Ml Of Phosphate-buffered Saline Research Articles

Toxoplasma gondii: A Possible Inducer of Oxidative Stress in Reproductive System of Male Rats.

Background:Toxoplasmosis is suspected to have adverse effects on the male reproductive system. We aimed to determine the possible role of Toxoplasma gondii in oxidative stress in reproductive system of male rats.Methods:This study was performed from 2018 until 2019 at the Parasitology Research Laboratory of Hamadan University of Medical Sciences, Hamadan, Iran. Eighty male Wistar rats were randomly divided to control and test groups. The animals in the test group were inoculated by 107 T. gondii RH strain tachyzoites and the control group were injected by 0.2 ml of phosphate-buffered saline. The both groups were following every 10 days until day 80 post inoculation. Oxidative stress markers (OSMs) including antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and nonenzymatic markers including malondialdehyde (MDA), reduced glutathione (GSH) and total antioxidant capacity (TAC) were assessed in testis and serum of T. gondii infected rats.Results:After post inoculation, the variations of the OSMs in the testis tissue of infected rats were as follows: a significant decrease of SOD on day 80 (P=0.03), and CAT activity were detected on day 60 and 80 (P=0.04 and P=0.01) respectively. In addition, GSH (P =0.01) and TAC (P =0.03) concentration were significantly reduced on day 80. On the contrary, the concentration of MDA (P =0.01) was increased 70 days after infection. In addition, consistent changes with the tissue testis were observed in the serum OSMs of infected rats.Conclusion:T. gondii infection caused oxidative stress in testis tissue. Thus, the adverse effects of oxidative stress may affect the male rat reproductive system.

Vasoactive Intestinal Peptide Derived From Liver Mesenchymal Cells Mediates Tight Junction Assembly in Mouse Intrahepatic Bile Ducts.

Formation of intrahepatic bile ducts (IHBDs) proceeds in accordance with their microenvironment. Particularly, mesenchymal cells around portal veins regulate the differentiation and ductular morphogenesis of cholangiocytes in the developing liver; however, further studies are needed to fully understand the arrangement of IHBDs into a continuous hierarchical network. This study aims to clarify the interaction between biliary and liver mesenchymal cells during IHBD formation. To identify candidate factors contributing to this cell–cell interaction, mesenchymal cells were isolated from embryonic day 16.5 matrix metalloproteinase 14 (MMP14)‐deficient (knockout [KO]) mice livers, in which IHBD formation is retarded, and compared with those of the wild type (WT). WT mesenchymal cells significantly facilitated the formation of luminal structures comprised of hepatoblast‐derived cholangiocytes (cholangiocytic cysts), whereas MMP14‐KO mesenchymal cells failed to promote cyst formation. Comprehensive analysis revealed that expression of vasoactive intestinal peptide (VIP) was significantly suppressed in MMP14‐KO mesenchymal cells. VIP and VIP receptor 1 (VIPR1) were mainly expressed in periportal mesenchymal cells and cholangiocytic progenitors during IHBD development, respectively, in vivo. VIP/VIPR1 signaling significantly encouraged cholangiocytic cyst formation and up‐regulated tight junction protein 1, cystic fibrosis transmembrane conductance regulator, and aquaporin 1, in vitro. VIP antagonist significantly suppressed the tight junction assembly and the up‐regulation of ion/water transporters during IHBD development in vivo. In a cholestatic injury model of adult mice, exogenous VIP administration promoted the restoration of damaged tight junctions in bile ducts and improved hyperbilirubinemia. Conclusion: VIP is produced by periportal mesenchymal cells during the perinatal stage. It supports bile duct development by establishing tight junctions and up‐regulating ion/water transporters in cholangiocytes. VIP contributes to prompt recovery from cholestatic damage through the establishment of tight junctions in the bile ducts.

Evaluation of the ameliorative effects of Phyllanthus niruri on the deleterious insecticide imidacloprid in the vital organs of chicken embryos

BackgroundInsecticides are widely used in agriculture to curb the loss caused by insects. These insecticides are incorporated into the food chain and accumulate in the human body, as well disturb the various metabolic pathways. Imidacloprid is an insect neurotoxin commonly used in agriculture to control the insect pests. P. niruri is a traditional medicinal shrub widely used as an anti-inflammatory, antipyretic, and anti-lethality agent. ObjectiveThe present study is designed to evaluate the ameliorative effects of Phyllanthus niruri (Bhumi amla) on the deleterious Insecticide imidacloprid in the vital organs of Chicken embryos. Materials and methodsThe embryonated chicken eggs were divided into the four groups (one control and three treated groups); the chorioallantoic membranes of control received 200 μl phosphate buffer saline, whereas group I and group II received 100 μg imidacloprid and 200 μl aqueous extract of P. niruri (PNE) respectively. Group III received both 100 μg imidacloprid and 200 μl PNE. The serum was collected on the 18th day its development; which was subjected to the biochemical analysis based on colorimetric assay in semi-automated biochemical analyzer using commercial kits. ResultsWe observed significant in ovo effects of imidacloprid on chicken embryos; the values of aspartate aminotransferase (AST), alanine aminotransferase (ALT), were increased in imidacloprid treated group I; histopathology also revealed damage to the liver (necrotic areas and dilated blood sinusoids). Alkaline phosphatase (ALP), amylase, cholesterol, triglycerides protein and albumin levels were also altered significantly (p < 0.05). ConclusionThe serum biochemicals were returned back to the nearly normal levels. PNE has ameliorated and overcome the effects of imidacloprid reasonably with the subsequent treatment among group III. Hence, P. niruri may be used to minimize the effects of an accidental exposure of imidacloprid.

Technical note: novel delivery methods for an enterotoxigenic Escherichia coli infection model in MUC4-locus sequenced weaner pigs1.

An infection model with enterotoxigenic Escherichia coli (ETEC) harboring the F4 fimbriae can be used to assess the impacts that various challenges associated with weaning (e.g., dietary, psychological, environmental) have on the expression of postweaning diarrhea. The objective of this study was to develop a novel inoculation method for administering an ETEC culture that would induce a higher proportion of ETEC-F4 diarrhea, in pigs that genetically showed ETEC-F4 susceptibility or resistance. The study was designed as a factorial arrangement of treatments with the factors being 1) partially susceptible or resistant to ETEC-F4 based on genetic testing, and 2) 4 challenge treatments, being a) a conventional liquid broth method using a drenching gun [Positive control (PC)], b) a Syringe method, c) a Capsule method, and d) Negative control [pigs not challenged (NC)]. At 21 ± 3 d of age (mean ± SEM), 48 male castrate pigs (Large White × Landrace) weighing approximately 7.0 ± 1.18 kg were allocated to 4 treatment groups in 2 replicate pens (6 pigs per pen). Initial ETEC-F4 susceptibility was based on a DNA marker test and each treatment group had 9 partially susceptible and 3 resistant pigs. On days 7 and 8 after weaning, pigs were challenged with ETEC (serotype O149:K88; toxins LT1, ST1, ST2, and EAST). On each inoculation day the PC pigs were orally dosed with 9 mL 7.12 × 109 colony-forming unit (CFU), the Syringe pigs with 0.8 mL 6.72 × 109 CFU, the Capsule pigs were orally administered 2 capsules containing 0.8 mL 3.28 × 109 CFU, and the NC pigs 1 mL of phosphate-buffered saline (PBS) solution. Approximately 72 h after infection, 44, 22, 78, and 0% of partially susceptible pigs in the PC, the Syringe, the Capsule, and the NC group had developed ETEC-F4 diarrhea (P = 0.007). Partially susceptible pigs had a higher diarrhea index (DI) compared to resistant pigs (31.5 vs. 4.8, P < 0.001). The NC group had a lower DI compared to the PC and Capsule pigs (3.9, 38.1, and 40.3, respectively, P < 0.005). Following infection, genetically resistant pigs in the Capsule group had a DI of zero and the partially susceptible pigs had a DI of 55.6 (P = 0.014). This study showed that genetically screening pigs and using a Capsule to deliver ETEC-F4 can increase cases of diarrhea and the efficiency of the challenge model. Taken together, these methods have the potential to reduce the number of pigs needed in future experimental infection studies.

Effect of supernatant of mesenchymal stem cells on tumor necrosis factor-α and apoptosis protein caspase-3 in diabetic skin ulcer rat model

Objective To investigate the supernatant of umbilical cord-derived mesenchymal stem cells (UCMSCs) on tumor necrosis factor-α (TNF-α) and apoptosis protein caspase-3 in diabetic rats model with skin ulcer. Methods 45 Sprague-Dawley rats were randomly divided into control group (acute wounds group), phosphate buffered saline (PBS) group and UCMSCs supernatant group. The diabetic rat model was constructed by injecting with alloxan by tail vein and feeding with high-fat diet. Diabetic skin ulcer (DSU) rat model was constructed by scratching a wound and infusing suspension of Staphylococcus aureus. In the control group, the diabetic rats (n=15) were scratched to form a wound and treated by tail vein injection of 100 μl PBS. In the PBS group, DSU rats (n=15) were treated by tail vein injection of 100 μl PBS, and then 100 μl PBS was dropped at the ulcer site. In the UCMSCs supernatant group, freeze-dried powder of UCMSCs supernatant was dissolved in 200 μl PBS, 100 μl of which was injected into the tail vein of DSU rats (n=15), and other 100 μl was dropped at the ulcer site. After 5 days of the treatments, the levels of serum TNF-α were detected by radioimmunoassay method, and the expression of TNF-α and caspase-3 in the ulcer tissues of rats was detected by polymerase chain reaction and Western Blot. Results The levels of TNF-α in the PBS group [(35.9±3.7) μg/L] were significantly higher than that of the control group [(11.4±4.9) μg/L] and the UCMSCs group [(14.7±6.6) μg/L] (all P 0.05). Conclusions UCMSCs supernatant treatments can effectively down-regulate the expression of TNF-α and caspase-3 in ulcer tissue of DSU rats, and play an anti-inflammatory and anti-apoptotic effect. Key words: Diabetes mellitus; Skin ulcer; Umbilical cord derived-mesenchymal stem cells; Tumor necrosis factor-α; Caspase-3

Comparison of Three Protocols to Preserve Leptospira spp. in Cat Urine for Efficient DNA Extraction and PCR Amplification

Background: The pathogenic leptospira infection in mammalian species can cause a range of acute or chronic manifestations and may result in a carrier state. Previous studies have suggested that cats were resistant to acute leptospirosis however, the description of some clinical cases suggests that Leptospira spp. may also be pathogenic to this species. Recent studies have shown that leptospires may be shed in the urine of infected cats. Endogenous substances present in urine may inhibit PCR and allow leptospires to evade detection. This study aims to compare three protocols for sample processing to optimize the detection of pathogenic leptospires in cat urine.Materials, Methods &amp; Results: Three protocols to optimize the detection of pathogenic leptospires in cat urine were tested. Aliquots of standard concentration of L interrogans serovar Canicola culture were added to urine samples to achieve concentrations of 1×105 to 1×102 leptospires/mL for each protocol. In protocols A and B the urine was neutralized by the addition of phosphate-buffered saline (PBS), pH 7.4, in a proportion of 1 PBS: 2.5 urine (v/v). In protocol A, PBS was added to neutralize the urine pH for the leptospiral organisms immediately after addition of leptospires. In protocol B, PBS was added just before DNA extraction. In protocol C, no PBS was added. DNA extraction was performed at 4, 24 and 48 h after addition of the leptospires using a modified protocol. Samples were incubated at 37ºC for 10 min. Samples were then centrifuged (850 g) for 15 min, at 25ºC. The supernatants were transferred to another tube, and the pellets were discarded. The supernatants were centrifuged (16060 g) for 20 min at 4ºC. The supernatants were then discarded, and the pellets resuspended and washed with 1000 µL of PBS. All the samples were centrifuged at 16060 g for an additional 20 min at 25ºC. The supernatants were discarded and the pellets were resuspended in 100 µL of PBS and incubated at 94ºC for 10 min. DNA was stored at -20ºC until the molecular analysis. The PCR detection limit was evaluated. In samples from protocol A, leptospires were detected in concentrations up to 1×103 (4 h) and 1×104 (24 and 48 h). In protocol C, leptospires were detected in concentrations up to 1×104 (4 h) and 1×105 (24 and 48 h). No leptospiral DNA was detected in samples from protocol B.Discussion: Leptospires are sensitive to acid conditions, at pH 6.8 or lower and the urine pH of cats may vary from 5 to 7. In the present study, we found best results for DNA amplification with the addition of PBS immediately after urine collection (protocol A). Previous studies have shown the importance of neutralizing urine samples immediately after collection to avoid loss of bacterial DNA during the extraction process. However, protocols B and C may be an alternative in clinical practice, when PBS cannot be added immediately after collection. The delay after urine collection before DNA extraction is one more factor that may interfere with the PCR sensitivity. This was observed in the samples from protocol A, because although these samples were neutralized immediately, there was a 10-fold decrease in the detection limit of the test at 24 and 48 h. Leptospires rapidly lose their integrity in urine and the detection limit declines considerably over time, so prompt extraction is essential. These results show that the in-house method of preserving cat urine is useful to maintain the viability of leptospiral DNA extraction. In addition, this study highlights the importance of neutralizing urine samples immediately after collection and the need for prompt DNA extraction to improve PCR detection limit. However, if PBS cannot be added to the collected sample immediately, it is better to process the sample without PBS and extract DNA as soon as possible to minimize the risk of false-negative results.

Effects of E.coli O157:H7 Experimental Infections on Rabbits

The study aimed to show the effect of experimental infection with E.coli O157:H7 on some liver and kidney function in adult rabbits. Twenty five domestic rabbits (males and females), aged 6-8 weeks and weighing 1500-2000g, had a negative fecal bacteriological culture of E.coli O157:H7 were used. The rabbits were randomly divided into two groups: infected group (15 rabbits), each animal was drenched orally with 1ml of Phosphate buffer saline containing (2×109CFU) of E.coli O157:H7, while the control group (10 rabbits) were drenched orally (1ml) of Phosphate buffer saline, the clinical signs were daily observed for 30 days, recording of body weight and blood samples were collected at (0, 3, 15 and 30) days for serum to evaluate biochemical tests including Alanine transaminase, Aspartate aminotransaminase and alkaline phosphatase, creatinine, urea and total serum protein. The results showed different clinical signs after inoculation, including diarrhea, lethargy, weight loss, inability to eat or drink, urine was cloudy or milky, while control group remains in normal condition. The serum biochemical tests in infected group showed a significant increase of three enzymes at 15, and 30 days, the highest values of Alanine transaminase; Aspartate aminotransaminase and alkaline phosphatase showed at 30 days post infection as compared with the control group. Serum creatinine and urea recorded a significant increase (P&lt; 0.05) at 30 day post infection as compared with control group. However, total protein revealed a significant decrease in the infected group, and a significant decrease was noticed at 15 day post infection, while the control group showed an increase in total protein co-ordinated with increase of body weight and progressing age. This study concluded that the infection with E. coli O157:H7may cause changes in liver and kidney functions which were more sever at 15,30 days post infection.

Aptamer-Anchored Rubrene-Loaded Organic Nanoprobes for Cancer Cell Targeting and Imaging

Aptamer-Anchored Rubrene-Loaded Organic Nanoprobes for Cancer Cell Targeting and Imaging

Commensal bacterial sharing does not predict host social associations in kangaroos.

Social network analysis has been postulated as a tool to study potential pathogen transmission in wildlife but is resource-intensive to quantify. Networks based on bacterial genotypes have been proposed as a cost-effective method for estimating social or transmission network based on the assumption that individuals in close contact will share commensal bacteria. However, the use of network analysis to study wild populations requires critical evaluation of the assumptions and parameters these models are founded on. We test (a) whether networks of commensal bacterial sharing are related to hosts' social associations and hence could act as a proxy for estimating transmission networks, (b) how the parameters chosen to define host associations and delineate bacterial genotypes impact inference and (c) whether these relationships change across time. We use stochastic simulations to evaluate how uncertainty in parameter choice affects network structure. We focused on a well-studied population of eastern grey kangaroos (Macropus giganteus), from Sundown National Park, Australia. Using natural markings, each individual was identified and its associations with other kangaroos recorded through direct field observations over 2years to construct social networks. Faecal samples were collected, Escherichia coli was cultured and genotyped using BOX-PCR, and bacterial networks were constructed. Two individuals were connected in the bacterial network if they shared at least one E.coli genotype. We determined the capacity of bacterial networks to predict the observed social network structure in each year. We found little support for a relationship between social association and dyadic commensal bacterial similarity. Thresholds to determine host associations and similarity cut-off values used to define E.coli genotypes had important ramifications for inferring links between individuals. In fact, we found that inferences can show opposite patterns based on the chosen thresholds. Moreover, no similarity in overall bacterial network structure was detected between years. Although empirical disease transmission data are often unavailable in wildlife populations, both bacterial networks and social networks have limitations in representing the mode of transmission of a pathogen. Our results suggest that caution is needed when designing such studies and interpreting results.

MiR-140-3p overexpression activates the Wnt signaling pathway to promote fracture healing.

To study the mechanism of micro ribonucleic acid (miR)-140-3p participating in the regulation of fracture healing in rats. A total of 50 male Sprague-Dawley (SD) rats were randomly divided into five groups, namely, group A [phosphate-buffered saline (PBS)] (n=10), group B (miR-140-3p mimics) (n=10), group C [ mimics negative control (NC)] (n=10), group D [antisense oligonucleotide (ASO)-miR-140-3p] (n=10), and group E (ASO NC) (n=10). A rat model of fracture was established on all the rats through the operation. From the successful establishment of the model, the rats in group A were intraperitoneally injected with 50 μL PBS (2 nmol) once a week for 6 weeks, and those in group B, C, D, and E were injected with equivalent volume of miR-140-3p mimics, mimics NC, ASO-miR-140-3p, and ASO NC, respectively, once a week since the successful establishment of model for 6 weeks. The fracture healing in the rats was evaluated via imaging. Meanwhile, Real Time-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression of miR-140-3p in the five groups. Wnt and β-catenin expressions in the five groups were detected by means of Western blotting (WB). Alkaline phosphatase (ALP) and its quantized statistical value in the five groups were detected through immunohistochemical staining. The expression of miR-140-3p was stimulated in miR-140-3p mimics group and inhibited in ASO-miR-140-3p group. The detection of the miR-140-3p expression level in the five groups via RT-PCR showed that miR-140-3p mimics group had a remarkably higher miR-140-3p expression than the other four groups. The differences were statistically significant (p<0.05). The WB assay verified that the Wnt and β-catenin expressions in miR-140-3p mimics group were notably higher than those in control groups, and there were statistically significant differences (p<0.05). Compared with those in the groups injected with PBS, ASO miR-140-3p, mimics NC, and ASO NC, there were evidently more callus tissues, better healed and more blurred fracture lines, as well as no translocation and looseness of internal fixation, in the group injected with miR-140-3p mimics, suggesting that the stimulation of the miR-140-3p expression promotes the fracture healing in the rats. The results of immunohistochemical staining indicated that the number of ALP-positive osteoblasts in the rats in miR-140-3p mimics group was increased markedly in comparison with that in the remaining groups (p<0.05), implying that the differentiation of osteoblasts in the rats was affected in miR-140-3p mimics group. The overexpressed miR-140-3p in the rats with fracture can promote fracture healing by activating the Wnt signaling pathway.

The activity of polyclonal IgY derived from Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in inhibiting colonization of Fusobacterium nucleatum and Streptococcus sanguinis

Background: Fusobacterium nucleatum (F. nucleatum) and Streptococcus sanguinis (S. sanguinis) play a role in dental plaque formation which leads to periodontitis. Immunoglobulin Y (IgY) is present in both serum and egg yolk and can bind to the surface components of bacteria. F. nucleatum and S. sanguinis feature the same type of IV pili as Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans). Saliva binding protein (SsaB) in S. sanguinis is a FimA homolog. FimA constitutes a surface element of Porphyromonas gingivalis (P. gingivalis). F. nucleatum and P. gingivalis possess the same outer membrane protein (OMP) molecular mass. Purpose: The study aimed to determine the activity of A. actinomycetemcomitans and P. gingivalis polyclonal IgY present in serum and egg yolk that can inhibit colonization of F. nucleatum and S. sanguinis. Methods: IgY samples were diluted with phosphate buffer saline (PBS). Several holes were made in the nutrient medium with 10 μl antigen (F. nucleatum/S. sanguinis) being inserted into the center hole. 10 μl PBS, 1:1, 1:2, 1:4, 1:8, 1:16 A. actinomycetemcomitans or P. gingivalis polyclonal IgY were subsequently introduced into the surrounding holes. The results of incubation at 37°C were observed after 24-48 hours. Kruskal Wallis and MannWhitney tests were administered to analyse the data. Results: A. actinomycetemcomitans and P. gingivalis polyclonal IgY groups in serum showed a precipitation line at dilution ratios of 1:1 and 1:2, whereas in egg yolk this occurred only at a 1:1 dilution ratio with F. nucleatum and S. sanguinis bacteria in this study. No significant differences were evident between each dilution (p&gt;0.05) and none existed between serum and egg yolk (p&gt;0.05). Conclusion: IgY polyclonal of A. actinomycetemcomitans and P. gingivalis in both serum and egg yolk initiate activities that can inhibit colonization of F. nucleatum and S. sanguinis.

Nondestructive Identification and Accurate Isolation of Single Cells through a Chip with Raman Optical Tweezers.

Raman optical tweezers (ROT) as a label-free technique plays an important role in single-cell study such as heterogeneity of tumor and microbial cells. Herein we designed a chip utilizing ROT to isolate a specific single cell. The chip was made from a polydimethylsiloxane (PDMS) slab and formed into a gourd-shaped reservoir with a connected channel on a cover glass. On the chip an individual cell could be isolated from a cell crowd and then extracted with ∼0.5 μL of phosphate-buffered saline (PBS) via pipet immediately after Raman spectral measurements of the same cell. As verification, we separated four different type of cells including BGC823 gastric cancer cells, erythrocytes, lymphocytes, and E. coli cells and quantifiably characterized the heterogeneity of the cancer cells, leukocyte subtype, and erythrocyte status, respectively. The average time of identifying and isolating a specific cell was 3 min. Cell morphology comparison and viability tests showed that the successful rate of single-cell isolation was about 90%. Thus, we believe our platform could further couple other single-cell techniques such as single-cell sequencing and become a multiperspective analytical approach at the level of a single cell.

Oral bovine serum albumin immune‐stimulating complexes improve the immune responses and resistance of large yellow croaker (Pseudosciaena crocea,Richardson, 1846) againstVibrio alginolyticus

This study aimed to investigate effects of bovine serum albumin immune-stimulating complexes (BSA ISCOMs) on immune-related genes expression, serum nonspecific immunity and disease resistance of large yellow croaker (Pseudosciaena crocea). Fish were fed diets containing 3.5 ml of BSA ISCOMs per kg feed (experimental group) or 3.5 ml of phosphate-buffered saline per kg feed (control group) for 1 week. The liver, spleen, head-kidney tissues were sampled for determining gene expression of myxovirus-resistant protein (Mx), major histocompatibility complex class II alpha chain (MHC II α), tumour necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) 30 and 90 days after feeding. Also, blood samples were collected for determining activities of serum superoxide dismutase (SOD), interferon alpha (IFN-α), TNF-α and alkaline phosphatase (ALP). TNF-α and MHCⅡα gene expression in the liver, spleen, head-kidney, as well as IFN-α, TNF-α and ALP activities in the serum, of experimental fish were significantly higher 30 days after feeding; while only TNF-α and MHC II gene expression in the head-kidney remained upregulated 90 days after feeding. The cumulative mortality of the experimental fish was significantly lower than control. This study indicated that BSA ISCOMs improved the immune response and induced protective immunity in large yellow croaker.

Comparison of biotechnological culture of hypoxia-conditioned rat mesenchymal stem cells with conventional in vitroculture of normoxia-conditioned rat mesenchymal stem cells for testicular failure therapy with low libido in rats.

Aim:Biotechnological culture of hypoxia-conditioned (CH) rat mesenchymal stem cells (rMSC-CH) for testicular failure therapy with low libido improves the functional outcome of the testicle for producing spermatogenic cells and repairs Leydig cells in rats (Rattus norvegicus).Materials and Methods:In the first group (T1), rats with testicular failure and low libido were injected with normoxia-conditioned (CN) rMSCs (21% oxygen); in the second group (T2), rats with testicular failure and low libido were injected with rMSC-CH (1% oxygen); in the negative control group (T−), rats with normal testis were injected with 0.1 mL phosphate-buffered saline (PBS); and in the sham group (TS), rats with testicular failure and low libido were injected with 0.1 mL of PBS.Results:Vascular endothelial growth factor expression, as the homing signal, in the groups T2, T−, T1, and TS was 2.00±0.5%, 2.95±0.4%, 0.33±0.48%, and 0±0%, respectively. The number of cluster of differentiation (CD)34+ and CD45+ cells in the groups T− and TS was <20%, whereas that in T1 and T2 groups was >30% and >80%, respectively, showing the mobilization of hematopoietic stem cells (HSCs). The number of spermatogenic cells (spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatid) decreased significantly (p<0.05) in TS compared with that in T−, T1, and T2, whereas that in T2 did not show a significant (p>0.05) decrease compared to that in T−. The improvement in libido, based on the number of Leydig cells producing the hormone testosterone for libido expression, did not increase in T1, whereas T2 was able to maintain the number of Leydig cells significantly compared to that between TS and T1.Conclusion:rMSC-CH culture for testicular failure with low libido showed improvement in the functional outcome of the testicle and in repairing Leydig cells.

A method for improving the efficiency of DNA extraction from clotted blood samples.

BackgroundThe efficient and rapid extraction of high‐quality genomic DNA from clotted blood samples, which normally have a low yield and poor quality, is an important factor in genomic research. The objective of this study was to develop a simple and safe technique for dispersing the blood clots by the ball bearing metal shots. Normally, such clot samples may not have an acceptable yield by conventional DNA extraction methods. Also, in the present study, we have further investigated to improve salting‐out DNA extraction methods.MethodsInitially, 500 µL phosphate‐buffered saline (PBS) (1×) and two ball bearing metal shots were added to each tube of the clotted blood sample and then were gently rotated in an electric laboratory rotator for 1 hour at room temperature (18‐25°C). Genomic DNA was then extracted from samples using a modified salting‐out method and a modified QIAamp® DNA Blood Midi Kit and was compared with QIAamp® DNA Blood Midi Kit as a control. An assessment of the concentration and quality of the extracted DNA was performed using the UV‐visible spectrophotometer. The isolated DNA proved amenable to PCR amplification and gel electrophoresis.ResultsThe yield and purity of DNA obtained by these three methods were significantly different (P < 0.001), with a higher yield in the modified salting‐out method.ConclusionsOur proposed modified salting‐out method is simple and efficient for the isolation of DNA from old blood clot samples. It is both easy to use and is of low cost in routine laboratory tasks.

Molecular genetic mechanisms of influence of laser radiation with 577 nm wavelength in a microimpulse mode on the condition of the retina

The aim of the studywas to investigate the molecular genetic mechanisms of the influence of laser radiation with 577 nm wavelength in a microimpulse mode on the retina in the experimental conditions after the intravitreal injection of VEGF. Materials and methodsThe study was performed on 4-5 week-old male mice of the line C57BL/6J. The animals were divided into 4 groups of 5 mice in each group, one eye was excremental, the contralateral eye remained intact. In the first group, intravitreal injection of PBS was performed; in the second group, intravitreal injection of 50 ng/ml of recombinant VEGF165 in 2 μL of phosphate-buffered saline (PBS) was performed; in the third and fourth groups, a day after the intravitreal injection of recombinant VEGF165, laser radiation with wavelength 577 nm was applied in the micropulse and continuous modes, respectively. Tissue samples (neuroepithelium, pigment epithelium) for the microarray transcription analysis in the animals from group 1 and 2 were taken 2 days after the injection of PBS and VEGF, in the animals from group 3 and 4 – a day after the retina was exposed to laser radiation. Results and conclusionMolecular genetic mechanisms of the influence of laser radiation with wavelength 577 nm in a microimpulse mode on the retina in experimental conditions were studied and the genes that significantly changed the level of expression (the genes that take part in the regulation of neoangiogenesis, structural cell functions, processes of cells proliferation, transcription, differentiation, transmembrane transport, signaling, synaptic transmission, etc.) were identified.

Uptake and Release of Polyvinyl Alcohol from Hydrogel Daily Disposable Contact Lenses.

Polyvinyl alcohol is a wetting agent that could reduce the symptoms of dry eye and contact lens discomfort. Currently, only one lens type, nelfilcon A (DAILIES AquaComfort Plus), releases polyvinyl alcohol. The concept of releasing this agent from contact lenses could be applied to other lens materials. The purpose of this study was to measure the release of polyvinyl alcohol from commercially available hydrogel daily disposable contact lenses using refractive index and iodine-borate methods. Etafilcon A, omafilcon A, and nelfilcon A were soaked in phosphate-buffered saline and 0.2% trifluoroacetic acid/acetonitile for 24 hours to remove residual blister pack components. The lenses were then incubated in a 10-mg/mL solution of polyvinyl alcohol for 24 hours. After the incubation period, the lenses were placed in 2 mL of phosphate-buffered saline. At specified time intervals, t = 0.5, 1, 2, 4, 8, 12, and 24 hours, the samples were evaluated using refractive index and an iodine-borate assay. Polyvinyl alcohol uptake was determined by extracting the lenses with methanol for 24 hours. There were no differences in the uptake of polyvinyl alcohol between lens types (P > .05). The release of this wetting agent for all lens types followed a burst-plateau profile after the first 30 minutes (P > .05). Nelfilcon A had a slightly higher release of polyvinyl alcohol (P < .05) than did etafilcon A but was similar to omafilcon A (P > .05). The results suggest that the contact lenses tested in this study have similar efficiency in delivering polyvinyl alcohol.

MiR-214-3p delays fracture healing in rats with osteoporotic fracture through inhibiting BMP/Smad signaling pathway.

The purpose of this study was to explore the mechanism of micro-ribonucleic acid (miR)-214-3p in regulating fracture healing in rats with osteoporosis. A total of 30 female Sprague-Dawley rats were selected and randomly divided into 3 groups, including group A [phosphate-buffered saline (PBS), n=10], group B (AntagomiR-NC, n=10), and group C (AntagomiR-214-3p, n=10). All rats underwent ovariectomy, and the osteoporosis rat model was verified by dual-energy X-ray absorptiometry 8 weeks after the operation. Then the osteoporotic fracture was established in rats via a second operation. From the successful modeling until the 6th week, 50 μL PBS (2 nmoL) was intraperitoneally injected in group A, an equal amount of AntagomiR-NC was injected in group B, and an equal amount of AntagomiR-214-3p was injected in group C once a week. At the 6th week, fracture healing of osteoporosis rats was evaluated. At the same time, the expression of miR-214-3p in the three groups was detected via reverse Transcription-Polymerase Chain Reaction (RT-PCR). Furthermore, the protein expressions of bone morphogenetic protein 2 (BMP2) and Smad4 in the three groups were detected via Western blotting (WB). After ovariectomy, the bone mineral density in each group was significantly lower than that before ovariectomy, and the differences were statistically significant (p<0.05). Imaging evaluation demonstrated that compared with group A and B, there were significantly more callus tissues in group C. Meanwhile, the fracture line healing was better and blurred, and the internal fixation had no displacement and loosening. RT-PCR results indicated that the expression level of miR-214-3p in group C was significantly lower than that of the other two groups (p<0.05). WB results showed that the protein expression levels of BMP2 and Smad4 in group C were significantly higher than those of group A and group B (p<0.05). MiR-214-3p delays fracture healing in rats with osteoporotic fracture by inhibiting the BMP/Smad signaling pathway.

Effects of IL-33 on renal tubular injury in lupus nephritis through the JAK/STAT signaling pathway

Objective To investigate the effects and mechanism of interleukin 33 (IL-33) on renal tubular injury in mice with lupus nephritis. Methods Twelve-week-old female MRL/lpr mice were randomly divided into model group, IL-33 group and solvent control group with 10 rats in each group. Ten female MRL/MP mice of the same age were used as normal control group. The mice in IL-33 group were intraperitoneally injected with 100 μL of phosphate buffer saline (PBS), containing 2 μg of recombinant mouse IL-33, once a day for 14 days. The mice in control group and the model group were intraperitoneally injected with the same dose of PBS. All the mice were sacrificed at 14 weeks of age. Serum creatinine (Cr) and urea nitrogen (BUN) concentrations were determined by serum separation. The urine in 24 hours was collected testing urinary protein creatinine ratio and urinary protein quantification. The contents of E-cadherin, α-SMA, and JAK/STAT pathway signaling proteins, including JAK2, p-JAK2, STAT1, and p-STAT1, were detected by Western blot. Results The BUN, urinary protein creatinine ratio and urine protein level of the IL-33 group were significantly higher than those of the model group (all P 0.05). Conclusions IL-33 can cause tubulointerstitial lesions in lupus mice, and its mechanism may be related to the activation of JAK/STAT pathway. Key words: Lupus nephritis; Tubular interstitial leison; JAK2; Signal transducer and activator of transcription 1

Lactobacillus rhamnosus GG modulates gastrointestinal absorption, excretion patterns, and toxicity in Holstein calves fed a single dose of aflatoxin B1

The aim of the present study was to evaluate the effects of Lactobacillus rhamnosus GG (LGG; ATCC 53013) on growth performance and hepatotoxicity in calves fed a single dose of aflatoxin B1 (AFB1) and to investigate the absorption, distribution, and elimination of AFB1 and the hydroxylated metabolite aflatoxin M1 (AFM1) in rumen fluid, blood, and excretions. Twenty-four male Holstein calves were blocked for body weight and age and were randomly assigned to 1 of 3 treatment groups: (1) untreated control, (2) treated with 4.80 mg of AFB1 (AFB1 only), or (3) treated with 1 × 1010 cfu of LGG suspension and 4.80 mg of AFB1 (AFB1 plus LGG). The calves received LGG suspension in 50 mL of phosphate-buffered saline daily via oral administration for 14 d before and on the day they received a single oral dose of AFB1. Body weight was recorded at the beginning of the study (before LGG administration), at the day of AFB1 administration, and at the end of the trial. Rumen fluid, blood, urine, and feces samples were collected continuously for 96 h after AFB1 administration. Average daily gain (ADG) and plasma biochemical parameters were analyzed, and concentrations of AFB1 and AFM1 in the samples were determined for monitoring excretion pattern and toxicokinetics. The results showed that ADG was lower in AFB1-treated animals; LGG administration partially mitigated the decrease in ADG (0.85 ± 0.08 vs. 0.76 ± 0.18 kg of gain/d). The AFB1 treatment increased plasma aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase levels. Administration of LGG alleviated the AFB1-induced increase in plasma enzymes activity. The excretion patterns of AFB1 and AFM1 were surprisingly regular; toxins were rapidly detected in all samples after a single oral dose of AFB1, and the peak of toxins concentrations was sequentially reached in rumen fluid, plasma, urine, and feces (except AFM1 in rumen fluid), followed by an exponential decrease. The excretion curves showed that AFB1 and AFM1 concentrations were the highest in feces and urine, respectively. Administration of LGG decreased the concentrations of free AFB1 and AFM1 in rumen fluid and reduced the release of toxins into plasma and urine. Toxicokinetic parameters (except for the time of maximum concentration and the terminal half-life) were reduced by LGG administration. In conclusion, the absorption, distribution, and excretion of AFB1 and AFM1 were rapid in calves fed a single dose of AFB1. Urine was the main route for the excretion of AFM1, and the clearance pattern from the peak of concentration was well fitted by exponential decreasing function. Administration of LGG reduced the absorption of AFB1 in the gastrointestinal tract by increasing the excretion via the feces, thus alleviating the hepatotoxic effect of AFB1.