Hybrid Sequencing-Based Genomic Analysis of Klebsiella pneumoniae from Urinary Tract Infections Among Inpatients at a Tertiary Hospital in Beijing.

Background: Urinary tract infection (UTI) associated with Klebsiella pneumoniae poses a serious threat for inpatients. This study aimed to describe the genomic characteristics of K. pneumoniae causing UTI in a tertiary-care hospital in Beijing, China. Methods: A total of 20 K. pneumoniae strains collected from 2020 to 2021 were performed whole-genome sequencing. The Antibiotic susceptibility of 19 common antimicrobial agents was tested against all strains. The multi-locus sequence types (MLSTs) and serotypes were determined from the WGS data. De novo assemblies were used to identify resistance and virulence genes. The presence and characteristics of the plasmids were detected using hybrid assembly of long and short-read data. Results: These K. pneumoniae strains were clustered into nine sequence types (STs) and twelve K-serotypes. All the carbapenem-resistant K. pneumoniae (CRKP) strains acquired carbapenemase blaKPC-2 (n=7). Two CRKP strains exhibited increased resistance to Polymyxin B with MIC ≥ 4 mg/L due to insertion of an IS5-like sequence in the mgrB gene, and they were also involved in a transmission event in Intensive Care Unit. Long-read assemblies identified many plasmids co-carrying multiple replicons. Acquisition of a new IncM2_1 type blaCTX-M-3 positive plasmid was observed after transfer from ICU to neurovascular surgery by comparing the two strains collected from the same patient. Conclusion: K. pneumoniae is a significant pathogen responsible for urinary tract infections. The ST11-KL47 strain, prevalent at our hospital, exhibits a combination of high drug resistance and hypervirulence. It is imperative to enhance ongoing genomic surveillance of urinary tract infection-causing pathogens.

In situ detection of dopamine in single living cells by molecularly imprinted polymer-functionalized nanoelectrodes.

In situ detection of dopamine (DA) at single-cell level is critical for exploring neurotransmitter-related biological processes and diseases. However, the low content of DA and a variety of distractors with similar oxidation potentials as DA in cells brought great challenges. Here, a sensitive and specific electrochemical nanosensor was proposed for in situ detection of DA in single living cells based on nanodiamond (ND) and molecularly imprinted polymer (MIP)-functionalized carbon fiber nanoelectrode (ND/MIP/CFNE). Due to its excellent electrocatalytic property, ND was modified to the surface of CFNE based on amide bonding. Compared with bare CFNE, ND-modified CFNE can enhance oxidation currents of DA by about 4-fold, improving signal-to-noise ratio and detection sensitivity. MIP was further electropolymerized on the surface of nanoelectrodes to achieve specific capture and recognition of DA, which could avoid the interference of complex matrix and analogs in cells. Taking advantage of the precise positioning capability of a single-cell analyzer and micromanipulator, ND/MIP/CFNE could be precisely inserted into different locations of single cells and monitor oxidation signal of DA. The concentration of DA in the cytoplasm of single pheochromocytoma (PC12) cell was measured to be about 0.4 µM, providing a sensitive and powerful method for single-cell detection. Furthermore, the nanoelectrodes can monitor the fluctuation of intracellular DA under drug stimulation, providing new ideas and methods for new drug development and efficacy evaluation.

GATA3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in breast cancer.

BACKGROUND: Inadequate DNA damage repair promotes aberrant differentiation of mammary epithelial cells. Mammary luminal cell fate is mainly determined by a few transcription factors including GATA3. We previously reported that GATA3 functions downstream of BRCA1 to suppress aberrant differentiation in breast cancer. How GATA3 impacts DNA damage repair preventing aberrant cell differentiation in breast cancer remains elusive. We previously demonstrated that loss of p18, a cell cycle inhibitor, in mice induces luminal-type mammary tumors, whereas depletion of either Brca1 or Gata3 in p18 null mice leads to basal-like breast cancers (BLBCs) with activation of epithelial-mesenchymal transition (EMT). We took advantage of these mutant mice to examine the role of Gata3 as well as the interaction of Gata3 and Brca1 in DNA damage repair in mammary tumorigenesis. RESULTS: Depletion of Gata3, like that of Brca1, promoted DNA damage accumulation in breast cancer cells in vitro and in basal-like breast cancers in vivo. Reconstitution of Gata3 improved DNA damage repair in Brca1-deficient mammary tumorigenesis. Overexpression of GATA3 promoted homologous recombination (HR)-mediated DNA damage repair and restored HR efficiency of BRCA1-deficient cells. Depletion of Gata3 sensitized tumor cells to PARP inhibitor (PARPi), and reconstitution of Gata3 enhanced resistance of Brca1-deficient tumor cells to PARP inhibitor. CONCLUSIONS: These results demonstrate that Gata3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in mammary tumorigenesis and progression. Our findings suggest that PARP inhibitors are effective for the treatment of GATA3-deficient BLBCs.

Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro.

BACKGROUND: Prohibitin 1 (PHB1) has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed, and it participates in a variety of essential cellular functions, including apoptosis, cell cycle regulation, proliferation, and survival. Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma (HCC). However, the role of PHB1 in HCC is controversial. AIM: To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro. METHODS: HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria; then, PHB1 levels in the sera and liver tissues of these participates were determined using ELISA, RT-PCR, and immunohistochemistry. Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA (shRNA-PHB1) for 24-72 h. Cell proliferation was analysed with an MTT assay. Cell cycle progression and apoptosis were analysed using flow cytometry (FACS). The mRNA and protein expression levels of the cell cycle-related molecules p21, Cyclin A2, Cyclin E1, and CDK2 and the cell apoptosis-related molecules cytochrome C (Cyt C), p53, Bcl-2, Bax, caspase 3, and caspase 9 were measured by real-time PCR and Western blot, respectively. RESULTS: Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals, and decreased PHB1 was positively correlated with low differentiation, TNM stage III-IV, and alpha-fetoprotein ≥ 400 µg/L. Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner. FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis. The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells. The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41% ± 1.06%, which was significantly greater than that of apoptotic control cells (3.65% ± 0.85%, P < 0.01) and empty vector-transfected cells (4.21% ± 0.52%, P < 0.01). Similar results were obtained with SMMC-7721 cells. Furthermore, the mRNA and protein expression levels of p53, p21, Bax, caspase 3, and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2, Cyclin E1, CDK2, and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells. However, when PHB1 was upregulated in human HCC cells, Cyt C expression levels were increased in the cytosol and decreased in the mitochondria, which indicated that Cyt C had been released into the cytosol. Conversely, these effects were reversed when PHB1 was knocked down. CONCLUSION: PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

Suggesting Dictyostelium as a Model for Disease-Related Protein Studies through Myosin II Polymerization Pathway.

Dictyostelium myosin II displays remarkable dynamism within the cell, continually undergoing polymerization and depolymerization processes. Under low-ion conditions, it assumes a folded structure like muscle myosins and forms thick filaments through polymerization. In our study, we presented intermediate structures observed during the early stages of polymerization of purified myosin via negative staining electron microscopy, immediately crosslinked with glutaraldehyde at the onset of polymerization. We identified folded monomers, dimers, and tetramers in the process. Our findings suggest that Dictyostelium myosin II follows a polymerization pathway in vitro akin to muscle myosin, with folded monomers forming folded parallel and antiparallel dimers that subsequently associate to create folded tetramers. These folded tetramers eventually unfold and associate with other tetramers to produce long filaments. Furthermore, our research revealed that ATP influences filament size, reducing it regardless of the status of RLC phosphorylation while significantly increasing the critical polymerization concentrations from 0.2 to 9 nM. In addition, we demonstrate the morphology of fully matured Dictyostelium myosin II filaments.

Improved physicochemical properties and in vitro digestion of walnut oil microcapsules with soy protein isolate and highly oxidized konjac glucomannan as wall materials.

This study investigated the effect of the oxidation degrees of oxidized konjac glucomannan (OKGM) on the encapsulation efficiency (EE), physicochemical and in vitro digestive properties of soy protein isolate (SPI)-based microcapsules walnut oil using experimental and computational approaches. Microcapsules had the highest EE when the ratio of OKGM and SPI to oil was 2.5:1. With increasing the oxidation degree of OKGM, the EE of microcapsules was increased and the hygroscopicity was decreased. Molecular dynamics simulation results showed that SPI/oil/highly OKGM had relatively low binding energy (-4.03 × 106 kJ/mol) and strong electrostatic interactions, which may contribute to a higher EE and lower hygroscopicity of microcapsules, respectively. The oxidative stability of the oil was markedly improved by SPI and OKGM, and microcapsules prepared with SPI and highly OKGM had the highest in vitro digestion. This study provided theoretical support for broadening the application of microcapsules prepared with SPI and OKGM.

A pH-Sensitive Nanoparticle as Reactive Oxygen Species Amplifier to Regulate Tumor Microenvironment and Potentiate Tumor Radiotherapy.

Background: Radiotherapy is a widely used clinical tool for tumor treatment but can cause systemic toxicity if excessive radiation is administered. Although numerous nanoparticles have been developed as radiosensitizers to reduce the required dose of X-ray irradiation, they often have limitations, such as passive reliance on radiation-induced apoptosis in tumors, and little consider the unique tumor microenvironment that contributes radiotherapy resistance. Methods: In this study, we developed and characterized a novel self-assembled nanoparticle containing dysprosium ion and manganese ion (Dy/Mn-P). We systematically investigated the potential of Dy/Mn-P nanoparticles (NPs) as a reactive oxygen species (ROS) amplifier and radiosensitizer to enhance radiation therapy and modulate the tumor microenvironment at the cellular level. Additionally, we evaluated the effect of Dy/Mn-P on the stimulator of interferon genes (STING), an innate immune signaling pathway. Results: Physicochemical analysis demonstrated the prepared Dy/Mn-P NPs exhibited excellent dispersibility and stability, and degraded rapidly at lower pH values. Furthermore, Dy/Mn-P was internalized by cells and exhibited selective toxicity towards tumor cells compared to normal cells. Our findings also revealed that Dy/Mn-P NPs improved the tumor microenvironment and significantly increased ROS generation under ionizing radiation, resulting in a ~70% increase in ROS levels compared to radiation therapy alone. This enhanced ROS generation inhibited ~92% of cell clone formation and greatly contributed to cytoplasmic DNA exposure. Subsequently, the activation of the STING pathway was observed, leading to the secretion of pro-inflammatory immune factors and maturation of dendritic cells (DCs). Conclusion: Our study demonstrates that Dy/Mn-P NPs can potentiate tumor radiotherapy by improving the tumor microenvironment and increasing endogenous ROS levels within the tumor. Furthermore, Dy/Mn-P can amplify the activation of the STING pathway during radiotherapy, thereby triggering an anti-tumor immune response. This novel approach has the potential to expand the application of radiotherapy in tumor treatment.

Chitosan/oxidized Konjac Glucomannan films incorporated with Zanthoxylum Bungeanum essential oil: A novel approach for extending the shelf life of meat.

In this study, a novel edible composite film was prepared by chitosan, konjac glucomannan oxidized with ozone for 60 min (OKGM), and Zanthoxylum Bungeanum essential oil (ZEO). The chitosan/OKGM film was fortified with ZEO to assess the physical properties, structure, antioxidant and antibacterial abilities, and pork preservation systematically. Compared to the control group, the addition of 1 % ZEO increased tensile strength by 18.92 % and decreased water solubility, water vapor permeability, and moisture content by 10.05 %, 6.60 %, and 1.03 %, respectively. However, the treatment with ZEO (1.5 % and 2 %) decreased mechanical properties and increased the water vapor permeability. The ultraviolet barrier, antioxidant, and antibacterial abilities of composite films were enhanced by increasing the ZEO addition. Moreover, the COZ-1 film was used to protect the freshness of pork with slow-release behavior of ZEO. The results showed that addition of ZEO significantly decreased the pH value, total viable count, redness, total volatile basic nitrogen, and thiobarbituric acid and increased the hardness of pork after preservation for 10 days. Therefore, the chitosan/OKGM loaded with ZEO film can potentially be used as food packaging, providing new ideas for the research on active packaging materials.

SLMO2 is a potential prognostic and immunological biomarker in human pan-cancer.

SLMO2 is a lipid transporter that transports phosphatidylserine to the interior of mitochondria, also known as PRELID3B, which plays an important role in lipid metabolism. It has also been reported to be involved in the growth process of breast and lung tumors. However, its functions and underlying mechanisms in cancer progress remain elusive, and the potential as pan-cancer biomarker and therapeutic target remains unexplored. Using the TCGA project and GEO database, we performed pan-cancer analysis of SLMO2, which including the expression pattern, prognostic value, mutation landscape, methylation modification, protein-protein interaction network and the relationship between SLMO2 expression and immune infiltration. KEGG enrichment analysis was also performed to predict function and relevant cellular pathways of SLMO2. In addition, proliferation and migration assays were performed to detect the proliferation and metastasis capacity of breast cancer and lung cancer cells. In our study, we found that SLMO2 was overexpressed in pan-cancer and the elevated expression of SLMO2 was correlated with poorer prognosis. SLMO2 mutations were distributed in a variety of tumors and correlated with prognosis. Promoter methylation analysis showed that SLMO2 methylation levels were lower in most tumors compared with normal tissues, while a few tumors showed increased methylation levels of SLMO2. SLMO2 expression was also positively correlated with immune infiltration of MDSCs. Further pathway enrichment analysis indicated that SLMO2 was involved in regulating of cytoplasmic transport and other oncogenic processes. In vitro experiments have shown that SLMO2 promotes the proliferation and migration of breast cancer and lung cancer cells. In conclusion, our findings suggested that SLMO2 was a potential prognostic and immunological marker in pan-cancer. This study suggested a potential strategy for targeting SLMO2 to treat tumors, including manipulating tumor growth or the tumor microenvironment, especially the infiltration of MDSC.

Nomogram incorporating Epstein-Barr virus DNA and a novel immune-nutritional marker for survival prediction in nasopharyngeal carcinoma.

BACKGROUND: Since Immune response, nutritional status and Epstein-Barr Virus (EBV) DNA status have been confirmed to be relevant to the prognosis of patients with nasopharyngeal carcinoma (NPC), we believe that the combination of these factors is of great value for improving the predictive ability. LA (lymphocytes × albumin), a novel indicator, had not been studied yet in NPC. We combined it with EBV DNA and used nomograms to increase the accuracy of prognosis. METHODS: A total of 688 NPC patients were retrospectively reviewed and further divided into training and validation cohort randomly. Kaplan-Meier analyses were used to to distinguish the different survival outcomes. Multivariate Cox analyses were used to identify the independent prognostic factors for progression-free survival (PFS) and overall survival (OS). Calibration curves, concordance indexes (C-indexes) and decision curve analyses (DCA) were used to evaluate the nomograms' predictive value. RESULTS: Patients with low LA and positive EBV DNA correlated with poorer 5-year PFS and OS (all P < 0.005). In multivariate Cox analyses, LA and EBV DNA were both confirmed to be independent prognostic factors for PFS and OS (all P < 0.05). Prognostic nomograms incorporating LA and EBV DNA achieved ideal C-indexes of 0.69 (95% CI: 0.65-0.73) and 0.77 (95% CI: 0.71-0.82) in the prediction of PFS and OS. Otherwise, the calibration curves and DCA curves also revealed that our nomograms had pleasant predictive power. CONCLUSIONS: LA is a novel and powerful biomarker for predicting clinical outcomes in NPC. Our nomograms based on LA and EBV DNA can predict individual prognosis more accurately and effectively.

Methylation of lncSHGL promotes adipocyte differentiation by regulating miR-149/Mospd3 axis.

Obesity poses significant health risks and can negatively impact an individual's quality of life. The human obesity phenotype results from the differentiation of pre-adipocytes into adipocytes, which leads to hypertrophy and hyperplasia in adipose tissue. The molecular mechanisms by which long non-coding RNAs (lncRNAs) modulate adipocyte differentiation, a process implicated in obesity development, remain poorly characterized. A lncRNA which suppressed the hepatic gluconeogenesis and lipogenesis (lncSHGL) was newly identified. Our research aims to elucidate the functional role and mechanistic underpinnings of suppressor of lncSHGL in adipocyte differentiation. We observed that lncSHGL expression progressively diminished during 3T3-L1 differentiation and was downregulated in the liver and perirenal adipose tissue of ob/ob mice. lncSHGL acts as a molecular sponge for miR-149, with Mospd3 identified as a target of miR-149.Overexpression of lncSHGL and inhibition of miR-149 led to suppressed 3T3-L1 proliferation, decreased lipid droplet accumulation, and attenuated promoter activity of PPARγ2 and C/EBPα. These changes consequently resulted in reduced expression of Cyclin D1, LPL, PPARγ2, AP2, and C/EBPα, as well as inhibited the PI3K/AKT/mTOR signaling pathway. In contrast, lncSHGL suppression yielded opposing outcomes. Moreover, the effects of lncSHGL overexpression and miR-149 inhibition on reduced expression of Cyclin D1, LPL, PPARγ2, AP2, and C/EBPα were reversible upon miR-149 overexpression and Mospd3 suppression. These findings were further validated in vivo. We also discovered a significant increase in methylation levels during 3T3-L1 differentiation, with lncSHGL highly expressed in the presence of a methylation inhibitor. In conclusion. lncSHGL methylation facilitates adipocyte differentiation by modulating the miR-149/Mospd3 axis. Targeting lncSHGL expression may represent a promising therapeutic strategy for obesity-associated adipogenesis, particularly in the context of fatty liver disease.

Bone marrow mesenchymal stem cell-derived exosomal microRNA regulates microglial polarization.

Objective: This study aimed to explore the effects of bone marrow mesenchymal stem cell (BMSC)-derived exosomal miR-146a-5p on microglial polarization and the potential underlying mechanisms in oxygen-glucose deprivation (OGD)-exposed microglial cells. Methods: Exosomes were isolated from BMSCs, and their characteristics were examined. The effects of BMSC-derived exosomes on microglial polarization were investigated in OGD-exposed BV-2 cells. Differentially expressed miRNAs were identified and their biological function was explored using enrichment analyses. The regulatory role of miR-146a-5p in microglial polarization was studied via flow cytometry. Finally, the downstream target gene Traf6 was validated, and the role of the miR-146a-5p/Traf6 axis in modulating microglial polarization was investigated in OGD-exposed BV-2 cells. Results: BMSC-derived exosomes were successfully isolated and characterized. A total of 10 upregulated and 33 downregulated miRNAs were identified. Exosomal treatment resulted in significant changes in microglial polarization markers. miR-146a-5p was found to be significantly downregulated in OGD-exposed microglial cells treated with exosomes. Manipulation of miR-146a-5p expression modulated microglial polarization. Moreover, the miR-146a-5p/Traf6 axis regulated microglial polarization. Conclusion: Our findings demonstrate that BMSC-derived exosomal via miR-146a-5p modulates microglial polarization by targeting Traf6, providing a potential thermal target for the treatment of neurological diseases involving microglial activation.

Impact of sex on treatment-related adverse effects and prognosis in nasopharyngeal carcinoma.

BACKGROUND: In nasopharyngeal cancer (NPC), women have a lower incidence and mortality rate than men. Whether sex influences the prognosis of NPC patients remains debatable. We retrospectively examined the influence of sex on treatment-related side effects and prognosis in NPC. METHODS: Clinical data of 1,462 patients with NPC treated at the Southern Hospital of Southern Medical University from January 2004 to December 2015 were retrospectively examined. Statistical analysis was performed to assess differences in overall survival (OS), distant metastasis-free survival (DMFS), local recurrence-free survival(LRFS), and progression-free survival(PFS), as well as treatment-related adverse effects, including myelosuppression, gastrointestinal responses, and radiation pharyngitis and dermatitis, between men and women. RESULTS: Women had better 5-year OS (81.5% vs. 87.1%, P = 0.032) and DMFS (76.2% vs. 83.9%, P = 0.004) than men. Analysis by age showed that the prognoses of premenopausal and menopausal women were better than those of men, whereas prognoses of postmenopausal women and men were not significantly different. Additionally, women had a better prognosis when stratified by treatment regimen. Furthermore, chemotherapy-related adverse effects were more severe in women than in men; however, the incidences of radiation laryngitis and dermatitis were not significantly different between the sexes. Logistic regression analysis revealed that the female sex was an independent risk factor for severe myelosuppression and gastrointestinal reactions. CONCLUSIONS: Chemotherapy-related side effects are more severe but the overall prognosis is better in women with NPC than in men with NPC. Patients may benefit from a personalized treatment approach for NPC. TRIAL REGISTRATION: This study was approved by the Medical Ethics Committee of Nanfang Hospital of the Southern Medical University (NFEC-201,710-K3).

UBE3B promotes breast cancer progression by antagonizing HIF-2α degradation.

Mutations in E3 ubiquitin ligase UBE3B have been linked to Kaufman Oculocerebrofacial Syndrome (KOS). Accumulating evidence indicates that UBE3B may play an important role in cancer. However, the precise role of UBE3B in cancer and the underlying mechanism remain largely uncharted. Here, we reported that UBE3B is an E3 ligase for hypoxia-inducible factor 2α (HIF-2α). Mechanically, UBE3B physically interacts with HIF-2α and promotes its lysine 63 (K63)-linked polyubiquitination, thereby inhibiting the Von Hippel-Lindau (VHL) E3 ligase complex-mediated HIF-2α degradation. UBE3B depletion inhibits breast cancer cell proliferation, colony formation, migration, and invasion in vitro and suppresses breast tumor growth and lung metastasis in vivo. We further identified K394, K497, and K503 of HIF-2α as key ubiquitination sites for UBE3B. K394/497/503R mutation of HIF-2α dramatically abolishes UBE3B-mediated breast cancer growth and lung metastasis. Intriguingly, the protein levels of UBE3B are upregulated and positively correlated with HIF-2α protein levels in breast cancer tissues. These findings uncover a critical mechanism underlying the role of UBE3B in HIF-2α regulation and breast cancer progression.

Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model.

Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies. Following single-dose CD117-ADC, a >99% depletion of bone marrow CD34 + CD90 + CD45RA- cells without lymphocyte reduction is observed, which results are not inferior to multi-day myeloablative busulfan conditioning. CD117-ADC, similarly to busulfan, allows efficient engraftment, gene marking, and vector-derived fetal hemoglobin induction. Importantly, ADC treatment is associated with minimal toxicity, and CD117-ADC-conditioned animals maintain fertility. In contrast, busulfan treatment commonly causes severe toxicities and infertility in humans. Thus, the myeloablative capacity of single-dose CD117-ADC is sufficient for efficient engraftment of gene-modified HSCs while preserving fertility and reducing adverse effects related to toxicity in non-human primates. This targeted conditioning approach thus provides the proof-of-principle to improve risk-benefit ratio in a variety of HSC-based gene therapy products in humans.

BAT6026, a novel anti-OX40 antibody with enhanced antibody dependent cellular cytotoxicity effect for cancer immunotherapy.

OX40 (CD134), a member of the TNF receptor superfamily, is a widely studied costimulatory immune checkpoint. Several OX40 agonistic antibodies are in the clinical stage for cancer treatment, among which PF-04518600 is the leader and currently in phase II trial. It has been recognized that one potential mode of action for anti-OX40 antibodies is the deletion of intratumoral Tregs. Thus, a novel human anti-OX40 antibody, BAT6026, was generated with enhanced antibody dependent cellular cytotoxicity (ADCC) via fucose deletion to strengthen its Treg depletion activity. This characteristic of BAT6026 differentiates it from other previously reported anti-OX40 antibodies in the field of tumor therapy. The affinity of BT6026 to OX40 was 0.28nM, approximately 8 times stronger than that of PF-04518600. BAT6026 effectively competed for the binding of ligand OX40L to OX40, whereas PF-04518600 only partially competed. Moreover, compared to PF-04518600, BAT6026 activated T cells more effectively when clustered by FcγRs engagement and stimulated SEB-pretreated PBMCs to secrete IL-2 cytokines in vitro. In addition, BAT6026 demonstrated stronger anti-tumor activity than PF-04518600 in an OX40-humanized mouse MC38 tumor model. BAT6026 also showed a significantly synergistic effect on tumor inhibition when combined treatment with PD-1 antibody. Analysis of tumor-infiltrating T cells revealed that BAT6026 treatment significantly reduced Treg cells and increased CD8+ T cells in tumor. Preclinical safety assessment in non-human primates demonstrated a good safety profile for BAT6026. Together these data warrant further development of BAT6026 into clinical trials for patients with cancer.

Insight into increased risk of portal vein thrombosis in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the leading chronic liver diseases with increased morbidity and mortality rates for extrahepatic diseases (including cardiovascular disease, portal vein thrombosis, etc.). There is an increased risk of thrombosis in both the portal and systemic circulation in patients with NAFLD, independent of traditional liver cirrhosis. However, increased portal pressure, the most critical factor, is frequently observed in NAFLD patients, predisposing them to portal vein thrombosis (PVT). It has been reported that there is an 8.5% incidence of PVT among patients with non-cirrhotic NAFLD in a prospective cohort study. Based on the prothrombotic status of NAFLD itself, patients combined with cirrhosis may accelerate the development of PVT and lead to a poor prognosis. Moreover, PVT has been shown to complicate the procedure and adversely affect the outcome during liver transplantation surgery. NAFLD is in a prothrombotic state, and its underlying mechanisms have not been fully understood so far. Particularly noteworthy is that gastroenterologists currently overlook the higher risk of PVT in NAFLD. We investigate the pathogenesis of NAFLD complicated with PVT from the perspective of primary, secondary, and tertiary hemostasis, and also summarize relevant studies in humans. Some treatment options that may affect NAFLD and its PVT are also explored to improve patient-oriented outcomes.

p18INK4C and BRCA1 inhibit follicular cell proliferation and dedifferentiation in thyroid cancer.

Only 3% of thyroid cancers are medullary thyroid carcinomas (MTCs), the rest are follicular epithelial cell derived non-MTCs (NMTCs). A dysfunctional INK4-CDK4-RB pathway is detected in most of NMTCs. DNA repair defects and genome instability are associated with NMTC dedifferentiation and aggressiveness. Whether inactivation of the INK4-CDK4-RB pathway induces NMTCs and how differentiation of NMTC cells is controlled remain elusive. In this study, we generated p18Ink4c and Brca1 singly and doubly deficient mice as well as p16Ink4a and Brca1 singly and doubly deficient mice. By using these mice and human thyroid carcinoma cell lines, we discovered that loss of p18Ink4c, not p16Ink4a, in mice stimulated follicular cell proliferation and induced NMTCs. Depletion of Brca1 alone or both p16Ink4a and Brca1 did not induce thyroid tumor. Depletion of Brca1 in p18Ink4c null mice results in poorly differentiated and aggressive NMTCs with epithelial-mesenchymal transition (EMT) features and enhanced DNA damage. Knockdown of BRCA1 in thyroid carcinoma cells activated EMT and promoted tumorigenesis whereas overexpression of BRCA1 inhibited EMT. BRCA1 and EMT marker expression were inversely related in human thyroid cancers. Our finding, for the first time, demonstrates that inactivation of INK4-CDK4-RB pathway induces NMTCs and that Brca1 deficiency promotes dedifferentiation of NMTC cells. These results suggest that BRCA1 and p18INK4C collaboratively suppress thyroid tumorigenesis and progression and CDK4 inhibitors will be effective for treatment of INK4-inactivated or cyclin D-overexpressed thyroid carcinomas.