A novel mouse line with epididymal initial segment-specific expression of Cre recombinase driven by the endogenous Lcn9 promoter.

Spermatozoa released from testes undergo a maturation process and acquire the capacity to fertilize ova through epididymal transit. The epididymis is divided into four regions, each with unique morphology, gene profile, luminal microenvironment and distinct function. To study the functions of relevant genes in the epididymal initial segment (IS), a novel IS-specific mouse model, Lcn9-Cre knock-in (KI) mouse line was generated via CRISPR/Cas9 technology. The TAG stop codon was replaced by a 2A-NLS-Cre cassette, resulting in the co-expression of Lcn9 and Cre recombinase. IS-specific Cre expression was first observed from postnatal day 17. Using the Rosa26tdTomato reporter mice, the Cre-mediated DNA recombination was detected exclusively in principal cells. The epididymal IS-specific Cre activity in vivo was further confirmed using Lcn9-Cre mice crossed with a mouse strain carrying Tsc1 floxed alleles (Tsc1flox/+). Cre expression did not affect either normal development or male fecundity. Different from any epididymis-specific Cre mice reported previously, the novel Lcn9-Cre mouse line can be used to introduce entire IS-specific conditional gene editing for gene functional study.

Electroacupuncture inhibits IL-17/IL-17R and post-receptor MAPK signaling pathways in a rat model of chronic obstructive pulmonary disease.

OBJECTIVE: Interleukin (IL)-17, as a T-helper 17 cell (Th17) cytokine, plays a key role in chronic obstructive pulmonary disease (COPD) pathophysiology including chronic inflammation and airway obstruction, which lead to decreased pulmonary function. The aim of this study was to investigate the effect of acupuncture on IL-17, its receptor (IL-17R) and the mitogen-activated protein kinase (MAPK) signaling pathway, in a rat model of COPD. METHODS: The COPD model was induced in Sprague Dawley rats by exposure to cigarette smoke for 12 weeks. The model rats were treated with electroacupuncture (EA) at BL13 and ST36. The lung function and histology of the rats were observed. IL-17, tumor necrosis factor (TNF)-α, and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and in plasma. The leukocytes and macrophages in the BALF were counted. The expression levels of IL-17R were assayed in lung tissue by real-time polymerase chain reaction (PCR), western blotting, and immunohistochemistry. MAPK signaling pathway molecules including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2 and p38, and their phosphorylated forms, were observed in the lung by western blotting. RESULTS: Compared with the control group rats, lung function decreased and there was a severe inflammatory infiltration of the pulmonary parenchyma in the COPD rats. EA effectively improved lung function and alleviated the inflammatory infiltration in the lungs of COPD rats. EA also reversed the elevated total leukocyte and macrophage counts, the high levels of IL-17 and TNF-α, and the low IL-10 content in COPD rats. Meanwhile, EA downregulated the increased mRNA and protein expression of IL-17R, and significantly inhibited the elevated levels of phosphorylated JNK, ERK1/2, and p38 in the lungs of COPD rats. CONCLUSION: Our results suggest that the protective effects of acupuncture therapy on the lungs of COPD rats are likely related to inhibition of IL-17/IL-17R and the post-receptor MAPK signaling pathways.

MiR-490-5p inhibits the metastasis of hepatocellular carcinoma by down-regulating E2F2 and ECT2.

We intended to evaluate miR-490-5p expression in hepatocellular carcinoma (HCC) tissues and detect the potential targets of miR-490-5p. In vitro experiments were conducted to further investigate the biological function of miR-490-5p on HCC cell metastasis. We investigated the abnormally expressed miRNAs in HCC tissues, and the miR-490-5p expression level was detected by qRT-PCR. E2F2 and ECT2 were proved to be the potential targets of miR-490-5p by luciferase reporter assay. The expression levels of E2F2 and ECT2 were determined using Western blot. Transwell assay was used to analyse the impact of miR-490-5p on metastasis of HCC cells. Four high-expressed miRNAs, and seven low-expressed miRNAs, including miR-490-5p, were detected in HCC tissues. The expression level of miR-490-5p was connected with the tumor size, tumor node metastasis (TNM) stage, and survival ratio of HCC patients. E2F2 and ECT2 were the targets of miR-490-5p, and miR-490-5p inhibited HCC cell metastasis through down-regulating the expressions of E2F2 and ECT2. The over-expressed miR-490-5p could restrain the metastasis of HCC cells by down-regulating E2F2 and ECT2 expression levels.

[Regulation of Thl/Th2 cells by T cell-mediated transcription factor in rats with chronic obstructive pulmonary disease].

OBJECTIVE: To determine T box expressed in T cells (T bet), GATA binding protein-3 (GATA 3), and retinoid-related orphan nuclear receptor gammat (RORgammat) in rats with chronic obstructive pulmonary disease (COPD). METHODS: Thirty rats were randomly divided into a control and a COPD group. The COPD model was established through smoking and lipopolysaccharide (LPS) tracheal instillation. Pulmonary function of the rats was measured 28 d after the establishment of the COPD model by a spirometer. Enzyme linked immunosorbent assay was performed to detect serum γ interferon (IFN-γ), interleukin (IL)-4, and IL-17. The expressions of T-bet, GATA-3, and RORgammat protein in lung tissues were determined by Western blot. RESULTS: Compared with the controls, the COPD rats had decreased pulmonary function and expression of serum IL-4, and increased INF-γ, IL- 17, Th1/Th2, T-bet, T bet /GATA-3, and RORgammat protein (P<0. 05). Forced expiratory volume in 0. 3 seconds (FEV 0.3) was negatively correlated with INF γ and T-bet/GATA-3. Forced vital capacity (FVC) was positively correlated with IL 4. FEV0.3/FVC was negatively correlated with Thl/Th2, T-bet and T-bet/GATA-3. Peak expiratory flow (PEF) was negatively correlated with IL-17, T bet, and RORgammat (P<0. 05). Thl/Th2 was positively correlated with T bet/GATA-3. IL-17 was positively correlated with RORgammat. T bet/GATA-3 was positively correlated with RORgammat (P<0. 05). CONCLUSION: Imbalanced regulation of T bet / GATA 3 and RORgammat on Th1/Th2 and Th17 cells is associated with the occurrence of COPD.

Early transcriptomic adaptation to Na2CO3 stress altered the expression of a quarter of the total genes in the maize genome and exhibited shared and distinctive profiles with NaCl and high pH stresses.

Sodium carbonate (Na2CO3) presents a huge challenge to plants by the combined damaging effects of Na⁺, high pH, and CO3²â». Little is known about the cellular responses to Na2CO3 stress. In this study, the transcriptome of maize (Zea mays L. cv. B73) roots exposed to Na2CO3 stress for 5 h was compared with those of NaCl and NaOH stresses. The expression of 8,319 genes, representing over a quarter of the total number of genes in the maize genome, was altered by Na2CO3 stress, and the downregulated genes (5,232) outnumbered the upregulated genes (3,087). The effects of Na2CO3 differed from those of NaCl and NaOH, primarily by downregulating different categories of genes. Pathways commonly altered by Na2CO3, NaCl, and NaOH were enriched in phenylpropanoid biosynthesis, oxidation of unsaturated fatty acids, ATP-binding cassette (ABC) transporters, as well as the metabolism of secondary metabolites. Genes for brassinosteroid biosynthesis were specifically upregulated by Na2CO3, while genes involved in ascorbate and aldarate metabolism, protein processing in the endoplasmic reticulum and by N-glycosylation, fatty acid biosynthesis, and the circadian rhythm were downregulated. This work provides the first holistic picture of early transcriptomic adaptation to Na2CO3 stress, and highlights potential molecular pathways that could be manipulated to improve tolerance in maize.

[Protective effect of electroacupuncture intervention on neurovascular unit in rats with focal cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention on behavior changes, expression of cerebral vascular endothelial growth factor (VEGF), nerve growth associated protein-43 (GAP-43), synaptophysin (SYN), myelin basic protein (MBP), neurite outgrowth inhibitor-A (Nogo-A) in cerebral focal ischemia-reperfusion injury (CI/RI) rats, so as to study its mechanism underlying improvement of ischemic cerebral vascular disease. METHODS: Sixty male SD rats were randomly divided into sham-operation group, model group and electroacupuncture (EA) group. CI/RI model was established by occlusion of the middle cerebral artery (MCAO) and reperfusion. EA was applied to bilateral "Neiguan" (PC 6), "Sanyinjiao" (SP 6), "Shuigou" (GV 26) and "Baihui" (GV 20) for 30 min, once a day for 14 days. The neurologic deficits were evaluated by Longa 5-grade standard (the higher the score, the severer the neurologic deficit). The immunoactivity of cerebral VEGF, GAP-43, SYN, MBP (important in the process of myelination of nerves in the nervous system) and Nogo-A (inhibiting axonal regeneration) in the focal ischemic cerebral tissue was detected by immunohistochemistry. RESULTS: The Longa's score of the model group was significantly increased after MCAO in comparison with the sham-operation group (P < 0.01). In comparison with the model group, Longa's score of the EA group was evidently lower on day 14 after CI/RI (P < 0.05), suggesting an improvement of the neurological deficits after EA intervention. In comparison with the sham-operation group, the immunoactivity of cerebral VEGF, GAP-43 and Nogo-A was significantly upregulated on day 7 and 14 in the model group (P < 0.01), while that of cerebral SYN was remarkably down-regulated in the model group on day 7 and 14 after CI/RI (P < 0.05). Compared with the model group, cerebral VEGF, GAP-43, SYN and MBP expression levels were considerably upregulated on day 7 and 14 following CI/RI in the EA group (P < 0.01, P < 0.05), while that of cerebral Nogo-A was significantly decreased at the two time-points in the EA group (P < 0.01). CONCLUSION: EA intervention can effectively improve neurological function in cerebral infarction rats, which is closely related to its effects in upregulating cerebral VEGF, GAP-43, SYN and MBP expression, and down-regulating Nogo-A protein, suggesting a protective effect on neurovascular unit.

[Wuzi yanzong pills increases sperm mitochondrial membrane potential and protects its ultrastructure in oligo-asthenozoospermia model rats].

OBJECTIVE: To study the effects of Wuzi Yanzong Pills (WYP) on sperm mitochondrial membrane potential (MMP) and its ultrastructure in oligo-asthenozoospermia model rats. METHODS: Oligo-asthenozoospermia models were made in 50 male rats weighing 200 - 220 g by intragastric administration of Tripterygium Glucosides at 30 mg per kg per d for 8 weeks, and then equally allocated to a model control, a Huangjing Zanyu Capsule (HZC) control, a low-dose WYP, a medium-dose WYP, and a high-dose WYP group. Another 10 age-matched normal male rats were included as normal controls. The rats in the model and normal control groups were given intragastrically distilled water at 10 ml/kg, those in the HZC group administered HZC at 3.01 g/kg, and those in the low-, medium- and high-dose WYP groups medicated with WYP at 2.30, 4.60 and 9.20 g/kg, respectively, once daily for 30 days. At 30 minutes after the last administration, we detected the sperm MMP by JC-1 fluorescent staining and flow cytometry, and examined the sperm ultrastructure under the JEM-1230 transmission electron microscope. RESULTS: JC-1 + % and its fluorescence intensity were (33.77 +/- 6.19)% and 1 468 +/- 496 in the model control, (56.34 +/- 10.35)% and 3 277 +/- 895 in the HZC control, (40.80 +/- 10.40)% and 2 016 +/- 767 in the low-dose WYP, (59.40 +/- 6.51)% and 3 897 +/- 643 in the medium-dose WYP, and (60.71 +/- 7.81)% and 3 371 +/- 647 in the high-dose WYP group, significantly reduced in comparison with (70.80 +/- 4.92)% and 4 360 +/- 945 in the normal control group (P < 0.05), but remarkably higher in the medium- and high-dose WYP groups than in the model controls (P < 0. 05). After modeling, the sperm membrane was loose and degenerated, the mitochondria swelling, variously sized and with incomplete membrane, and the axonemal structure unclear or ruptured. After 30 days of WYP administration, compared with the model control group, the rats exhibited integrated sperm membrane and mitochondrial membrane, reduced mitochondrial swelling and basically normal axonemal and microtubular structures. CONCLUSION: Tripterygium Glucosides could decrease the sperm mitochondrial membrane potential and damage the mitochondrial structure, while WYP could significantly increase the sperm mitochondrial membrane potential and reduce the sperm mitochondrial structure damage. The protection of the integrity of sperm mitochondrial structure and function is one of the mechanisms of WYP acting on oligo-asthenozoospermia.

[PI 3 K/Akt signaling pathway contributed to the protective effect of acupuncture intervention on epileptic seizure-induced injury of hippocampal pyramidal cells in epilepsy rats].

OBJECTIVE: To observe the protective effect of acupuncture stimulation on pyramidal cells in hippocampal CA 1 and CA 3 regions and to analyze the involvement of phosphatidy linositol-3-kinase (PI 3 K)/protein kinase B(PKB or Akt) signaling pathway in the acupuncture effect in epilepsy rats. METHODS: A total of 120 SD rats were randomly divided into normal control group, model group, LY 294002 (a specific antagonist for PI 3 K/Akt signaling) group, acupuncture+ LY 294002 group and acupuncture group (n = 24 in each group, 12 for H. E. staining, and 12 for electron microscope observation). Epilepsy model was established by intraperitoneal injection of pentylenetetrazol (PTZ, 5 microL). Manual acupuncture stimulation was applied to "Baihui" (GV 20) and "Dazhui" (GV 14) once daily for 5 days. Dimethyl Sulfoxide (DMSO, 5 microL, a control solvent) was given to rats of the normal, model and acupuncture groups, and LY294002 (5 microL, dissolved in DMSO) given to rats of the LY 294002 and acupuncture+ LY 294002 groups by lateral ventricular injection. Four hours and 24 h after modeling, the hippocampus tissues were sampled for observing pathological changes of CA 1 and CA 3 regions after H. E. staining under light microscope and for checkin ultrastructural changes of the pyramidal cells under transmission electron microscope. RESULTS: In comparison with the normal control group, the numbers of pyramidal cells of hippocampal CA 3 region in the model group were decreased significantly 4 h and 24 h after epileptic seizure (P < 0.01). While compared to the model group, the pyramidal cells of hippocampal CA 3 region in the acupuncture group were increased considerably in the number at both 4 h and 24 h after seizure (P < 0.01). No significant differences were found between the LY 294002 and model groups, and between the acupuncture+ LY 294002 and model groups in the numbers of pyramidal cells at 4 h and 24 h after seizure (P > 0.05). Findings of the light microscope and electron microscope showed that the injury severity of pyramidal cells of hippocampal CA 1 and CA 3 regions was moderate 4 h after epileptic seizure and even worse 24 h after seizure in the model group, LY 294002 group and acupuncture+ LY 294002 group, but relatively lighter in the acupuncture group. These results suggested an elimination of the acupuncture effect after blocking the PI 3 K/Akt signaling pathway by lateral ventricular injection of LY 294002 in epilepsy rats. CONCLUSION: Acupuncture intervention has a protective effect on pyramidal cells of hippocampal CA 1 and CA 3 regions in epilepsy rats, which is associated with the normal function of intracellular PI 3 K/Akt signaling pathway.

A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants.

High-resolution melting (HRM) analysis relies on the use of fluorescent dyes, such as LCGreen, ResoLight, and SYTO9, which bind in a saturated manner to the double-stranded DNAs. These dyes are expensive in use and may not be affordable when dealing with a large quantity of samples. EvaGreen is a much cheaper DNA helix intercalating dye and has been used in quantitative real-time polymerase chain reaction (PCR) and post-PCR DNA melt curve analysis. Here we report on the development of an EvaGreen-based HRM analysis and its performance, in comparison with the popular LCGreen-based HRM analysis, in detection of DNA polymorphism in plants. We found that various polymorphisms ranged from single nucleotide polymorphisms (SNPs) to Indels were equally detected by using EvaGreen- or LCGreen-based HRM. EvaGreen dye was sensitive enough in discovery of SNPs in fivefold pooled samples. Using this economical dye we successfully identified multiple novel mutant alleles of Gln1-3 gene, which produces a cytosolic glutamine synthetase isoenzyme (GS1), in a maize ethyl methanesulfonate (EMS)-mutagenized library, and genotyped rice mapping populations with SNP markers. The current results suggest that EvaGreen is a promising dye for HRM analysis for its ease to use and cost effectiveness.

Ca2+-binding protein S100A11: a novel diagnostic marker for breast carcinoma.

To evaluate whether S100A11 could be considered to be a novel diagnostic marker in breast carcinoma, the method of differential proteomics, Western blotting, and immunohistochemistry were used to detect the expression pattern and subcellular localization of S100A11. Statistical analyses indicated that specific up-regulated of A100A11 did not correlate with other prognostic factors such as age, tumor size, grade and stage, ER, PR, HER-2 and nodal status. Our data support that S100A11 is a novel diagnostic marker in breast carcinoma. Analysis of S100A11 expression in breast cancer may be an effective tool help in detection of early-stage breast cancer.

[Effect of electroacupuncture on epileptic EEG and intracellular Ca2+ content in the hippocampus in epilepsy rats].

OBJECTIVE: To study the underlying mechanism of electroacupuncture (EA) in relieving epilepsy in pentylenetetrazole (PTZ)-induced epilepsy rats. METHODS: Twenty SD rats were randomly divided into normal control, model, EA, Nimodipine groups, with 5 cases in each. Epilepsy model was established by intraperitoneal injection of PTZ (32.0 mg/kg), once daily for 28 days. EA (100 Hz, 0.6 mA) was applied to "Baihui" (GV 20) and "Dazhui" (GV 14), once daily for 7 days. For Nimodipine group, the rats were given with nimodipine (0.25 mg/kg, i.p.), once daily for 7 days. Electroencephalogram (EEG) was recorded and the fluorescence intensity of Ca2+ of the hippocampus tissue sections was detected by laser scanning confocal microscope (LSCM) after incubation in artificial cerebrospinal fluid containing Flou-3/AM (10 micromol/L) and pluronic F-127 (5 microl). RESULTS: Compared with model group, the latencies of epileptic EEG seizure prolonged obviously (P < 0.05), and epileptic EEG seizure frequencies decreased significantly (P < 0.05) in EA and Nimodipine groups. The fluorescence intensity of intracellular Ca2+ in hippocampus tissue in model group was obviously higher than that in control group (P < 0.01). In comparison to model group, Ca2+ levels in EA and Nimodipine groups lowered considerably (P < 0.05, P < 0.01). No significant differences were found between EA and Nimodipine groups in the aforementioned 3 indexes (P > 0.05). CONCLUSION: EA has an obvious anti-epileptic effect, which may be closely related to its effect in downregulating the increased hippocampal Ca2+ level in PTZ-kindled epilepsy rats.

Anti-T-cell humoral and cellular responses in healthy BALB/c mice following immunization with ovalbumin or ovalbumin-specific T cells.

It is known that T-cell vaccination (TCV) elicits cellular immune responses against the immunizing T cells in vivo. However, it is still unclear whether similar anti-T-cell responses are also induced in individuals responding to foreign antigen (Ag) challenge. This study was undertaken to characterize and compare anti-T-cell cellular and humoral responses of BALB/c mice after ovalbumin (OVA) immunization or TCV. Splenocytes from OVA-primed BALB/c mice proliferated in response to stimulation with a syngeneic OVA-specific T-cell clone, OVA-T3, and secreted interferon-gamma (IFN-gamma) but not interleukin-4 (IL-4). Cytotoxic T-lymphocyte (CTL) activity against the T-cell clone was also observed. Serum samples from these animals discriminated between activated and resting murine T cells, as determined by indirect immunofluorescence staining. Vaccination of BALB/c mice with OVA-T3 cells induced similar, but stronger, cellular and humoral responses. TCV-induced antibodies were not only able to distinguish between activated and resting syngeneic T cells but also positively stained allogeneic T cells from BXSB mice. Furthermore, TCV resulted in hyporesponsiveness of BALB/c mice to subsequent Ag challenge, and antisera from the immunized animals inhibited T-cell proliferation in vitro. Our data suggest that anti-T-cell antibodies, and CD4+ and CD8+ regulatory T lymphocytes may form a complex and co-ordinated regulatory network that plays an important role in regulating the adaptive immune responses in vivo. Implications of this observation for our understanding of the immunoregulation and peripheral tolerance are discussed.

Nonsense mutations of replicase and movement protein genes contribute to the attenuation of an avirulent tomato mosaic virus.

Three recovery mutants of an avirulent Tomato mosaic virus genus: (Tobamovirus) (ToMV-K) with back mutations of the replicase and/or movement protein (MP) genes, have been constructed by site-directed mutagenesis, and infectious plasmids (pToMV-K) were obtained. The rescued phenotypes of the progeny viruses showed that the replicase and MP recovery mutant (ToMV-K(rase-mp)) induced severe symptoms on both systemic and necrotic plants similar to those induced by the virulent strain. The replicase back mutant (ToMV-K(rase)) produced chlorosis and mosaic symptoms on N. tabacum cv. Huangmiaoyu (systemic host), while the MP recovery mutant (ToMV-K(mp)) produced no systemic symptoms on Huangmiaoyu tobacco. Sequencing of the cDNA of progeny viruses revealed that the "back mutants" maintained these mutation sites during infection. Protein immunoblots indicated that the 98 and 126 kDa proteins were expressed in the plants systemically infected by ToMV-K and pToMV-K, whereas no 98 kDa protein was detected in the plants infected by ToMV. The MPs (27 kDa) of ToMV-K and pToMV-K in the plants were smaller in size than those (30 kDa) of ToMV and pToMVK(rase-mp). These data suggest that ToMV-K replicates and spreads by expressing the truncated 98 and 126 kDa replicases and 27 kDa MP in plants. The opal mutation at nucleotides (nt) 2670-2672 of the replicase gene mainly contributes to the attenuation of ToMV-K, whereas the mutations at nt 5632-5664 of the MP gene attenuate the induced symptoms.

Large quantity of ribosomal RNA exists extracellularly in mouse spleen.

When BALB/c mouse spleens were gently homogenized in saline, the resultant supernatant (without cells and tissue debris) contained significant amount of 28S and 18S ribosomal RNA, reaching up to 70% of the total spleen RNA. Haemoglobin assays indicated that less than 15% of the spleen cells were lysed during the homogenization process, indicating that the majority of the spleen 'supernatant RNA' was from the extracellular space of the organ rather than released by the splenocytes as a consequence of grinding. Quantitative RNA analysis showed that the ratio of spleen supernatant RNA/total RNA of BALB/c mice was inversely correlated with age (from approximately 70% at 3 weeks to 45% at 6 months), but that of BXSB mice (an animal model for systemic lupus erythematosus) remained at about 70% irrespective of age. Methyl Green-Pyronin Y staining of paraffin sections of mouse spleen revealed that extracellular RNA was distributed mainly in the sinuses of the organ. Culture supernatants of apoptotic splenocytes contained significant amounts of RNA, suggesting that the extracellular RNA in the spleen might have come from apoptotic lymphocytes. This is supported by the fact that 'thymus supernatant' also contained significant amount of RNA. A possible correlation between spleen extracellular RNA and autoimmune diseases is discussed.