Locating, tracing and sequencing multiple expanded genetic letters in complex DNA context via a bridge-base approach.

A panel of unnatural base pairs is developed to expand genetic alphabets. One or more unnatural base pairs (UBPs) can be inserted to enlarge the capacity, diversity, and functionality of canonical DNA, so monitoring the multiple-UBPs-containing DNA by simple and convenient approaches is essential. Herein, we report a bridge-base approach to repurpose the capability of determining TPT3-NaM UBPs. The success of this approach depends on the design of isoTAT that can simultaneously pair with NaM and G as a bridge base, as well as the discovering of the transformation of NaM to A in absence of its complementary base. TPT3-NaM can be transferred to C-G or A-T by simple PCR assays with high read-through ratios and low sequence-dependent properties, permitting for the first time to dually locate the multiple sites of TPT3-NaM pairs. Then we show the unprecedented capacity of this approach to trace accurate changes and retention ratios of multiple TPT3-NaM UPBs during in vivo replications. In addition, the method can also be applied to identify multiple-site DNA lesions, transferring TPT3-NaM makers to different natural bases. Taken together, our work presents the first general and convenient approach capable of locating, tracing, and sequencing site- and number-unlimited TPT3-NaM pairs.

Bioinspired Heterogeneous Construction of Lignocellulose-Reinforced COF Membranes for Efficient Proton Conduction.

The exponential interest in covalent organic frameworks (COFs) arises from the direct correlation between their diverse and intriguing properties and the modular design principle. However, the insufficient interlamellar interaction among COF nanosheets greatly hinders the formation of defect-free membranes. Therefore, developing a methodology for the facile fabrication of these materials remains an enticing and highly desirable objective. Herein, ultrahigh proton conductivity and superior stability are achieved by taking advantage of COF composite membranes where 2D TB-COF nanosheets are linked by 1D lignocellulosic nanofibrils (LCNFs) through π-π and electrostatic interactions to form a robust and ordered structure. Notably, the high concentration of -SO3 H groups within the COF pores and the abundant proton transport paths at COFs-LCNFs interfaces impart composite membranes ultrahigh proton conductivity (0.348 S cm-1 at 80 °C and 100% RH). Moreover, the directional migration of protons along the stacked nanochannels of COFs is facilitated by oxygen atoms on the keto groups, as demonstrated by density functional theory (DFT) calculations. The simple design concept and reliable operation of the demonstrated mixed-dimensional composite membrane are expected to provide an ideal platform for next-generation conductive materials.

Hirudin ameliorates myocardial ischemia-reperfusion injury in a rat model of hemorrhagic shock and resuscitation: roles of NLRP3-signaling pathway.

Severe hemorrhage shock and resuscitation (HSR) has been reported to induce myocardial ischemia-reperfusion injury (MIRI), resulting in a poor prognosis. Hirudin, an effective thrombin inhibitor, can offer protection against MIRI. This study aimed to determine if hirudin administration ameliorates HSR-induced MIRI and the underlying mechanism. A rat model of HSR was established by bleeding rats to a mean arterial blood pressure of 30-35 mmHg for 45 min and then resuscitating them with all the shed blood through the left femoral vein. After HSR, 1 mg/kg of hirudin was administrated immediately. At 24 h after HSR, the cardiac injury was assessed using serum CK-MB, cTnT, hematoxylin-eosin (HE) staining, echocardiography, M1-polarized macrophages, and pyroptosis-associated factors, including cleaved caspase-1, Gasdermin D (GSDMD) N-terminal, IL-1ß, and IL-18 were measured by immunofluorescence and western blot assays. Nigericin, a unique agonist, was utilized to evaluate the responsibilities of NLRP3 signaling. Under the HSR condition, rats exhibited a significant increase in myocardial injury score, an elevation of serum cTnT, CK-MB levels, an aggrandization of M1-polarized macrophages, an upregulation of pyroptosis-associated factors, including cleaved caspase-1, GSDMD N-terminal, IL-1ß, and IL-18, but a significant decrease in left ventricular ejection fraction (EF%) and a reduction of left ventricular fractional shortening (FS%), while hirudin administration partially restored the changes. However, the NLRP3 agonist nigericin reversed the cardioprotective effects of hirudin. We determined the cardioprotective effects of hirudin against HSR-induced MIRI. The mechanism may involve the inhibition of NLRP3-induced pyroptosis.

Exploration of the optimal time to discontinue propranolol treatment in infantile hemangiomas: A prospective study.

BACKGROUND: Relapse of infantile hemangiomas after withdrawal from propranolol treatment is common. Early withdrawal is believed to increase the risk of relapse. OBJECTIVE: The objective of this study was to determine the optimal time to discontinue propranolol treatment for infantile hemangiomas. METHODS: A prospective study conducted at a tertiary referral center. RESULTS: Compared to withdrawal after 1-month maintenance treatment, withdrawal after 3-month maintenance, corresponding achieving maximum regression of infantile hemangiomas, was associated with a lower major relapse rate (P = .041). The relapse (P = .055) and adverse event rates (P = .154) between the 2 withdrawal modes were not statistically significant. Compared with direct withdrawal, the relapse (P = .396), major relapse (P = .963), and adverse event rates (P = .458) of gradual withdrawal were not statistically different. Patients with/without relapse could be best distinguished according to whether withdrawal followed a 3-month maintenance and age >13 months (area under the receiver operating characteristic curve = 0.603). Patients with/without major relapse could be best distinguished according to whether withdrawal was accompanied by 3-month maintenance (area under the receiver operating characteristic curve = 0.610). LIMITATIONS: The limitations of this study are nonrandomization and single-center design. CONCLUSIONS: The optimal propranolol withdrawal time to avoid relapse is when the patient is aged >13 months and the lesion has maintained for 3 months after reaching maximum regression, while the optimal time to prevent major relapse is after 3 months of maintenance.

Risk assessment of antimony-arsenic contaminated soil remediated using zero-valent iron at different pH values combined with freeze-thaw cycles.

Soil in mining wastelands is seriously polluted with heavy metals. Zero-valent iron (ZVI) is widely used for remediation of heavy metal-polluted soil because of its excellent adsorption properties; however, the remediation process is affected by complex environmental conditions, such as acid rain and freeze-thaw cycles. In this study, the effects of different pH values and freeze-thaw cycles on remediation of antimony (Sb)- and arsenic (As)-contaminated soil by ZVI were investigated in laboratory simulation experiments. The stability and potential human health risks associated with the remediated soil were evaluated. The results showed that ZVI has a significant stabilizing effect on Sb and As in both acidic and alkaline soils contaminated with dual levels of Sb and As, and the freeze-thaw process in different pH value solution systems further enhances the ability of ZVI to stabilize Sb and As, especially in acidic soils. However, it should be noted that apart from the pH=1.0 solution environment, ZVI's ability to stabilize As is attenuated under other circumstances, potentially leading to leaching of its unstable form and thereby increasing contamination risks. This indicates that the F1 (2% ZVI+pH=1 solution+freeze-thaw cycle) processing exhibits superior effectiveness. After F1 treatment, the bioavailability of Sb and As in both soils also significantly decreased during the gastric and intestinal stages (about 60.00%), the non-carcinogenic and carcinogenic risks of Sb and As in alkaline soils are eliminated for children and adults, with a decrease ranging from 60.00% to 70.00%, while in acidic soil, the non-carcinogenic and carcinogenic risks of As to adults and children is acceptable, but Sb still poses non-carcinogenic risks to children, despite reductions of about 65.00%. These findings demonstrate that soil pH is a crucial factor influencing the efficacy of ZVI in stabilizing Sb and As contaminants during freeze-thaw cycles. This provides a solid theoretical foundation for utilizing ZVI in the remediation of Sb- and As-contaminated soils, emphasizing the significance of considering both pH levels and freeze-thaw conditions to ensure effective and safe treatment.

KIAA1429 promotes infantile hemangioma regression by facilitating the stemness of hemangioma endothelial cells.

Infantile hemangiomas are common vascular tumors with a specific natural history. The proliferation and regression mechanism of infantile hemangiomas may be related to the multilineage differentiation ability of hemangioma stem cells, but the specific mechanism is not well elucidated. KIAA1429 is an N6 -methyladenosine methylation-related protein that can also exert its role in a methylation-independent manner. This study aims to explore the function of KIAA1429 in infantile hemangiomas. qRT-PCR, western blotting, and immunostaining were performed to verify the expression of KIAA1429. The endothelial and fibroblast-like phenotypes of hemangioma endothelial cells were detected after KIAA1429 knockdown and overexpression. The stemness properties of hemangioma endothelial cells and the underlying mechanism of KIAA1429 in hemangiomas were also investigated. Nude mouse models of infantile hemangiomas were conducted to ascertain the effects of KIAA1429 in vivo. The results showed that KIAA1429 was highly expressed in infantile hemangiomas, particularly in involuting hemangiomas. In vitro experiments confirmed that KIAA1429 inhibited the endothelial phenotype, enhanced the differentiation ability, and promoted the fibroblast-like phenotype of hemangioma endothelial cells by inducing endothelial cell transition to facultative stem cells. However, the effect of KIAA1429 on the potential target was shown to be independent of N6 -methyladenosine methylation modification. Mouse models further revealed that KIAA1429 could inhibit the proliferation and promote the regression of hemangiomas. In conclusion, this study found that KIAA1429 played an important role in the regression of infantile hemangiomas by enhancing the stemness of hemangioma endothelial cells and could be a potential treatment target for infantile hemangiomas.

NLRP3-GABA signaling pathway contributes to the pathogenesis of impulsive-like behaviors and cognitive deficits in aged mice.

BACKGROUND: Perioperative neurocognitive disorders (PND), such as delirium and cognitive impairment, are commonly encountered complications in aged patients. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is aberrantly synthesized from reactive astrocytes following inflammatory stimulation and is implicated in the pathophysiology of neurodegenerative diseases. Additionally, the activation of NOD-like receptor protein 3 (NLRP3) inflammasome is involved in PND. Herein, we aimed to investigate whether the NLRP3-GABA signaling pathway contributes to the pathogenesis of aging mice's PND. METHODS: 24-month-old C57BL/6 and astrocyte-specific NLRP3 knockout male mice were used to establish a PND model via tibial fracture surgery. The monoamine oxidase-B (MAOB) inhibitor selegiline (1 mg/kg) was intraperitoneally administered once a day for 7 days after the surgery. PND, including impulsive-like behaviors and cognitive impairment, was evaluated by open field test, elevated plus maze, and fear conditioning. Thereafter, pathological changes of neurodegeneration were explored by western blot and immunofluorescence assays. RESULTS: Selegiline administration significantly ameliorated TF-induced impulsive-like behaviors and reduced excessive GABA production in reactive hippocampal astrocytes. Moreover, astrocyte-specific NLRP3 knockout mice reversed TF-induced impulsive-like and cognitive impairment behaviors, decreased GABA levels in reactive astrocytes, ameliorated NLRP3-associated inflammatory responses during the early stage, and restored neuronal degeneration in the hippocampus. CONCLUSIONS: Our findings suggest that anesthesia and surgical procedures trigger neuroinflammation and cognitive deficits, which may be due to NLRP3-GABA activation in the hippocampus of aged mice.

Mediation effect of pregnancy-induced hypertension on the association between assisted reproductive technology and adverse neonatal outcomes: a population-based study.

BACKGROUND: Assisted reproductive technology (ART) has been widely used in the treatment of infertility, and is associated with adverse maternal and neonatal outcomes. However, the potential pathways by which ART affects adverse neonatal outcomes are unclear. We aimed to investigate the role of pregnancy-induced hypertension (PIH) in the association between ART and adverse neonatal outcomes. METHODS: Adult women (aged ≥ 18 years) with a singleton pregnancy in the National Vital Statistics System (NVSS) 2020 were enrolled in this retrospective cohort study. Study outcomes were adverse neonatal outcomes, including premature birth, low birth weight, and admission to the neonatal intensive care unit (NICU). Logistic regression models were utilized to investigate the association between ART, PIH, and adverse neonatal outcomes, expressed as odds ratio (OR) and 95% confidence interval (CI). The distribution-of-the-product method was used to explore whether there was a mediating effect of PIH between ART and adverse neonatal outcomes, and the 95% CI of the distribution-of-the-product did not contain 0 indicating a mediating effect. RESULTS: This study included 2,824,418 women, of whom 35,020 (1.24%) women used ART, 239,588 (8.48%) women had PIH, and 424,741 (15.04%) neonates had any adverse neonatal outcomes. The use of ART was associated with higher odds of PIH (OR = 1.42; 95%CI: 1.37-1.46) and any adverse neonatal outcomes (OR = 1.47; 95%CI: 1.43-1.51). The distribution-of-the-product was 0.31 (95%CI: 0.28-0.34), and 8.51% of the association between ART and adverse neonatal outcomes was mediated through PIH. Among different adverse neonatal outcomes, PIH mediated 29.17% of the association between ART and low birth weight, 9.37% of the association between ART and premature birth, and 12.20% of the association between ART and NICU admission. The mediating effect of PIH was found in women of different ages (< 35 years and ≥ 35 years) and parities (primipara and multipara). CONCLUSION: This study supports a mediating role for PIH in the association between ART and adverse neonatal outcomes. Further studies are needed to determine the mechanisms by which AR affects PIH so that interventions to reduce PIH can be developed to reduce adverse neonatal outcomes associated with ART.

Chlamydia Deficient in Plasmid-Encoded Glycoprotein 3 (pGP3) as an Attenuated Live Oral Vaccine.

Despite the extensive efforts, there is still a lack of a licensed vaccine against Chlamydia trachomatis in humans. The mouse genital tract infection with Chlamydia muridarum has been used to both investigate chlamydial pathogenic mechanisms and evaluate vaccine candidates due to the C. muridarum's ability to induce mouse hydrosalpinx. C. muridarum mutants lacking the entire plasmid or deficient in only the plasmid-encoded pGP3 are highly attenuated in inducing hydrosalpinx. We now report that intravaginal immunization with these mutants as live attenuated vaccines protected mice from hydrosalpinx induced by wild type C. muridarum. However, these mutants still productively infected the mouse genital tract. Further, the mutant-infected mice were only partially protected against the subsequent infection with wild type C. muridarum. Thus, these mutants as vaccines are neither safe nor effective when they are delivered via the genital tract. Interestingly, these mutants were highly deficient in colonizing the gastrointestinal tract. Particularly, the pGP3-deficient mutant failed to shed live organisms from mice following an oral inoculation, suggesting that the pGP3-deficient mutant may be developed into a safe oral vaccine. Indeed, oral inoculation with the pGP3-deficient mutant induced robust transmucosal immunity against both the infection and pathogenicity of wild type C. muridarum in the genital tract. Thus, we have demonstrated that the plasmid-encoded virulence factor pGP3 may be targeted for developing an attenuated live oral vaccine.

Characterization of Pathogenic CD8+ T cells in Chlamydia-Infected OT1 Mice.

Chlamydia trachomatis is a leading infectious cause of infertility in women due to its induction of lasting pathology such as hydrosalpinx. Chlamydia muridarum induces mouse hydrosalpinx because C. muridarum can both invade tubal epithelia directly (as a first hit) and induce lymphocytes to promote hydrosalpinx indirectly (as a second hit). In the current study, a critical role of CD8+ T cells in chlamydial induction of hydrosalpinx was validated in both wild type C57BL/6J mice and OT1 transgenic mice. OT1 mice failed to develop hydrosalpinx partially due to the failure of their lymphocytes to recognize chlamydial antigens. CD8+ T cells from naive C57BL/6J mice rescued the ability of recipient OT1 mice to develop hydrosalpinx when naive CD8+ T cells were transferred at the time of infection with Chlamydia. However, when the transfer was delayed for 2 weeks or longer after the Chlamydia infection, naive CD8+ T cells no longer promoted hydrosalpinx. Nevertheless, CD8+ T cells from mice immunized against Chlamydia still promoted significant hydrosalpinx in the recipient OT1 mice even when the transfer was delayed for 3 weeks. Thus, CD8+ T cells must be primed within 2 weeks after Chlamydia infection to be pathogenic, but, once primed, they can promote hydrosalpinx for >3 weeks. However, Chlamydia-primed CD4+ T cells failed to promote chlamydial induction of pathology in OT1 mice. This study optimized an OT1 mouse-based model for revealing the pathogenic mechanisms of Chlamydia-specific CD8+ T cells.

Clinical analysis of high-intensity focused ultrasound (HIFU) combined with hysteroscopy-guided suction curettage (HGSC) in patients with cervical pregnancy.

OBJECTIVE: To evaluate the effectiveness of high-intensity focused ultrasound (HIFU) combined with hysteroscopy-guided suction curettage (HGSC) in treating cervical pregnancy. MATERIALS AND METHODS: This is a retrospective study. Seven patients with cervical pregnancy who visited the Third Xiangya Hospital of Central South University from January 2015 to December 2020 were enrolled in the current study. All seven patients were treated with HIFU under conscious sedation. All of them underwent HGSC at an average of 2 ± 1 days (range: 1-3 days) after HIFU. Before the therapy, the patient's clinical characteristics were collected, including duration of amenorrhea, gravidity and parity, the patient history of cesarean section and miscarriage, and the size of the gestational sac. The levels of ß-hCG and hemoglobin in serum were also reviewed. To assess the clinical outcomes of this combined treatment, the suction time of HGSC, bleeding volume, the clearance time of ß-hCG, and the time with returning of menstruation were evaluated. RESULTS: All seven patients (average age: 31 ± 6 years) have experienced amenorrhea (duration range, 48 ± 8 days) before the treatment of HIFU. The average number of pregnancies was four, and the number of deliveries was one. Previous medical history showed six patients had cesarean sections, and five patients have been miscarriages. After HIFU treatment, the fetal heartbeats were stopped in all seven patients based on the diagnosis by doppler ultrasound. The bleeding of gestational tissue decreased significantly. All patients had only mild lower abdominal pain, no fever, intestinal damage, or other complications were reported. The average operation time of operative suction curettage was 21 ± 9 min (range: 9-32 min), and the median bleeding volume was 10 ± 8 mL (range: 2-20 mL). Follow-up observations showed that the menstruations were returned in patients at an average of 38 ± 9 days (range: 30-50 days) after the treatment. The ß-hCG decreased from 41773 ± 32242 mIU/mL to 13101 ± 8454 mIU/mL in 29 ± 10 days after surgery. CONCLUSION: Based on these results with small subjects, we concluded that HIFU combined with HGSC might be an effective and safe treatment for patients with cervical pregnancy.

Chlamydia Spreads to the Large Intestine Lumen via Multiple Pathways.

Chlamydia in the genital tract is known to spread via the blood circulation system to the large intestine lumen to achieve long-lasting colonization. However, the precise pathways by which genital Chlamydia accesses the large intestine lumen remain unclear. The spleen was recently reported to be critical for chlamydial spreading. In the current study, it was found that following intravaginal inoculation with Chlamydia, mice with and without splenectomy both yielded infectious Chlamydia on rectal swabs, indicating that the spleen is not essential for genital Chlamydia to spread to the gastrointestinal tract. This conclusion was validated by the observation that intravenously inoculated Chlamydia was also detected on the rectal swabs of mice regardless of splenectomy. Careful comparison of the tissue distribution of live chlamydial organisms following intravenous inoculation revealed redundant pathways by which Chlamydia can reach the large intestine lumen. The intravenously inoculated Chlamydia was predominantly recruited to the spleen within 12 h and then detected in the stomach lumen by 24 h, in the intestinal lumen by 48 h, and on rectal swabs by 72 h. These observations suggest a potential spleen-to-stomach pathway for hematogenous Chlamydia to reach the large intestine lumen. This conclusion was supported by the observation made in mice under coprophagy-free condition. However, in the absence of spleen, hematogenous Chlamydia was predominantly recruited to the liver and then simultaneously detected in the intestinal tissue and lumen, suggesting a potential liver-to-intestine pathway for Chlamydia to reach the large intestine lumen. Thus, genital/hematogenous Chlamydia may reach the large intestine lumen via multiple redundant pathways.

Gastrointestinal Chlamydia-Induced CD8+ T Cells Promote Chlamydial Pathogenicity in the Female Upper Genital Tract.

Chlamydia is known to both ascend to the upper genital tract and spread to the gastrointestinal tract following intravaginal inoculation. Gastrointestinal Chlamydia was recently reported to promote chlamydial pathogenicity in the genital tract since mice intravaginally inoculated with an attenuated Chlamydia strain, which alone failed to develop pathology in the genital tract, were restored to develop hydrosalpinx by intragastric coinoculation with wild-type Chlamydia. Gastrointestinal Chlamydia promoted hydrosalpinx via an indirect mechanism since Chlamydia in the gut did not directly spread to the genital tract lumen. In the current study, we further investigated the role of CD8+ T cells in the promotion of hydrosalpinx by gastrointestinal Chlamydia. First, we confirmed that intragastric coinoculation with wild-type Chlamydia promoted hydrosalpinx in mice that were inoculated with an attenuated Chlamydia strain in the genital tract 1 week earlier. Second, the promotion of hydrosalpinx by intragastrically coinoculated Chlamydia was blocked by depleting CD8+ T cells. Third, adoptive transfer of gastrointestinal Chlamydia-induced CD8+ T cells was sufficient for promoting hydrosalpinx in mice that were intravaginally inoculated with an attenuated Chlamydia strain. These observations have demonstrated that CD8+ T cells induced by gastrointestinal Chlamydia are both necessary and sufficient for promoting hydrosalpinx in the genital tract. The study has laid a foundation for further revealing the mechanisms by which Chlamydia-induced T lymphocyte responses (as a 2nd hit) promote hydrosalpinx in mice with genital Chlamydia-triggered tubal injury (as a 1st hit), a continuing effort in testing the two-hit hypothesis as a chlamydial pathogenic mechanism.

Integration of RNA-seq and RNAi reveals the contribution of znuA gene to the pathogenicity of Pseudomonas plecoglossicida and to the immune response of Epinephelus coioides.

Pseudomonas plecoglossicida is an important pathogen in aquaculture and causes serious economic losses. Our previous study indicated that znuA gene might play an important role in the pathogenicity of P. plecoglossicida. Five shRNAs were designed and synthesized to silence the znuA gene of P. plecoglossicida. Two of the five mutants of P. plecoglossicida exhibited significant reduction in the expression level of znuA mRNA with different efficiencies. The mutant with the highest silencing efficiency of 89.2% was chosen for further studies. Intrapleural injection of the znuA-RNAi strain at a dose of 105  cfu/fish did not cause the death of Epinephelus coioides, and no significant signs were observed at the spleen surface of infected E. coioides, while the counterpart E. coioides infected by the same dose of wild-type strain of P. plecoglossicida all died in 5 days post-infection (dpi). The expression of znuA gene of znuA-RNAi strain in E. coioides was always lower than that in wild-type strain of P. plecoglossicida. The pathogen load in the early stage of infection was higher than that in the later stage of infection. Although the infection of the znuA-RNAi strain of P. plecoglossicida could induce the production of antibodies in E. coioides, it failed to produce a good immune protection against the infection of wild-type strain of P. plecoglossicida. Compared with the transcriptome data of E. coioides infected by the wild-type strain of P. plecoglossicida, the transcriptome data of E. coioides infected by the znuA-RNAi strain of P. plecoglossicida have altered significantly. Among them, KEGG enrichment analysis showed that the focal adhesion pathway was significantly enriched and exhibited the largest number of 302 DEMs (differentially expressed mRNAs). These results showed that the immune response of E. coioides to P. plecoglossicida infection was significantly affected by the RNAi of znuA gene.

Lactobacillus rhamnosus GG attenuates the pathology of Chlamydial muridarium in the upper genital tract in mice. / å£æœé¼ æŽç³–ä¹³æ†èŒGGå‡è½»é¼ è¡£åŽŸä½“æ„ŸæŸ“å¼•èµ·çš„ä¸Šç”Ÿæ®–é“ç— å˜.

OBJECTIVES: Chlamydia trachomatis is a pathogen which can cause hydrosalpinx and tubal fibrosis when infecting the urogenital tract. However, the mechanism is still not clear. There is growing evidence that the gut microbiota is associated with the pathogenesis of both intestinal and extra-intestinal disorders, such as cardiovascular disease, hepatocirrhosis, allergy, respiratory tract infection, polycystic ovary syndrome, endometriosis, and bacterial vaginitis. Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria, the benefits of LGG including the treatment in gastrointestinal disorders and immunomodulation are well demonstrated, and it can also alleviate hypersensitivity reaction and diarrhoea, inhibit a variety of respiratory and urogenital diseases. Chlamydia muridarium (Cm) infection is a good model for the study on human Chlamydia pathogenicity in genitourinary tract. The mice infected with Cm were used as animal models to preliminarily explore the mechanism for the effect of LGG on upper reproductive tract infection in the mice, and to provide experimental basis for the pathogenesis of Chlamydia trachomatis genitourinary tract infection and the new idea for the treatment of Chlamydia trachomatis infection. METHODS: Five to six weeks-old C57BL/6J mice were divided into 2 groups: An experimental group and a control group. The experimental group were administrated with 5×108 colony forming units (CFU) LGG for 19 consecutive days, while the control group were feed PBS. The mice in the 2 group were subcutaneously injected with 2.5 mg progesterone on Day 9 and infected with 1×105 inclusion body forming unit of Cm via the vaginal tract on Day 14. Vaginal and rectal swabs were taken every 7 days to infect HeLa cells for 24 hours, then the indirect immunofluorescence assay was used and the number of inclusion bodies of Chlamydia were calculated. Mice were euthanized on Day 14 and Day 63 after Cm inoculation, the vaginal tracts were dissected, and the tissue homogenates were prepared to culture the pathogens for 24 hours. The Cm bearing capacity in the bilateral uterine horn, tubal ovary, and cervical vaginal tissues in the 2 groups were calculated. The spleen cells were harvested to assay the intracellular IFN-γ, IL-5, and IL-17 by flow cytometry. On Day 63 after the Chlamydia infection, the pathology injury in the bilateral uterine horn and oviduct was observed, and the pathological sections and HE staining in the various part of genital tract were performed. The inflammatory cell infiltration and lumen dilatation was assessed. The specific IgM and IgG in sera were detected by indirect ELISA on Day 14 and 63 after infection. RESULTS: There was no effect of LGG on the clearing of Cm from the urogenital tract, the Chlamydia ascending to fallopian tube or the uterine horn, and the organism dissemination and colonization to the gastrointestinal tract (all P>0.05). On Day 14 after Cm infection via the vagina, the IL-17 expression level in the experimental group was significant decreased than that in the control group (t=2.486, P<0.05), but there was no significant difference between the 2 groups in the CD4+ T rate in spleen and IgM and IgG levels in serum after Cm intravaginal infection (all P>0.05). On Day 63 after Cm infection, there was no difference in the severity of inflammation in the uterine horns and fallopian tubes between the 2 groups (P>0.05), but the dilation of the fallopian tubes and hydrosalpinx was attenuated in the experimental group compared with the control group (P<0.05). CONCLUSIONS: Oral administration of LGG has no effect on inhibiting Cm ascending to upper genital tract and preventing the dissemination and colonization of Cm to the gastrointestinal tract, which also cannot affect the secretion of specific IgM and IgG in sera. Oral administration of LGG can suppress the production of IL-17 in the spleen cells and attenuate hydrosalpinx development when following Cm intravaginal infection in mice.

Effects of Immunomodulatory Drug Fingolimod (FTY720) on Chlamydia Dissemination and Pathogenesis.

Fingolimod (FTY720), an FDA-approved immunomodulatory drug for treating multiple sclerosis, is an agonist of sphingosine-1-phosphate receptor (S1PR), which has been used as a research tool for inhibiting immune cell trafficking. FTY720 was recently reported to inhibit Chlamydia dissemination. Since genital Chlamydia spreading to the gastrointestinal tract correlated with its pathogenicity in the upper genital tract, we evaluated the effect of FTY720 on chlamydial pathogenicity in the current study. Following an intravaginal inoculation, live chlamydial organisms were detected in mouse rectal swabs. FTY720 treatment significantly delayed live organism shedding in the rectal swabs. However, FTY720 failed to block chlamydial spreading to the gastrointestinal tract. The live chlamydial organisms recovered from rectal swabs reached similar levels between mice with or without FTY720 treatment by day 42 in C57BL/6J and day 28 in CBA/J mice, respectively. Thus, genital Chlamydia is able to launch a 2nd wave of spreading via an FTY720-resistant pathway after the 1st wave of spreading is inhibited by FTY720. As a result, all mice developed significant hydrosalpinx. The FTY720-resistant spreading led to stable colonization of chlamydial organisms in the colon. Consistently, FTY720 did not alter the colonization of intracolonically inoculated Chlamydia Thus, we have demonstrated that, following a delay in chlamydial spreading caused by FTY720, genital Chlamydia is able to both spread to the gastrointestinal tract via an FTY720-resistant pathway and maintain its pathogenicity in the upper genital tract. Further characterization of the FTY720-resistant pathway(s) explored by Chlamydia for spreading to the gastrointestinal tract may promote our understanding of Chlamydia pathogenic mechanisms.

Gastrointestinal Coinfection Promotes Chlamydial Pathogenicity in the Genital Tract.

Sexually transmitted Chlamydia, which can cause fibrotic pathology in women's genital tracts, is also frequently detected in the gastrointestinal tract. However, the medical significance of the gastrointestinal Chlamydia remains unclear. A murine Chlamydia readily spreads from the mouse genital tract to the gastrointestinal tract while inducing oviduct fibrotic blockage or hydrosalpinx. We previously proposed a two-hit model in which the mouse gastrointestinal Chlamydia might induce the second hit to promote genital tract pathology, and we are now providing experimental evidence for testing the hypothesis. First, chlamydial mutants that are attenuated in inducing hydrosalpinx in the genital tract also reduce their colonization in the gastrointestinal tract, leading to a better correlation of chlamydial induction of hydrosalpinx with chlamydial colonization in the gastrointestinal tract than in the genital tract. Second, intragastric coinoculation with a wild-type Chlamydia rescued an attenuated Chlamydia mutant to induce hydrosalpinx, while the chlamydial mutant infection in the genital tract alone was unable to induce any significant hydrosalpinx. Finally, the coinoculated gastrointestinal Chlamydia failed to directly spread to the genital tract lumen, suggesting that gastrointestinal Chlamydia may promote genital pathology via an indirect mechanism. Thus, we have demonstrated a significant role of gastrointestinal Chlamydia in promoting pathology in the genital tract possibly via an indirect mechanism. This study provides a novel direction/dimension for further investigating chlamydial pathogenic mechanisms.

Multiple and sensitive SERS detection of cancer-related exosomes based on gold-silver bimetallic nanotrepangs.

Exosomes are endogenous vesicles of cells, and can be used as important biomarkers for cancers. In this work, we developed a sensitive and reliable SERS sensor for simultaneous detection of multiple cancer-related exosomes. The SERS detection probes were made of bimetallic SERS-active nanotags, gold-silver-silver core-shell-shell nanotrepangs (GSSNTs), which were composed of bumpy surface nanorod (gold nanotrepang, GNT) cores and bilayer silver shells, and decorated with linker DNAs, which were complementary to the aptamer targeting exosomes. Three kinds of SERS detection probes were designed via the adoption of different Raman reporter molecules and linker DNAs. The capture probes were prepared by modifying specific aptamers of the target exosomes on magnetic beads (MBs). In the absence of target exosomes, SERS detection probes were coupled with MBs via specific DNA hybridization for use as aptamer-based SERS sensors. In the presence of target exosomes, the aptamer specifically recognized and captured the exosomes, and GSSNTs were subsequently released into the supernatant. Therefore, attenuated SERS signals were detected on the MBs, indicating the presence of target exosomes. The proposed aptamer-based SERS sensor is expected to be a facile and sensitive method for the multiplex detection of cancer biomarkers and has potential future applications in clinical diagnosis.

Topography-Induced Cell Self-Organization from Simple to Complex Aggregates.

Self-organization is a fundamental and indispensable process in a living system. To understand cell behavior in vivo such as tumorigenesis, 3D cellular aggregates, instead of 2D cellular sheets, have been employed as a vivid in vitro model for self-organization. However, most focus on the macroscale wetting and fusion of cellular aggregates. In this study, it is reported that self-organization of cells from simple to complex aggregates can be induced by multiscale topography through confined templates at the macroscale and cell interactions at the nanoscale. On the one hand, macroscale templates are beneficial for the organization of individual cells into simple and complex cellular aggregates with various shapes. On the other hand, the realization of these macro-organizations also depends on cell interactions at the nanoscale, as demonstrated by the intimate contact between nanoscale pseudopodia stretched by adjacent frontier cells, much like holding hands and by the variation in the intermolecular interactions based on E-cadherin. Therefore, these findings may be very meaningful for clarifying the organizational mechanism of tumor development, tissue engineering and regenerative medicine.