Parallel evolution of fluconazole resistance and tolerance in Candida glabrata.

Introduction: With the growing population of immunocompromised individuals, opportunistic fungal pathogens pose a global health threat. Candida species, particularly C. albicans and non-albicans Candida species such as C. glabrata, are the most prevalent pathogenic fungi. Azoles, especially fluconazole, are widely used therapeutic options. Objective: This study investigates how C. glabrata adapts to fluconazole, with a focus on understanding the factors regulating fluconazole tolerance and its relationship to resistance. Methods: This study compared the factors regulating fluconazole tolerance between C. albicans and C. glabrata. We analyzed the impact of temperature on fluconazole tolerance, and requirement of calcineurin and Hsp90 for maintenance of fluconazole tolerance. We isolated colonies from edge, inside and outside of inhibition zone in disk diffusion assays. And we exposed C. glabrata strain to high concentrations of fluconazole and investigated the mutants for development of fluconazole resistance and tolerance. Results: We found temperature modulated tolerance in the opposite way in C. albicans strain YJB-T1891 and C. glabrata strain CG4. Calcineurin and Hsp90 were required for maintenance of fluconazole tolerance in both species. Colonies from inside and outside of inhibition zones did not exhibited mutated phenotype, but colonies isolated from edge of inhibition zone exhibited diverse phenotype changes. Moreover, we discovered that high concentrations (16-128 µg/mL) of fluconazole induce the simultaneous but parallel development of tolerance and resistance in C. glabrata, unlike the sole development of tolerance in C. albicans. Conclusion: This study highlights that while tolerance to fluconazole is a common response in Candida species, the specific molecular mechanisms and evolutionary pathways that lead to this response vary between species. Our findings emphasize the importance of understanding the regulation of fluconazole tolerance in different Candida species to develop effective therapeutic strategies.

Acid-Promoted Self-Photocatalyzed Regioselective Oxidation: A Novel Strategy for Accessing Quinoxaline-2,3-diones.

Disclosed here is an efficient approach for the preparation of quinoxaline-2,3-diones using air (O2) as a green oxidant via acid-promoted self-photocatalyzed regioselective oxidation of quinoxalin-2(1H)-ones at C-3 position. This protocol presents a novel synthetic route for the preparation of quinoxaline-2,3-dione derivatives, featuring mild reaction conditions, simple operation, and a wide range of substrates, without the need for external photocatalysts, metal reagents, and strong oxidants.

Ketoconazole induces reversible antifungal drug tolerance mediated by trisomy of chromosome R in Candida albicans.

Background: The emergence of tolerance to antifungal agents in Candida albicans complicates the treatment of fungal infections. Understanding the mechanisms underlying this tolerance is crucial for developing effective therapeutic strategies. Objective: This study aims to elucidate the genetic and molecular basis of ketoconazole tolerance in C. albicans, focusing on the roles of chromosomal aneuploidy, Hsp90, and calcineurin. Methods: The wild-type C. albicans strain SC5314 was exposed to increasing concentrations of ketoconazole (0.015-32 µg/mL) to select for tolerant adaptors. Disk diffusion and spot assays were used to assess tolerance. Whole-genome sequencing identified chromosomal changes in the adaptors. The roles of Hsp90 and calcineurin in maintaining and developing ketoconazole tolerance were investigated using specific inhibitors and knockout strains. Results: Adaptors exhibited tolerance to ketoconazole concentrations up to 16 µg/mL, a significant increase from the parent strain's inhibition at 0.015 µg/mL. All tolerant adaptors showed amplification of chromosome R, with 29 adaptors having trisomy and one having tetrasomy. This aneuploidy was unstable, reverting to euploidy and losing tolerance in drug-free conditions. Both Hsp90 and calcineurin were essential for maintaining and developing ketoconazole tolerance. Inhibition of these proteins resulted in loss of tolerance. The efflux gene CDR1 was not required for the development of tolerance. Chromosome R trisomy and tetrasomy induce cross-tolerance to other azole antifungal agents, including clotrimazole and miconazole, but not to other antifungal classes, such as echinocandins and pyrimidines, exemplified by caspofungin and 5-flucytosine. Conclusion: Ketoconazole tolerance in C. albicans is mediated by chromosomal aneuploidy, specifically chromosome R amplification, and requires Hsp90 and calcineurin. These findings highlight potential targets for therapeutic intervention to combat antifungal tolerance and improve treatment outcomes.

Pornographic Video Consumption and Partner Preference Among Chinese Male Sexual Minorities: The Moderating Role of Perceived Realism.

A growing body of research documents that the use of pornography is becoming more frequent among male sexual minorities. According to the sexual script acquisition, activation, application model of mediated sexual socialization (3AM), pornography can be considered a potential factor influencing an individual's partner preference. In addition, perceived realism could moderate the link between pornography consumption and partner preferences. This study explored the relationship between pornographic video consumption and partner preference among male sexual minorities and the moderating role of perceived realism. A total of 595 male sexual minorities aged 18 to 47 years (M = 21.70, SD = 3.83), including 82.9% gay men and 17.1% bisexual men, were enrolled in the study. Our results revealed the following: (1) The frequency of pornographic video consumption and male sexual minorities' preferences for appearance and sexual ability in partner selection were positively correlated (r = .06/.07, ps < .05). (2) When perceived realism was low, pornographic video consumption was not significantly associated with appearance and sexual ability preference. (3) Conversely, when perceived realism was high, pornographic video consumption was positively associated with appearance and sexual ability preference (ß = .17/.16, ps < .01). Findings highlight the mechanisms behind pornographic video consumption and partner preference in sexual minority males.

Miconazole induces aneuploidy-mediated tolerance in Candida albicans that is dependent on Hsp90 and calcineurin.

Antifungal resistance and antifungal tolerance are two distinct terms that describe different cellular responses to drugs. Antifungal resistance describes the ability of a fungus to grow above the minimal inhibitory concentration (MIC) of a drug. Antifungal tolerance describes the ability of drug susceptible strains to grow slowly at inhibitory drug concentrations. Recent studies indicate antifungal resistance and tolerance have distinct evolutionary trajectories. Superficial candidiasis bothers millions of people yearly. Miconazole has been used for topical treatment of yeast infections for over 40 years. Yet, fungal resistance to miconazole remains relatively low. Here we found different clinical isolates of Candida albicans had different profile of tolerance to miconazole, and the tolerance was modulated by physiological factors including temperature and medium composition. Exposure of non-tolerant strains with different genetic backgrounds to miconazole mainly induced development of tolerance, not resistance, and the tolerance was mainly due to whole chromosomal or segmental amplification of chromosome R. The efflux gene CDR1 was required for maintenance of tolerance in wild type strains but not required for gain of aneuploidy-mediated tolerance. Heat shock protein Hsp90 and calcineurin were essential for maintenance as well as gain of tolerance. Our study indicates development of aneuploidy-mediated tolerance, not resistance, is the predominant mechanism of rapid adaptation to miconazole in C. albicans, and the clinical relevance of tolerance deserves further investigations.

The Pattern of Physique Preferences on Gay Dating App "Blued" in China: A Multiple Group Latent Class Analysis.

Previous studies have indicated homogamy in partner preferences among gay and bisexual men. Individuals on Blued (the most popular gay dating app in China) could identify their body type and preferred physiques according to six categories: monkey (thin gay men), average, muscularity, bear (heavy and hairy gay men), baboon (smaller than bears), and pig (plumper than bears). This study examined patterns of physique preferences across body types as well as sexual self-labels (tops, bottoms, versatiles, and others). In total, 188,716 Blued profiles (Mage = 31.1, SD = 7.1) were obtained. Multiple group latent class analysis was used to examine the patterns of physique preferences across the body type groups. The results revealed seven classes of physique preference. The four common physique preferences shared by all body type groups were "like averages," "like monkeys," "like muscularity," and "like bears." Individuals with a monkey or average body type exhibited a specific pattern of physique preference: "dislike bears." The two physique preferences specific to gay bear men were "like averages and muscularity," and "dislike monkeys." The highest preference for each physique was observed among individuals of the same body type. Furthermore, tops were more likely to have a muscular body and prefer a monkey physique, whereas bottoms were more likely to have a monkey body and prefer a muscular physique. These findings indicate body type-specific physique preferences based on an individual's own body type and sexual self-label identification, thus partially supporting the concept of homogamy in the physique preferences of gay men.

RENIN AND ANGIOTENSIN (1-7) OFFER PREDICTIVE VALUE IN PEDIATRIC SEPSIS: FINDINGS FROM PROSPECTIVE OBSERVATIONAL COHORTS.

ABSTRACT: Background: Pediatric sepsis is a common and complex syndrome characterized by a dysregulated immune response to infection. Aberrations in the renin-angiotensin system (RAS) are factors in several infections of adults. However, the precise impact of RAS dysregulation in pediatric sepsis remains unclear. Methods: Serum samples were collected from a derivation cohort (58 patients with sepsis, 14 critically ill control subjects, and 37 healthy controls) and validation cohort (50 patients with sepsis, 37 critically ill control subjects, and 46 healthy controls). Serum RAS levels on day of pediatric intensive care unit admission were determined and compared with survival status and organ dysfunction. Results: In the derivation cohort, the serum renin concentration was significantly higher in patients with sepsis (3,678 ± 4,746) than that in healthy controls (635.6 ± 199.8) ( P < 0.0001). Meanwhile, the serum angiotensin (1-7) was significantly lower in patients with sepsis (89.7 ± 59.7) compared to that in healthy controls (131.4 ± 66.4) ( P < 0.01). These trends were confirmed in a validation cohort. Nonsurvivors had higher levels of renin (8,207 ± 7,903) compared to survivors (2,433 ± 3,193) ( P = 0.0001) and lower levels of angiotensin (1-7) (60.9 ± 51.1) compared to survivors (104.0 ± 85.1) ( P < 0.05). A combination of renin, angiotensin (1-7) and procalcitonin achieved a model for diagnosis with an area under the receiver operating curve of 0.87 (95% CI: 0.81-0.92). Conclusion: Circulating renin and angiotensin (1-7) have predictive value in pediatric sepsis.

Evaluation of the novel endophytic fungus Chaetomium ascotrichoides 1-24-2 from Pinus massoniana as a biocontrol agent against pine wilt disease caused by Bursaphelenchus xylophilus.

BACKGROUND: Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD), is an ever-increasing threat to Pinus forests worldwide. This study aimed to develop biological control of PWD by the application of endophytic fungi isolated from healthy pine trees. RESULTS: We successfully isolated a novel endophytic fungal strain 1-24-2 from branches of healthy Pinus massoniana. The culture filtrates (CFs) of strain 1-24-2 exhibited strong nematicidal activity against Bursaphelenchus xylophilus, with a corrected mortality rate of 99.00%. Based on the morphological and molecular characteristics, the isolated strain 1-24-2 was identified as Chaetomium ascotrichoides. In the in-planta assay, pine seedlings (2-years-old) treated with 1-24-2 CFs + pine wood nematode (T2) showed a significant control effect of 80%. A total of 24 toxic compounds were first identified from 1-24-2 CFs through gas chromatography-mass spectrometry (GC-MS) analysis, from which O-methylisourea, 2-chlorobenzothiazole, and 4,5,6-trihydroxy-7-methylphthalide showed robust binding sites at Tyr119 against phosphoethanolamine methyltransferase (PMT) protein of Bursaphelenchus xylophilus by molecular docking approach and could be used as potential compounds for developing effective nematicides. Interestingly, strain 1-24-2 produces toxic volatile organic compounds (VOCs), which disturb the natural development process of B. xylophilus, whose total number decreased by up to 83.32% in the treatment group as compared to control and also reduced Botrytis cinerea growth by up to 71.01%. CONCLUSION: Our results highlight the potential of C. ascotrichoides 1-24-2 as a promising biocontrol agent with solid nematicidal activity against B. xylophilus. This is the first report of C. ascotrichoides isolated from P. massoniana exhibiting strong biocontrol potential against B. xylophilus in the world. © 2024 Society of Chemical Industry.

The giant panda gut harbors a high diversity of lactic acid bacteria revealed by a novel culturomics pipeline.

Some lactic acid bacteria (LAB) can provide significant health benefits, which are critically important for the conservation of endangered animals, such as giant pandas. However, little is known about the diversity and culturability of LAB in the giant panda gut microbiota. To understand the roles of LAB in giant panda conservation, it is critical to culture bacterial strains of interest. In this study, we established a pipeline to culture bacterial strains using enrichment of target bacteria with different liquid media and growth conditions. Then, the strains were isolated in solid media to study their functions. Using 210 samples from the culture enrichment method and 138 culture-independent samples, we obtained 1120 amplicon sequencing variants (ASVs) belonging to Lactobacillales. Out of the 1120 ASVs, 812 ASVs from the culture enrichment approach were twofold more diverse than 336 ASVs from the culture-independent approach. Many ASVs of interest were not detected in the culture-independent approach. Using this pipeline, we isolated many relevant bacterial strains and established a giant panda gut bacteria strain collection that included strains with low-abundance in culture-independent samples and included most of the giant panda LAB described by other researchers. The strain collection consisted of 60 strains representing 35 species of 12 genera. Thus, our pipeline is powerful and provides guidance in culturing gut microbiota of interest in hosts such as the giant panda.IMPORTANCECultivation is necessary to screen strains to experimentally investigate microbial traits, and to confirm the activities of novel genes through functional characterization studies. In the long-term, such work can aid in the identification of potential health benefits conferred by bacteria and this could aid in the identification of bacterial candidate strains that can be applied as probiotics. In this study, we developed a pipeline with low-cost and user-friendly culture enrichment to reveal the diversity of LAB in giant pandas. We compared the difference between culture-independent and culture enrichment methods, screened strains of interest that produced high concentrations of short-chain fatty acids (SCFAs), and we investigated the catalog of virulence factors, antibiotic resistance, butyrate and lactate synthesis genes of the strains at a genomic level. This study will provide guidance for microbiota cultivation and a foundation for future research aiming to understand the functions of specific strains.

Association Between Pornography Use, Sexism, and Sexual Violence Myth Acceptance in Chinese Men: The Moderating Effect of Perceived Realism.

Pornography is spreading more and more widely due to websites, applications, and social media. It has attracted the attention of a large number of researchers who are sometimes divided on the impact of pornography. However, the relationship between pornography and sexual violence myths has received little scholarly attention in China. Based on the 3AM model and previous research, the study examined hostile sexism (HS) as a mediator and perceived realism as a moderator in the links between pornography use frequency and sexual violence myths in a sample of Chinese men (N = 376). The results showed that although pornography use and sexual violence myths did not directly correlate with one another, there was an indirect correlation through HS. Further, perceived realism moderated the relationship between pornography use frequency and HS. When participants' perceived realism was high (i.e. +1 SD), the indirect effect of HS was strong; when participants' perceived realism was low (i.e. -1 SD), the indirect effect of HS was not significant. Taken together, the findings reveal the cross-cultural consistency of the 3AM theory in China, and the findings provide new insight into the potential impact of pornography on sexism. At the same time, the results suggest an increase in appropriate education and interventions to reduce the incidence of sexual violence.

The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. Here, to explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3 and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely, thymosin beta 4, thymosin beta 10 and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

Copper Quantum Dot/Polyacrylamide Composite Nanospheres: Spreading on Quartz Flake Surfaces and Displacing Crude Oil in Microchannel Chips.

Polyacrylamide, silica, and other nanoparticles have all been realized in the field of enhanced oil recovery. Researchers often explore the mechanisms of spreading behavior and simulated displacement to develop more efficient types of nanoparticles. In this study, copper quantum dots were introduced into a acrylamide copolymerization system to obtain composite nanospheres and its structure, topographic, and application performance were characterized. The results show that the composite nanospheres have a particle size of around 25 nm, are uniformly loaded with copper particles, and have good temperature resistance. The spreading ability on the quartz flake surfaces and displacement effect in microchannels of composite nanospheres, acrylamide copolymer nanospheres, and copper quantum dots were compared by nanofluid spreading experiments and microchannel chip oil displacement experiments. The results indicate that the composite nanospheres can effectively reduce the water contact angle, promote the spreading of aqueous phase, and accelerate the oil droplet removal process; the accelerating effect is stronger than other samples. Its oil displacement effect is also the strongest, and it is minimized by the influence of channel size, temperature, and dispersing medium, with better stratigraphic adaptability. This work supports the practical application of copper quantum dot/polyacrylamide composite nanospheres in the oilfield.

MiR-4319 targets tuftelin 1 to reduce malignancy of cervical cancer cells.

Cervical cancer (CC) is the most common malignant tumor of female reproductive system. MiR-4319 has been identified as an anti-oncogene in various cancers. In the present study, role of miR-4319 in CC was identified. Colony formation, flow cytometer, wound healing, and transwell assays were used to detect CC cell proliferation, apoptosis, migration, and invasion. The expression of miR-4319 was decreased in clinical CC tissues and CC cell lines. Upregulation of miR-4319 suppressed cell viability, proliferation, migration, and invasion, and induced cell apoptosis in CC cells. Moreover, tuftelin 1 (TUFT1) was verified as a direct target of miR-4319, as confirmed by dual-luciferase reporter assay. Additionally, TUFT1 expression was remarkably increased in clinical CC tissues and CC cell lines and was negatively associated with miR-4319 expression. Furthermore, overexpression of TUFT1 partially restored the effects of miR-4319 mimic on cell viability, proliferation, migration, invasion, and cell apoptosis in CC cells. To conclude, miR-4319 played an anti-cancer role in the occurrence and development of CC, which might be achieved by targeting TUFT1.

The subcommissural organ regulates brain development via secreted peptides.

The subcommissural organ (SCO) is a gland located at the entrance of the aqueduct of Sylvius in the brain. It exists in species as distantly related as amphioxus and humans, but its function is largely unknown. To explore its function, we compared transcriptomes of SCO and non-SCO brain regions and found three genes, Sspo, Car3, and Spdef, that are highly expressed in the SCO. Mouse strains expressing Cre recombinase from endogenous promoter/enhancer elements of these genes were used to genetically ablate SCO cells during embryonic development, resulting in severe hydrocephalus and defects in neuronal migration and development of neuronal axons and dendrites. Unbiased peptidomic analysis revealed enrichment of three SCO-derived peptides, namely thymosin beta 4, thymosin beta 10, and NP24, and their reintroduction into SCO-ablated brain ventricles substantially rescued developmental defects. Together, these data identify a critical role for the SCO in brain development.

Intact lung tissue and bronchoalveolar lavage fluid are both suitable for the evaluation of murine lung microbiome in acute lung injury.

BACKGROUND: Accumulating clinical evidence suggests that lung microbiome is closely linked to the progression of pulmonary diseases; however, it is still controversial which specimen type is preferred for the evaluation of lung microbiome. METHODS AND RESULTS: To address this issue, we established a classical acute lung injury (ALI) mice model by intratracheal instillation of lipopolysaccharides (LPS). We found that the bacterial DNA obtained from the bronchoalveolar lavage fluid (BALF), intact lung tissue [Lung(i)], lung tissue after perfused [Lung(p)], and feces of one mouse were enough for 16S rRNA sequencing, except the BALF of mice treated with phosphate buffer saline (PBS), which might be due to the biomass of lung microbiome in the BALF were upregulated in the mice treated with LPS. Although the alpha diversity among the three specimens from lungs had minimal differences, Lung(p) had higher sample-to-sample variation compared with BALF and Lung(i). Consistently, PCoA analysis at phylum level indicated that BALF was similar to Lung(i), but not Lung(p), in the lungs of mice treated with LPS, suggesting that BALF and Lung(i) were suitable for the evaluation of lung microbiome in ALI. Importantly, Actinobacteria and Firmicutes were identified as the mostly changed phyla in the lungs and might be important factors involved in the gut-lung axis in ALI mice. Moreover, Actinobacteria and Proteobacteria might play indicative roles in the severity of lung injury. CONCLUSION: This study shows both Lung(i) and BALF are suitable for the evaluation of murine lung microbiome in ALI, and several bacterial phyla, such as Actinobacteria, may serve as potential biomarkers for the severity of ALI. Video Abstract.

Reversible Carbon Dioxide/Lithium Oxalate Regulation toward Advanced Aprotic Lithium Carbon Dioxide Battery.

Li-CO2 batteries have received significant attention owing to their advantages of combining greenhouse gas utilization and energy storage. However, the high kinetic barrier between gaseous CO2 and the Li2CO3 product leads to a low operating voltage (<2.5 V) and poor energy efficiency. In addition, the reversibility of Li2CO3 has always been questioned owing to the introduction of more decomposition paths caused by its higher charging plateau. Here, a novel "trinity" Li-CO2 battery system was developed by synergizing CO2, soluble redox mediator (2,2,6,6-tetramethylpiperidoxyl, as TEM RM), and reduced graphene oxide electrode to enable selective conversion of CO2 to Li2C2O4. The designed Li-CO2 battery exhibited an output plateau reaching up to 2.97 V, higher than the equilibrium potential of 2.80 V for Li2CO3, and an ultrahigh round-trip efficiency of 97.1 %. The superior performance of Li-CO2 batteries is attributed to the TEM RM-mediated preferential growth mechanism of Li2C2O4, which enhances the reaction kinetics and rechargeability. Such a unique design enables batteries to cope with sudden CO2-deficient environments, which provides an avenue for the rationally design of CO2 conversion reactions and a feasible guide for next-generation Li-CO2 batteries.

Self-Objectification and Self-Sexualizing Appearance Behaviors in Chinese Lesbian and Bisexual Females: Moderating Effect of Femme/Butch/Androgyne Identity.

In heterosexual populations, self-objectification is associated with self-sexualizing appearance behaviors. This study examined the relationship between self-objectification and self-sexualizing appearance behaviors and the moderating effect of butch/androgynous/femme sexual self-label identification in Chinese bisexual and lesbian females. We recruited 637 bisexual and lesbian females to complete an online questionnaire that asked about demographic information and feminine and masculine self-sexualizing appearance behaviors and measured the Objectified Body Consciousness Scale. Feminine self-sexualizing appearance behaviors include wearing high heels, short skirts, low-cut outfits, skinny clothes and makeup. Masculine self-sexualizing appearance behaviors include wearing short hair and binding breasts. Femme-identified females scored higher on body surveillance than did butch- and androgynous-identified females. Butch-identified females reported having more masculine self-sexualizing behaviors, whereas femme-identified females reported having more feminine self-sexualizing appearance behaviors. Sexual self-label identification moderated the relationship between self-objectification and feminine self-sexualizing behaviors. Body surveillance was significantly associated with feminine self-sexualizing behaviors in femme- and androgynous-identified females but not in butch-identified females. Body shame was negatively associated with feminine self-sexualizing appearance behaviors in androgynous-identified females. The current findings highlight the role of sexual self-label identification in self-objectification among Chinese bisexual and lesbian females. The findings imply the heterogeneousness of self-objectification among Chinese bisexual and lesbian females.

Implications of neonatal absence of innate immune mediated NFκB/AP1 signaling in the murine liver.

BACKGROUND: The developmental immaturity of the innate immune system helps explains the increased risk of infection in the neonatal period. Importantly, innate immune signaling pathways such as p65/NFκB and c-Jun/AP1 are responsible for the prevention of hepatocyte apoptosis in adult animals, yet whether developmental immaturity of these pathways increases the risk of hepatic injury in the neonatal period is unknown. METHODS: Using a murine model of endotoxemia (LPS 5 mg/kg IP x 1) in neonatal (P3) and adult mice, we evaluated histologic evidence of hepatic injury and apoptosis, presence of p65/NFκB and c-Jun/AP1 activation and associated transcriptional regulation of apoptotic genes. RESULTS: We demonstrate that in contrast to adults, endotoxemic neonatal (P3) mice exhibit a significant increase in hepatic apoptosis. This is associated with absent hepatic p65/NFκB signaling and impaired expression of anti-apoptotic target genes. Hepatic c-Jun/AP1 activity was attenuated in endotoxemic P3 mice, with resulting upregulation of pro-apoptotic factors. CONCLUSIONS: These results demonstrate that developmental absence of innate immune p65/NFκB and c-Jun/AP1 signaling, and target gene expression is associated with apoptotic injury in neonatal mice. More work is needed to determine if this contributes to long-term hepatic dysfunction, and whether immunomodulatory approaches can prevent this injury. IMPACT: Various aspects of developmental immaturity of the innate immune system may help explain the increased risk of infection in the neonatal period. In adult models of inflammation and infection, innate immune signaling pathways such as p65/NFκB and c-Jun/AP1 are responsible for a protective, pro-inflammatory transcriptome and regulation of apoptosis. We demonstrate that in contrast to adults, endotoxemic neonatal (P3) mice exhibit a significant increase in hepatic apoptosis associated with absent hepatic p65/NFκB signaling and c-Jun/AP1 activity. We believe that these results may explain in part hepatic dysfunction with neonatal sepsis, and that there may be unrecognized developmental and long-term hepatic implications of early life exposure to systemic inflammatory stress.

GSK3ß/NF-κB -dependent transcriptional regulation of homeostatic hepatocyte Tnf production.

Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-α (TNFα) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 ß (GSK3ß) and nuclear factor κB (NF-κB) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3ß and NF-κB inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-κB activation. Pharmacological inhibition of GSK3ß resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3ß inhibition decreased nuclear levels of the NF-κB subunits p65 and p50. We determined that NF-κB transcription factor subunit p65 binds to consensus sequence elements present in the murine TNFα promoter and inhibition of GSK3ß decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3ß and NF-κB inhibition. These results demonstrate that GSK3ß and NF-κB are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNFα mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver.NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-α (TNFα)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.

Proteomics and phosphoproteomics to study Tuina reverses capsule fibrosis in frozen shoulder: a research report based on rats.

Frozen shoulder (FS) is a common disorder often treated with Tuina, but the mechanisms involved remain unclear. We employed proteomics and phosphoproteomics to investigate the mechanisms associated with the treatment of capsule fibrosis in FS rats. We used a method composed of three weeks of cast immobilization to establish a model of FS. We then administered Tuina once daily for 14 days, evaluated glenohumeral range of motion (ROM), assessed histological changes, and identified differentially expressed proteins (DEPs) using proteomics and phosphoproteomics. This study demonstrated that Tuina could improve glenohumeral ROM and reserve capsule fibrosis in FS rats. Proteomics revealed proteins regulated by Tuina belonging to the PI3K-AKT and ECM receptor interaction signaling pathways. Phosphoproteomics detected differentially phosphorylated proteins regulated by Tuina to be enriched in the MAPK signaling pathway. The combination of proteomics and phosphoproteomics for Protein-Protein Interaction (PPI) network analysis revealed that the phosphorylation of Myh3 and Srsf1 with a node degree larger than the average degree were considered the central regulatory protein modulated by Tuina to reverse capsule fibrosis. Thbs1, Vtn, and Tenascin-W were significantly enriched in PI3K-AKT and ECM receptor interaction signaling pathways and highly expressed in model rats. Tuina resulted in reduced expression of these proteins. Our findings demonstrated some of mechanisms behind the reversal of FS capsule fibrosis following Tuina, a scientific medical therapy for FS patients.