Single-cell RNA sequencing reveals transcriptomic landscape and potential targets for human testicular ageing.

STUDY QUESTION: What is the molecular landscape underlying the functional decline of human testicular ageing? SUMMARY ANSWER: The present study provides a comprehensive single-cell transcriptomic atlas of testes from young and old humans and offers insights into the molecular mechanisms and potential targets for human testicular ageing. WHAT IS KNOWN ALREADY: Testicular ageing is known to cause male age-related fertility decline and hypogonadism. Dysfunction of testicular cells has been considered as a key factor for testicular ageing. STUDY DESIGN, SIZE, DURATION: Human testicular biopsies were collected from three young individuals and three old individuals to perform single-cell RNA sequencing (scRNA-seq). The key results were validated in a larger cohort containing human testicular samples from 10 young donors and 10 old donors. PARTICIPANTS/MATERIALS, SETTING, METHODS: scRNA-seq was used to identify gene expression signatures for human testicular cells during ageing. Ageing-associated changes of gene expression in spermatogonial stem cells (SSCs) and Leydig cells (LCs) were analysed by gene set enrichment analysis and validated by immunofluorescent and functional assays. Cell-cell communication analysis was performed using CellChat. MAIN RESULTS AND THE ROLE OF CHANCE: The single-cell transcriptomic landscape of testes from young and old men was surveyed, revealing age-related changes in germline and somatic niche cells. In-depth evaluation of the gene expression dynamics in germ cells revealed that the disruption of the base-excision repair pathway is a prominent characteristic of old SSCs, suggesting that defective DNA repair in SSCs may serve as a potential driver for increased de novo germline mutations with age. Further analysis of ageing-associated transcriptional changes demonstrated that stress-related changes and cytokine pathways accumulate in old somatic cells. Age-related impairment of redox homeostasis in old LCs was identified and pharmacological treatment with antioxidants alleviated this cellular dysfunction of LCs and promoted testosterone production. Lastly, our results revealed that decreased pleiotrophin signalling was a contributing factor for impaired spermatogenesis in testicular ageing. LARGE SCALE DATA: The scRNA-seq sequencing and processed data reported in this paper were deposited at the Genome Sequence Archive (https://ngdc.cncb.ac.cn/), under the accession number HRA002349. LIMITATIONS, REASONS FOR CAUTION: Owing to the difficulty in collecting human testis tissue, the sample size was limited. Further in-depth functional and mechanistic studies are warranted in future. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide a comprehensive understanding of the cell type-specific mechanisms underlying human testicular ageing at a single-cell resolution, and suggest potential therapeutic targets that may be leveraged to address age-related male fertility decline and hypogonadism. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2022YFA1104100), the National Natural Science Foundation of China (32130046, 82171564, 82101669, 82371611, 82371609, 82301796), the Natural Science Foundation of Guangdong Province, China (2022A1515010371), the Major Project of Medical Science and Technology Development Research Center of National Health Planning Commission, China (HDSL202001000), the Open Project of NHC Key Laboratory of Male Reproduction and Genetics (KF202001), the Guangdong Province Regional Joint Fund-Youth Fund Project (2021A1515110921, 2022A1515111201), and the China Postdoctoral Science Foundation (2021M703736). The authors declare no conflict of interest.

MSC-derived exosomes mitigate cadmium-induced male reproductive injury by ameliorating DNA damage and autophagic flux.

Cadmium, an environmental toxicant, severely impairs male reproductive functions and currently lacks effective clinical treatments. Mesenchymal stem cell-derived exosomes (MSC-Exos) are increasingly recognized as a potential alternative to whole-cell therapy for tissue injury and regeneration. This study aims to investigate the protective effects of MSC-Exos against cadmium toxicity on male reproduction. Our findings reveal that MSC-Exos treatment significantly promotes spermatogenesis, improves sperm quality, and reduces germ cell apoptosis in cadmium-exposed mice. Mechanistically, MSC-Exos dramatically mitigate cadmium-induced cell apoptosis in a spermatogonia cell line (GC-1 spg) in vitro by reducing DNA damage and promoting autophagic flux. These results suggest that MSC-Exos have a protective effect on cadmium-induced germ cell apoptosis by ameliorating DNA damage and autophagy flux, demonstrating the therapeutic potential of MSC-Exos for cadmium toxicity on male reproduction.

Effects of chlorantraniliprole on the development, fecundity and prey consumption of a non-specific predator, Rhynocoris fuscipes (Hemiptera: Reduviidae).

Transplant treatment with chlorantraniliprole (CAP) is a proactive approach to protect transplanted plants from pests during early establishment and has been comprehensively applied in tobacco fields in Guangdong Province, China. However, it is not known whether the high dose of CAP in transplant treatments has lethal or sublethal effects on the generalist predator Rhynocoris fuscipes Fabricius (Hemiptera: Reduviidae). To address this concern, the mortalities of R. fuscipes were assessed when 2nd instar larvae of R. fuscipes were in direct contact with or consuming CAP and when their eggs were exposed to CAP. Furthermore, 2nd instar nymphs R. fuscipes were long-term exposed to CAP until they reached adulthood, and their life table parameters were determined. After exposure to CAP, the activity of detoxification enzymes (P450, CaeE and GST) and the functional respond of R. fuscipes to their preys Agrotis ipsilon larvae were determined. In this study, CAP at all concentrations did not significantly increase the mortality of 2nd instar of R. fuscipes nymphs in comparison with the control. The detoxification enzyme (P450, CarE and GST) activities and the number of A. ipsilon larvae consumed by R. fuscipes in the transplant treatment were not affected by CAP after 3-d or long-term exposure. These results indicated that CAP was harmless to R. fuscipes according to IOBC protocols. However, during the treatment of 2nd instar nymphs with a label rate of 15 g AI/ha and a 5× label rate of 75 g AI/ha, CAP significantly prolonged the pre-adult and pre-oviposition periods, and treated adults had lower oviposition. Attention should be given to the time interval between transplant treatment and the release of this biocontrol agent into the field to minimize the impact of CAP on the predator R. fuscipes.

First Report of Bacterial Soft Rot of Potato Caused by Pectobacterium aroidearum strains in China.

Potato (Solanum tuberosum L.) is a globally important staple crop, and China has contributed more than 20% of the world's production. In Guangdong Province, potato has become one of the most important winter crops, increased the farmer income. However, the potato were seriously affected by soft rot disease caused by Pectobacterium spp. in the past decade. In February 2024, typical symptoms of potato soft rot were observed on the plants of cultivar "Daxiyang" in Enping City, Guangdong Province, where the disease incidences ranged 3%-5% in the investigated fields. The stems adjacent to the tubes showed typical inky black symptoms, and the plants appeared yellow. The interior of the tubes appeared water-soaked and had developed to overall decay, with an evident smell. Eleven symptomatic plants were collected and used to isolate the causal agents. Pieces of tube tissue (about 5×5 mm) were removed between the rot-symptomatic and non-symptomatic margins, and surface sterilized using 75% ethanol for 30 s and 2% NaClO for 1 min. The pieces were rinsed in sterile water for three times, and then plated on Luria-Bertani (LB) medium agar for 36 h at 30°C. The produced colonies were selected for hypersensitive test on tobacco, and the colonies with a positive reaction were subcultured three times. Two representative strains, EP51-2 and EP51-3, were processed for gene sequencing, including house-keeping genes, danX, leuS, and recA (Portier et al. 2019). The gene sequences (GenBank accessions no. PP870934 to PP870939) of these two representative strains were matched with Pectobacterium aroidearum strain NCPPB 929 (CP166097.1) with identities ranging from 97.15 to 100% and the coverage of 100%. The sequences of three genes were concatenated and used for phylogenetic analyses, along with representative strains from 19 other Pectobacterium species. Phylogenetic analyses supported that EP51-2 and EP51-3 were grouped into P. aroidearum with the representative strains. Koch's postulates were applied to determine the pathogenicity of P. aroidearum strain EP51-2. According to the external inoculation method on potato stems (Czajkowski et al. 2010), each of six pots of healthy potato plants was inoculated at the basal stem with 100 µl of bacterial suspension (108 CFU/ml), while six pots inoculated with LB served as controls. The plants were then kept at 30°C and maintained at 95% humidity. After 2 days, all six plants inoculated with the bacteria exhibited typical symptoms on the stems, which progressed to the collapse of the whole plant; the controls remained symptom-free during the observation. Single colonies reisolated from the symptomatic stem of the inoculated plants were confirmed using PCR and sequencing with recA primers as described above, and the causal agent was identified as P. aroidearum strains, fulfilling Koch's postulates. P. aroidearum has been reported on Chinese cabbage (Xie et al. 2018) and leaf mustard (Chu et al. 2024) in China, as well as on crops in the family Araceae, such as konjac (Wei et al. 2021), taro (Zhou et al. 2022), and Pinellia ternata (Du et al. 2024). To our knowledge, this is the first report of P. aroidearum causing potato soft rot in China. This pathogen has been prevalent in the taro fields in Guangdong Province and has caused severe losses. This report highlights an expansion of the host range for this pathogen. Attention should be focused on this newly emerging pathogen affecting potatoes, and immediate measures should be implemented to control its spread.

First Report of Fusarium Root Rot of Tobacco Caused by Fusarium fujikuroi in Guangdong Province, China.

From March to June 2022, Fusarium tobacco root rot broke out in Shaoguan Guangdong Province, China, affecting approximately 15% of tobacco production fields, with an incidence of 24% to 66%. In the early stage, the lower leaves showed chlorosis, and the roots became black. In the later stage, the leaves became browned and withered, the root cortices were broken and shed, and only a small number of roots were left. Eventually, the entire plant died. Six diseased plant samples (cv. yueyan 97) from Shaoguan (113.8°E, 24.8°N) were collected as test materials. The diseased root tissues (4×4 mm) were surface-sterilized using 75% ethanol for 30 s and 2% NaOCl for 10 min, rinsed 3 times with sterile water and incubated for 4 days on potato dextrose agar (PDA) medium at 25 °C. Fungal colonies were subcultured on fresh PDA, grown for the next 5 d and purified by single-spore separation. Eleven isolates with similar morphological characteristics were obtained. Their colonies were white and fluffy, and the bottoms of the culture plates were pale pink after 5 days of incubation. The macroconidia were slender, slightly curved and measured 18.54~45.85 µm×2.35~3.84 µm (n=50), with 3 to 5 septa. The microconidia were oval or spindle shaped, with one to two cells, and measured 5.56~16.76 µm×2.32~3.86 µm (n=50). Chlamydospores were absent. Such characteristics are typical of the genus Fusarium (Booth C, 1971). The SGF36 isolate was chosen for further molecular analysis. The TEF-1α and ß-tubulin genes (Pedrozo et al.2015) were amplified. Based on a phylogenetic tree (neighbor-joining method and 1,000 bootstrap values) obtained using multiplex alignments of concatenations of these two genes from 18 Fusarium species, SGF36 was grouped into a clade with Fusarium fujikuroi strain 12-1 (MK443268.1/MK443267.1) and F. fujikuroi isolate BJ-1 (MH263736.1/MH263737.1). To further identity the isolate, five additional gene sequences (rDNA-ITS (OP862807.1), RPB2, histone 3, calmodulin, and mitochondrial small subunit) (Pedrozo et al.2015), were subjected to BLAST searches in GenBank, and the results indicated that they were most similar to F. fujikuroi sequences, with sequence identities greater than 99%. The phylogenetic tree obtained using six genes except mitochondrial small subunit gene showed that SGF36 was grouped together with four F. fujikuroi strains to form a single clade. Pathogenicity was determined by the inoculation of wheat grains with fungi in potted tobacco plants. The SGF36 isolate was inoculated onto sterilized wheat grains, which were then incubated at 25 °C for 7 d. Thirty wheat grains with fungi were added to 200 g of sterilized soil, which was then mixed well and placed into pots. One six-leaf-stage tobacco seedling (cv. yueyan 97) was planted in each pot. A total of 20 tobacco seedlings were treated. Another 20 control seedlings were treated with wheat grains without fungi. All seedlings were placed in a greenhouse at 25 °C with 90% relative humidity. After 5 d, the leaves of all inoculated seedlings showed chlorosis, and the roots became discolored. No symptoms were observed in the controls. The fungus was reisolated from symptomatic roots and confirmed to be F. fujikuroi based on the TEF-1α gene sequence. No F. fujikuroi isolates were recovered from control plants. F. fujikuroi was previously reported to be associated with rice bakanae disease (Ram et al., 2018), soybean root rot (Zhao et al., 2020) and cotton seedling wilt (Zhu et al., 2020). To our knowledge, this is the first report of F. fujikuroi causing root wilt on tobacco in China. The identification of the pathogen may help to establish appropriate measures for controlling this disease.

Petunia hybrida is a New Host of Pectobacterium brasiliense Associated with Soft Rot Disease in China.

Petunia hybrida is commonly cultivated for ornamental use in urban parks greening and street embellishment in China. In March 2022, 60% of P. hybrida plants cv. Wave Purple (n≈1800) from an ornamental plant nursery under natural conditions in Tianhe district (N 113°21'21", E 23°9'3.5"), Guangzhou, Guangdong Province, China, were affected with soft rot disease. The distribution of the disease was generally uniform. Infected plants initially exhibit small water-soaked lesions at the base of the stem, which then extended to the leaves. Eventually the diseased plant collapsed and died. Nine diseased plants were collected, and affected tissues cut into small fragments (5 × 5 mm), which were disinfested in 75% ethanol (30 s) and 2% sodium hypochlorite (60 s), followed by three rinses with sterile distilled water. The sterilized sections were macerated in 200 µl sterile water, and streaked on Luria-Bertani (LB) agar medium and incubated at 28°C for 48 h. Single colonies were restreaked three times to obtain purified isolation. Sixteen bacterial strains with similar morphology were isolated, and their colonies were yellowish white, round, and convex with smooth surfaces on LB agar plate. The representative strain BDQ1 was selected for further analyses and the 16S rDNA gene (GenBank Accession ON982467) were amplified using primer pair 27F/1492R, revealed above 99% sequence identity with some Pectobacterium brasiliense isolates (GenBank Accession Nos. CP046380(1421/1422), MN393966(1419/1422), and CP020350(1419/1422)) using BLASTn. A multilocus phylogenetic analysis by neighbor-joining method (1,000 bootstrap values) based on six housekeeping gene sequences of gyrA (GenBank Accession No. ON995454), icdA (ON995455), mdh (ON995456), mtlD (ON995457), proA (ON995458), and rpoS genes (ON995459) (Ma et al. 2007; Waleron et al., 2008). The results of phylogenetic analysis showed BDQ1 strain belong to the P. brasiliense clade. Pathogenicity tests were performed on ten healthy P. hybrida cv. Wave Purple plants by injecting 10 µl of bacterial suspensions of BDQ1 (108 CFU/ml) into the stems; another 10 healthy control plants were injected with 10 µl of sterile water. All plants were grown at 25-30°C and 60% humidity in natural light/dark cycle. After 3 d, all inoculated plants showed soft rot symptoms resembling to those observed in the nursery, while control plants remained healthy. Bacteria were successfully reisolated from the symptomatic tissues and identified to be P. brasiliense by PCR mentioned above. P. brasiliense is considered a very aggressive pathogen, which has been reported in Eurasia and Africa (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing bacterial soft rot on P. hybrida in China. This pathogen may pose threat to P. hybrida production in area with warmand humid climate in China. The current study expands the known host range of P. brasiliense and helped raise attention on controlling pathogen spread.

[Human urine-derived stem cell-derived exosomes improve diabetic erectile dysfunction in rats].

Objective: To investigate the improving effect of human urine-derived stem cell-derived exosomes (USC-Exo) on the endothelial function and erectile function of male rats with diabetic ED (DED) and explore their action mechanism. METHODS: USC-Exo were extracted from the culture medium of USC by ultracentrifugation and identified. Cavernous sinus endothelial cells (CCEC) were collected from SD male rats and cultured in endothelial cell growth medium-2 (EGM-2) (the normal control group), EGM-2 + L-glucose at 25 mM (the high glucose group), EGM-2 + L-glucose at 25 mmol/L) + USC-Exo at 10 µg/ml (the Exo group), and EGM-2 + L-glucose at 25 mmol/L + USC-Exo at 10 µg/ml) + 3-methyladenine at 2 mmol/L (the 3-MA group), respectively. Changes of the autophagic flux in the CCECs transfected with mRFP-GFP-LC3 adenovirus were detected under the fluorescence microscope. The proliferation and tube-forming ability of the cells were assessed by CCK8 and Matrigel assays, respectively. DED was induced by intraperitoneal injection of streptozotocin in 10 of the rats, which were equally and randomly divided into a DED and an Exo group, and another 5 normal male rats were taken as controls. The rats in the normal and DED groups were injected intracavernously with 100 µl of PBS, and those in the Exo group with 100 µl of USC-Exo at the concentration of 1 µg/µl. Four weeks after treatment, the maximum intracavernous pressure (ICPmax) and mean arterial pressure (MAP) were measured, the endothelial marker CD31 detected by immunofluorescence assay, the expressions of the CD31, Beclin1 and LC3 I/II proteins examined by Western blot, and the number of autophagosomes in the cavernous endothelial cells determined under the transmission electron microscope. RESULTS: USC-Exo significantly increased the number of autophagosomes in the CCEC in the high glucose group compared with that in the normal controls (39.5 ± 6.2 vs 12.5 ± 5.4, P < 0.05). The expression of Beclin1 and proliferation of the CCEC were significantly higher in the Exo than in the high glucose group (P < 0.05). The autophagy inhibitor 3-MA evidently reversed the increasing effect of USC-Exo on the proliferation of the CCEC. The tube-forming ability of the CCEC was significantly increased in the Exo group compared with that in the high glucose group (15.3 ± 3.2 vs 6.3 ± 2.1, P < 0.05), which was also reversed in the 3-MA group. Both ICPmax and the ICPmax/MAP ratio were significantly higher in the Exo than in the DED group (ï¼»86.6 ± 12.6ï¼½ vs ï¼»37.9 ± 10.9ï¼½ mmHg, P < 0.05; 89.3 ± 14.1 vs 41.7 ± 11.5, P < 0.05), and so were the expressions of CD31, Beclin1 and LC3 I/II (P< 0.05) and the number of autophagosomes in the cavernosal endothelial cells (3.7 ± 0.6 vs 1.0 ± 1.0, P < 0.05). CONCLUSIONS: USC-Exo can significantly improve the endothelial and erectile functions of DED rats by increasing the autophagy of cavernosal endothelial cells.

[Compound Chamomile and Lidocaine Hydrochloride Gel applied at different time-windows for premature ejaculation: A prospective single-center randomized controlled study].

OBJECTIVE: To investigate the efficacy and safety of Compound Chamomile and Lidocaine Hydrochloride Gel (CCLH) (Kamistad) applied at different time-windows on premature ejaculation (PE). METHODS: This prospective study included 72 PE patients treated by application of CCLH to the glans and penile body in our hospital from February to October 2021. According to the time of drug administration before insertion into the vagina, we randomly divided the patients into a 5-minute group (n = 39) and a 15-minute group (n = 33). Before and after 1 and 2 weeks of treatment, we compared the intravaginal ejaculation latency time (IELT), PE diagnostic tool (PEDT) score, quality of life, and adverse reactions between the two groups of patients. RESULTS: Totally 62 of the patients completed the follow-up, 35 in the 5-minute group and 27 in the 15-minute group, and all showed significant improvement in IELT (P < 0.01) and PEDT score (P < 0.05) after treatment compared with the baseline. No allergic reactions, such as redness and swelling, developed at the application site in any of the patients, and no adverse significant effect was observed on the erectile hardness in 61 of the cases. Six cases showed increased erectile hardness instead. Fifty-seven of the patients experienced no obvious penile numbness or reduced sexual satisfaction, and all could complete their sexual activities. CONCLUSION: Compound Chamomile and Lidocaine Hydrochloride Gel applied at different time-windows is effective on PE, with a 5-minute rapid onset of action before intercourse, and no obvious adverse effects.

Antimicrobial Activity of Natural Plant Compound Carvacrol Against Soft Rot Disease Agent Dickeya zeae.

Dickeya zeae is a globally important bacterial pathogen that has been reported to cause severe soft rot diseases in several essential food crops, including bananas, rice, maize, and potatoes. Carvacrol, a hydrophobic terpene component, is found in aromatic plants of the Labiatae family and various essential oils. However, little work has been done on its antimicrobial potential against D. zeae. This study aimed to evaluate the antimicrobial activity and the functional mechanism of carvacrol against D. zeae. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of carvacrol against D. zeae were 0.1 mg/mL and 0.2 mg/mL, respectively. Carvacrol affected the cell membrane of D. zeae, as revealed by decreased intracellular ATP concentration, nucleic acid leakage, and decreased membrane potential. Scanning electron microscopy (SEM) micrographs confirmed that D. zeae cell membranes were damaged by carvacrol. Furthermore, a significant inhibition of D. zeae swimming motility and biofilm formation was observed following treatments with carvacrol at sub-inhibitory concentrations, indicating a significantly negative effect on these virulence factors. Accordingly, the tissue infection test revealed that carvacrol significantly reduced the pathogenicity of D. zeae. In a pot experiment, inoculated banana seedlings displayed remarkably lesser disease symptoms following treatment with carvacrol, and the control efficiency for banana soft rot was 32.0% at 14 days post-inoculation. To summarize, carvacrol exhibits strong antimicrobial activity against D. zeae and great potential applications in the control of D. zeae-associated crop diseases.

Comparative study of different transplantation methods of adipose tissue-derived stem cells in the treatment of erectile dysfunction caused by cavernous nerve injury.

We aimed to compare intracavernosal injection (ICI), tail vein injection (IV), and periprostatic injection (PPI) of adipose-derived stem cells (ADSCs) for their ability to improve erectile function in cavernous nerve injury-induced erectile dysfunction (CNIED) rats and to explore the possible mechanism. Eighty-four male SD rats were divided into the sham group (n = 6), BCNI group (bilateral CN crush injury, n = 6), PBS-ICI group (n = 6), PBS-IV group (n = 6), PBS-PPI group (n = 6), ADSC-ICI group (n = 18), ADSC-IV group (n = 18) and ADSC-PPI group (n = 18). ADSCs were labelled with 5-ethynyl-2'-deoxyuridine (EdU), and six rats each in the ADSC-ICI group, ADSC-IV group, and ADSC-PPI group were sacrificed 2, 7, and 28 days after injection. EdU-labelled ADSCs were tracked by immunofluorescence staining. The intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio, neuronal nitric oxide synthase (nNOS)-positive nerve fibres in the dorsal penile nerve and the smooth muscle/collagen ratio in the cavernosum between groups were also evaluated. ADSCs can significantly improve erectile function through ICI or IV. The two are similar in efficacy and superior to PPI. The mechanism may be that after CN injury, ADSCs are recruited to around the MPG and secrete a variety of neurotrophic factors that promote the repair of the CN, thereby improving erectile function.

First report of Curvularia gladioli causing leaf spots on Gladiolus gandavensis in China.

Gladiolus (Gladiolus gandavensis Van Houtte) is a perennial plant in the family Iridaceae, which shows sword-shaped leaves and spikes of brilliantly colored irregular flowers arising from corms. It is one of the most important fresh cut flowers and is widely cultivated worldwide, including in China. In September 2020, white pinpoints were first observed on gladiolus leaves in Jiangmen City, Guangdong Province, China. The white spots eventually turned brown. The lesions then developed into oval to circular spots, which were surrounded with an obvious yellow halo. The spots expanded and coalesced, causing leaf blight. These symptoms were observed on approximately 10% of gladiolus plants in fields measuring ca. 70 ha. Symptomatic leaves were sampled from fields, surface sterilized in 75% ethanol for 30 s, submerged in a 2% NaOCl solution for 10 min, and rinsed three times with sterile water. The samples were then cut into pieces (5 × 5 mm) and incubated for 4 d on potato dextrose agar (PDA) at 25°C. A representative fungal colony was subcultured onto new PDA and grown for another 7 d, and its mycelium appeared to be grayish-black and villiform. This strain was named as Cg_TS. Its conidiophores were simple, septate, cylindrical in shape, and moderate brown in color. They occurred singly or in groups. They were straight or slightly flexuous and ranged in size from 57.0 to 80.0 µm × 4.0 to 8.0 µm. Conidia were 3-distoseptate and curved at the third cell from the base. The third cell was swollen to one side and larger than other cells. These conidia ranged in size from 23.5 to 32.0 µm × 11.5 to 16.0 µm. These morphological characteristics were consistent with the description of Curvularia gladioli Boerema & Hamers (Boerema and Hamers 1989). Using primer pair ITS1 and ITS4, PCR was applied to amplify the internal transcribed spacer (ITS) region of rDNA. This sequence (GenBank accession No. MW426196.1) was subjected to BLAST in GenBank, suggesting that it was most similar to C. gladioli sequences, LT631345.1 and HG778987.1, with both of 99.49% of similarity. To fulfill Koch's postulates, healthy two-month-old gladiolus plants were used for pathogenicity testing, and the leaves were wounded by pressing slightly with a pipette tip. Mycelium disks (3 mm diameter) were applied onto wounded leaves of 10 plants. Another 10 healthy plants were inoculated with PDA disks which served as control. Inoculated samples were placed in a greenhouse at 25°C and 90% relative humidity. After 3 d, brown leaf spots appeared on all of pathogen-inoculated leaves. The symptoms were consistent with those initially observed and C. gladioli was re-isolated from the symptomatic tissue. Identification was confirmed by morphological observation and ITS sequencing. Control leaves remained symptomless. The curvularia fungus was firstly reported on gladiolus in Florida in 1947 and spread globally via infected corms (Torre et al. 2015), it was also reported to cause leaf spots on gladiolus in Brazil in 2013 (Torres et al. 2013). Although C. gladioli had been recorded as a Curvularia species occurring in China (Zhang et al. 2006), it was not reported to cause leaf spots on gladiolus in Guangdong Province and elsewhere in China. To our knowledge, this is the first report of Curvularia gladioli causing leaf spots on gladiolus in China. Identification of this pathogen will help develop diagnostic methods for corms and seedlings, and may lead to the development of appropriate chemical management strategies.

Intratunical injection of human urine-derived stem cells derived exosomes prevents fibrosis and improves erectile function in a rat model of Peyronie's disease.

We aimed to evaluate the effects of intratunical injection of exosomes derived from human urine-derived stem cells (USC-exo) on plaque formation and erectile function in a transforming growth factor-ß1 (TGF-ß1) induced Peyronie's disease (PD) rat model. Twenty-four SD rats were randomly assigned equally to three groups: (I) Sham group (50 µl phosphate-buffered saline [PBS] injected into the tunica albuginea [TA]), (II) PD group (0.5 µg TGF-ß1 in 50 µl PBS injected into the TA) and (III) USC-exo group (0.5 ug TGF-ß1 plus 100 µg USC-exo injected into the TA at the same day). The maximum intracavernous pressure (ICPmax ) and mean arterial pressure (MAP) of each group were evaluated 4 weeks after injection. The plaque formation, fibrosis, matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs (TIMPs) in the TA were evaluated. Four weeks after injection, USC-exo group showed more significantly enhanced ICPmax and ICPmax /MAP than PD group (p < .05). USC-exo could significantly ameliorate the TA fibrosis that could be associated with the inhibition of transdifferentiation of fibroblasts into myofibroblasts, decreased expression of TIMPs (TIMP-1, 2, 3) and increased activity of MMPs (MMP-1, 3, 9) in the TA. According to these findings, USC-exo can be a new candidate for the prevention of PD.

In Vitro Formation of Dickeya zeae MS1 Biofilm.

Bacterial soft rot caused by Dickeya zeae MS1 (Erwinia chrysanthemi) is one of the most devastating banana diseases worldwide. However, knowledge of the development and ecological interactions of D. zeae MS1 biofilm is limited. Here, we visualized the development and architecture of D. zeae MS1 biofilm using confocal laser scanning microscopy, and we evaluated the ability of D. zeae MS1 to form biofilms under different environmental conditions (carbon sources, temperatures, pH levels and mineral elements) using a microtiter plate assay. We found that the development of D. zeae MS1 biofilm could be categorized into four phases and that mature biofilm consisted of a highly organized architecture of both bacterial cells and a self-produced matrix of extracellular polysaccharides. Furthermore, sucrose was the most suitable carbon source for supporting the growth of biofilm cells and that 32 °C and pH 7.0 were the most favorable of the temperatures and pH levels examined. Meanwhile, the addition of Ca2+, Fe2+, K+ and Na+ enhanced the formation of biofilm in minimal medium cultures, whereas 2.5 mM Cu2+ and Mn2+ was inhibitory. A better understanding of biofilm formation under different environmental parameters will improve our knowledge of the growth kinetics of D. zeae MS1 biofilm.

Sperm DNA fragmentation index influences assisted reproductive technology outcome: A systematic review and meta-analysis combined with a retrospective cohort study.

Studies have explored the influence of DNA damage in assisted reproductive technology (ART), but the outcome remains controversial. To determine whether sperm DNA fragmentation index (DFI) has any effect on ART outcomes, we collected detailed data regarding 1,333 IVF cycles performed at our centre, and the data of our retrospective cohort study were extracted for this meta-analysis. We searched PubMed, Web of Science, EMBASE and Google Scholar and performed a systemic review and meta-analysis. Primary meta-analysis of 10 studies comprising 1,785 couples showed that live birth rate was no significantly different between low-DFI group and high-DFI group (p > 0.05). Secondary meta-analysis of 25 studies comprising 3,992 couples showed a higher miscarriage rate in high-DFI group than in low-DFI group (RR=1.57 [1.18, 2.09], p < 0.01). Meta-analysis of eight studies comprising 17,879 embryos revealed a lower good-quality embryo rate (RR=0.65 [0.62, 0.68], p < 0.01). Meta-analysis of 23 studies comprising 6,771 cycles showed that the high-DFI group had a lower clinical pregnancy rate than low-DFI group (RR=0.85 [0.75, 0.96], p < 0.01). Heterogeneity of included studies weakened our conclusions. Our study showed that DFI has adverse effects on ART outcome. More well-designed studies exploring the association between DFI and ART outcome are desired.

[Oxidative stress induces damage to epididymal epithelial tight junction protein ZO-1 and impairs epididymal function in varicocele rats].

OBJECTIVE: To investigate oxidative stress-mediated damage to the epididymal epithelial tight junction protein ZO-1 and its impact on epididymal function in varicocele rats. METHODS: We randomly divided 45 male adolescent SD rats into three groups of equal number: sham operation (left renal vein exposed and isolated), experimental (left renal vein constricted and collaterals of the left spermatic vein fully ligated), and treatment (60-day intragastric administration of vitamin E at 150 mg/kg/d after modeling). At 60 days after modeling, we observed the histological changes in the left epididymis, detected the expressions of ZO-1 and other tight junction-related proteins by real-time quantitative PCR, immunohistochemistry, immunofluorescence staining and Western blotting, determined sperm motility, and measured the levels of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), methylene dioxyamphetamine (MDA) and α-glucosidase (α-Glu) in the epididymal tissue of the rats. RESULTS: Compared with the rats of the sham operation group, those of the experimental group showed disorganized epithelial structure and decreased number of epithelial cells in the left epididymis, with some epithelial cells desquamated into the lumen. The expression of ZO-1 was significantly lower in the experimental than in the sham operation group (P < 0.05) but markedly upregulated after VE treatment (P < 0.05). In comparison with the sham operation group, the animals in the experimental group exhibited remarkably increased content of MDA in the epididymal tissue (ï¼»0.41 ± 0.05ï¼½ vs ï¼»1.21 ± 0.18ï¼½ nmol/mg prot, P < 0.05) but decreased levels of SOD (ï¼»814.65 ± 73.64ï¼½ vs ï¼»298.62 ± 67.84ï¼½ U/mg prot, P < 0.05), T-AOC (ï¼»0.84 ± 0.07ï¼½ vs ï¼»0.24 ± 0.04ï¼½ nmol/mg prot, P < 0.05) and α-Glu (ï¼»11.72 ± 2.72ï¼½ vs ï¼»5.82 ± 1.24ï¼½ U/mg prot, P < 0.05). VE treatment, however, remarkably reduced the content of MDA (ï¼»0.69 ± 0.12ï¼½ nmol/mg prot) and elevated the levels of SOD (ï¼»497.73 ± 48.03ï¼½ U/mg prot), T-AOC (ï¼»0.42 ± 0.06ï¼½ nmol/mg prot) and α-Glu (ï¼»9.11 ± 1.91ï¼½ U/mg prot) as compared with those in the experimental group (all P < 0.05). The percentage of progressively motile sperm was significantly lower in the experimental than in the sham operation group (ï¼»31.33 ± 6.32ï¼½% vs ï¼»71.21 ± 5.21ï¼½%, P < 0.05), but markedly increased after VE treatment (ï¼»60.68 ± 5.31ï¼½%, P < 0.05). CONCLUSIONS: Varicocele reduces the expression of the EETJ protein ZO-1 and impairs epididymal function via oxidative stress, while vitamin E can effectively upregulate the ZO-1 expression and improve epididymal function by decreasing oxidative stress in the epididymis of varicocele rats.

Genomic analysis of the Phalaenopsis pathogen Dickeya sp. PA1, representing the emerging species Dickeya fangzhongdai.

BACKGROUND: Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a novel species, Dickeya fangzhongdai, and only the orchid-associated strains have been shown to cause soft rot symptoms. METHODS: We constructed the complete PA1 genome sequence and used comparative genomics to explore the differences in genomic features between D. fangzhongdai and other Dickeya species. RESULTS: PA1 has a 4,979,223-bp circular genome with 4269 predicted protein-coding genes. D. fangzhongdai was phylogenetically similar to Dickeya solani and Dickeya dadantii. The type I to type VI secretion systems (T1SS-T6SS), except for the stt-type T2SS, were identified in D. fangzhongdai. The three phylogenetically similar species varied significantly in terms of their T5SSs and T6SSs, as did the different D. fangzhongdai strains. Genomic island (GI) prediction and synteny analysis (compared to D. fangzhongdai strains) of PA1 also indicated the presence of T5SSs and T6SSs in strain-specific regions. Two typical CRISPR arrays were identified in D. fangzhongdai and in most other Dickeya species, except for D. solani. CRISPR-1 was present in all of these Dickeya species, while the presence of CRISPR-2 varied due to species differentiation. A large polyketide/nonribosomal peptide (PK/NRP) cluster, similar to the zeamine biosynthetic gene cluster in Dickeya zeae rice strains, was discovered in D. fangzhongdai and D. solani. The D. fangzhongdai and D. solani strains might recently have acquired this gene cluster by horizontal gene transfer (HGT). CONCLUSIONS: Orchid-associated strains are the typical members of D. fangzhongdai. Genomic analysis of PA1 suggested that this strain presents the genomic characteristics of this novel species. Considering the absence of the stt-type T2SS, the presence of CRISPR loci and the zeamine biosynthetic gene cluster, D. fangzhongdai is likely a transitional form between D. dadantii and D. solani. This is supported by the later acquisition of the zeamine cluster and the loss of CRISPR arrays by D. solani. Comparisons of phylogenetic positions and virulence determinants could be helpful for the effective quarantine and control of this emerging species.

[A preliminary investigation on sexual arousal in blind males].

OBJECTIVE: To explore the perception of the female partner's beauty, stimulating factors for sexual arousal, and the acquisition of sexual knowledge in blind males and analyze the related influencing factors. METHODS: Using a self-designed questionnaire and with the help of the Disabled Persons' Federation and the Association for the Blind of Ganzhou, we conducted an investigation among 54 blind males at an average age of 33.5 years, who met the inclusion criteria and were enrolled according to the rosters provided by the above institutions. We convened the subjects in the appointed hospitals or other places for telephone or face-to-face question-and-answer interviews with qualified investigators trained previously. RESULTS: In the first phase, as the preliminary investigation, open-ended questions were asked of 21 of the subjects on the telephone or at a one-to-one interview. In the second phase, as the formal investigation, closed-ended questions were asked of the other 33 blind males at a face-to-face interview. The blind males perceived the beauty of their sexual partners mainly by the female's mild disposition and sweet voice (63.6% ï¼»21/33ï¼½), soft and satiny skin (51.5% ï¼»17/33ï¼½), sexy and well-shaped body (54.5% ï¼»18/33ï¼½), and intoxicating bodily scent (18.2% ï¼»6/33ï¼½). The stimulating factors for sexual arousal of the subjects included hearing the partner's soft and sweet voice (39.4% ï¼»13/33ï¼½), feeling her satiny skin (54.5% ï¼»18/33ï¼½), touching her sexy and well-shaped body (66.7% ï¼»22/33ï¼½), and the pleasant mood of the males (30.3% ï¼»10/33ï¼½). CONCLUSIONS: The blind males in Ganzhou perceive the beauty of their sexual partners mainly by their senses of hearing, touch and smell, and the stimulating factors for sexual arousal chiefly include the partner's sweet voice, satiny skin and well-shaped body as well as the pleasant mood of the males.

[Protective effect of urine-derived stem cells on erectile dysfunction in rats with cavernous nerve injury].

OBJECTIVE: To investigate the protective effect of human urine-derived stem cells (USCs) on erectile function and cavernous structure in rats with cavernous nerve injury (CNI). METHODS: Sixty adult male SD rats with normal sexual function were randomly divided into four groups of equal number: sham operation, bilateral CNI (BCNI) model control, phosphate buffered saline (PBS), and USC. The BCNI model was established in the latter three groups of rats by clamping the bilateral cavernous nerves. After modeling, the rats in the PBS and USC groups were treated by intracavernous injection of PBS at 200 µl and USCs at 1×106/200 µl PBS respectively for 28 days. Then, the maximum intracavernous pressure (mICP) and the ratio of mICP to mean arterial pressure (mICP/MAP) of the rats were calculated by electrical stimulation of the major pelvic ganglions, the proportion of nNOS- or NF200-positive nerve fibers in the total area of penile dorsal nerves determined by immunohistochemical staining, the levels of endothelial cell marker eNOS, smooth muscle marker α-SMA and collagen I detected by Western blot, and the smooth muscle to collagen ratio and the cell apoptosis rate in the corpus cavernosum measured by Masson staining and TUNEL, respectively. RESULTS: After 28 days of treatment, the rats in the USC group, as compared with those in the PBS and BCNI model control groups, showed significant increases in the mICP (ï¼»81 ± 9.9ï¼½ vs ï¼»31 ± 8.3ï¼½ and ï¼»33 ± 4.2ï¼½ mmHg, P <0.05), mICP/MAP ratio (0.72 ± 0.05 vs 0.36 ± 0.03 and 0.35 ± 0.04, P <0.05), the proportions of nNOS-positive nerve fibers (ï¼»11.31 ± 4.22ï¼½% vs ï¼»6.86 ± 3.08ï¼½% and ï¼»7.29 ± 4.84ï¼½% , P <0.05) and NF200-positive nerve fibers in the total area of penile dorsal nerves (ï¼»27.31 ± 3.12ï¼½% vs ï¼»17.38 ± 2.87ï¼½% and ï¼»19.49 ± 4.92ï¼½%, P <0.05), the eNOS/GAPDH ratio (0.52 ± 0.08 vs 0.31 ± 0.06 and 0.33 ± 0.07, P <0.05), and the α-SMA/GAPDH ratio (1.01 ± 0.09 vs 0.36 ± 0.05 and 0.38 ± 0.04, P <0.05), but a remarkable decrease in the collagen I/GAPDH ratio (0.28 ± 0.06 vs 0.68 ± 0.04 and 0.70 ± 0.10, P <0.05). The ratio of smooth muscle to collagen in the corpus cavernosum was significantly higher in the USC than in the PBS and BCNI model control groups (17.91 ± 2.86 vs 7.70 ± 3.12 and 8.21 ± 3.83, P <0.05) while the rate of cell apoptosis markedly lower in the former than in the latter two (3.31 ± 0.83 vs 9.82 ± 0.76, P <0.01; 3.31 ± 0.83 vs 9.75 ± 0.91, P <0.05). CONCLUSIONS: Intracavernous injection of USCs can protect the erectile function of the rat with cavernous nerve injury by protecting the nerves, improving the endothelial function, alleviating fibrosis and inhibiting cell apoptosis in the cavernous tissue.

[Anatomical variability of the left spermatic vein and establishment of the experimental left varicocele model in adolescent rats].

OBJECTIVE: To identify the anatomical variability of the left spermatic vein (LSV) and determine its effect on the induction of experimental left varicocele (ELV) in adolescent rats. METHODS: We equally randomized 30 adolescent male SD rats to groups A (LSV collaterals fully ligated and the left renal vein constricted), B (only the left renal vein constricted), and C (sham operation), observed the courses of the LSVs and measured their diameters. At 30 days after operation, we analyzed the changes in the left kidneys and the diameters of the LSVs. RESULTS: Irregular collaterals were observed in 90% of the LSVs and no abnormal changes were found in the left kidneys after surgery. The postoperative LSV diameter was remarkably increased in group A as compared with the baseline ([1.47 +/- 0.15 ] vs [0.16 +/- 0.08] mm, P < 0.01), but showed no significant difference in group B ([0.31 +/- 0.49] vs [0.15 +/- 0.07] mm, P > 0.05) and C ([0.17 +/- 0.07] vs [0.16 +/- 0.06] mm, P > 0.05), and it was significantly longer in A than in B (P < 0.01). The success rate of ELV induction was 100% in group A and 10% in group B, but no varicocele was observed in group C. CONCLUSION: Correct identification of the anatomical course of the LSV and ligation of its irregular collaterals are essential for the establishment of a stable and consistent ELV model.

Dickeya fangzhongdai was prevalent and caused taro soft rot when coexisting with the Pectobacterium complex, with a preference for Araceae plants.

Bacterial soft rot caused by coinfection with Dickeya spp. and Pectobacterium spp. in hosts can cause successive changes in fields, and it is difficult to prevent the spread of and control the infection. Pectobacterium spp. are prevalent in the growing areas of tuberous crops, including taro and potato. Recently, Dickeya fangzhongdai has emerged as a virulent pathogen in taro. To determine the prevalence status of the causal agents and evaluate the potential spreading risks of D. fangzhongdai, screening and taxonomic classification were performed on phytopathogenic bacteria collected from different taro-growing areas in Guangdong Province, China, and biological and genomic characteristics were further compared among typical strains from all defined species. The causative agents were verified to be phytobacterial strains of D. fangzhongdai, Pectobacterium aroidearum and Pectobacterium colocasium. P. aroidearum and P. colocasium were found to form a complex preferring Araceae plants and show intensive genomic differentiation, indicating their ancestor had adapted to taro a long time prior. Compared with Pectobacterium spp., D. fangzhongdai was more virulent to taro corms under conditions of exogenous infection and more adaptable at elevated temperatures. D. fangzhongdai strains isolated from taro possessed genomic components of additional T4SSs, which were accompanied by additional copies of the hcp-vgrG genes of the T6SS, and these contributed to the expansion of their genomes. More gene clusters encoding secondary metabolites were found within the D. fangzhongdai strains than within the Pectobacterium complex; interestingly, distinct gene clusters encoding zeamine and arylpolyene were both most similar to those in D. solani that caused potato soft rot. These comparisons provided genomic evidences for that the newly emerging pathogen was potentially equipped to compete with other pathogens. Diagnostic qPCR verified that D. fangzhongdai was prevalent in most of the taro-growing areas and coexisted with the Pectobacterium complex, while the plants enriching D. fangzhongdai were frequently symptomatic at developing corms and adjacent pseudostems and caused severe symptoms. Thus, the emerging need for intensive monitoring on D. fangzhongdai to prevent it from spreading to other taro-growing areas and to other tuberous crops like potato; the adjustment of control strategies based on different pathopoiesis characteristics is recommended.