ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/ß-Catenin Signaling Pathway.

OBJECTIVE: Uterine corpus endometrial carcinoma (UCEC), a kind of gynecologic malignancy, poses a significant risk to women's health. The precise mechanism underlying the development of UCEC remains elusive. Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein superfamily, was reported to be dysregulated in various illnesses, including malignant tumors. This study aimed to examine the involvement of ZNF554 in the development of UCEC. METHODS: The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay. Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection. CCK-8, wound healing, and Transwell invasion assays were employed to assess cell proliferation, migration, and invasion. Propidium iodide (PI) staining combined with fluorescence-activated cell sorting (FACS) flow cytometer was utilized to detect cell cycle distribution. qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels. Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5 (RBM5). RESULTS: The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines. Decreased expression of ZNF554 was associated with higher tumor stage, decreased overall survival, and reduced disease-free survival in UCEC. ZNF554 overexpression suppressed cell proliferation, migration, and invasion, while also inducing cell cycle arrest. In contrast, a decrease in ZNF554 expression resulted in the opposite effect. Mechanistically, ZNF554 transcriptionally regulated RBM5, leading to the deactivation of the Wingless (WNT)/ß-catenin signaling pathway. Moreover, the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression on ß-catenin and p-glycogen synthase kinase-3ß (p-GSK-3ß). Similarly, the deliberate activation of RBM5 reduced the increase in ß-catenin and p-GSK-3ß caused by the suppression of ZNF554. In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown. Additionally, when RBM5 was overexpressed, it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels. CONCLUSION: ZNF554 functions as a tumor suppressor in UCEC. Furthermore, ZNF554 regulates UCEC progression through the RBM5/WNT/ß-catenin signaling pathway. ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.

Inhibition of cisplatin-induced Acsl4-mediated ferroptosis alleviated ovarian injury.

Given that the severity of the chemotherapy-induced ovarian damage, effective fertility preservation is a necessary part of the treatment process. Ferroptosis is a regulated cell death triggered by excessive phospholipid peroxidation caused by iron and the role of ferroptosis in chemotherapy-induced ovarian damage remains unclear. In this study, we demonstrated that cisplatin treatment caused the accumulation of iron ions which induced ferroptosis in ovarian tissue. And our results show that ferrostatin-1 was able to suppress the ovarian injury and granulosa cell death caused by cisplatin (Cis) in vivo and in vitro. At the same time, we observed significant changes in the expression levels of Acyl-CoA synthetase long-chain family member 4 (Acsl4) and glutathione peroxidase 4 (GPX4). Similarly, Rosiglitazone, an inhibitor of Acsl4, administration alleviated the ovary damage of the mice undergoing chemotherapy. Further mechanistic investigation showed that cisplatin increased the expression level of specificity protein 1 (SP1), and SP1 could bind to the promoter of Acsl4 to increased Acsl4 transcription. Overall, ferroptosis plays an important role in Cis induced ovarian injury, and inhibition of ferroptosis protects ovarian tissues from damage caused by cisplatin, and for the first time, we have identified the potential of Fer-1 and Rosi to protect ovarian function in female mice undergoing chemotherapy.

Self-assembly of H2S-responsive nanoprodrugs based on natural rhein and geraniol for targeted therapy against Salmonella Typhimurium.

Salmonellosis is a globally extensive food-borne disease, which threatens public health and results in huge economic losses in the world annually. The rising prevalence of antibiotic resistance in Salmonella poses a significant global concern, emphasizing an imperative to identify novel therapeutic agents or methodologies to effectively combat this predicament. In this study, self-assembly hydrogen sulfide (H2S)-responsive nanoprodrugs were fabricated with poly(α-lipoic acid)-polyethylene glycol grafted rhein and geraniol (PPRG), self-assembled into core-shell nanoparticles via electrostatic, hydrophilic and hydrophobic interactions, with hydrophilic exterior and hydrophobic interior. The rhein and geraniol are released from self-assembly nanoprodrugs PPRG in response to Salmonella infection, which is known to produce hydrogen sulfide (H2S). PPRG demonstrated stronger antibacterial activity against Salmonella compared with rhein or geraniol alone in vitro and in vivo. Additionally, PPRG was also able to suppress the inflammation and modulate gut microbiota homeostasis. In conclusion, the as-prepared self-assembly nanoprodrug sheds new light on the design of natural product active ingredients and provides new ideas for exploring targeted therapies for specific Enteropathogens. Graphical  illustration for construction of self-assembly nanoprodrugs PPRG and its antibacterial and anti-inflammatory activities on experimental Salmonella infection in mice.

WNT4 promotes macrophage polarization via granulosa cell M-CSF and reduces granulosa cell apoptosis in endometriosis.

BACKGROUND: WNT4 gene polymorphism are common in endometriosis and may functionally link estrogen and estrogen receptor signaling. Previous study confirmed estrogen and estrogen receptor signaling recruit macrophage to promote the pathogenesis of endometriosis. To investigate the effect of WNT4 in endometriosis involved in macrophage polarization and whether WNT4 could reduce the apoptosis of granulosa cells. METHODS: An observational study consisting of 8 cases of women with endometriosis (diagnosed by surgery and histology) and 22 mice of endometriosis animal model was conducted. Granulosa cells were isolated from 16 patients with endometriosis and co-cultured with macrophage under WNT4 treatment using TUNEL assay, quantitative reverse transcription PCR, flow cytometry and ELISA analysis. 22 mice of endometriosis animal model confirmed the WNT4 treatment effects using histology and immunohistochemistry, Western blot and flow cytometry. RESULTS: We observed that the apoptotic proportion of granulosa cells was significantly decreased and M2 macrophage was significantly increased after WNT4 treatment during the granulosa cell and macrophage co-culture system. To reveal the underlying mechanism for this, we conducted a series of experiments and found that high expression of granulosa cell M-CSF led to the M2 polarization of macrophages. The animal model also suggested that the anti-apoptotic effect of WNT4 on granulosa cells were conducted by the M2 polarized macrophage. CONCLUSIONS: WNT4 could reduce granulosa cell apoptosis and improve ovarian reserve by promoting macrophage polarization in endometriosis. M-CSF secreted by granulosa cell after WNT4 treatment was the main mediator of macrophage polarization.

CpxA/R-Controlled Nitroreductase Expression as Target for Combinatorial Therapy against Uropathogens by Promoting Reactive Oxygen Species Generation.

The antibiotic resistances emerged in uropathogens lead to accumulative treatment failure and recurrent episodes of urinary tract infection (UTI), necessitating more innovative therapeutics to curb UTI before systematic infection. In the current study, the combination of amikacin and nitrofurantoin is found to synergistically eradicate Gram-negative uropathogens in vitro and in vivo. The mechanistic analysis demonstrates that the amikacin, as an aminoglycoside, induced bacterial envelope stress by introducing mistranslated proteins, thereby constitutively activating the cpxA/R two-component system (Cpx signaling). The activation of Cpx signaling stimulates the expression of bacterial major nitroreductases (nfsA/nfsB) through soxS/marA regulons. As a result, the CpxA/R-dependent nitroreductases overexpression generates considerable quantity of lethal reactive intermediates via nitroreduction and promotes the prodrug activation of nitrofurantoin. As such, these actions together disrupt the bacterial cellular redox balance with excessively-produced reactive oxygen species (ROS) as "Domino effect", accelerating the clearance of uropathogens. Although aminoglycosides are used as proof-of-principle to elucidate the mechanism, the synergy between nitrofurantoin is generally applicable to other Cpx stimuli. To summarize, this study highlights the potential of aminoglycoside-nitrofurantoin combination to replenish the arsenal against recurrent Gram-negative uropathogens and shed light on the Cpx signaling-controlled nitroreductase as a potential target to manipulate the antibiotic susceptibility.

Prevalence of Fundus Tessellation and Its Screening Based on Artificial Intelligence in Chinese Children: the Nanjing Eye Study.

INTRODUCTION: To investigate the prevalence of fundus tessellation (FT), and the threshold for screening FT using an artificial intelligence (AI) technology in Chinese children. METHODS: The Nanjing Eye Study was a population-based cohort study conducted in children born between September 2011 and August 2012 in Yuhuatai District of Nanjing. The data presented in this paper were obtained in 2019, when these children were 7 years old and underwent 45° non-mydriatic fundus photography. FT in whole fundus, macular area, and peripapillary area was manually recognized from fundus photographs and classified into three grades. Fundus tessellation density (FTD) in these areas was obtained by calculating the average exposed choroid area per unit area using artificial intelligence (AI) technology based on fundus photographs. The threshold for screening FT using FTD was determined using receiver operating characteristic (ROC) curve analysis. RESULTS: Among 1062 enrolled children (mean [± standard deviation] spherical equivalent: - 0.28 ± 0.70 D), the prevalence of FT was 42.18% in the whole fundus (grade 1: 36.53%; grade 2: 5.08%; grade 3: 0.56%), 45.57% in macular area (grade 1: 43.5%; grade 2: 1.60%; grade 3: 0.50%), and 49.72% in peripapillary area (grade 1: 44.44%; grade 2: 4.43%; grade 3: 0.85%), respectively. The threshold value of FTD for screening severe FT (grade ≥ 2) was 0.049 (area under curve [AUC] 0.985; sensitivity 98.3%; specificity 92.3%) in the whole fundus, 0.069 (AUC 0.987; sensitivity 95.5%; specificity 96.2%) in the macular area, and 0.094 (AUC 0.980; sensitivity 94.6%; specificity 94.2%) in the peripapillary area, respectively. CONCLUSION: Fundus tessellation affected approximately 40 in 100 children aged 7 years in China, indicating the importance and necessity of early FT screening. The threshold values of FTD provided by this study had high accuracy for detecting severe FT and might be applied for rapid screening.

Fundus Tessellated Density Assessed by Deep Learning in Primary School Children.

Purpose: To explore associations of fundus tessellated density (FTD) and compare characteristics of different fundus tessellation (FT) distribution patterns, based on artificial intelligence technology using deep learning. Methods: Comprehensive ocular examinations were conducted in 577 children aged 7 years old from a population-based cross-sectional study, including biometric measurement, refraction, optical coherence tomography angiography, and 45° nonmydriatic fundus photography. FTD was defined as the average exposed choroid area per unit area of the fundus, and obtained by artificial intelligence technology. The distribution of FT was classified into the macular pattern and the peripapillary pattern according to FTD. Results: The mean FTD was 0.024 ± 0.026 in whole fundus. Multivariate regression analysis showed that greater FTD was significantly correlated with thinner subfoveal choroidal thickness, larger parapapillary atrophy, greater vessel density inside the optic disc, larger vertical diameter of optic disc, thinner retinal nerve fiber layer, and longer distance from optic disc center to macular fovea (all P < 0.05). The peripapillary distributed group had larger parapapillary atrophy (0.052 ± 0.119 vs 0.031 ± 0.072), greater FTD (0.029 ± 0.028 vs 0.015 ± 0.018), thinner subfoveal choroidal thickness (297.66 ± 60.61 vs 315.33 ± 66.46), and thinner retinal thickness (285.55 ± 10.89 vs 288.03 ± 10.31) than the macular distributed group (all P < 0.05). Conclusions: FTD can be applied as a quantitative biomarker to estimate subfoveal choroidal thickness in children. The role of blood flow inside optic disc in FT progression needs further investigation. The distribution of FT and the peripapillary pattern correlated more with myopia-related fundus changes than the macular pattern. Translational Relevance: Artificial intelligence can evaluate FT quantitatively in children, and has potential value for assisting in myopia prevention and control.

Natural flavonoids disrupt bacterial iron homeostasis to potentiate colistin efficacy.

In the face of the alarming rise in global antimicrobial resistance, only a handful of novel antibiotics have been developed in recent decades, necessitating innovations in therapeutic strategies to fill the void of antibiotic discovery. Here, we established a screening platform mimicking the host milieu to select antibiotic adjuvants and found three catechol-type flavonoids-7,8-dihydroxyflavone, myricetin, and luteolin-prominently potentiating the efficacy of colistin. Further mechanistic analysis demonstrated that these flavonoids are able to disrupt bacterial iron homeostasis through converting ferric iron to ferrous form. The excessive intracellular ferrous iron modulated the membrane charge of bacteria via interfering the two-component system pmrA/pmrB, thereby promoting the colistin binding and subsequent membrane damage. The potentiation of these flavonoids was further confirmed in an in vivo infection model. Collectively, the current study provided three flavonoids as colistin adjuvant to replenish our arsenals for combating bacterial infections and shed the light on the bacterial iron signaling as a promising target for antibacterial therapies.

Associations between Plasma Essential Metals Levels and the Risks of All-Cause Mortality and Cardiovascular Disease Mortality among Individuals with Type 2 Diabetes.

Epidemiological evidence regarding the possible link between multiple essential metals levels and all-cause mortality and cardiovascular disease (CVD) mortality among type 2 diabetes (T2D) patients is sparse. Here, we aimed to evaluate the longitudinal associations between 11 essential metals levels in plasma and all-cause mortality and CVD mortality among T2D patients. Our study included 5278 T2D patients from the Dongfeng-Tongji cohort. LASSO penalized regression analysis was used to select the all-cause and CVD mortality-associated metals from 11 essential metals (iron, copper, zinc, selenium, manganese, molybdenum, vanadium, cobalt, chromium, nickel, and tin) measured in plasma. Cox proportional hazard models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Results: With a median follow-up of 9.8 years, 890 deaths were documented, including 312 deaths of CVD. LASSO regression models and the multiple-metals model revealed that plasma iron and selenium were negatively associated with all-cause mortality (HR: 0.83; 95%CI: 0.70, 0.98; HR: 0.60; 95%CI: 0.46, 0.77), whereas copper was positively associated with all-cause mortality (HR: 1.60; 95%CI: 1.30, 1.97). Only plasma iron has been significantly associated with decreased risk of CVD mortality (HR: 0.61; 95%CI: 0.49, 0.78). The dose-response curves for the association between copper levels and all-cause mortality followed a J shape (Pfor nonlinear = 0.01). Our study highlights the close relationships between essential metals elements (iron, selenium, and copper) and all-cause and CVD mortality among diabetic patients.

Changes in Nutritional Status of Cancer Patients Undergoing Proton Radiation Therapy Based on Real-World Data.

Methods: Observational study on 47 adult hospitalized cancer patients including 27 males and 20 females who received proton beam radiotherapy during December 2021 and August 2022. Nutritional assessments, 24 h dietary survey, handgrip strength (HGS) test, anthropometrical measurements, and hematological parameters were conducted or collected at the beginning and the completion of treatment. Results: The rate of nutritional risk and malnutrition among the total of 47 enrolled patients was 4.3% and 12.8% at the onset of proton radiation and raised up to 6.4% and 27.7% at the end of the treatment. 42.6% of patients experienced weight loss during the proton radiotherapy, and 1 of them had weight loss over 5%, and in general, the average body weight was stable over radiotherapy. The changes in patients' 24 h dietary intakes, HGS, and anthropometrical parameters, including triceps skinfold thickness (TSF), midupper arm circumference (MUAC), and midupper arm muscle circumference (MAMC), were statistically insignificant over the treatment (all p values > 0.05). The changes in patients' hematological parameters, including total protein (TP) and serum albumin (ALB), were not statistically significant over the treatment (all p values >0.05), and the level of hemoglobin (HGB) at the end of treatment was higher than that at the onset (p < 0.05). Conclusion: The results of this study demonstrated that proton radiotherapy might have a lighter effect on the nutritional status of cancer patients.

Bacterial Vaginosis Decreases the Risk of Cervical Cytological Abnormalities.

Genital tract infections, including vulvovaginal candidiasis and bacterial vaginosis, have emerged as potential modulators of persistent human papillomavirus (HPV) infections causing cervical cytologic abnormalities and cervical cancer. This study aimed to investigate whether vulvovaginal candidiasis or bacterial vaginosis had an additional effect on HPV infection and thus caused such abnormalities. ThinPrep cytologic tests were used to detect cytologic abnormalities, vulvovaginal candidiasis, and bacterial vaginosis in 14,679 women. Cytologic abnormalities included atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, atypical squamous cells-cannot exclude HSIL, and squamous cell carcinoma. Logistic regression Model 1 (univariate regression) and Model 2 (multivariate logistic regression analysis adjusted for age combined with HPV infection) were used to analyze the association between bacterial vaginosis and cytologic abnormalities, or vulvovaginal candidiasis and cytologic abnormalities, alone or in the presence of HPV infection. Bacterial vaginosis infection rates were found to be significantly higher in the cytology-negative group among all participants and those with HPV infection (P = 0.003, P < 0.001, respectively). Analyses using Model 1 and Model 2 both pointed to bacterial vaginosis as a protective factor against cytologic abnormalities for all participants (OR = 0.36, 0.17, respectively, P < 0.05) and for HPV-infected participants (OR = 0.17, 0.16, respectively, P < 0.05). Neither vulvovaginal candidiasis nor vulvovaginal candidiasis + HPV was significantly associated with the incidence of cytologic abnormalities based on Model 1 (OR = 0.94, 0.71, respectively, P > 0.05) and Model 2 (OR = 0.91, 0.74, respectively, P > 0.05). Furthermore, neither vulvovaginal candidiasis nor bacterial vaginosis increased the incidence of cytologic abnormalities regardless of HPV infection status, while bacterial vaginosis might possibly prevent cytologic abnormalities in women coinfected by HPV. PREVENTION RELEVANCE: Neither vulvovaginal candidiasis nor bacterial vaginosis was found to increase the incidence of cervical cytologic abnormalities with or without the presence of HPV. On the contrary, bacterial vaginosis may play a role in preventing cytologic abnormalities in women with HPV coinfection.

A Generic Pixel Pitch Calibration Method for Fundus Camera via Automated ROI Extraction.

Pixel pitch calibration is an essential step to make the fundus structures in the fundus image quantitatively measurable, which is important for the diagnosis and treatment of many diseases, e.g., diabetes, arteriosclerosis, hereditary optic atrophy, etc. The conventional calibration approaches require the specific parameters of the fundus camera or several specially shot images of the chess board, but these are generally not accessible, and the calibration results cannot be generalized to other cameras. Based on automated ROI (region of interest) and optic disc detection, the diameter ratio of ROI and optic disc (ROI-disc ratio) is quantitatively analyzed for a large number of fundus images. With the prior knowledge of the average diameter of an optic disc in fundus, the pixel pitch can be statistically estimated from a large number of fundus images captured by a specific camera without the availability of chess board images or detailed specifics of the fundus camera. Furthermore, for fundus cameras of FOV (fixed field-of-view), the pixel pitch of a fundus image of 45° FOV can be directly estimated according to the automatically measured diameter of ROI in the pixel. The average ROI-disc ratio is approximately constant, i.e., 6.404 ± 0.619 in the pixel, according to 40,600 fundus images, captured by different cameras, of 45° FOV. In consequence, the pixel pitch of a fundus image of 45° FOV can be directly estimated according to the automatically measured diameter of ROI in the pixel, and results show the pixel pitches of Canon CR2, Topcon NW400, Zeiss Visucam 200, and Newvision RetiCam 3100 cameras are 6.825 ± 0.666 µm, 6.625 ± 0.647 µm, 5.793 ± 0.565 µm, and 5.884 ± 0.574 µm, respectively. Compared with the manually measured pixel pitches, based on the method of ISO 10940:2009, i.e., 6.897 µm, 6.807 µm, 5.693 µm, and 6.050 µm, respectively, the bias of the proposed method is less than 5%. Since our method doesn't require chess board images or detailed specifics, the fundus structures on the fundus image can be measured accurately, according to the pixel pitch obtained by this method, without knowing the type and parameters of the camera.

Associations of multiple plasma metals with chronic kidney disease in patients with diabetes.

As common contaminants, metals are non-negligible risk factors for diabetes and chronic kidney disease. However, whether there is an association between multiple metals exposure and incident chronic kidney disease (CKD) risk in patients with diabetes is unclear. We conducted a prospective study to evaluate these associations. In total, 3071 diabetics with baseline estimated glomerular filtration rate (eGFR) ≥ 60 mL/min/1.73 m2 from the Dongfeng-Tongji cohort were included. We measured baseline plasma concentrations of 23 metals and investigated the associations between plasma metal concentrations and CKD in diabetics using logistic regression, the least absolute shrinkage and selection operator (LASSO), and the Bayesian Kernel Machine Regression (BKMR) models. During average 4.6 years of follow-up, 457 diabetics developed CKD (14.9 %). The three models consistently found plasma levels of zinc, arsenic, and rubidium had a positive association with incident CKD risk in patients with diabetes, while titanium, cadmium, and lead had an inverse correlation. The results of BKMR showed a significant and positive overall effect of 23 metals on the risk of CKD, when all of the metals were above the 50th percentile as compared to the median value. In addition, potential interactions of zinc and arsenic, zinc and cadmium, zinc and lead, titanium and arsenic, and cadmium and lead on CKD risk were observed. In summary, we found significant associations of plasma titanium, zinc, arsenic, rubidium, cadmium, and lead with CKD in diabetes and interactions between these metals except for rubidium. Co-exposure to multiple metals was associated with increased CKD risk in diabetics.

Anlotinib improved the reactive cutaneous capillary endothelial proliferation induced by camrelizumab: a case report.

Background: Programmed cell death protein-1 (PD-1) or its ligand PD-L1 monoclonal antibodies, opening a new era of tumor immunotherapy, and they have significantly improved the overall survival of many patients with advanced solid tumors. However, in addition to its effectiveness, we should also pay attention to its adverse effects. The instructions of the PD-1 inhibitor camrelizumab clearly indicate that reactive cutaneous capillary endothelial proliferation (RCCEP) is the most common adverse reaction; it is common for many immune checkpoint inhibitors (ICIs). Here we describe a case that anlotinib improved RCCEP induced by anti-PD-1 blockade camrelizumab with some focus on further management of this symptoms. Case Description: A 57-year-old man with squamous cell carcinoma of the upper lobe of the left lung, and with mediastinal lymphocyte and liver metastasis, received the fifth cycle of chemotherapy and immunotherapy with camrelizumab (200 mg, every 3 weeks). Four days after treatment with camrelizumab, the patient's face, head, neck, and chest skin had multiple scattered bright red round papules, which were diagnosed as RCCEP. The patient was treated with oral anlotinib (8 mg, once a day). After 5 days of treatment, the symptoms of RCCEP gradually eased, and the patient was discharged. Conclusions: In conclusion, we have reported a case of RCCEP induced by anti-PD-1 blockade camrelizumab. The patient was given oral anlotinib to relieve the symptoms of RCCEP. Suggesting that anlotinib could be a potential management to reduce the adverse reactions who are treated with camrelizumab. The risk for RCCEP should always be kept in mind during camrelizumab treatment.

A Trade-Off for Maintenance of Multidrug-Resistant IncHI2 Plasmids in Salmonella enterica Serovar Typhimurium through Adaptive Evolution.

Understanding the fitness costs associated with plasmid carriage is a key to better understanding the mechanisms of plasmid maintenance in bacteria. In the current work, we performed multiple serial passages (63 days, 627.8 generations) to identify the compensatory mechanisms that Salmonella enterica serovar Typhimurium ATCC 14028 utilized to maintain the multidrug-resistant (MDR) IncHI2 plasmid pJXP9 in the presence and absence of antibiotic selection. The plasmid pJXP9 was maintained for hundreds of generations even without drug exposure. Endpoint evolved (the endpoint of evolution) S. Typhimurium bearing evolved plasmids displayed decreased growth lag times and a competitive advantage over ancestral pJXP9 plasmid-carrying ATCC 14028 strains. Genomic and transcriptomic analyses revealed that the fitness costs of carrying pJXP9 were derived from both specific plasmid genes and particularly the MDR regions and conjugation transfer region I and conflicts resulting from chromosome-plasmid gene interactions. Correspondingly, plasmid deletions of these regions could compensate for the fitness cost that was due to the plasmid carriage. The deletion extent and range of large fragments on the evolved plasmids, as well as the trajectory of deletion mutation, were related to the antibiotic treatment conditions. Furthermore, it is also adaptive evolution that chromosomal gene mutations and altered mRNA expression correlated with changed physiological functions of the bacterium, such as decreased flagellar motility, increased oxidative stress, and fumaric acid synthesis but increased Cu resistance in a given niche. Our findings indicated that plasmid maintenance evolves via a plasmid-bacterium adaptative evolutionary process that is a trade-off between vertical and horizontal transmission costs along with associated alterations in host bacterial physiology. IMPORTANCE The current idea that compensatory evolution processes can account for the "plasmid paradox" phenomenon associated with the maintenance of large costly plasmids in host bacteria has attracted much attention. Although many compensatory mutations have been discovered through various plasmid-host bacterial evolution experiments, the basis of the compensatory mechanisms and the nature of the bacteria themselves to address the fitness costs remain unclear. In addition, the genetic backgrounds of plasmids and strains involved in previous research were limited and clinical drug resistance such as the poorly understood compensatory evolution among clinically dominant multidrug-resistant plasmids or clones was rarely considered. The IncHI2 plasmid is widely distributed in Salmonella Typhimurium and plays an important role in the emergence and rapid spread of its multidrug resistance. In this study, the predominant multidrug-resistant IncHI2 plasmid pJXP9 and the standard Salmonella Typhimurium ATCC 14028 bacteria were used for evolution experiments under laboratory conditions. Our findings indicated that plasmid maintenance through experimental evolution of plasmid-host bacteria is a trade-off between increasing plasmid vertical transmission and impairing its horizontal transmission and bacterial physiological phenotypes, in which compensatory mutations and altered chromosomal expression profiles collectively contribute to alleviating plasmid-borne fitness cost. These results provided potential insights into understanding the relationship of coexistence between plasmids encoding antibiotic resistance and their bacterial hosts and provided a clue to the adaptive forces that shaped the evolution of these plasmids within bacteria and to predicting the evolution trajectory of antibiotic resistance.

NRF2 Genetic Polymorphism Modifies the Association of Plasma Selenium Levels With Incident Coronary Heart Disease Among Individuals With Type 2 Diabetes.

Plasma selenium and NRF2 promoter variants (e.g., rs6721961) are associated with cardiovascular disease risk in the general population. However, epidemiological evidence on the interaction between plasma selenium and NRF2 genetic susceptibility in relation to incident coronary heart disease (CHD) risk remains scarce, especially among individuals with type 2 diabetes (T2D). Thus, we examined whether rs6721961 in the NRF2 gene might modify the association between plasma selenium levels and incident CHD risk among people with T2D. During a mean (SD) follow-up period of 6.90 (2.96) years, 798 incident CHD cases were identified among 2,251 T2D cases. Risk-allele carriers of rs6721961 had a higher risk of incident CHD among people with T2D (adjusted hazard ratio [HR] 1.17; 95% CI 1.02-1.35) versus nonrisk-allele carriers. Each 22.8-µg/L increase in plasma selenium levels was associated with a reduced risk of incident CHD among risk-allele carriers with T2D (HR 0.80; 95% CI 0.71-0.89), whereas no association was found in those without risk alleles (P for interaction = 0.004), indicating that the NRF2 promoter polymorphism might modify the association between plasma selenium levels and incident CHD risk among people with T2D. Our study findings suggest redox-related genetic variants should be considered to identify populations that might benefit most from selenium supplementation. More mechanistic studies are warranted.

Sonosensitizer nanoplatform-mediated sonodynamic therapy induced immunogenic cell death and tumor immune microenvironment variation.

Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic malignancies, and effective treatments are still lacking due to drug tolerance and tumor recurrence. In this study, we aimed to investigate the effects of sonodynamic therapy (SDT) on ovarian cancer and its potential mechanism. Folate receptor-targeted and ultrasound-responsive nanoparticles (NPs) were constructed using PLGA-PEG-FA (PLGA: poly (lactic-co-glycolic) acid, polyethylene glycol (PEG), FA: folate), the reactive oxygen species (ROS)-generating sonosensitizer IR780 and the oxygen-carrying material perfluorohexane (PFH), termed IRO@FA NPs. The antitumor effect of NPs triggered by ultrasound (US) was measured by an apoptosis assay in a C57/BL6 mouse model. Immunochemistry and flow cytometry were used to detect the proportion of CD3+ T, CD4+ T, CD8+ T cells and activated dendritic cells (DCs) in spleens and tumor tissues to assess variation in the immune response. Moreover, endoplasmic reticulum (ER) stress and immunogenic cell death (ICD) markers (high mobility group protein box-1, ATP and calreticulin) were detected to identify potential mechanisms. The results showed that IRO@FA NP-mediated SDT promoted ID8 cell apoptosis both in vitro and in vivo. The densities of CD3+ and CD8+ T lymphocytes and inflammatory markers were upregulated in tumor tissues. IRO@FA NP-mediated SDT prompted DC maturation and T lymphocyte infiltration by inducing ID8 cell ICD.

Anti-fibrosis attributes: UHPLC-MS/MS-based pharmacokinetics profiling of a novel autotaxin inhibitor with excellent in vivo efficacy in rat.

3,4-Difluorobenzyl(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)-methyl)thiazol-2-yl)-1H-indol-3-yl)carbamate (NAI59), a small molecule with outstanding therapeutic effectiveness to anti-pulmonary fibrosis, was developed as an autotaxin inhibitor candidate compound. To evaluate the pharmacokinetics and plasma protein binding of NAI59, a UPLC-MS/MS method was developed to quantify NAI59 in plasma and phosphate-buffered saline. The calibration curve linearity ranged from 9.95 to 1990.00 ng/mL in plasma. The accuracy was -6.8 to 5.9%, and the intra- and inter-day precision was within 15%. The matrix effect and recovery, as well as dilution integrity, were within the criteria. The chromatographic and mass spectrometric conditions were also feasible to determine phosphate-buffered saline samples, and it has been proved that this method exhibits good precision and accuracy in the range of 9.95-497.50 ng/mL in phosphate-buffered saline. This study is the first to determine the pharmacokinetics, absolute bioavailability, and plasma protein binding of NAI59 in rats using this established method. Therefore, the pharmacokinetic profiles of NAI59 showed a dose-dependent relationship after oral administration, and the absolute bioavailability in rats was 6.3%. In addition, the results of protein binding showed that the combining capacity of NAI59 with plasma protein attained 90% and increased with the increase in drug concentration.

Tumor-associated macrophages induced spheroid formation by CCL18-ZEB1-M-CSF feedback loop to promote transcoelomic metastasis of ovarian cancer.

BACKGROUND: Ovarian cancer (OvCa)-tumor-associated macrophages (TAMs) spheroids are abundantly present within ascites of high malignant patients. This study investigated the mutual interaction of OvCa cells and TAMs in the spheroids. METHODS: Three-dimensional coculture system and transwell coculture system were created to mimic the OvCa and TAMs in spheroids and in disassociated state. Transwell-migration assay and scratch wound healing assay were used to measure the invasive and migratory capacity. Western blot, quantitative reverse transcription-PCR and immunostaining were used to measure the mesenchymal and epithelial markers. Flow cytometry was used to assess the polarization of TAMs. Also, the differential gene expression profile of OvCa cells and OvCa cells from spheroids were tested by RNA-sequence. Finally, the ovarian mice models were constructed by intraperitoneal injection of ID8 or OvCa-TAMs spheroids. RESULTS: Our results indicated that the formation of OvCa-TAMs spheroids was positive related to the malignancy of OvCa cells. M2-TAMs induced the epithelial-mesenchymal transition of OvCa cells by releasing chemokine (C-C motif) ligand 18 (CCL18) in the spheroids. While, CCL18 induced macrophage colony-stimulating factor (M-CSF) transcription in OvCa cells through zinc finger E-box-binding homeobox 1 (ZEB1). This study further indicated that M-CSF secreted by OvCa cells drived the polarization of M2-TAMs. Therefore, a CCL18-ZEB1-M-CSF interacting loop between OvCa cells and TAMs in the spheroids was identified. Moreover, with blocking the expression of ZEB1 in the OvCa cell, the formation of OvCa-TAMs spheroids was impeded. In the ovarian mice models, the formation of OvCa-TAMs spheroids in the ascites was promoted by overexpressing of ZEB1 in OvCa cells, which resulted in faster and earlier transcoelomic metastasis. CONCLUSION: These findings suggested that the formation of OvCa-TAMs spheroids resulted in aggressive phenotype of OvCa cells, as a specific feedback loop CCL18-ZEB1-M-CSF in it. Inhibition of ZEB1 reduced OvCa-TAMs spheroids in the ascites, impeding the transcoelomic metastasis and improving the outcome of ovarian patients.

Re-engineering a mobile-CRISPR/Cas9 system for antimicrobial resistance gene curing and immunization in Escherichia coli.

OBJECTIVES: In this study, we developed an IS26-based CRISPR/Cas9 system as a proof-of-concept study to explore the potential of a re-engineered bacterial translocatable unit (TU) for curing and immunizing against the replication genes and antimicrobial resistance genes. METHODS: A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guide RNAs were designed to target the replication gene of IncX4, IncI2 and IncHI2 plasmids, and the antibiotic resistance genes mcr-1, blaKPC-2 and blaNDM-5. Through conjugation and induction, the transposition efficiency and plasmid-curing efficiency in each recipient were tested. In addition, we examined the efficiency of the IS26-CRISPR/Cas9 system of cell immunity against the acquisition of the exogenous resistant plasmids by introducing this system into antimicrobial-susceptible hosts. RESULTS: This study aimed to eliminate the replication genes and antimicrobial resistance genes using pIS26-CRISPR/Cas9. Three plasmids with different replicon types, including IncX4, IncI2 and IncHI2 in three isolates, two pUC19-derived plasmids, pUC19-mcr-1 and pUC19-IS26mcr-1, in two lab strains, and two plasmids bearing blaKPC-2 and blaNDM-5 in two isolates were all successfully eliminated. Moreover, the IS26-based CRISPR/Cas9 system that remained in the plasmid-cured strains could efficiently serve as an immune system against the acquisition of the exogenous resistant plasmids. CONCLUSIONS: The IS26-based CRISPR/Cas9 system can be used to efficiently sensitize clinical Escherichia coli isolates to antibiotics in vitro. The single-guide RNAs targeted resistance genes or replication genes of specific incompatible plasmids that harboured resistance genes, providing a novel means to naturally select bacteria that cannot uptake and disseminate such genes.