [ESCs]
Results: Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus
Conclusion: While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies
Subject(s)
Gene Knock-In Techniques , Embryonic Stem Cells , Gene Targeting , Genetic Engineering/methods , Homologous Recombination/genetics , MiceABSTRACT
Background: Nepeta dschuparensis Bornm [NP] is used as a medicinal herb in Iran. In traditional medicine, this herb is extensively employed for curing ailments such as cardiovascular diseases. NP has antioxidant and anti-inflammatory properties. This project examined the effects of the NP extract on cyclooxygenase-2 [COX-2] and interleukin-1 +/- [IL-1 +/- ] protein levels and its efficacy in neuroprotection in a cerebral ischemiareperfusion model
Methods: Twenty-six male rats were randomly divided into 3 groups: 1] sham [n=6]: no middle cerebral artery occlusion [MCAO] procedure, 2] control [n=10]: MCAO procedure and treatment with normal saline, and 3] NP extract [n=10]: MCAO procedure and treatment with the NP extract [20 mg/kg, i.p.] at the beginning of reperfusion. To examine the injury caused by cerebral ischemia, we measured motor coordination and the infarct area using the rotarod test and triphenyl tetrazolium chloride staining, respectively. IL-1 +/- and COX-2 protein levels, as inflammatory markers, were measured by immunoblotting assay. The statistical analyses were performed using SPSS, version 16, and the data are expressed as means +/- SEMs. Statistical difference was evaluated using the one-way ANOVA, followed by the post hoc LSD test [P<0.01]
Results: Treatment with the NP extract significantly diminished the infarct volume and alleviated the motor coordination disorder induced by cerebral ischemia. The NP extract administration significantly attenuated the increase in IL-1 +/- and COX-2 protein levels too [P<0.01]
Conclusion: The beneficial effects of the NP extract are related to its ability to decrease the levels of IL-1 +/- and COX-2
ABSTRACT
Objective: following traumatic brain injury, disruption of blood-brain-barrier and consequent brain edema are critical events which might lead to increasing intracranial pressure [ICP], and nerve damage. The current study assessed the effects of aqueous date fruit extract [ADFE] on the aforementioned parameters
Materials and Methods: in this experimental study, diffused traumatic brain injury [TBI] was generated in adult male rats using Marmarou's method. Experimental groups include two pre-treatment [oral ADFE, 4 and 8 mL/kg for 14 days], vehicle [distilled water, for 14 days] and sham groups. Brain edema and neuronal injury were measured 72 hours after TBI. Veterinary coma scale [VCS] and ICP were determined at -1, 4, 24, 48 and 72 hours after TBI. Differences among multiple groups were assessed using ANOVA. Turkey's test was employed for the ANOVA post-hoc analysis. The criterion of statistical significance was sign at P<0.05
Results: brain water content in ADFE-treated groups was decreased in comparison with the TBI+vehicle group. VCS at 24, 48 and 72 hours after TBI showed a significant increase in ADFE groups in comparison with the TBI+vehicle group. ICP at 24, 48 and 72 hours after TBI, was decreased in ADFE groups, compared to the TBI+vehicle. Brain edema, ICP and neuronal injury were also decreased in ADFE group, but VCS was increased following on TBI
Conclusion: ADFE pre-treatment demonstrated an efficient method for preventing traumatic brain deterioration and improving pathological parameters after TBI
ABSTRACT
Objective: alcohol consumption is habitually accompanied by the use of other psychoactive substances, mostly tobacco. Nicotine and alcohol affect male accessory reproductive glands function. Most studies have been done on pathologic features of prostate, but there has been no systematic study on the seminal vesicle. Therefore, the aim of current study was to investigate the distribution of androgen receptor [AR] and estrogen receptors-beta [ER-beta] immune reactivities following long-term treatment of alcohol, nicotine or a combination of both substances
Materials and Methods: in this experimental study, a total of 40 adult Wistar rats, nine weeks of age, were used. Animals were randomly divided into four groups, including: i. Control group receiving normal saline 0.09%, ii. Ethanol group receiving ethanol 20% [2 ml/kg, via gavage], iii. Nicotine group receiving nicotine [0.1 mg/kg, subcutaneous injection], and iv. Ethanol-nicotine group receiving simultaneous ethanol 20% [2 ml/kg] and nicotine [0.1 mg/kg] treatment. All treatment lasted for eight weeks. Prior to intracardiac perfusion, blood sample was collected from left ventricle. The seminal vesicles were isolated and processed for paraffin blocking. The sample tissues were then studied for dis-tribution of AR and ER-beta immunereactivities using immunohistochemical [IHC] staining method. One way analysis of variance [ANOVA] and Tukey's test were performed for data analysis. A value of P<0.05 was considered significant
Results: our results revealed that the lowest mean number of positive cells belonged to the animals of ethanol-nicotine group that was followed by the ethanol, nicotine, and control groups, respectively. However, there was no significant difference regarding serum testosterone level among experimental groups
Conclusion: it was concluded that combination of both ethanol and nicotine may be a crucial factor in the expression levels of AR and ER-beta
ABSTRACT
Stroke is one of the main leading causes of mortality and disability in many countries. In the absence of definitive treatment search for, neuroprotective agents with minimal side effects should be continued. Natural nutrients can be ideal sources to produce safe and valuable agents for the management of stroke. Walnut kernel [WK] has numerous beneficial components with antioxidant and anti-inflammatory properties. The goal of this study was to investigate the protective effects of WK on neuronal injury and astrocyte reactivity after induction of focal cerebral ischemia in male rats. In this experimental study, Wistar male rats were divided into three groups: sham, control [fed with ordinary food] and walnut [fed with WK]. Each group consisted of 6 rats. The right middle cerebral artery was occluded for 15 min in the control and walnut groups. Forty-eight hours after reperfusion the animals were killed and their brains were processed for histological [Hematoxylin and Eosin staining] and immunohistochemical [Glial Fibrillary Acidic Protein, GFAP], and histochemical [TUNEL] studies. The results showed that WK significantly decreased neuronal death induced by cerebral ischemia; however, the density of GFAP positive astrocytes has been increased at the site of injury in the treatment group compared to the other 2 groups. In addition, WK significantly decreased the mortality rate of the animals due to cerebral ischemia. The results suggested that wk might provide protection against the cerebral ischemia-induced injuries in the rat brain through antioxidant and anti-inflammatory mechanisms
Subject(s)
Animals, Laboratory , Neurons/drug effects , Astrocytes/drug effects , Infarction, Middle Cerebral Artery , Protective Agents , Rats, WistarABSTRACT
The effects of cigarette and alcohol on male reproductive system have been studied mainly on the testis and prostate but studies on their co-administration on the seminal vesicle which produces about 70% of the semen volume are scarce. Therefore, the present study evaluated the effects of nicotine and/ or alcohol on the glandular epithelial cells of seminal vesicle in adult rats. In this study, 50 adult Wistar rats, aged 9 weeks, were randomly divided into five groups, including: sham, control [0.09% normal saline], 20% ethanol [2 ml/kg], nicotine [0.1 mg/kg] and ethanol-nicotine. Ethanol was given via oral gavage but nicotine was administered subcutaneously for 50 days. Blood samples were collected prior to intracardiac perfusion. The seminal vesicles were later dissected and tissue samples were stained by H and E for morphologic and morphometric studies. One-way ANOVA and Tukey's post-hoc test were used for data analysis. A pvalue <0.05 was considered statistically significant. The height of glandular epithelial cells of the seminal vesicle was reduced remarkably in rats in ethanol versus the control group [p<0.0001]. However, testosterone concentrations were not significantly different in the two groups. Semen volume, as well as its acidophilic properties in the lumen of most acini were lower in the ethanol comparative to the control group. Ethanol had the most negative effects on the cells and tissue structure of seminal vesicle compared to nicotine. The minimal effects seen by the simultaneous use of alcohol and nicotine on seminal vesicle structure might be attributed to the reduction of alcohol absorption following nicotine administration
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Clostridium Botulinum Type E neurotoxin heavy chain consists of two domains: the translocation domain as the N-terminal half and the binding domain as the Cterminal half [Hc]. One effective way to neutralize botulinum neurotoxin is to inhibit binding of this toxin to neuromuscular synapses with antibodies against binding domain. Two synthetic genes, coding for Hc [the full length binding domain] and the c-terminal quarter of binding domain [HcQ], were cloned in pET-28a vector and over-expressed in E. coli BL21 [DE3] cells. These recombinant proteins were purified by affinity Ni-NTA column [under native condition]. Mice were vaccinated with 2 microg of purified proteins, respectively; at step one with complete adjuvant, steps two and three with incomplete adjuvant and step four only with phosphate buffered saline [PBS]. Enzyme-linked immunosorbent assay [ELISA] has been performed with mice serum samples 14 days following their third and final vaccination. Binding activity of the purified proteins to ganglioside and synaptotagmin II was analyzed by ELISA. The results showed that HcQ and Hc could bind with ganglioside. Based on challenge experiments it was revealed that HcQ, Hc and BoNT/E toxoid could give protections in mice challenged with 10[2], 10[4] and 10[5] minimum lethal dose [MLD] dose of BoNT/E
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Helicobacter pylori multiplies and causes infection in human gastric mucosal layer. New approaches have focused on using specific treatments, such as immunotherapy, to limit this infection. Urease, as one of the most important virulent and antigenic factors of the bacterium, is a suitable target for this purpose. In order to prepare recombinant proteins, the synthetic genes for total ureC protein [UreCt] and its N [ureCn] and C [ureCc] terminal fragments were ligated into pET28a. The recombinant proteins were expressed in E. coli BL21[DE3]. White leghorn hens were injected with the purified recombinant proteins. IgY recovered from egg yolk, using PEG precipitation. Finally, urease neutralizing ability of the antibodies was evaluated by urease activity assay in presence of the purified IgY. SDS-PAGE analysis revealed a good expression and purification of the recombinant proteins. Indirect ELISA observation demonstrated high antibody titer in sera and egg yolks and high ability of IgY Anti-UreCt and IgY Anti-UreCc antibodies in recognition of urease subunit C. Anti-UreCT and Anti-UreCc IgYs were more potential H. pylori urease inhibitors than Anti-UreCn. While all three UreC fragments induce prophylactic responses. UreCt and UreCc possess almost equal responses. Anti-UreCc IgY has advantage of smaller size and is preferred for its activity and easier protein recovery and purification process. These features emphasize on importance of simpler, easier and cost effective antibody production