Improving linolenic acid content in rapeseed oil by overexpression of CsFAD2 and CsFAD3 genes.

With the increasing public attention to the health benefit of polyunsaturated fatty acids (PUFAs) and demand for linolenic acid (C18:3), it is of great significance to increase the C18:3 content in our meal. As an oil crop with high content of C18:3, Camelina sativa has three homologous copies of FAD2 and three homologous copies FAD3. In this study, we seed-specifically overexpressed two Camelina sativa fatty acid desaturase genes, CsFAD2 and CsFAD3, in rapeseed cultivar Zhongshuang 9. The results show that C18:3 content in CsFAD2 and CsFAD3 overexpressed seeds is increased from 8.62% in wild-type (WT) to 10.62-12.95% and 14.54-26.16%, respectively. We crossed CsFAD2 and CsFAD3 overexpression lines, and stable homozygous digenic crossed lines were obtained. The C18:3 content was increased from 8.62% in WT to 28.46-53.57% in crossed overexpression lines. In addition, we found that the overexpression of CsFAD2 and CsFAD3 had no effect on rapeseed growth, development, and other agronomic traits. In conclusion, we successfully generated rapeseed germplasms with high C18:3 content by simultaneously overexpressing CsFAD2 and CsFAD3, which provides a feasible way for breeding high C18:3 rapeseed cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01445-0.

Nanofiber-based wound dressing in wound healing of elderly patients.

Introduction: With the aging of the population, it has become a serious problem that the wounds of elderly patients are not easy to heal. Aim: To explore the application of nanofiber wound dressing in wound healing of elderly patients. Material and methods: In this article, 86 elderly patients with chronic wounds admitted to the Affiliated Hospital of Shaanxi University of Chinese Medicine from January 2023 to September 2023 were enrolled, and they were randomly divided into control and experimental groups, with 43 cases in each group. Controls used traditional wound dressings in care. The experimental group used nanofiber wound dressings. The wound healing efficacy, inflammatory factors, side effects, wound infection degree, healing time, and pain were compared between the two groups. Results: The total effective rate of the experimental group (97.67%) was higher as against controls (86.04%). Wound germiculture positive (7.14 ±2.76%) was lower in the experimental group as against controls (22.13 ±3.27%. The wound healing time of the experimental group (18.68 ±5.78 d) was shorter as against controls (30.24 ±6.19 d). The visual analogue scale (VAS) score of the experimental group (2.68 ±0.41 s) was lower as against controls (3.57 ±0.89 s) after 16 days of care (p < 0.05). Conclusions: The use of nanofiber dressings is an effective means to promote wound healing in elderly patients, which is worthy of application in practical care.

Cadmium induces apoptosis of mouse spermatocytes through JNK activation and disruption of autophagic flux.

Cadmium has been reported to accumulate primarily in spermatogonia and spermatocytes. Exposure to cadmium results in male reproductive toxicity via germ-cell apoptosis and impaired autophagy. Apoptosis and autophagy are two physiologically conserved events that maintain cellular homeostasis. However, the precise role of autophagy in cadmium-induced apoptosis of male germ cells has yet to be addressed. The present study aimed to investigate the impact of cadmium exposure on the cytotoxicity of GC-2 spd cells, a mouse spermatocyte cell line. The results showed that cadmium exposure caused apoptotic cell death and the accumulation of autophagosomes, along with the up-regulation of ATG proteins in GC-2 spd cells. It was demonstrated that the cadmium-induced accumulation of autophagosomes contributes to the apoptosis of GC-2 spd cells. This notion is supported by the findings that the autophagy inhibitor 3-MA reduced accumulation of autophagosomes and apoptotic cell death. Conversely, the apoptosis inhibitor Z-VAD-FMK inhibited apoptosis but had little effect on the accumulation of autophagosomes. Cadmium may impede the fusion of autophagosomes with lysosomes, leading to the autophagosome buildup. Additionally, we found that the JNK pathway mediates transcriptional induction of several autophagy-related (ATG) genes involved in autophagosome formation. The cadmium-activated JNK pathway regulates apoptosis by mediating the autophagosome formation. Treatment of cells with the JNK inhibitor SP600125 attenuated the accumulation of autophagosomes, the upregulated expression of autophagosome-associated proteins and apoptotic cell death induced by cadmium. Overall, these findings suggest that cadmium enhances apoptosis of GC-2 spd cells by activating the JNK pathway and inhibiting autophagic flux.

[The role of PERK-eIF2α-ATF4-CHOP pathway in the apoptosis of TM4 cells induced by bisphenol A].

OBJECTIVE: To investigate the effects of bisphenol A(BPA) on the proliferation and apoptosis of mouse testicular sertoli cells(TM4 cells) and the role of PERK-eIF2α-ATF4-CHOP pathway. METHODS: TM4 cells were treated with different concentrations of BPA(0, 25, 50, 100 µmol/L) and 100 µmol/L BPA combined with protein kinase R-like ER kinase(PERK) inhibitor GSK2656157 for 24 h, and the apoptosis of TM4 cells was observed by TUNEL staining. The expression levels of Bax, Bcl-2, cleaved Caspase-3, GRP78 and PERK-eIF2α-ATF4-CHOP pathway-related proteins were detected by Western blot. RESULTS: The apoptosis rate of TM4 cells in 25, 50 and 100 µmol/L BPA exposed groups was increased to 3.31%±0.34%, 7.51%±1.10% and 14.58%±0.91%, respectively, which was significantly higher than that in control group(0.73%±0.03%, P<0.05). Compared with the control group(1.00), cleaved Caspase-3 protein expression of TM4 cells in the 25, 50 and 100 µmol/L BPA exposed groups increased to 1.49±0.11, 1.59±0.12, 2.42±0.24, respectively; the ratio of Bax/Bcl-2 increased to 2.06±0.19, 3.94±0.034, 6.14±0.71, respectively; the protein expression of GRP78 increased to 1.29±0.06, 1.39±0.06, 1.92±0.17, respectively; the expression of p-PERK protein was increased to 1.64±0.03, 2.52±0.09, 2.80±0.11, respectively; the expression of p-eIF2α protein was increased to 1.79±0.05, 2.48±0.10, 4.77±0.32, respectively; ATF4 protein expression was increased to 2.51±0.03, 3.24±0.14 and 7.45±0.51, respectively; CHOP protein expression was increased to 1.44±0.01, 3.20±0.11 and 3.80±0.11, respectively, and all the differences were statistically significant(P<0.05). Compared to 100 µmol/L BPA group, the expression level of p-PERK, p-eIF2α, ATF4, CHOP, cleaved Caspase-3 protein and the ratio of Bax/Bcl-2 in 100 µmol/L BPA+10 µmol/L GSK2656157 group were decreased to 2.17±0.11, 1.81±0.13, 1.71±0.23, 2.18±0.22, 1.43±0.03, 2.22±0.13, respectively; the apoptosis rate of TM4 cells was also decreased to 7.28%±0.47%, all the differences were statistically significant(P<0.05). CONCLUSION: BPA can induce apoptosis of TM4 cells by activating endoplasmic reticulum stress and regulating PERK-eIF2α-ATF4-CHOP pathway.

[KEAP1/PGAM5/AIFM1 mediated oxeiptosis pathway in TDCIPP-induced reduction of TM4 cell viability].

OBJECTIVE: To investigate the toxic effects and potential mechanisms of tri(1, 3-dichloro-2-propyl) phosphate(TDCIPP) exposure on the mouse testicular supporting cell line(TM4 cells). METHODS: TM4 cells were treated with different concentrations of TDCIPP(0, 12.5, 25 and 50 µmol/L), or 50 µmol/L TDCIPP combined with antioxidant N-acetylcysteine(NAC) for 24 h. Cell viability was assessed using the CCK8 assay, intracellular ROS levels were detected using the DCFH-DA probe, and the protein levels of oxeiptosis-related proteins, such as KEAP1, PGAM5, AIFM1 and phosphorylated AIFM1(p-AIFM1), were detected using Western blot. RESULTS: TDCIPP dose-dependently reduced TM4 cell viability(P<0.05). ROS levels in TM4 cells treated with 12.5, 25 and 50 µmol/L TDCIPP were 9.44±1.42, 17.25±1.81 and 18.38±2.66, respectively, significantly higher than the control group's 5.08±0.90(P<0.05). ROS levels in the 5 mmol/L NAC+50 µmol/L TDCIPP group were 14.70±0.50, significantly lower than the corresponding TDCIPP group's 26.44±0.73(P<0.05). The activity of TM4 cells in KEAP1siRNA+TDCIPP group and PGAM5siRNA+TDCIPP group were 77.00±1.73 and 76.67±1.53, respectively, significantly higher than TDCIPP group 68.67±1.53(P<0.05). The relative expression of KEAP1 protein in TM4 cells treated with 25 and 50 µmol/L TDCIPP were 0.77±0.04 and 0.82±0.02, respectively, significantly higher than the control group's 0.57±0.01(P<0.05). The relative expression of PGAM5 protein in TDCIPP-treated TM4 cells were 1.17±0.04, 1.38±0.03 and 1.41±0.03, respectively, significantly higher than the control group's 0.81±0.02(P<0.05). The relative expression of AIFM1 protein were 0.42±0.01, 0.63±0.01 and 0.68±0.02, respectively, significantly higher than the control group's 0.34±0.02(P<0.05). The relative expression of p-AIFM1 protein were 1.73±0.02, 1.52±0.02 and 0.73±0.01, respectively, significantly lower than the control group's 2.25±0.02(P<0.05). In the 5 mmol/L NAC+50 µmol/L TDCIPP group, the relative expression of KEAP1, PGAM5 and AIFM1 proteins in TM4 cells were 0.61±0.01, 0.58±0.01 and 0.48±0.03, respectively, significantly lower than the TDCIPP group's 0.86±0.12(P<0.05), 0.74±0.02(P<0.05) and 0.92±0.01(P<0.05). The relative expression of p-AIFM1 protein in the 5 mmol/L NAC+50 µmol/L TDCIPP group was 0.45±0.11, significantly higher than the TDCIPP group's 0.23±0.01(P<0.05). CONCLUSION: The reduction of TM4 cell viability induced by TDCIPP may be related to ROS-mediated regulation of the KEAP1/PGAM5/AIFM1 pathway, leading to oxeiptosis.

[Effect of reactive oxygen species in cadmium chloride induced apoptosis of mouse Leydig cells].

OBJECTIVE: To study the effect of reactive oxygen species(ROS) in cadmium chloride-induced apoptosis of mouse Leydig cells(TM3 cells) and explore the underlying molecular mechanisms. METHODS: TM3 cells were used as an in vitro model for studying reproductive toxicity induced by cadmium exposure. The cells were treated with different concentrations of CdCl_2(0, 5 and 10 µmol/L) for 24 h. CCK-8 assay was used to detect the effect of CdCl_2 on TM3 cell activity. Hoechst33342 staining was performed to explore the formation of apoptotic bodies. DCFH-DA probe was used to detect the level of ROS in the cells. TM3 cells were pretreated with 1 mmol/L NAC for 1 h and then treated with 10 µmol/L CdCl_2 for 24 h. The protein expression levels of pro-apoptotic proteins Caspase-9 and cleaved Caspase-3 were detected by Western blot; RT-qPCR was used to measure the expression of anti-apoptotic gene Bcl-2 and pro-apoptotic genes Caspase-9 and Caspase-3. RESULTS: After exposure to CdCl_2 for 24 h, viability of TM3 cells decreased and the number of apoptotic bodies increased. Western blot result showed that the protein level of Caspase-9 in the 10 µmol/L CdCl_2 treatment group was increased to 0.86±0.10(P<0.05) compared with the control group(0.56±0.07). Compared with the control group(0.37±0.11), the protein level of cleaved Caspase-3 in the 5 and 10 µmol/L CdCl_2 treatment groups were increased to 0.65±0.03 and 1.05±0.13(P<0.05). Compared with the control group(46.80±1.24), the intracellular ROS content in the 5 and 10 µmol/L treatment groups increased to 60.47±1.39 and 80.63±1.34(P<0.05). Compared with the cadmium-treated group, NAC inhibited Caspase-9(CdCl_2 group: 0.89±0.07; CdCl_2+NAC group: 0.28±0.02)and cleaved Caspase-3(CdCl_2 group: 1.53±0.21; CdCl_2+NAC group: 0.66 ±0.07), the difference was statistically significant(P<0.05). At the same time, NAC decreased the ROS level(62.64±0.93) in the CdCl_2 exposure group(80.13±0.94)(P<0.05). In addition, RT-qPCR result showed that the Caspase-9 mRNA levels in the 5 and 10 µmol/L CdCl_2 treatment groups were 1.40±0.14 and 1.90±0.12(P<0.05), compared with the control group(0.97±0.10). Compared with the control group(0.88±0.08), the cleaved Caspase-3 mRNA levels in the 5 and 10 µmol/L CdCl_2 treatment groups were increased to 1.42±0.11 and 1.59±0.12(P<0.05). While in the 5 and 10 µmol/L CdCl_2-treated group, compared with the control group(0.94±0.02), the Bcl-2 mRNA level were decreased to 0.60±0.02 and 0.50±0.09(P<0.05). Compared with the cadmium treatment group(0.57±0.06), NAC could significantly improve the cadmium-induced Bcl-2 mRNA expression level(0.92±0.03), and Caspase-9(CdCl_2 group: 1.96±0.07; CdCl_2+NAC group: 1.04±0.02) and Caspase-3(CdCl_2 group: 1.65±0.02; CdCl_2+NAC group: 0.66±0.04) were decreased(P<0.05). CONCLUSION: The Caspase cascade in mouse Leydig cells can be activated by excessive ROS induced by CdCl_2, and inhibition of ROS production can significantly reduce the CdCl_2-induced apoptosis of TM3 cells.

[Role of autophagy in cadmium-induced damage to the blood-testis barrier in mice].

OBJECTIVE: To investigate the role of autophagy in cadmium chloride (CdCl2)-induced damage to the blood-testis barrier (BTB) in mice. METHODS: Twenty four-week-old male C57BL/6 mice were randomly divided into four groups and intraperitoneally injected with CdCl2 at 0 mg/kg/d (the control), 0.5 mg/kg/d (low-dose), 1.0 mg/kg/d (medium-dose) and 2.0 mg/kg/d (high-dose) respectively for 28 consecutive days. Then the morphological changes of the testis tissue was observed by HE staining, the integrity of BTB measured with the biotracer, and the expressions of the BTB components ZO-1 and N-Cadherin proteins detected by Western blot. The TM4 Sertoli cells were treated with CdCl2at 0, 2.5, 5 and 10 µmol/L respectively for 24 hours, followed by determination of the expression levels of ZO-1 and N-Cadherin as well as the autophagy-related proteins LC3II and p62. Then the cells were again treated with CdCl2 in the presence of the autophagy inhibitor chloroquine (CQ) at 5 µmol/L or the autophagy inducer rapamycin (Rap) at 50 nmol/L for 24 hours, followed by measurement of the expressions of LC3II, p62, ZO-1 and N-Cadherin by Western blot. RESULTS: Compared with the control group, the cadmium-exposed mice showed increased interstitial space in the seminiferous tubules, formation of intracellular cavitation in the germ cells with decreased layers and disordered arrangement, and damaged integrity of the BTB. The expressions of the ZO-1 and N-Cadherin proteins were significantly down-regulated in the testis tissue of the mice in the medium- and high-dose CdCl2 groups (P < 0.05), and even more significantly in the CdCl2-exposed cells in comparison with those in the control mice (P < 0.01), while the expressions of the LC3II and p62 proteins were remarkably up-regulated (P < 0.05). The expressions of ZO-1, N-Cadherin, LC3II and p62 were also up-regulated in the cells co-treated with CQ and CdCl2 (P < 0.01), those of ZO-1, N-Cadherin and p62 down-regulated (P< 0.05) and that of LC3II up-regulated (P < 0.05) in the cells co-treated with Rap and CdCl2. CONCLUSION: CdCl2 can damage the integrity of the mouse BTB, which may be attributed to its ability to enhance the autophagy in Sertoli cells and regulate the expressions of BTB proteins.

[HFD-induced obesity triggers testicular cell senescence and endoplasmic reticulum stress in male mice].

OBJECTIVE: To investigate high-fat diet-induced obesity-triggered testicular cell senescence and endoplasmic reticulum stress. METHODS: We randomly and equally divided 10 four-week-old male C57BL/6J mice into a control and a high-fat group, the former fed with a diet of 10% fat content while the latter with a diet of 60% fat content to establish an obesity model. After eight weeks of feeding, we observed the pathological changes in the testis tissue of the mice by HE staining, detected the serum T content by ELISA, measured the telomere length in the testis cells by RT-PCR, and examined the activity of senescence-associated ß-galactosidase (SA-ß-gal) by histochemical staining. Using RT-qPCR and Western blot, we determined the protein and mRNA expressions of the cell senescence markers p16 and p21 as well as the protein expressions of the endoplasmic reticulum stress markers GRP78 and CHOP in the testis tissue. RESULTS: Compared with the controls, the animals of the high-fat group showed a 45% increase in the body weight, disordered structure of the spermatogenic cells, reduced level of serum T and shortened telomere length of the testis cells (P < 0.01). The mRNA and protein expressions of p16 and p21 were dramatically higher in the high-fat than in the control group (P<0.01), so were the intracellular SA-ß-gal activity and the protein expressions of CHOP and GRP78 (P<0.01). CONCLUSION: High-fat diet-induced obesity triggers testicular cell senescence and endoplasmic reticulum stress in male mice.

The pheromone affects reproductive physiology and behavior by regulating hormone in juvenile mice.

Pheromones could promote hormone secretions and regulate sexual behavior. It was unclear whether multiparous pheromone could induce variations in puberty. The aim was to ascertain whether pheromone in urine of multiparous females induced central precocious puberty (CPP) in juvenile C57BL/6J females. The precocious puberty was examined by vaginal smear, lordosis reaction, HE stain, and ELISA analysis. Results suggested that the first vaginal opening and the first estrus were significantly earlier. The time interval of the first vaginal opening and estrus was significantly shortened. It was interesting that the first estrus was significantly correlated with the first vaginal opening and the time interval of the first estrus. In the first estrus, female lordosis reaction, the number of mature follicles, and the weight of the ovary and uterus significantly increased. The level of luteinizing hormones also significantly increased. Thus, multiparous pheromone can regulate sex hormone to induce CPP in juvenile C57BL/6J females.

Pyruvate transporter BnaBASS2 impacts seed oil accumulation in Brassica napus.

Bile acid: sodium symporter family protein 2 (BASS2) is a sodium-dependent pyruvate transporter, which transports pyruvate from cytosol into plastid in plants. In this study, we investigated the function of chloroplast envelope membrane-localized BnaBASS2 in seed metabolism and seed oil accumulation of Brassica napus (B. napus). Four BASS2 genes were identified in the genome of B. napus. BnaA05.BASS2 was overexpressed while BnaA05.BASS2 and BnaC04.BASS2-1 were mutated by CRISPR in B. napus. Metabolite analysis revealed that the manipulation of BnaBASS2 caused significant changes in glycolysis-, fatty acid synthesis-, and energy-related metabolites in the chloroplasts of 31 day-after-flowering (DAF) seeds. The analysis of fatty acids and lipids in developing seeds showed that BnaBASS2 could affect lipid metabolism and oil accumulation in developing seeds. Moreover, the overexpression (OE) of BnaA05.BASS2 could promote the expression level of multiple genes involved in the synthesis of oil and the formation of oil body during seed development. Disruption of BnaA05.BASS2 and BnaC04.BASS2-1 resulted in decreasing the seed oil content (SOC) by 2.8%-5.0%, while OE of BnaA05.BASS2 significantly promoted the SOC by 1.4%-3.4%. Together, our results suggest that BnaBASS2 is a potential target gene for breeding B. napus with high SOC.

Associations between home renovation and asthma, allergic rhinitis, and eczema among preschool children in Wuhan, China.

To investigate the potential associations between household renovation and allergic diseases among preschool children in Wuhan, we conducted a large cross-sectional questionnaire survey among 9455 preschool children aged 3-6 years in Wuhan during November to December 2019. Data on demographics, health status, and home decoration conditions were analysed based on a questionnaire. Compared with tiles/stone/cement floor covering, the use of composite floor significantly increased the risk of diagnosed rhinitis and eczema among children (rhinitis: AOR, 95% CI: 1.36, 1.06-1.73; eczema: AOR, 95% CI: 1.47, 1.17-1.85). Household renovation had significant associations with diagnosed eczema (within 1 year before pregnancy: AOR, 95% CI: 1.34, 1.20-1.50; during pregnancy: AOR, 95% CI: 1.25, 1.08-1.44). This study suggests that use of artificial synthetic materials in home renovation during early childhood and pregnancy may be potential risk factors for childhood asthma, allergic rhinitis, and eczema.

Mouse spermatogenesis-associated protein 1 (SPATA1), an IFT20 binding partner, is an acrosomal protein.

BACKGROUND: Intraflagellar transport is a motor-driven trafficking system that is required for the formation of cilia. Intraflagellar transport protein 20 (IFT20) is a master regulator for the control of spermatogenesis and male fertility in mice. However, the mechanism of how IFT20 regulates spermatogenesis is unknown. RESULTS: Spermatogenesis associated 1 (SPATA1) was identified to be a major potential binding partner of IFT20 by a yeast two-hybrid screening. The interaction between SPATA1 and IFT20 was examined by direct yeast two-hybrid, co-localization, and co-immunoprecipitation assays. SPATA1 is highly abundant in the mouse testis, and is also expressed in the heart and kidney. During the first wave of spermatogenesis, SPATA1 is detectable at postnatal day 24 and its expression is increased at day 30 and 35. Immunofluorescence staining of mouse testis sections and epididymal sperm demonstrated that SPATA1 is localized mainly in the acrosome of developing spermatids but not in epididymal sperm. IFT20 is also present in the acrosome area of round spermatids. In conditional Ift20 knockout mice, testicular expression level and acrosomal localization of SPATA1 are not changed. CONCLUSIONS: SPATA1 is an IFT20 binding protein and may provide a docking site for IFT20 complex binding to the acrosome area.

[Effects of down-regulating Spag6L expression on the proliferation and apoptosis of mouse spermatocytes].

OBJECTIVE: To explore the expression of the Spag6L gene during spermatogenesis and the effects of Spag6L silencing on the proliferation and apoptosis of mouse GC-2 spd cells. METHODS: Using reverse-transcription PCR and real-time qPCR, we detected the expression of the Spag6L gene in the testis tissue collected from the mice at 8, 16, 20, 28 and 42 postnatal days. We prepared lentiviral particles inhibiting the expression of Spag6L and transfected them into the GC-2 spd cells. Then we screened the stably transfected cell lines with the Spag6L expression effectively down-regulated by real-time qPCR, analyzed the effects of Spag6L silencing on the proliferation, activity, cell cycle and apoptosis of the GC-2 spd cells by cell counting and flow cytometry, and on the expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 by Western blot. RESULTS: The Spag6L gene was slightly expressed in the testis tissue of the mice at 8 postnatal days and gradually up-regulated with the development of the testis. Inhibition of the Spag6Lexpression significantly decreased the activity of the GC-2 spd cells (P < 0.01), leading to cell arrest in the G1 phase. The expression of the Bax protein was dramatically up-regulated (P < 0.01) while that of Bcl-2 remarkably down-regulated (P < 0.01) in the Spag6L shRNA- transfected cells, inducing the apoptosis of the cells. CONCLUSIONS: The Spag6L gene is involved in the spermatogenesis of mice by regulating the cell cycle, proliferation and apoptosis of spermatocytes.

Murine germ cell-specific disruption of Ift172 causes defects in spermiogenesis and male fertility.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. IFT172 is a component of the IFT complex. Global disruption of mouse Ift172 gene caused typical phenotypes of ciliopathy. Mouse Ift172 gene appears to translate two major proteins; the full-length protein is highly expressed in the tissues enriched in cilia and the smaller 130 kDa one is only abundant in the testis. In male germ cells, IFT172 is highly expressed in the manchette of elongating spermatids. A germ cell-specific Ift172 mutant mice were generated, and the mutant mice did not show gross abnormalities. There was no difference in testis/body weight between the control and mutant mice, but more than half of the adult homozygous mutant males were infertile and associated with abnormally developed germ cells in the spermiogenesis phase. The cauda epididymides in mutant mice contained less developed sperm that showed significantly reduced motility, and these sperm had multiple defects in ultrastructure and bent tails. In the mutant mice, testicular expression levels of some IFT components, including IFT20, IFT27, IFT74, IFT81 and IFT140, and a central apparatus protein SPAG16L were not changed. However, expression levels of ODF2, a component of the outer dense fiber, and AKAP4, a component of fibrous sheath, and two IFT components IFT25 and IFT57 were dramatically reduced. Our findings demonstrate that IFT172 is essential for normal male fertility and spermiogenesis in mice, probably by modulating specific IFT proteins and transporting/assembling unique accessory structural proteins into spermatozoa.

Dissecting the SPAG6 domain that mediates interaction with Snapin.

SPAG6 domain encompassing amino acids 199-280 mediates an interaction between SPAG6 and Snapin.

The sperm-associated antigen 6 interactome and its role in spermatogenesis.

Mammalian SPAG6, the orthologue of Chlamydomonas reinhardtii PF16, is a component of the central apparatus of the '9 + 2' axoneme that controls ciliary/flagellar motility, including sperm motility. Recent studies revealed that SPAG6 has functions beyond its role in the central apparatus. Hence, we reexamined the role of SPAG6 in male fertility. In wild-type mice, SPAG6 was present in cytoplasmic vesicles in spermatocytes, the acrosome of round and elongating spermatids and the manchette of elongating spermatids. Spag6-deficient testes showed abnormal spermatogenesis, with abnormalities in male germ cell morphology consistent with the multi-compartment pattern of SPAG6 localization. The armadillo repeat domain of mouse SPAG6 was used as a bait in a yeast two-hybrid screen, and several proteins with diverse functions appeared multiple times, including Snapin, SPINK2 and COPS5. Snapin has a similar localization to SPAG6 in male germ cells, and SPINK2, a key protein in acrosome biogenesis, was dramatically reduced in Spag6-deficient mice which have defective acrosomes. SPAG16L, another SPAG6-binding partner, lost its localization to the manchette in Spag6-deficient mice. Our findings demonstrate that SPAG6 is a multi-functional protein that not only regulates sperm motility, but also plays roles in spermatogenesis in multiple cellular compartments involving multiple protein partners.

[The role of SPAG6/SPINK2 protein complex in the formation of sperm acrosome in mice].

OBJECTIVE: To explore the expression and regulatory function of sperm-associated antigen 6 (SPAG6) in the formation of the sperm acrosome in mice. METHODS: The expression of SPAG6 during the first wave of spermatogenesis on postnatal days (PN) 8, 12, 16, 20, 24, 28, 30 and 35 was examined by Western blot and the localization of SPAG6 in the testicular germ cells was determined by immunofluorescence. The expression plasmids of SPAG6 and serine protease inhibitor Kazal-type 2 (SPINK2) were constructed, the interaction between SPAG6 and SPINK2 in the AH109 and CHO cells examined by yeast two-hybrid and co-localization assays, and the expression and localization of SPINK2 in the testicular germ cells of the SPAG6-knockout (SPAG6 KO) mice detected by immunofluorescence. RESULTS: SPAG6 was highly expressed between PN 16 and 28 and localized in the acrosome of the round spermatids. Yeast two-hybrid assay showed the growth of SPAG6 and SPINK2 in the selective culture medium SD/-Leu/-Trp/-His, and the transfection of the CHO cells revealed the co-localization of SPAG6 and SPINK2 around the nuclei. The expression and acrosomal localization of SPINK2 were not found in the testicular germ cells of the SPAG6-KO mice. CONCLUSIONS: SPAG6 interacts with SPINK2 and probably participates in the formation of the sperm acrosome by stabilizing the expression of SPINK2 during spermatogenesis.

Unravelling the complexity and redundancy of carbon catabolic repression in Pseudomonas fluorescens SBW25.

The two-component system CbrAB is the principal regulator for cellular metabolic balance in Pseudomonas fluorescens SBW25 and is necessary for growth on many substrates including xylose. To understand the regulatory linkage between CbrAB and genes for xylose utilization (xut), we performed transposon mutagenesis of ΔcbrB to select for Xut+ suppressors. This led to identification of crc and hfq. Subsequent genetic and biochemical analysis showed that Crc and Hfq are key mediators of succinate-provoked carbon catabolite repression (CCR). Specifically, Crc/Hfq sequentially bind to mRNAs of both the transcriptional activator and structural genes involved in xylose catabolism. However, in the absence of succinate, repression is relieved through competitive binding by two ncRNAs, CrcY and CrcZ, whose expression is activated by CbrAB. These findings provoke a model for CCR in which it is assumed that crc and hfq are functionally complementary, whereas crcY and crcZ are genetically redundant. Inactivation of either crcY or crcZ produced no effects on bacterial fitness in laboratory media, however, results of mathematical modelling predict that the co-existence of crcY and crcZ requires separate functional identity. Finally, we provide empirical evidence that CCR is advantageous in nutrient-complex environments where preferred carbon sources are present at high concentrations but fluctuate in their availability.

Spirostaphylotrichin X from a Marine-Derived Fungus as an Anti-influenza Agent Targeting RNA Polymerase PB2.

A new spirocyclic γ-lactam, named spirostaphylotrichin X (1), and three related known spirostaphylotrichins (2-4) were isolated from the marine-derived fungus Cochliobolus lunatus SCSIO41401. Their structures were determined by spectroscopic analyses. Spirostaphylotrichin X (1) displayed obvious inhibitory activities against multiple influenza virus strains, with IC50 values from 1.2 to 5.5 µM. Investigation of the mechanism showed that 1 inhibited viral polymerase activity and interfered with the production of progeny viral RNA. Homogeneous time-resolved fluorescence, surface plasmon resonance assays, and a molecular docking study revealed that 1 could inhibit polymerase PB2 protein activity by binding to the highly conserved region of the cap-binding domain of PB2. These results suggest that 1 inhibits the replication of influenza A virus by interfering with the activity of PB2 protein and that 1 represents a new type of potential lead compound for the development of anti-influenza therapeutics.

Transcriptional regulation of human sperm-associated antigen 16 gene by S-SOX5.

BACKGROUND: The mammalian sperm-associated antigen 16 gene (Spag16) uses alternative promoters to produce two major transcript isoforms (Spag16L and Spag16S) and encode proteins that are involved in the cilia/flagella formation and motility. In silico analysis of both mouse and human SPAG16L promoters reveals the existence of multiple putative SOX5 binding sites. Given that the SOX5 gene encodes a 48-kDa transcription factor (S-SOX5) and the presence of putative SOX5 binding sites at the SPAG16L promoter, regulation of SPAG16L expression by S-SOX5 was studied in the present work. RESULTS: S-SOX5 activated human SPAG16L promoter activity in the human bronchial epithelia cell line BEAS-2B cells. Mutation of S-SOX5 binding sites abolished the stimulatory effect. Overexpression of S-SOX5 resulted in a significant increase in the abundance of SPAG16L transcripts whereas silencing of S-SOX5 by RNAi largely reduced the SPAG16L expression. Chromatin immunoprecipitation assays showed that S-SOX5 directly interacts with the SPAG16L promoter. CONCLUSION: S-SOX5 regulates transcription of human SPAG16L gene via directly binding to the promoter of SPAG16L. It has been reported that expression of sperm-associated antigen 6 (SPAG6), encoding another axonemal protein, is activated by S-SOX5. Therefore, S-SOX5 may regulate formation of motile cilia/flagella through globally mediating expression of genes encoding axonemal proteins.