Quantitative detection of the Ralstonia solanacearum species complex in soil by qPCR combined with a recombinant internal control strain.

IMPORTANCE: DNA-based detection and quantification of soil-borne pathogens, such as the Ralstonia solanacearum species complex (RSSC), plays a vital role in risk assessment, but meanwhile, precise quantification is difficult due to the poor purity and yield of the soil DNA retrieved. The internal sample process control (ISPC) strain RsPC we developed solved this problem and significantly improved the accuracy of quantification of RSSC in different soils. ISPC-based quantitative PCR detection is a method especially suitable for the quantitative detection of microbes in complex matrices (such as soil and sludge) containing various PCR inhibitors and for those not easy to lyse (like Gram-positive bacteria, fungi, and thick-wall cells like resting spores). In addition, the use of ISPC strains removes additional workload on the preparation of high-quality template DNA and facilitates the development of high-throughput quantitative detection techniques for soil microbes.

A network pharmacology-based strategy for predicting the protective mechanism of Ginkgo biloba on damaged retinal ganglion cells.

Hallmarks of the pathophysiology of glaucoma are oxidative stress and apoptotic death of retinal ganglion cells (RGCs). Ginkgo biloba extract (EGb) with multi-target, multi-pathway functions has been reported to exert positive pharmacological effects on oxidative stress and damaged RGCs. However, the ingredients and anti-apoptotic targets of EGb in the treatment of open-angle glaucoma (OAG) have not been fully elucidated. Therefore, in-depth analysis is necessary for further research. Ginkgo biloba-related and anti-apoptotic targets were identified and then combined to obtain the intersection, representing the potential anti-apoptotic targets of Ginkgo biloba. In addition, compound-anti-apoptotic target and OAG-target protein-protein interaction network were merged to obtain five core genes and compound-OAG-anti-apoptotic target protein-protein interaction network. Consequently, the active compounds and anti-apoptotic targets of Ginkgo biloba in the treatment of OAG were identified, namely luteolin, ß-sitosterol, kaempferol, stigmasterol, quercetin, and p53, Bax, Bcl-2, Caspase-3 and Caspase-9, respectively. For the anti-apoptotic targets of Ginkgo biloba in the treatment of OAG, Gene Ontology (GO) and pathway analysis were executed to confirm the gene functions of Ginkgo biloba in antagonizing apoptosis of RGCs. The pathway enrichment was mainly involved in transcriptional activation of p53 responsive genes, activation of caspases and apoptotic processes. Finally, we confirmed the results of the network analysis by H2O2 treated RGC-5 cells in vitro. The results demonstrated that EGb protection can effectively diminish H2O2-induced apoptosis by inhibiting p53 acetylation, reducing the ratio of Bax/Bcl-2 and suppressing the expression of specific cleavage of Caspase-9 and Caspase-3.

Pathogenesis of acephalic spermatozoa syndrome caused by SUN5 variant.

Acephalic spermatozoa syndrome (ASS) is a rare teratozoospermia that leads to male infertility. Previous work suggested a genetic origin. Variants of Sad1 and UNC84 domain containing 5 (SUN5) are the main genetic cause of ASS; however, its pathogenesis remains unclear. Here, we performed whole-exome sequencing in 10 unrelated ASS and identified 2 homozygous variants, c.381delA[p.V128Sfs7*] and c.675C>A[p.Y225X], and 1 compound variant, c.88 C > T[p.R30X] and c.381 delA [p.V128Sfs7*], in SUN5 in 4 patients. The c.381delA variant had been identified as pathogenic in previous reports, while c.675C>A and c.88 C > T were two novel variants which could lead to a premature termination codon (PTC) and resulted in loss of SUN5, and may also be pathogenic. SUN5 mRNA and protein were present at very low levels in ASS patients with SUN5 nonsense mutation. Furthermore, the distribution of outer dense fiber protein 1 (ODF1) and Nesprin3 was altered in sperm of ASS patients with SUN5 variants. The co-immunoprecipitation analysis indicated that SUN5 and ODF1, SUN5 and Nesprin3, and ODF1 and Nesprin3 interacted with each other in transfected HEK293T cells. Thus, we propose that SUN5, Nesprin3, and ODF1 may form a 'triplet' structure through interactions at neck of sperm. When gene variants resulted in a loss of SUN5, the 'triplet' structure disappears and then the head-tail junction becomes fragile, leading to the occurrence of ASS.

Sequential interval micro-droplet loading in closed hemi-straw carrier system: A convenient and efficient method for ultra-rapid cryopreservation in extreme oligozoospermia.

Cryopreservation of human spermatozoa with low concentration while maintaining adequate post-thawing motility remains a major challenge for male fertility preservation. A convenient and efficient ultra-rapid freezing method for small amounts of human spermatozoa in a closed Hemi-Straw carrier system (CHS) was developed. Spermatozoa from 60 healthy men were involved in a parameter refining test and another 15 extreme oligozoospermic specimens were assigned to a verification test. A commercialized sperm freezing medium, Quinn's Advantage® Sperm Freeze medium (glycerol and sucrose as the cryoprotective agent) was used in the study. The results showed that the highest recovery rates would be obtained via the method of 2 µl single droplet sequential interval loading, by placing the straw at 1 cm above the liquid nitrogen (LN2) surface for 60 s during freezing and 2 cm above the LN2 for 2 min during thawing. This method was applied in cryopreservation for the normozoospermic specimens and compared with a conventional slow freezing method. The results were better than those in the control group in the total motility recovery rate (77.8 ± 11.2% vs 56.6 ± 11.9%, P < 0.01), progressive motility recovery rate (77.6 ± 13.2% vs 47.7 ± 14.6%, P < 0.01), 24 h survival index (60.9 ± 13.4% vs 42.1 ± 14.1%, P < 0.01) and the sperm DNA fragment index (4.2 ± 3.7% vs 5.8 ± 3.7%, P = 0.126). This method was applied to the oligozoospermic specimens. Motile spermatozoa could be found in 12 of 15 cases in the ultra-rapid freezing group, while only in 7 cases in control group. The results indicated that this freezing method was simple, convenient and bio-safe for cryopreservation of severe oligozoospermic specimens.

Persistence of Metarhizium (Hypocreales: Clavicipitaceae) and Beauveria bassiana (Hypocreales: Clavicipitaceae) in Tobacco Soils and Potential as Biocontrol Agents of Spodoptera litura (Lepidoptera: Noctuidae).

Entomopathogenic fungi (EPF), such as Metarhizium spp. and Beauveria bassiana, are widely used in the biocontrol of many species of insect pests. Tobacco is an economically important crop in Guangdong Province of China, but insect pests, such as Spodoptera litura Fabricius, are a major threat to production. Here, we tested the persistence of five Metarhizium species and B. bassiana in glasshouse pot and field experiments and assessed their long-term efficacy against S. litura. We found that the colony forming units of these EPF decreased by c. 93% by 180 d in the pot soils declines tended to be exponential. In contrast, declines of c. 99% in field soils were more gradual (linear), occurring throughout the 360 d experiment. Metarhizium anisopliae Ma09 had the longest estimated half-life of 41 d, while that of B. bassiana was the shortest (9 d). Fungal density in the upper soil layer (0-5 cm) decreased rapidly and was undetectable after 150 d, whereas density was consistently greatest in the mid-layer (10-15 cm). At 180 d after inoculation, strain Ma09 elicited highest rates of mortality in S. litura. We conclude that soils in Guangdong Province are all suitable for the use of Metarhizium as a biocontrol agent, where M. anisopliae Ma09 offers greatest residual activity.

[Effect of moxibustion at Sanyinjiao (SP 6) on delivery stage and postpartum bleeding of primipara].

OBJECTIVE: To observe the effect of moxibustion at Sanyinjiao (SP 6) on delivery stage of primipara. METHODS: Sixty cases of primipara were divided into a moxibustion at Sanyinjiao group, a non-acupoint group and a blank group. When the diameter of wormb mouth opened to 2-3 cm, showing that it entered into active stage of uterine contraction, moxibustion at Sanyinjiao (SP 6) for primipara was applied in moxibustion at Sanyinjiao group; moxibustion was applied at 1 cun towards radialis from the region where 2 cun above Kongzui (LU 6) in non-acupoint group, 30 minutes treatment was for both groups. There was no intervention in blank group. After delivery, the time of first,second and third delivery stages and postpartum bleeding 2 hours after delivery were compared among three groups. RESULTS: The active duration of first delivery stage in moxibustion at Sanyinjiao group was significantly shorter than that in the other two groups (P < 0.05, P < 0.01); the duration of the second delivery stage in moxibustion at Sanyinjiao group was significantly shorter than that in the other two groups (both P < 0.05) as well; the postpartum bleeding 2 hours after delivery in moxibustion at Sanyinjiao group was less than other two groups and the difference was statistic significant between moxibustion at Sanyinjiao group and blank group (P < 0.05). CONCLUSION: By moxibustion at Sanyinjiao (SP 6), the active duration of first delivery stage and the duration of second delivery stage are markedly shortened, postpartum bleeding 2 hours after delivery is obviously reduced, and the safety of vaginal delivery is increased.

Effect of acupoint Sanyinjiao (SP6) moxibustion on the first stage of labor and uterine contractive pain in primiparae.

OBJECTIVE: To observe the effect of acupoint Sanyinjiao (SP6) moxibustion (S-Mox) on the duration of the first labor stage and uterine contractive pain in primiparae. METHODS: Sixty primipara women in labor were equally assigned according to their choice to three groups: women in the S-Mox group received bilateral S-Mox for 30 min, women in the non-acupoint group received moxibustion (Mox) applied on non-acupoints for 30 min, and those in the control group did not receive Mox intervention. The duration of the first labor stage was recorded and the degree of labor pain was estimated by a visual analogue scale (VAS) before and after Mox. RESULTS: The duration of the first stage active phase in the S-Mox group was significantly shorter than that in the other two groups (P<0.05, P<0.01); the VAS score after Mox was lower in the S-Mox group, showing a statistical difference in comparison with the control group (P<0.05). CONCLUSIONS: Applying S-Mox could markedly shorten the active phase of the first stage of labor and lower the VAS score of uterine contractive pain, which means alleviating the pain caused by vaginal delivery. Its mechanism is worthy of further study.

[Novel synthetic method and analgesic activity of tepoxalin].

Tepoxalin is a potent inhibitor of both the cyclooxygenase and lipoxygenase pathways of the arachidonic acid cascade, as well as a potent anti-inflammatory and control-pain (postoperation, arthritis et. al.) agent. The new method about the use of novel synthesis reagents and the first using ionic liquid as reactive solvent to synthesize tepoxalin were presented in this paper. The ionic liquid can be easily recycled and reused for several runs efficiently. The analgesic activity of tepoxalin was detected by acetic acid test on mice. The analysis of variance showed that oral administration of tepoxalin could significantly inhibit the number of writhing response within 1 hour and prolong the latent time in a dose dependent manner as compared with CMC control group (P < 0.05). At the same time, tepoxalin had the same analgesic activity as diclofenac sodium.

[Study on the circular dichroism of (S)- and (R)- Wuweizisu C].

The circular dichroism (CD) is an excellent method for determining the absolute stereochemistry of organic compounds. The CD spectra of four biphenyl compounds were determined by using CD spectrometer, and two pairs of symmetrical CD spectra were obtained. The absolute configuration of biphenyl bond was confirmed by the Cotton effect according to the CD rule. The CD spectrum of compound 3 shows a negative Cotton effect at 256 nm, and a positive Cotton effect at 220 nm, which predicts an S-configuration of biphenyl according to an experimental CD rule. Conversely, the CD spectrum of compound 3' displays a positive Cotton effect at 256 nm, and a negative Cotton effect at 219 nm, which predicts an R-configuration of biphenyl. And the regularity of oxazoline-mediated Ullmann reaction was obtained, too.

Controlled fabrication of highly oriented ZnO microrod/microtube arrays on a zinc substrate and their photoluminescence properties.

Well-aligned zinc oxide microrod and microtube arrays with high aspect ratios were fabricated on zinc foil by a simple solution-phase approach in an aqueous solution of ethylenediamine (en). The shape of the ZnO microstructures can be easily modulated from rods to tubes by adding cetyl trimethyl ammonium bromide (CTAB) into the reaction system. Control experiments demonstrate that some reaction parameters, such as the concentration of ethylenediamine, the kind of surfactant, reaction time, and the temperature, all have direct influences on the morphology of the products. Based on the early structure arising from arrested growth (nanosheets), a reasonable mechanism for the growth of ZnO microrods and microtubes has been proposed. The products were characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and photoluminescence emission.

Microarray analysis of gene-expression profile in hepatocellular carcinoma cell, BEL-7402, with stable suppression of hLRH-1 via a DNA vector-based RNA interference.

To establish a cell line with a permanent suppression of hLRH-1 in this study, a stable RNAi vector (pSineohLRH-1) targeting hLRH-1 was constructed and introduced into hepatocellular carcinoma cell, BEL-7402. By semiquantitative RT-PCR analysis, the expression of hLRH-1 in BEL-7402 cells carrying pSineohLRH-1 was shown to be significantly suppressed by up to approximately 60%. In addition, microarray analysis was carried out to assess the extent of altered gene expression in BEL-7402 cells with stable knockdown of hLRH-1. Direct comparison of gene-expression profiles of more than 18,000 genes showed that 405 of the expressed genes in hLRH-1-knockdown cells differed dramatically in expression levels from those in controls, which suggested the even extensive biological functions of hLRH-1. Interestingly, among those differentially expressed genes, some are cancer-associated such as Gadd45beta and PTEN, and their expressions were further validated. Although the identification of the exact relationship between these genes and hLRH-1 awaits intensive investigation, the findings of this study provide new insights into the mechanism by which hLRH-1 is involved in tumorigenesis.