Immunodeviation towards T cell-mediated immune response in the testes of LPS-induced mouse epididymo-orchitis.

The testicular consequences of acute epididymo-orchitis remain largely unelucidated in long-term damage, which might be a neglected factor for male infertility. In this study, the differential phenotype of testicular immune cell subpopulations in lipopolysaccharide (LPS)-induced mouse epididymo-orchitis were analyzed by flow cytometry on day 1, day 7, and day 28. The number of macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs) steadily decreased in the testes with inoculation. Total F4/80-CD11c+ dendritic cells (DCs) maintained a relatively stable level, whereas conventional type 1 dendritic cells (cDC1) increased gradually from day 1 to day 28. There was a lower number of CD4+ and CD8+ T cells at day 1 and day 7, and they had similar results with a ceiling level at day 28. The testes displayed a higher level of CD3+ T cells but a lower frequency of macrophages, cDC2, and neutrophils at 28 days post-inoculation compared with the epididymis. In summary, our data indicates acute epididymo-orchitis could lead to long-term damage in the testes, which is characterized by CD3+ T cell (including CD4+ and CD8+ T cells)-mediated immune responses.

Early changes of bone metabolites and lymphocyte subsets may participate in osteoporosis onset: a preliminary study of a postmenopausal osteoporosis mouse model.

Purpose: Metabolic and immune changes in the early stages of osteoporosis are not well understood. This study aimed to explore the changes in bone metabolites and bone marrow lymphocyte subsets and their relationship during the osteoporosis onset. Methods: We established OVX and Sham mouse models. After 5, 15, and 40 days, five mice in each group were sacrificed. Humeri were analyzed by microCT. The bone marrow cells of the left femur and tibia were collected for flow cytometry analysis. The right femur and tibia were analyzed by LC-MS/MS for metabolomics analysis. Results: Bone microarchitecture was significantly deteriorated 15 days after OVX surgery. Analysis of bone metabolomics showed that obvious metabolite changes had happened since 5 days after surgery. Lipid metabolism was significant at the early stage of the osteoporosis. The proportion of immature B cells was increased, whereas the proportion of mature B cells was decreased in the OVX group. Metabolites were significantly correlated with the proportion of lymphocyte subsets at the early stage of the osteoporosis. Conclusion: Lipid metabolism was significant at the early stage of the osteoporosis. Bone metabolites may influence bone formation by interfering with bone marrow lymphocyte subsets.

From Waterloo to the Great Wall: A retrospective, multicenter study on the clinical practice and cultural attitudes in the management of premature ejaculation, in China.

Premature ejaculation (PE), despite its wide prevalence, is largely underdiagnosed and undertreated. Being a multifactorial dysfunction with strong cultural characteristics, PE requires skillful attitudes in the psychosexological support, necessary to manage the patient's and the couple's expectations, as well as in the medical treatment. Dapoxetine is a short-acting selective serotonin reuptake inhibitor approved for use in lifelong and acquired PE in a number of countries. Opinions, not always generated by the evidence-based medicine, impacted the attitudes of Western andrologists, as a nocebo effect which produced a drug's Waterloo, characterized by low prescription rates much more built on the patients' and doctors' expectations than on costs, side effects, and efficacy. In the present study, we retrospectively reviewed real-life data from eight Andrology and Sexual Medicine Public Centers in China to assess the prevalence of PE among attending patients, its association with erectile dysfunction, its subtype, and the proposed treatments. In 2019, among 156,486 patients coming to the centers, 32,667 visits having PE as the chief complaint were performed (20.9%). Almost all patients received treatment prescriptions (32,641 patients, 99.92%); 23,273 patients came back for a follow-up visit in the subsequent 12 months (71.2% of those who initially received treatment). Dapoxetine, either alone or in combination with another therapy, was the most prevalent treatment, prescribed to 22,767 patients (69.7% of treated patients), followed by traditional Chinese medicine (TCM) (39.4%). At follow-up, 8174 patients were unsatisfied with treatment, and a new treatment was proposed (35.12%). Dapoxetine was the best treatment, with an overall 27.1% switching rate when used either alone or in combination: Although the switching rate for Dapoxetine alone was 44.2%, the association of the same drug with psychotherapy resulted in much lower rates (19.5%) and reached a minimum of 12% when also combined with TCM demonstrating how cultural aspects and medical attitudes may dramatically impact on the therapy of a multifaceted, complex, and culture-grounded sexual symptom such as PE. In conclusion, taking switching rates as surrogate markers of treatment failure, this real-life study-the largest in the field-shows that in a more patient-oriented (as in Chinese medical culture), and less symptom-oriented (as in Western medical attitudes), Dapoxetine is a successful treatment for PE patients, with higher reliability when used alone or as part of combined and integrated therapies.

Alterations of small non-coding RNA in the spermatozoa of mice with paternal experimental autoimmune epididymo-orchitis are associated with metabolic dysfunction in offspring.

BACKGROUND: Infection and inflammation of the genital tract are major potentially treatable factors contributing to male infertility. The profile of small non-coding RNA (sncRNAs) in spermatozoa can be altered by environmental exposures and inflammatory conditions. OBJECTIVES: Experimental autoimmune epididymo-orchitis (EAEO) is a well-established model of autoimmune-induced chronic testicular and epididymal inflammation. This model investigates the effect of chronic inflammation on sperm sncRNA profiles and offspring phenotypes. MATERIALS AND METHODS: Regarding the EAEO model, mice were immunized with testis homogenates thrice. Subsequently, flow cytometry and histological analyses were conducted on EAEO mice. Next-generation sequencing was used to profile small RNA of spermatozoa from the caput, corpus, and cauda epididymis. We performed a comprehensive integrative analysis of sperm sncRNAs and chronic epididymitis and identified their molecular signatures. The metabolic functions of the first-generation (F1) offspring were evaluated using a glucose tolerance test (GTT). RESULTS: Body weight and metabolic function were significantly altered in F1 offspring from EAEO sperm donors. The analysis of cauda sperm sncRNA profiles revealed that the proportions of miRNAs and tsRNAs increased and decreased, respectively, after autoimmunization. Three differentially expressed miRNAs and seven differentially expressed tsRNAs were significantly correlated with F1 metabolic dysfunction. The expression patterns of miRNAs and tsRNAs in mice partially overlapped with those observed in the spermatozoa from human patients with chronic epididymitis. DISCUSSION AND CONCLUSIONS: We revealed that autoimmune epididymo-orchitis alters sncRNA profiles in mouse spermatozoa. Offspring from mice with autoimmune orchitis develop metabolic disorders. A comprehensive analysis of human and mouse inflammation data revealed an association between alterations in the miRNA and tsRNA profiles of epididymal spermatozoa and offspring phenotypes.

Myd88 Signaling Is Involved in the Inflammatory Response in LPS-Induced Mouse Epididymitis and Bone-Marrow-Derived Dendritic Cells.

Epididymitis is an epididymal inflammation that may lead to male infertility. Dendritic cells (DCs) and myeloid differentiation primary response gene 88 (Myd88) were associated with epididymitis in rodents. However, the functions of Myd88 on epididymal DCs remain unclear. This study investigated the role of Myd88 in DCs for epididymitis. The Myd88 signaling pathway, phenotypes of DC subsets, and cytokines were investigated in lipopolysaccharide (LPS)-induced epididymitis in mice. CRISPR-Cas9 was used to knockout Myd88 in bone-marrow-derived dendritic cells (BMDCs) and immortalized mouse epididymal (DC2) cell line. In the vivo experiments, levels of the proinflammatory cytokines IL-1α, IL-6, IL-17A, TNF-α, IL-1ß, MCP-1, and GM-CSF, mRNA for MyD88 related genes, and the percentages of monocyte-derived DCs (Mo-DCs) were significantly elevated in mice with epididymitis. In the vitro experiments, LPS significantly promoted the apoptosis of BMDCs. In addition, the concentration of inflammatory cytokines in BMDCs and DC2s were increased in the LPS group, while decreasing after the knockout of Myd88. These findings indicate that Myd88 on DCs is involved in the inflammation of epididymitis in mice, which may be a potential target for better strategies regarding the treatment of immunological male infertility.

Recent advances in hierarchical MoS2/graphene-based materials for supercapacitor applications.

Hierarchical MoS2/graphene (MoS2/G) has been widely researched in energy storage via supercapacitors. The combination of MoS2 with graphene not only provides high conductivity but also enhances the structural stability, which are critical factors determining the electrochemical performance for energy storage. In this review, the recent development of various hierarchical MoS2/G nanostructures in supercapacitor applications is summarized by classifying the materials into MoS2/G nanospheres, MoS2/G nanosheets, and MoS2/G-based ternary composite. The description of the structural characteristics and electrochemical performance gives a clear and profound understanding of hierarchical MoS2/G nanostructures as a supercapacitor material. In addition, further research prospects of hierarchical MoS2/G are suggested.

Effect of pretreatment approach on the ELISA-based detection of cyanotoxins in water: Analysis and application.

Common cyanotoxins, such as microcystins and nodularins, are produced by frequently occurring harmful cyanobacterial algal blooms in freshwater systems. The required routine monitoring of microcystins and nodularins in drinking water and ambient water demands cost-efficient and reliable enzyme-linked immunosorbent assay kits. We validated the performance of a self-produced broad-spectrum enzyme-linked immunosorbent assay kit and investigated two different methods of mitigating the matrix effects to elucidate the effect of the respective pretreatment approaches recommended by China and the United States on the quantitative detection of cyanotoxins in surface water. We found that the enzyme-linked immunosorbent assay kit achieved a detection limit of 0.15 µg/L with a linear detection range from 0.27 µg/L to 1.87 µg/L for microcystin-LR (the most studied and widely distributed cyanotoxin). The matrix effects could be mitigated both by dilution of water samples with an optimal dilution ratio and dilution of antibody with the buffer containing phosphate buffer solution (10×), bovine serum albumin (1 %) and ethylene diamine tetraacetic acid (0.5 %). In terms of the surface water samples being tested, the concentrations of microcystins and nodularins measured based on pretreatment approach recommended by the United States were 1- 5 times that measured based on pretreatment approach recommended by China, indicating that the pretreatment approach of China overlooks cyanotoxins. In addition, all the measured total microcystins and nodularins of the surface water samples were below the health advisory limit (1.6 µg/L) for microcystins in drinking water proposed by the United States Environmental Protection Agency for school-age children and adults. Our research could provide significant information for outbreak warnings and risk management of harmful cyanobacterial algal blooms.

Role of small RNAs harbored by sperm in embryonic development and offspring phenotype.

BACKGROUND: RNA harbored by mammalian sperm is increasingly considered to be an additional source of paternal hereditary information, beyond DNA. Recent studies have demonstrated the role of sperm small noncoding RNAs (sncRNAs) in modulating early embryonic development and offspring phenotype. The biogenesis of the sperm sRNA payload of mammalian sperm has been explored in many studies. AIMS: To summarize the possible mechanisms underpinning sperm sncRNAs regulating embryonic development and offspring phenotypes. MATERIALS AND METHODS: PubMed database (papers published from 2002 to 2022) was searched for studies reporting the impact of sperm sncRNAs on early embryonic development and offspring phenotype. RESULTS: The sncRNAs categories and source (such as tRNA-derived small RNAs, ribosomal RNA-derived small RNAs, microRNAs, and PIWI-interacting RNAs), and RNA modification upon different types of environmental exposure or by paternally-acquired factors were summarized. The potential mechanisms whereby the modifications of sperm sncRNAs modulate embryonic development and offspring phenotype under normal and pathological conditions (such as obesity, altered glucose metabolism, and psychological stress) were discussed. DISCUSSION AND CONCLUSION: Sperm sncRNAs modulate embryo development and offspring phenotype, and the resulting modifications may be transgenerationally inherited.

Integrative Analysis of Small RNA and mRNA Expression Profiles Identifies Signatures Associated With Chronic Epididymitis.

Chronic epididymitis (CE) refers to a long-lasting inflammatory condition of the epididymis, which is considered the most common site of intrascrotal inflammation and an important aetiological factor of male infertility. Recent studies demonstrate that small RNAs secreted from epididymal epithelium modulate embryo development and offspring phenotypes via sperm transmission, and the resulting modifications may lead to transgenerational inheritance. However, to date, the genome-wide analysis of small RNA together with the transcriptomic expression profiles of human epididymis and CE is still lacking. In this study, we facilitated next-generation sequencing and bioinformatics to comprehensively analyze the small RNA and mRNA in an integrative way and identified signatures associated with CE. Both of the small RNA and mRNA expression data demonstrated relatively larger molecular differences among the segmental region of the epididymides, including caput, corpus, and cauda, than that of the inflammatory conditions. By comparing the inflamed caputs to the controls, a total of 1727 genes (1220 upregulated and 507 downregulated; 42 most significant genes, adjusted P <0.05) and 34 miRNAs (23 upregulated and 11 downregulated) were identified as differentially expressed. In silico functional enrichment analysis showed their roles in regulating different biological activities, including leukocyte chemotaxis, extracellular milieu reconstruction, ion channel and transporter-related processes, and nervous system development. Integrative analysis of miRNA and mRNA identified a regulatory network consisting of 22 miRNAs and 31 genes (miRNA-mRNA) which are strong candidates for CE. In addition, analysis about other species of small RNA, including (miRNA), piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), Y RNA, and rsRNA identified the distinct expression pattern of tsRNA in CE. In summary, our study performed small RNA and miRNA profiling and integrative analysis in human CE. The findings will help to understand the role of miRNA-mRNA in the pathogenesis of CE and provide molecular candidates for the development of potential biomarkers for human CE.

Design, Synthesis and Cytotoxicity of Thiazole-Based Stilbene Analogs as Novel DNA Topoisomerase IB Inhibitors.

A series of new thiazole-based stilbene analogs were designed, synthesized and evaluated for DNA topoisomerase IB (Top1) inhibitory activity. Top1-mediated relaxation assays showed that the synthesized compounds possessed variable Top1 inhibitory activity. Among them, (E)-2-(3-methylstyryl)-4-(4-fluorophenyl)thiazole (8) acted as a potent Top1 inhibitor with high Top1 inhibition of ++++ which is comparable to that of CPT. A possible binding mode of compound 8 with Top1-DNA complex was further provided by molecular docking. An MTT assay against human breast cancer (MCF-7) and human colon cancer (HCT116) cell lines revealed that the majority of these compounds showed high cytotoxicity, with IC50 values at micromolar concentrations. Compounds 8 and (E)-2-(4-tert-butylstyryl)-4-(4-fluorophenyl)thiazole (11) exhibited the most potent cytotoxicity with IC50 values of 0.78 and 0.62 µM against MCF-7 and HCT116, respectively. Moreover, the preliminary structure-activity relationships of thiazole-based stilbene analogs was also discussed.

Nanozyme enhanced paper-based biochip with a smartphone readout system for rapid detection of cyanotoxins in water.

Cyanobacterial harmful algal blooms in freshwater systems can produce cyanotoxins, such as microcystins (MCs) and nodularins (NODs), presenting serious threats to human health and ecosystems. Required routine monitoring of cyanotoxins in water samples, as posed by U.S. EPA drinking water contaminant candidate list 5 (CCL5), demands for cost-effective, reliable and sensitive MCs/NODs detection methods. We report the development of a colorimetric paper-based immunochip assisted by nanozyme catalysis with a smartphone readout system for rapid detection of cyanotoxins in water. We show that the introduction of biorthogonal click reaction enables in situ facile self-assembly of multi-layers of peroxidase-like nanozyme onto the anti-MCs/NODs monoclonal antibody. We can detect 13 variants of MCs/NODs even in the sub-microgram per liter range with detection limit of below 0.7 µg/L and satisfactory recovery percentages between 88 and 120% in different water matrices. Our technology shows a good correlation with the well-developed ELISA technology, demonstrating its great potential applications in resource-limited or less-developed regions for on-site and large-scale screening of cyanotoxins in water environment.

Titanium-Enriched Slag Prepared by Atmospheric Hydrochloric Acid Leaching of Mechanically Activated Vanadium Titanomagnetite Concentrates.

The titanium-enriched slag was obtained via atmospheric hydrochloric acid leaching of mechanically activated vanadium titanomagnetite concentrates (VTMCs). Under the influence of mechanical activation, specific physicochemical changes were observed via X-ray diffractometry, scanning electron microscopy, and granulometric laser diffraction analysis. Experimental findings revealed that the mechanical activation of VTMCs resulted in a decrease in the median volume particle diameter (d50) and an increase in the specific surface area (SA) with an increased milling time. The results of the leaching experiment revealed that the mechanical activation treatment favors the extraction of iron (Fe) and titanium dioxide (TiO2) from the VTMCs. The Fe and TiO2 extractions from the mechanically activated sample after 10 h compared with the unactivated sample were increased by 12.82% and 4.73%, respectively. The presence of the ilmenite phase in the titanium-enriched slag was confirmed by X-ray diffractometry and EDS patterns, and the content of the TiO2 in the enriched slag can get as high as 43.75%.

ELISA-Based Method for Variant-Independent Detection of Total Microcystins and Nodularins via a Multi-immunogen Approach.

Required routine monitoring of microcystins (MCs) and nodularins (NODs) in water samples, as posed by U.S. EPA Unregulated Contaminant Monitoring Rule 4, demands cost-effective, reliable, and sensitive detection methods. To target as many MC and NOD variants as possible, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) with group-specific monoclonal antibodies for variant-independent detection of total MCs and NODs. In this ELISA method, the mice monoclonal antibodies presenting both high affinities and broad-spectrum recognition capabilities against MCs and NODs were self-produced by designing MC hapten-based multi-immunogens to minimize specificity for the particular variant. Their high affinities and variant-independent binding capabilities against MCs and NODs were validated by both wet lab and in silico methods. The developed ELISA method achieved a limit of detection of below 0.3 µg/L for 13 MC/NOD variants, well with the reported best cross-reactivities of 60-127% relative to MC-LR. As a case study, this ELISA method was used to map the variations of intracellular and extracellular total MCs/NODs in the Luoma Lake drinking water source, China, in July, 2020. Its capability to measure total MCs/NODs with high sensitivity and high throughput in a simple and affordable way would truly be a disruptive technology capable of changing our understanding of bloom/toxin dynamics and having obvious implications for monitoring efforts.

Protective Role of microRNA-31 in Acetaminophen-Induced Liver Injury: A Negative Regulator of c-Jun N-Terminal Kinase (JNK) Signaling Pathway.

BACKGROUND & AIMS: Sustained c-Jun N-terminal kinase (JNK) activation plays a major role in drug-induced liver injury (DILI). Stress-responsive microRNA-31 (miR-31) has been implicated in regulating different cellular damage, and JNK activation could induce miR-31 expression. However, the regulatory role of miR-31 in DILI has not been studied previously. We aimed to investigate whether miR-31 could ameliorate DILI and ascertain potential molecular mechanism. METHODS: miR-31 gene knockout (31-KO) and wild-type C57BL/6J mice were used to construct an acetaminophen (APAP)-induced DILI model. Primary mouse hepatocytes, as well as alpha mouse liver 12 (AML-12) cell lines, were used for in vitro experiments. Argonaute 2-associated RNA immunoprecipitation combined with high-throughput sequencing were performed to identify specific targets of miR-31. RESULTS: 31-KO mice showed a higher mortality rate, liver transaminase levels, and hepatic necrosis compared with those in wild-type mice after APAP-induced hepatotoxicity. The protective role of miR-31 on hepatocytes has been analyzed via constructing bone marrow chimeric mice. Mechanistically, we found that hepatic JNK phosphorylation increased significantly in 31-KO mice. This caused mitochondrial phosphorylated Src (p-Src) inactivation and more reactive oxygen species production, which directly amplifies hepatocyte necrotic cell death, while administration of JNK-specific inhibitor SP600125 could abrogate the differences. Moreover, bioinformatics analysis of RNA immunoprecipitation combined with high-throughput sequencing identified that guanosine triphosphatase, cell division cycle protein 42 (Cdc42), the upstream molecule of JNK signaling, was the specific target of miR-31 and could form a miR-31/Cdc42/phosphorylated mixed-lineage kinase 3 (p-MLK3) negative feedback loop to restrict JNK overactivation. Clinically, both miR-31 and phosphorylated JNK (p-JNK) were highly increased in liver tissues of DILI patients with different etiologies. CONCLUSIONS: miR-31 can down-regulate Cdc42 to restrict overactivation of reactive oxygen species/JNK/mitochondria necrotic death loop in hepatocytes of APAP-induced DILI, which might provide a new therapeutic target for alleviating JNK overactivation-based liver injury.

A CRISPR-based and post-amplification coupled SARS-CoV-2 detection with a portable evanescent wave biosensor.

The continuing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), has spread globally and its reliable diagnosis is one of the foremost priorities for protecting public health. Herein a rapid (<1 h), easy-to-implement, and accurate CRISPR-based evanescent wave fluorescence biosensing platform for detection of SARS-CoV-2 is reported. The collateral effect of Cas13a is combined with a universal autonomous enzyme-free hybridization chain reaction (HCR) by designing a cleavage hairpin reporter, which is cleaved upon target recognition, and hence releasing the initiator sequence to trigger the downstream HCR circuits. Detection of HCR assemblies is accomplished by first adsorbing to the desthiobiotin-modified optical fiber, followed by fluorescence emission induced by an evanescent field. Three Cas13a crRNAs targeting the genes of S, N and Orf1ab of SARS-CoV-2 are programmed to specifically target SARS-CoV-2 or broadly detect related coronavirus strains, such as MERS-CoV and SARS-CoV. The HCR amplification coupled Cas13a-based biosensing platform is capable of rapid detection of SARS-CoV-2 with attomolar sensitivity. This method is further validated by adding target RNA of SARS-CoV-2 in negative oropharyngeal swabs. The good discrimination capability of this technique demonstrates its promising potential for point-of-care diagnosis of COVID-19.

Bile Acids Impair Vaccine Response in Children With Biliary Atresia.

Background: Vaccination is the best way to protect children under 5 years from death or disability. Children with biliary atresia (BA), which is the most common pediatric cholestatic end-stage liver disease (PELD), are more vulnerable to infectious diseases. However, the vaccination coverage and factors modulating vaccine responses in children with BA are largely unknown. Methods: In this study, 288 children (median age: 7 months) diagnosed with BA before liver transplantation were enrolled for the evaluation of vaccination status and the factors affecting the immune response to the hepatitis B (HBV) vaccine. Moreover, 49 BA children (median age: 4 months) were enrolled for flow cytometric analysis of CD4+ T cells and CD19+ B cell subsets and correlations with serum bile acid levels. Results: Generally, these children had very low routine vaccination rates for the meningococcal serogroup AC (Men AC) (41.2%), measles-mumps-rubella (MMR) (31.3%), poliomyelitis (Polio) (25.3%), hepatitis A (HAV) (25.0%), Japanese encephalitis (JE) (15.0%), diphtheria-tetanus-pertussis (DTP) (14.2%), meningococcal serogroup A (Men A) (13.5%) and varicella (VAR) (10.8%) vaccines, but not for the HBV (96.2%) and bacillus Calmette-Guérin (BCG) (84.7%) vaccines. Remarkably, 19.8% (57/288) of the patients had HBV infection. Out of 220 patients vaccinated for HBV, 113 (51.4%), 85 (38.6%) and 22 (10%) had one, two or three doses of the HBV vaccine, respectively. Furthermore, logistic regression analysis revealed that the bile acid level was an independent factor associated with poor HBV vaccine response (p = 0.03; OR = 0.394; 95% CI = 0.170-0.969). Immunophenotyping showed that bile acids were only negatively correlated with the CD19+CD27+IgG+ post-class-switched memory B cell ratio (p = 0.01). Conclusion: This study reveals the overall vaccination rates of routine vaccines in Chinese BA children are very low and the poor HBV vaccine responses are associated with bile acids, possibly via the inhibition of CD19+CD27+IgG+ post-class-switched memory B cell response. Clinical Trial Registration: http://www.chictr.org.cn, identifier ChiCTR1800019165.

Light-sheet skew rays enhanced U-shaped fiber-optic fluorescent immunosensor for Microcystin-LR.

A novel U-shaped fiber-optic evanescent-wave fluorescent immunosensor was designed that exploits light-sheet excitation of skew rays in a passive fiber for sensitive microcystin-LR (MC-LR) detection in real-time. In particular, a light sheet comprising a thin plane of light can be concentrated into exciting the optimum ray group, resulting in enhanced interaction between light and fluorophores. Meanwhile, skew rays excited by transmitting light into an optical fiber with an angle offset allow a much higher number of total-internal-reflections with increased interaction length along the fiber interface, which strengthens the light-matter interactions. Under the optimal angle offset, the proposed evanescent wave fluorescent immunosensor is the first demonstration of integrating light-sheet skew rays and a U-shaped fiber-optic probe for enhanced sensitivity. The results show that fluorescence sensitivity of the U-shaped fiber-optic probe with light-sheet skew rays excitation is 16 times higher than that of collimated skew rays excitation. Combined with this newly designed light-sheet skew rays enhanced U-shaped fiber-optic fluorescent immunosensor, a sensitive and real-time MC-LR detection method was established based on the indirect competitive immunoassay principle. Real environmental water samples spiked with MC-LR were determined by the immunosensor with recovery rates between 85% and 112%. The present system could be an alternative tool for the on-site environmental monitoring, in-field food safety assurance and clinical diagnostics. It also advances the fiber-optic sensors field in terms of experimental design.

Multiple microRNAs regulate tacrolimus metabolism through CYP3A5.

The CYP3A5 gene polymorphism accounts for the majority of inter-individual variability in tacrolimus pharmacokinetics. We found that the basal expression of CYP3A5 in donor grafts also played a significant role in tacrolimus metabolism under the same genetic conditions after pediatric liver transplantation. Thus, we hypothesized that some potential epigenetic factors could affect CYP3A5 expression and contributed to the variability. We used a high-throughput functional screening for miRNAs to identify miRNAs that had the most abundant expression in normal human liver and could regulate tacrolimus metabolism in HepaRG cells and HepLPCs. Four of these miRNAs (miR-29a-3p, miR-99a-5p, miR-532-5p, and miR-26-5p) were selected for testing. We found that these miRNAs inhibited tacrolimus metabolism that was dependent on CYP3A5. Putative miRNAs targeting key drug-metabolizing enzymes and transporters (DMETs) were selected using an in silico prediction algorithm. Luciferase reporter assays and functional studies showed that miR-26b-5p inhibited tacrolimus metabolism by directly regulating CYP3A5, while miR-29a-5p, miR-99a-5p, and miR-532-5p targeted HNF4α, NR1I3, and NR1I2, respectively, in turn regulating the downstream expression of CYP3A5; the corresponding target gene siRNAs markedly abolished the effects caused by miRNA inhibitors. Also, the expression of miR-29a-3p, miR-99a-5p, miR-532-5p, and miR-26b-5p in donor grafts were negatively correlated with tacrolimus C/D following pediatric liver transplantation. Taken together, our findings identify these miRNAs as novel regulators of tacrolimus metabolism.

Impact of EBV infection and immune function assay for lymphoproliferative disorder in pediatric patients after liver transplantation: A single-center experience.

BACKGROUND: Post-transplant lymphoproliferative disorder (PTLD) is a lethal complication after pediatric liver transplantation, but information regarding risk factors for the development of PTLD remains unclear. This study was to identify characteristics and risk factors of PTLD. METHODS: A total of 705 pediatric patients who underwent liver transplantation between January 2017 and October 2018 were studied. Impact of clinical characteristics and Epstein-Barr virus (EBV) infection on the development of PTLD was evaluated. In addition, ImmuKnow assay was adopted in partial patients to analyze the immune status. RESULTS: Twenty-five (3.5%) patients suffered from PLTD with a median time of 6 months (3-14 months) after transplantation. Extremely high tacrolimus (TAC) level was found in 2 fatal cases at PTLD onset. EBV infection was found in 468 (66.4%) patients. A higher peak EBV DNA loads (>9590 copies/mL) within 3 months was a significant indicator for the onset of PTLD. In addition, the ImmuKnow assay demonstrated that overall immune response was significantly lower in patients with EBV infection and PTLD (P<0.0001). The cumulative incidence of PTLD was also higher in patients with lower ATP value (≤187 ng/mL, P<0.05). CONCLUSIONS: A careful monitoring of EBV DNA loads and tacrolimus concentration might be supportive in prevention of PTLD in pediatric patients after liver transplantation. In addition, application of the ImmuKnow assay may provide guidance in reducing immunosuppressive agents in treatment of PTLD.

Optical biosensors based on refractometric sensing schemes: A review.

Biosensor technology is an active field of research and development presenting rapid progress in recent decades, and the subfield of optical biosensors based on refractometric sensing schemes has developed dramatically during this time. This review focuses on advances in the refractometric sensing-based guided-wave optical biosensors particularly in the last two decades. It starts with a concise discussion on the underlying principles of label-free refractometric biosensor. Subsequently, advances in biosensor design, especially the transducer configuration and the integration of the sensing device are reviewed, highlighting the challenges and efforts dedicated to improving this technology. Various surface functionalization strategies designed to produce well-defined and reproducible surface properties are introduced for evaluation. Refractometric sensing scheme-based optical biosensors have found versatile applications varying from environmental monitoring and food safety to clinical diagnostics, together with advances in these applications and others are described. This paper concludes with a brief discussion on the outlook for integrating biosensors with emerging technologies.