Face presentation at term: incidence, risk factors and influence on maternal and neonatal outcomes.

OBJECTIVES: The incidence, diagnosis, management and outcome of face presentation at term were analysed. METHODS: A retrospective, gestational age-matched case-control study including 27 singletons with face presentation at term was conducted between April 2006 and February 2021. For each case, four women who had the same gestational age and delivered in the same month with vertex position and singletons were selected as the controls (control group, n = 108). Conditional logistic regression was used to assess the risk factors of face presentation. The maternal and neonatal outcomes of the face presentation group were followed up. RESULTS: The incidence of face presentation at term was 0.14‰. After conditional logistic regression, the two factors associated with face presentation were high parity (adjusted odds ratio [aOR] 2.76, 95% CI 1.19-6.39)] and amniotic fluid index > 18 cm (aOR 2.60, 95% CI 1.08-6.27). Among the 27 cases, the diagnosis was made before the onset of labor, during the latent phase of labor, during the active phase of labor, and during the cesarean section in 3.7% (1/27), 40.7% (11/27), 11.1% (3/27) and 44.4% (12/27) of cases, respectively. In one case of cervical dilation with a diameter of 5 cm, we innovatively used a vaginal speculum for rapid diagnosis of face presentation. The rate of cesarean section and postpartum haemorrhage ≥ 500 ml in the face presentation group was higher than that of the control group (88.9% vs. 13.9%, P < 0.001, and 14.8% vs. 2.8%, P = 0.024), but the Apgar scores were similar in both sets of newborns. Among the 27 cases of face presentation, there were three cases of adverse maternal and neonatal outcomes, including one case of neonatal right brachial plexus injury and two cases of severe laceration of the lower segment of the uterus with postpartum haemorrhage ≥ 1000 ml. CONCLUSIONS: Face presentation was rare. Early diagnosis is difficult, and thus easily neglected. High parity and amniotic fluid index > 18 cm are risk factors for face presentation. An early diagnosis and proper management of face presentation could lead to good maternal and neonatal outcomes.

Ling-Gui-Zhu-Gan decoction ameliorates nonalcoholic fatty liver disease via modulating the gut microbiota.

Numerous studies have supported that nonalcoholic fatty liver disease (NAFLD) is highly associated with gut microbiota dysbiosis. Ling-Gui-Zhu-Gan decoction (LG) has been clinically used to treat NAFLD, but the underlying mechanism remains unknown. This study investigated the therapeutic effect and mechanisms of LG in mice with NAFLD induced by a high-fat diet (HD). An HD-induced NAFLD mice model was established to evaluate the efficacy of LG followed by biochemical and histopathological analysis. Metagenomics, metabolomics, and transcriptomics were used to explore the structure and metabolism of the gut microbiota. LG significantly improved hepatic function and decreased lipid droplet accumulation in HD-induced NAFLD mice. LG reversed the structure of the gut microbiota that is damaged by HD and improved intestinal barrier function. Meanwhile, the LG group showed a lower total blood bile acids (BAs) concentration, a shifted BAs composition, and a higher fecal short-chain fatty acids (SCFAs) concentration. Furthermore, LG could regulate the hepatic expression of genes associated with the primary BAs biosynthesis pathway and peroxisome proliferator-activated receptor (PPAR) signaling pathway. Our study suggested that LG could ameliorate NAFLD by altering the structure and metabolism of gut microbiota, while BAs and SCFAs are considered possible mediating substances. IMPORTANCE: Until now, there has still been no study on the gut microbiota and metabolomics of Ling-Gui-Zhu-Gan decoction (LG) in nonalcoholic fatty liver disease (NAFLD) mouse models. Our study is the first to report on the reshaping of the structure and metabolism of the gut microbiota by LG, as well as explore the potential mechanism underlying the improvement of NAFLD. Specifically, our study demonstrates the potential of gut microbial-derived short-chain fatty acids (SCFAs) and blood bile acids (BAs) as mediators of LG therapy for NAFLD in animal models. Based on the results of transcriptomics, we further verified that LG attenuates NAFLD by restoring the metabolic disorder of BAs via the up-regulation of Fgf15/FXR in the ileum and down-regulation of CYP7A1/FXR in the liver. LG also reduces lipogenesis in NAFLD mice by mediating the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which then contributes to reducing hepatic inflammation and improving intestinal barrier function to treat NAFLD.

Knowledge, attitudes, and practices towards Kawasaki disease from caregivers of children with Kawasaki disease: a cross-sectional study.

PURPOSE: To examine the knowledge, attitudes, and practices (KAP) of caregivers of children with Kawasaki disease toward Kawasaki disease. METHODS: This cross-sectional study was conducted at four hospitals in China from March 2023 to June 2023. The KAP scores were evaluated using a self-designed questionnaire (Cronbach's α = 0.840; KMO = 0.7381). Correlations between dimension scores were evaluated by Pearson correlation analysis. A structural equation model (SEM) was used to examine the relationships among factors. RESULTS: Of 643 surveyed, 49.50% were male caregivers. The mean knowledge, attitude, and practice scores were 7.12 ± 2.34 (possible range, 0-11), 29.23 ± 5.67 (possible range, 12-60), and 21.57 ± 5.34 (possible range, 6-30). Knowledge correlated with attitude (r = 0.172, P < 0.001) and practice (r = 0.280, P < 0.001). Attitude was significantly related to practice (r = 0.598, P < 0.001). SEM showed knowledge had a positive effect on attitudes (ß = 0.581, P < 0.001) and practices (ß = 0.786, P < 0.001). In addition, attitudes also positively affected practices (ß = 0.554, P < 0.001). Occupation type (ß = 0.598, P = 0.025) and monthly per capita income (ß=-0.750, P = 0.020) had different effects on attitudes, while monthly per capita income also had negative effects on practices (ß=-0.410, P = 0.021). CONCLUSION: Caregivers of children with Kawasaki disease have moderate knowledge and unfavorable attitudes but proactive practices toward this disease. The results could help design an educational intervention to improve KAP, which could translate into better patient management and outcomes. TRIAL REGISTRATION: Not applicable.

Protective role of forsythoside B in Kawasaki disease-induced cardiac injury: Inhibition of pyroptosis via the SIRT1-NF-κB-p65 signaling pathway.

Kawasaki disease (KD), an acute exanthematous febrile pediatric illness involving systemic non-specific inflammatory reactions in small- and medium-sized arteries, poses a significant risk of coronary artery and myocardial inflammatory injury. Developing new KD treatments with improved safety and fewer side-effects is highly desirable. Forsythoside B (FTS-B), extracted from the Forsythia suspensa plant, exerts anti-inflammatory activity by inhibiting NF-κB, which is regulated by SIRT1, the reduced expression of which is strongly associated with cardiovascular disease. However, it has yet to be established whether FTS-B influences KD-related inflammatory damage. In this study, we investigated the effects of FTS-B on inflammation in cellular and murine models of KD. Our findings revealed that KD is associated with cardiac dysfunction and inflammatory injury to myocardial and human coronary artery endothelial cells (HCAECs), resulting in a pyroptosis-feedback loop. Both cellular and KD models were characterized by reduced SIRT1 expression and increased NF-κB p65 expression. Contrastingly, the rates of pyroptosis in both murine model myocardial tissues and HCAECs were significantly alleviated in response to FTS-B treatment. Also in both models, we detected an increase of SIRT1 expression and a decrease in the expression of p65. Further examination of the protective mechanism of FTS-B using the SIRT1-specific inhibitor, EX 527, revealed that this inhibitor blocked the palliative effects of FTS-B on inflammatory injury-induced pyroptosis. These results highlight the potential utility of the SIRT1-NF-κB-p65 pathway as a therapeutic target for KD treatment and demonstrate that FTS-B can alleviate KD-induced cardiac and HCAEC inflammatory injury via inhibition of pyroptosis.

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis.

IMPORTANCE: Kaposi's sarcoma (KS) is the most common cancer in HIV-infected patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Hyperinflammation is the hallmark of KS. In this study, we have shown that KSHV mediates hyperinflammation by inducing IL-1α and suppressing IL-1Ra. Mechanistically, KSHV miRNAs and vFLIP induce hyperinflammation by activating the NF-κB pathway. A common anti-inflammatory agent dexamethasone blocks KSHV-induced hyperinflammation and tumorigenesis by activating glucocorticoid receptor signaling to suppress IL-1α and induce IL-1Ra. This work has identified IL-1-mediated inflammation as a potential therapeutic target and dexamethasone as a potential therapeutic agent for KSHV-induced malignancies.

Activation of glucocorticoid receptor signaling inhibits KSHV-induced inflammation and tumorigenesis.

Hyperinflammation is the hallmark of Kaposi's sarcoma (KS), the most common cancer in AIDS patients caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. However, the role and mechanism of induction of inflammation in KS remain unclear. In a screening for inhibitors of KSHV-induced oncogenesis, over half of the identified candidates were anti-inflammatory agents including dexamethasone functions by activating glucocorticoid receptor (GR) signaling. Here, we examined the mechanism mediating KSHV-induced inflammation. We found that numerous inflammatory pathways were activated in KSHV-transformed cells. Particularly, interleukin-1 alpha (IL-1α) and IL-1 receptor antagonist (IL-1Ra) from the IL-1 family were the most induced and suppressed cytokines, respectively. We found that KSHV miRNAs mediated IL-1α induction while both miRNAs and vFLIP mediated IL-1Ra suppression. Furthermore, GR signaling was inhibited in KSHV-transformed cells, which was mediated by vFLIP and vCyclin. Dexamethasone treatment activated GR signaling, and inhibited cell proliferation and colony formation in soft agar of KSHV-transformed cells but had a minimal effect on matched primary cells. Consequently, dexamethasone suppressed the initiation and growth of KSHV-induced tumors in mice. Mechanistically, dexamethasone suppressed IL-1α but induced IL-1Ra expression. Treatment with recombinant IL-1α protein rescued the inhibitory effect of dexamethasone while overexpression of IL-1Ra caused a weak growth inhibition of KSHV-transformed cells. Furthermore, dexamethasone induced IκBα expression resulting in inhibition of NF-κB pathway and IL-1α expression. These results reveal an important role of IL-1 pathway in KSHV-induced inflammation and oncogenesis, which can be inhibited by dexamethasone-activated GR signaling, and identify IL-1-mediated inflammation as a potential therapeutic target for KSHV-induced malignancies.

Molecular and immune signatures, and pathological trajectories of fatal COVID-19 lungs defined by in situ spatial single-cell transcriptome analysis.

Despite intensive studies during the last 3 years, the pathology and underlying molecular mechanism of coronavirus disease 2019 (COVID-19) remain poorly defined. In this study, we investigated the spatial single-cell molecular and cellular features of postmortem COVID-19 lung tissues using in situ sequencing (ISS). We detected 10 414 863 transcripts of 221 genes in whole-slide tissues and segmented them into 1 719 459 cells that were mapped to 18 major parenchymal and immune cell types, all of which were infected by SARS-CoV-2. Compared with the non-COVID-19 control, COVID-19 lungs exhibited reduced alveolar cells (ACs) and increased innate and adaptive immune cells. We also identified 19 differentially expressed genes in both infected and uninfected cells across the tissues, which reflected the altered cellular compositions. Spatial analysis of local infection rates revealed regions with high infection rates that were correlated with high cell densities (HIHD). The HIHD regions expressed high levels of SARS-CoV-2 entry-related factors including ACE2, FURIN, TMPRSS2 and NRP1, and co-localized with organizing pneumonia (OP) and lymphocytic and immune infiltration, which exhibited increased ACs and fibroblasts but decreased vascular endothelial cells and epithelial cells, mirroring the tissue damage and wound healing processes. Sparse nonnegative matrix factorization (SNMF) analysis of niche features identified seven signatures that captured structure and immune niches in COVID-19 tissues. Trajectory inference based on immune niche signatures defined two pathological routes. Trajectory A primarily progressed with increased NK cells and granulocytes, likely reflecting the complication of microbial infections. Trajectory B was marked by increased HIHD and OP, possibly accounting for the increased immune infiltration. The OP regions were marked by high numbers of fibroblasts expressing extremely high levels of COL1A1 and COL1A2. Examination of single-cell RNA-seq data (scRNA-seq) from COVID-19 lung tissues and idiopathic pulmonary fibrosis (IPF) identified similar cell populations consisting mainly of myofibroblasts. Immunofluorescence staining revealed the activation of IL6-STAT3 and TGF-ß-SMAD2/3 pathways in these cells, likely mediating the upregulation of COL1A1 and COL1A2 and excessive fibrosis in the lung tissues. Together, this study provides a spatial single-cell atlas of cellular and molecular signatures of fatal COVID-19 lungs, which reveals the complex spatial cellular heterogeneity, organization, and interactions that characterized the COVID-19 lung pathology.

Preparation of thrombin-loaded calcium alginate microspheres with dual-mode imaging and study on their embolic properties in vivo.

Transcatheter arterial embolization (TAE) has played a huge role in the interventional treatment of organ bleeding and accidental bleeding. Choosing bio-embolization materials with good biocompatibility is an important part of TAE. In this work, we prepared a calcium alginate embolic microsphere using high-voltage electrostatic droplet technology. The microsphere simultaneously encapsulated silver sulfide quantum dots (Ag2S QDs) and barium sulfate (BaSO4), and fixed thrombin on its surface. Thrombin can achieve an embolic effect while stopping bleeding. The embolic microsphere has good near-infrared two-zone (NIR-II) imaging and X-ray imaging effects, and the luminous effect of NIR-II is better than that of X-rays. This breaks the limitations of traditional embolic microspheres that only have X-ray imaging. And the microspheres have good biocompatibility and blood compatibility. Preliminary application results show that the microspheres can achieve a good embolization effect in the ear arteries of New Zealand white rabbits, and can be used as an effective material for arterial embolization and hemostasis. This work realizes the clinical embolization application of NIR-II combined with X-ray multimodal imaging technology in biomedical imaging, achieving complementary advantages and excellent results, more suitable for studying biological changes and clinical applications.

SARS-CoV-2 infection of kidney tissues from severe COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests diverse clinical pathologies involving multiple organs. While the respiratory tract is the primary SARS-CoV-2 target, acute kidney injury is common in COVID-19 patients, displaying as acute tubular necrosis (ATN) resulting from focal epithelial necrosis and eosinophilia, glomerulosclerosis, and autolysis of renal tubular cells. However, whether any renal cells are infected by SARS-CoV-2 and the mechanism involved in the COVID-19 kidney pathology remain unclear. METHODS: Kidney tissues obtained at autopsy from four severe COVID-19 patients and one healthy subject were examined by hematoxylin and eosin staining. Indirect immunofluorescent antibody assay was performed to detect SARS-CoV-2 spike protein S1 and nonstructural protein 8 (NSP8) together with markers of different kidney cell types and immune cells to identify the infected cells. RESULTS: Renal parenchyma showed tissue injury comprised of ATN and glomerulosclerosis. Positive staining of S1 protein was observed in renal parenchymal and tubular epithelial cells. Evidence of viral infection was also observed in innate monocytes/macrophages and NK cells. Positive staining of NSP8, which is essential for viral RNA synthesis and replication, was confirmed in renal parenchymal cells, indicating the presence of active viral replication in the kidney. CONCLUSIONS: In fatal COVID-19 kidneys, there are SARS-CoV-2 infection, minimally infiltrated innate immune cells, and evidence of viral replication, which could contribute to tissue damage in the form of ATN and glomerulosclerosis.

N6-Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections.

Mitogen-activated protein kinases (MAPKs) play critical roles in the induction of numerous cytokines, chemokines, and inflammatory mediators that mobilize the immune system to counter pathogenic infections. Dual-specificity phosphatase 1 (DUSP1) is a member of the dual-specificity phosphatases that inactivates MAPKs through a negative-feedback mechanism. Here, we report that in response to viral and bacterial infections, not only the DUSP1 transcript but also its N6-methyladenosine (m6A) levels rapidly increase together with that of the m6A reader protein YTHDF2, resulting in enhanced YTHDF2-mediated DUSP1 transcript degradation. The knockdown of DUSP1 promotes p38 and Jun N-terminal kinase (JNK) phosphorylation and activation, thus increasing the expression of innate immune response genes, including the interleukin-1ß (IL-1ß), colony-stimulating factor 3 (CSF3), transglutaminase 2 (TGM2), and proto-oncogene tyrosine-protein kinase Src (SRC) genes. Similarly, the knockdown of the m6A eraser ALKBH5 increases the DUSP1 transcript m6A level, resulting in accelerated transcript degradation, the activation of p38 and JNK, and the enhanced expression of IL-1ß, CSF3, TGM2, and SRC. These results demonstrate that m6A and the reader protein YTHDF2 orchestrate optimal innate immune responses during viral and bacterial infections by downregulating the expression of a negative regulator, DUSP1, of the p38 and JNK pathways that are central to innate immune responses against pathogenic infections. IMPORTANCE Innate immunity is central to controlling pathogenic infections and maintaining the homeostasis of the host. In this study, we have revealed a novel mechanism regulating innate immune responses during viral and bacterial infections. We have found that N6-methyladenosine (m6A) and the reader protein YTHDF2 regulate dual-specificity phosphatase 1, a negative regulator of the mitogen-activated protein kinases p38 and JNK, to maximize innate immune responses during viral and bacterial infections. These results provide novel insights into the mechanism regulating innate immunity, which could help in the development of novel approaches for controlling pathogenic infections.

Infectivity of pseudotyped SARS-CoV-2 variants of concern in different human cell types and inhibitory effects of recombinant spike protein and entry-related cellular factors.

Since the report of the first COVID-19 case in 2019, SARS-CoV-2 variants of concern (VOCs) have continued to emerge, manifesting diverse infectivity, evasion of host immunity and pathology. While ACE2 is the predominant receptor of SARS-CoV-2, TMPRSS2, Kim-1, NRP-1, CD147, furin, CD209L, and CD26 have also been implicated as viral entry-related cofactors. To understand the variations in infectivity and pathogenesis of VOCs, we conducted infection analysis in human cells from different organ systems using pseudoviruses of VOCs including Alpha, Beta, Gamma, and Delta. Recombinant spike S1, RBD, ACE2, Kim-1, and NRP-1 proteins were tested for their ability to block infection to dissect their roles in SARS-CoV-2 entry into cells. Compared with wild type SARS-CoV-2 (WT), numerous VOCs had significant increases of infectivity across a wide spectrum of cell types. Recombinant ACE2 protein more effectively inhibited the infection of VOCs including Delta and Omicron (BA.1 and BA.2) than that of WT. Interestingly, recombinant S1, RBD, Kim-1, and NRP-1 proteins inhibited the infection of all pseudoviruses in a manner dependent on the levels of ACE2 expression in different cell types. These results provide insights into the diverse infectivity of SARS-CoV-2 VOCs, which might be helpful for managing the emergence of new VOCs.

DRR1 promotes neuroblastoma cell differentiation by regulating CREB expression.

BACKGROUND: Neuroblastoma is the most common cancer in infants and the most common extracranial solid tumor in childhood. DRR1 was identified to be downregulated in poorly differentiated ganglion cells from neuroblastoma model mice. However, the roles of DRR1 in neuroblastoma remain largely unclear. METHODS: The neuroblastoma cells were induced to differentiate, and the expression of DRR1 was detected. The expression of the neuroblastoma cell differentiation markers was analyzed in DRR1 shRNA- or DRR1-expressing vector-treated neuroblastoma cells. The downstream genes of DRR1 were screened with ChIP-seq assay. Finally, TNB1 cells were infected with DRR1 shRNA and CREB expressing vector containing lentivirus, and the expression of the cell differentiation markers, cell cycle distribution and tumor growth were analyzed. RESULTS: The expression of DRR1 was increased in differentiated neuroblastoma cells, and downregulation of DRR1 expression inhibited the differentiation of neuroblastoma cells. Further experiments indicated that CREB is a candidate downstream gene of DRR1, and it mediates neuroblastoma cell differentiation. Moreover, overexpression of CREB rescued the effect of DRR1 shRNA on cell differentiation, cell cycle distribution and tumor growth in neuroblastoma. CONCLUSIONS: DRR1-CREB axis modulates the differentiation of neuroblastoma cells and is associated with the outcome of neuroblastoma patients. IMPACT: DRR1 is involved in regulation of the differentiation of neuroblastoma. Binding with actin is essential for DRR1 to regulate neuroblastoma cell differentiation. CREB is a candidate downstream gene of DRR1 in regulating of the differentiation of neuroblastoma.

Deploying Indocyanine Green Fluorescence-Guided Navigation System in Precise Laparoscopic Resection of Pediatric Hepatoblastoma.

Background: Hepatoblastoma (HB) is the most common form of liver cancer in children. To date, complete tumor resection is still the gold standard for treating HB. Indocyanine green (ICG) has been identified as a sensitive adjunct that is highly effective in the identification and surgical management of local and metastatic HB. It has thus becomes an increasingly popular choice among surgeons in HB resection surgeries that are fluorescence-guided. However, laparotomy remains the preferred choice in most cases since the applications and limitations of fluorescence-guided laparoscopic surgery in treating HB remain unclear. In this study, the characteristics and outcomes of laparoscopic HB resections that were guided by intraoperative ICG fluorescent imaging were investigated. Methods: Seven HB patients underwent ICG-guided laparoscopic HB resection surgery from August 2019 to December 2021. ICG was intravenously administered to the patients at a dosage of 0.5 mg/kg 48 h prior to the scheduled operation. During operation, tumor localization and resection boundary were guided by fluorescence visualization. The data on surgical and clinical features were collected retrospectively. Results: The resection area and tumor boundary could be clearly viewed in real-time under the ICG fluorescence imaging navigation system during operation, except for one patient who had received interventional chemoembolization before surgery. The image produced by laparoscopic fluorescence navigation was clear since it was not affected by ambient light. All tumors were completely resected as confirmed by negative margins for HB during postoperative pathological examination. No residual or recurrence were also found through computed tomography during follow-up visits from 9 to 37 months. Conclusions: ICG fluorescence-guided laparoscopic surgery is safe and effective in treating HB due to its ability to provide clear information on tumor localization and delineate tumor margins in real-time.

Identification of hub genes and construction of prognostic nomogram for patients with Wilms tumors.

Background: In children, Wilms' tumors are the most common urological cancer with unsatisfactory prognosis, but few molecular prognostic markers have been discovered for it. With the rapid development of high-throughput quantitative proteomic and transcriptomic approaches, the molecular mechanisms of various cancers have been comprehensively explored. This study aimed to uncover the molecular mechanisms underlying Wilms tumor and build predictive models by use of microarray and RNA-seq data. Methods: Gene expression datasets were downloaded from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and Gene Expression Omnibus (GEO) databases. Bioinformatics methods wereutilized to identified hub genes, and these hub genes were validated by experiment. Nomogram predicting OS was developed using genetic risk score model and clinicopathological variables. Results: CDC20, BUB1 and CCNB2 were highly expressed in tumor tissues and able to affect cell proliferation and the cell cycle of SK-NEP-1 cells. This may reveal molecular biology features and a new therapeutic target of Wilms tumour.7 genes were selected as prognostic genes after univariate, Lasso, and multivariate Cox regression analyses and had good accuracy, a prognostic nomogram combined gene model with clinical factors was completed with high accuracy. Conclusions: The current study discovered CDC20,BUB1 and CCNB2 as hub-genes associated with Wilms tumor, providing references to understand the pathogenesis and be considered a novel candidate to target therapy and construct novel nomogram, incorporating both clinical risk factors and gene model, could be appropriately applied in preoperative individualized prediction of malignancy in patients with Wilms tumor.

CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy.

BRAF V600E mutation drives uncontrolled cell growth in most melanomas. While BRAF V600E tumors are initially responsive to BRAF inhibitors, prolonged treatment results in inhibitor resistance and tumor regrowth. Clinical data have linked the NRAS Q61K, KRAS G13D and MEK1 Q56P mutations to the BRAF inhibitor resistance. However, development of novel therapeutics is hindered by the lack of relevant isogeneic cell models. We employed CRISPR/Cas9 genome engineering to introduce NRAS Q61K, KRAS G13D and MEK1 Q56P mutations into the A375 melanoma cell line with endogenously high expression of BRAF V600E. The resulting isogenic cell lines are resistant to BRAF inhibitors. The A375 MEK1 Q56P isogenic cells are additionally resistant to MEK inhibitors as single agent, but interestingly, these cells become sensitive to MEK/BRAF inhibitor combo. Our results suggest that resistance in the NRAS and MEK isogenic lines is driven by constitutive MEK/ERK signaling, while the resistance in the KRAS isogenic line is driven by EGFR overexpression. Interestingly, the KRAS G13D isogenic line displays elevated PD-L1 expression suggesting the KRAS G13D mutation could be a potential indication for immunotherapy. Overall, these three novel isogenic cell models with endogenous level RAS and MEK1 point mutations provide direct bio-functional evidence demonstrating that acquiring a drug-resistant gene drives tumor cell survival and may simultaneously introduce new indications for combo therapy or immunotherapy in the clinic.

[Comparative analysis of different fecal DNA extraction methods].

The community structure and diversity of the gut microbiota are associated with human diseases. However, the analysis of different community structure might be influenced by experimental approaches such as the quality of DNA extraction. Therefore, evaluating the efficiency of different DNA extraction methods for specific intestinal species is a guideline for obtaining a comprehensive human gut microbial profile, which may assist the in-depth investigation into the structure of the gut microbial community. The aim of this study was to perform a comparative analysis of five different DNA extraction methods. With the aid of qPCR, the efficiency of five DNA extraction kits was evaluated in terms of the purity of the extracted DNA, the DNA concentration, and the abundance of genomic DNA extracted from specific intestinal species. The results showed that the kit Q gave the best extraction results, especially for Gram-positive bacteria such as Lactobacillus and Bifidobacterium. The average DNA concentration of the N kit was lower than that of the Q kit, but there was no significant difference between the two in terms of the purity. Compared to the other three commercial kits (M, PSP, TG), the efficiency of the N kit in extracting the genomic DNA of the specified microorganisms were the least different from those of the Q kit. In contrast, the DNA extracted by the M kit was of higher quality but of lower concentration, and was not very efficient for Gram-positive bacteria. The DNA extracted by the TG and PSP kits was inferior to the other validated kits in terms of the concentration, quality and bacterial abundance. These results provide a basis for the selection of genomic DNA extraction methods in microecological research experiments.

Neutralizing antibodies to SARS-CoV-2 variants of concern including Delta and Omicron in subjects receiving mRNA-1273, BNT162b2, and Ad26.COV2.S vaccines.

SARS-CoV-2 vaccines have contributed to the control of COVID-19 in some parts of the world. However, the constant emergence of variants of concern (VOCs) challenges the effectiveness of SARS-CoV-2 vaccines over time. In particular, Omicron contains a high number of mutations in the spike (S) protein gene, on which most vaccines were developed. In this study, we quantitated neutralizing antibodies in vaccine recipients at various times postvaccination using S protein-based pseudoviruses derived from wild type (WT) SARS-CoV-2 and five VOCs including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). We found that two-dose mRNA-1273 and BNT162b2 vaccines elicited robust neutralizing antibodies against WT, Alpha, Beta, Gamma, and Delta, but wanned after 6 months with a faster decline observed for BNT162b2. Both mRNA-1273 and BNT162b2 elicited weak neutralizing antibodies against Omicron. One dose of Ad26.COV2.S vaccine induced weaker neutralizing antibodies against WT and most VOCs than mRNA-1273 and BNT162b2 did but moderate neutralizing antibodies against Delta and Omicron, which lasted for 6 months. These results support current recommendations of the Centers for Disease Control and Prevention for a booster 5 months after full immunization with an mRNA-based vaccine and the use of an mRNA-based vaccine 2 months after Ad26.COV2.S vaccination.

Microencapsulation of blueberry anthocyanins by spray drying with soy protein isolates/high methyl pectin combination: Physicochemical properties, release behavior in vitro and storage stability.

Eleven anthocyanins in the blueberry anthocyanins powders (BAP) were identified and quantified by HPLC-DAD-ESI-MS. BAP microcapsules (MBAP) were produced by spray drying using high methyl pectin (HMP) combined with whey protein isolates (WPI) or soy protein isolates (SPI) in different proportions as wall materials. Generally, SPI/HMP combination was more efficient in increasing the encapsulation efficiency and Tg, and in decreasing the particle size and hygroscopicity of the microcapsules than WPI or HMP or WPI/HMP combination. Microcapsules created with 4% SPI + 2% HMP combination (MBAPc), possessed superior anthocyanin release behavior and antioxidant stability to those produced with 4% SPI alone (MBAPs). Both MBAPc and MBAPs had continuous release of anthocyanins throughout the simulated gastrointestinal digestion, and exhibited two first-order kinetics, but MBAPc exhibited higher stability than MBAPs and BAP, because it showed the longest half-life and the lowest anthocyanin degradation rate at 25 °C and 35 °C during 6-months' storage.

pH/Thermal-Sensitive Nanoplatform Capable of On-Demand Specific Release to Potentiate Drug Delivery and Combinational Hyperthermia/Chemo/Chemodynamic Therapy.

Therapeutic platforms with spatiotemporal control were recently of considerable interest. However, the site-specific regulation of chemotherapeutics release remains an enormous challenge. Herein, a versatile nanoplatform capable of tumor-specific delivery and controlled drug release, coined as PDDFe, was constructed for elevating cancer theranostics. Iron-oxide nanoparticles (IONPs) and doxorubicin (Dox) were encapsulated in pH/thermal-sensitive micelles composed of poly(ethylene)glycol-poly(ß-amino esters) and dipalmitoyl phosphatidylcholine to obtain tumor-targeted dual-responsive nanoplatforms. With remarkable magnetic targeting effects, PDDFe specifically accumulated at tumor locations. After internalization by cancer cells, the acidic environment and localized heat generated by hyperthermia therapy would spur PDDFe to become loose and collapse to liberate its payload. In addition to boosting the release, the increased temperature also resulted in direct tumor damage. Meanwhile, the released Dox and IONPs, respectively, stimulated chemotherapy and chemodynamic therapy to jointly destroy cancer, thus leading to a pronounced therapeutic effect. In vivo magnetic resonance/fluorescence/photoacoustic imaging experiments validated that the dual-sensitive nanoplatforms were able to accumulate at the tumor sites. Treatment with PDDFe followed by alternating magnetic field and laser irradiation could prime hyperthermia/chemo/chemodynamic therapy to effectively retard tumor growth. This work presents a nanoplatform with a site-specific controlled release characteristic, showing great promises in potentiating drug delivery and advancing combinational cancer therapy.

Copper-based theranostic nanocatalysts for synergetic photothermal-chemodynamic therapy.

Chemodynamic therapy (CDT) has aroused extensive attention as a potent therapeutic modality. However, its practical application is severely restricted by the strong acidity requirement for Fenton reaction and upregulated antioxidant defense within metastatic breast cancer. Herein, a copper-based single-site nanocatalyst functionalized with carbonic anhydrase inhibitor (CAI) was constructed for magnetic resonance/photoacoustic imaging (MRI/PA)-guided synergetic photothermal therapy (PTT) and CDT. Once reaching tumor sites, the nanocatalyst can be recognized by tumor cell membranes-overexpressed carbonic anhydrase IX (CA IX). Subsequently, the single-site CuII can be reduced to CuI by the tumor-overexpressed glutathione (GSH), which simultaneously impaired the tumor antioxidant defense system and triggered CAI release for inducing intracellular H+ accumulation. Further, the decreased intracellular pH can accelerate the nanocatalyst biodegradation to release more CuII and CAI to participate in next-cycle GSH-depletion and cytoplasm acidification, respectively, thereby continuously supplying CuI and H+ for self-cyclically amplified CDT. Upon laser irradiation, the nanocatalyst can generate local heat, which not only permits PTT but also enhances the nanocatalyst-mediated CDT. Moreover, the suppression of CA IX can hinder the tumor extracellular matrix degradation to prevent tumor metastasis. Overall, this work highlighted the great application prospect in enhancing CDT via tumor acidic/redox microenvironment remodeling, and provides an insightful paradigm for inhibiting breast cancer metastasis. STATEMENT OF SIGNIFICANCE: The practical application of chemodynamic therapy (CDT) is severely restricted by the strong acidity requirement for Fenton reaction and upregulated antioxidant defense within cancer. Herein, we developed a carbonic anhydrase inhibitor (CAI)-functionalized Cu-based nanocatalyst. Once reaching tumor sites, the CuII can be reduced to CuI by the tumor-overexpressed glutathione (GSH), which simultaneously impaired the tumor antioxidant system and triggered CAI release for inducing intracellular H+ accumulation. Further, the decreased intracellular pH can accelerate the nanocatalyst biodegradation to release more CuII and CAI to participate in next-cycle GSH-depletion and cytoplasm acidification, respectively, thus continuously supplying CuI and H+ for self-cyclically amplified CDT. Upon laser irradiation, the nanocatalyst not only permits PTT but also enhances the CDT.