iPSC-derived NK cells with site-specific integration of CAR19 and IL24 at the multi-copy rDNA locus enhanced antitumor activity and proliferation.

The generation of chimeric antigen receptor-modified natural killer (CAR-NK) cells using induced pluripotent stem cells (iPSCs) has emerged as one of the paradigms for manufacturing off-the-shelf universal immunotherapy. However, there are still some challenges in enhancing the potency, safety, and multiple actions of CAR-NK cells. Here, iPSCs were site-specifically integrated at the ribosomal DNA (rDNA) locus with interleukin 24 (IL24) and CD19-specific chimeric antigen receptor (CAR19), and successfully differentiated into iPSC-derived NK (iNK) cells, followed by expansion using magnetic beads in vitro. Compared with the CAR19-iNK cells, IL24 armored CAR19-iNK (CAR19-IL24-iNK) cells showed higher cytotoxic capacity and amplification ability in vitro and inhibited tumor progression more effectively with better survival in a B-cell acute lymphoblastic leukaemia (B-ALL) (Nalm-6 (Luc1))-bearing mouse model. Interestingly, RNA-sequencing analysis showed that IL24 may enhance iNK cell function through nuclear factor kappa B (NFκB) pathway-related genes while exerting a direct effect on tumor cells. This study proved the feasibility and potential of combining IL24 with CAR-iNK cell therapy, suggesting a novel and promising off-the-shelf immunotherapy strategy.

Tyrosine phosphatase SHP2 aggravates tumor progression and glycolysis by dephosphorylating PKM2 in gastric cancer.

Gastric cancer (GC) is among the most lethal human malignancies, yet it remains hampered by challenges in fronter of molecular-guided targeted therapy to direct clinical treatment strategies. The protein tyrosine phosphatase Src homology 2 domain-containing phosphatase 2 (SHP2) is involved in the malignant progression of GC. However, the detailed mechanisms of the posttranslational modifications of SHP2 remain poorly understood. Herein, we demonstrated that an allosteric SHP2 inhibitor, SHP099, was able to block tumor proliferation and migration of GC by dephosphorylating the pyruvate kinase M2 type (PKM2) protein. Mechanistically, we found that PKM2 is a bona fide target of SHP2. The dephosphorylation and activation of PKM2 by SHP2 are necessary to exacerbate tumor progression and GC glycolysis. Moreover, we demonstrated a strong correlation between the phosphorylation level of PKM2 and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in GC cells. Notably, the low phosphorylation expression of AMPK was negatively correlated with activated SHP2. Besides, we proved that cisplatin could activate SHP2 and SHP099 increased sensitivity to cisplatin in GC. Taken together, our results provide evidence that the SHP2/PKM2/AMPK axis exerts a key role in GC progression and glycolysis and could be a viable therapeutic approach for the therapy of GC.

Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A.

Hemophilia A (HA) is a common X-linked recessive hereditary bleeding disorder. Coagulation factor VIII (FVIII) is insufficient in patients with HA due to the mutations in the F8 gene. The restoration of plasma levels of FVIII via both recombinant B-domain-deleted FVIII (BDD-FVIII) and B-domain-deleted F8 (BDDF8) transgenes was proven to be helpful. FVIII-Padua is a 23.4 kb tandem repeat mutation in the F8 associated with a high F8 gene expression and thrombogenesis. Here we screened a core enhancer element in FVIII-Padua for improving the F8 expression. In detail, we identified a 400 bp efficient enhancer element, C400, in FVIII-Padua for the first time. The core enhancer C400 extensively improved the transcription of BDDF8 driven by human elongation factor-1 alpha in HepG2, HeLa, HEK-293T and induced pluripotent stem cells (iPSCs) with different genetic backgrounds, as well as iPSCs-derived endothelial progenitor cells (iEPCs) and iPSCs-derived mesenchymal stem cells (iMSCs). The expression of FVIII protein was increased by C400, especially in iEPCs. Our research provides a novel molecular target to enhance expression of FVIII protein, which has scientific value and application prospects in both viral and nonviral HA gene therapy strategies.

TIM22 and TIM29 inhibit HBV replication by up-regulating SRSF1 expression.

Hepatitis B virus (HBV) infection is a serious global health problem. After the viruses infect the human body, the host can respond to the virus infection by coordinating various cellular responses, in which mitochondria play an important role. Evidence has shown that mitochondrial proteins are involved in host antiviral responses. In this study, we found that the overexpression of TIM22 and TIM29, the members of the inner membrane translocase TIM22 complex, significantly reduced the level of intracellular HBV DNA and RNA and secreted HBV surface antigens and E antigen. The effects of TIM22 and TIM29 on HBV replication and transcription is attributed to the reduction of core promoter activity mediated by the increased expression of SRSF1 which acts as a suppressor of HBV replication. This study provides new evidence for the critical role of mitochondria in the resistance of HBV infection and new targets for the development of treatment against HBV infection.

The microprotein encoded by exosomal lncAKR1C2 promotes gastric cancer lymph node metastasis by regulating fatty acid metabolism.

Lymph node metastasis (LNM) is the prominent route of gastric cancer dissemination, and usually leads to tumor progression and a dismal prognosis of gastric cancer. Although exosomal lncRNAs have been reported to be involved in tumor development, whether secreted lncRNAs can encode peptides in recipient cells remains unknown. Here, we identified an exosomal lncRNA (lncAKR1C2) that was clinically correlated with lymph node metastasis in gastric cancer in a VEGFC-independent manner. Exo-lncAKR1C2 secreted from gastric cancer cells was demonstrated to enhance tube formation and migration of lymphatic endothelial cells, and facilitate lymphangiogenesis and lymphatic metastasis in vivo. By comparing the metabolic characteristics of LN metastases and primary focuses, we found that LN metastases of gastric cancer displayed higher lipid metabolic activity. Moreover, exo-lncAKR1C2 encodes a microprotein (pep-AKR1C2) in lymphatic endothelial cells and promotes CPT1A expression by regulating YAP phosphorylation, leading to enhanced fatty acid oxidation (FAO) and ATP production. These findings highlight a novel mechanism of LNM and suggest that the microprotein encoded by exosomal lncAKR1C2 serves as a therapeutic target for advanced gastric cancer.

Ethylene-induced improvement in photosynthetic performance of Zanthoxylum armatum under reoxygenation conditions.

In this study, we evaluated the photosynthetic performance of Zanthoxylum armatum seedlings to test the tolerance to reoxygenation after waterlogging. The experiment included a control group without waterlogging (NW) and three reoxygenation groups with reoxygenation after 1day (WR1), 2days (WR2) and 3days (WR3). Seedlings were pretreated with concentrations of 0, 200 and 400µmolL-1 of ethylene. The results showed that reoxygenation after waterlogging for 1-3days decreased photosynthetic pigments content, enzymes activity, stomatal conductance (G s ), net photosynthetic rate (P n ), transpiration rate (T r ) and water-use efficiency (WUE). However, pretreatment with ethylene increased photosynthetic pigments content, enzymes activity and gas exchange parameters under both NW and WR3 treatments. The chlorophyll fluorescence results showed that the maximum quantum yield of PSII (F v /F m ) and actual photochemical efficiency of PSII (Φ PSII ) remained no significant changes under the NW and WR1 treatments, while they were significantly reduced with an increase in waterlogging days followed by reoxygenation under WR2 and WR3 treatments. Exogenous ethylene inhibited F v /F m and the non-photochemical quenching coefficient (NPQ), while enhanced Φ PSII and electron transfer efficiency (ETR) under WR2 treatments. Moreover, the accumulation of exogenous ethylene reduced photosynthetic ability. These findings provide insights into the role of ethylene in enhancing the tolerance of Z. armatum to reoxygenation stress, which could help mitigate the impact of continued climate change.

CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B.

Hemophilia B (HB) is an X-linked recessive disease caused by F9 gene mutation and functional coagulation factor IX (FIX) deficiency. Patients suffer from chronic arthritis and death threats owing to excessive bleeding. Compared with traditional treatments, gene therapy for HB has obvious advantages, especially when the hyperactive FIX mutant (FIX-Padua) is used. However, the mechanism by which FIX-Padua works remains ambiguous due to a lack of research models. Here, in situ introduction of F9-Padua mutation was performed in human induced pluripotent stem cells (hiPSCs) via CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs). The hyperactivity of FIX-Padua was confirmed to be 364% of the normal level in edited hiPSCs-derived hepatocytes, providing a reliable model for exploring the mechanism of the hyperactivity of FIX-Padua. Moreover, the F9 cDNA containing F9-Padua was integrated before the F9 initiation codon by CRISPR/Cas9 in iPSCs from an HB patient (HB-hiPSCs). Integrated HB-hiPSCs after off-target screening were differentiated into hepatocytes. The FIX activity in the supernatant of integrated hepatocytes showed a 4.2-fold increase and reached 63.64% of the normal level, suggesting a universal treatment for HB patients with various mutations in F9 exons. Overall, our study provides new approaches for the exploration and development of cell-based gene therapy for HB.

Establishment of a human cell line with a surface display system for screening and optimizing Na+-taurocholate cotransporting polypeptide-binding peptides.

One of the most desirable targets for HBV medications is the sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for the hepatitis B virus (HBV). N-myristoylated preS1 2-48 (Myrcludex B or Hepcludex), an NTCP-binding peptide from the large surface protein of HBV, has been developed as the first-in-class entry inhibitor. However, its relatively large molecular weight contributes to increased immunogenicity and antibody production. As a result, it is preferable to look for an NTCP-binding peptide with a smaller size. To do this, we developed a human cell surface display strategy and screened peptides based on preS1-21. PreS1-21 (genotype D) was extended by 7 random amino acids and fused with mCherry and FasL transmembrane domain. The pooled constructs were transfected into HEK293 cells by using the transposon/transposase system to create a library displaying various peptides on the cell surface with red fluorescence. On the other hand, we expressed NTCP protein fused with EGFP on HEK293 and used the membrane lysate containing NTCP-GFP as the bait protein to select peptides with increased NTCP affinity. After 7 cycles of selection, the deep sequencing results revealed that some polypeptides were more than 1,000 times enriched. Further screening of the mostly enriched 10 peptides yields the peptide preS1-21-pep3. Replacing the preS1-21 sequence of preS1-21-pep3 with those from different genotypes demonstrated that the consensus sequence of genotype A-F had the best performance. The peptide (Myr-preS1-21-pep3) was synthesized and tested on the HepG2-NTCP cell model. The results showed that Myr-preS1-21-pep3 is approximately 10 times more potent than the initial peptide Myr-preS1-21 in preventing HBV infection. In conclusion, we developed a new strategy for screening peptides binding to membrane proteins and identified a new NTCP-binding peptide with a much smaller size than Hepcludex.

Full-Length Dystrophin Restoration via Targeted Exon Addition in DMD-Patient Specific iPSCs and Cardiomyocytes.

Duchenne muscular dystrophy (DMD) is the most common fatal muscle disease, with an estimated incidence of 1/3500-1/5000 male births, and it is associated with mutations in the X-linked DMD gene encoding dystrophin, the largest known human gene. There is currently no cure for DMD. The large size of the DMD gene hampers exogenous gene addition and delivery. The genetic correction of DMD patient-derived induced pluripotent stem cells (DMD-iPSCs) and differentiation into suitable cells for transplantation is a promising autologous therapeutic strategy for DMD. In this study, using CRISPR/Cas9, the full-length dystrophin coding sequence was reconstructed in an exon-50-deleted DMD-iPSCs by the targeted addition of exon 50 at the junction of exon 49 and intron 49 via homologous-directed recombination (HDR), with a high targeting efficiency of 5/15, and the genetically corrected iPSCs were differentiated into cardiomyocytes (iCMs). Importantly, the full-length dystrophin expression and membrane localization were restored in genetically corrected iPSCs and iCMs. Thus, this is the first study demonstrating that full-length dystrophin can be restored in iPSCs and iCMs via targeted exon addition, indicating potential clinical prospects for DMD gene therapy.

Clinical and Microbiological Characteristics of Klebsiella pneumoniae Bloodstream Infection in a Chinese Hospital: Hypervirulent and Multiclonal.

Purpose: Hypervirulent Klebsiella pneumoniae (hvKP) is emerging globally and can cause various infections. This study aimed to investigate the clinical and microbiological characteristics of bloodstream infection (BSI) caused by hvKP. Patients and Methods: The clinical data of hospitalized patients with K. pneumoniae BSI were retrospectively analyzed. The K. pneumoniae strains were collected and re-identified, and antimicrobial susceptibility testing was performed using the broth microdilution method. Capsular serotypes and virulence genes were detected using polymerase chain reaction, and hvKP was defined as aerobactin positive. Molecular typing was done by multilocus sequence typing. The hvKP and classic K. pneumoniae (cKP) subgroups were compared. Results: Of the 66 nonrepetitive BSI K. pneumoniae strains included, 29 (43.9%) were hvKP. In these BSI hvKP strains, salmochelin and yersiniabactin accounted for 86.2% and 72.4%, respectively. The prevalence of rmpA, iroBCD cluster, ybtS, clbA, and allS was 89.7%, 86.2%, 72.4%, 51.7%, and 41.4%, respectively, which were all significantly different between the hvKP and cKP subgroups. Serotypes K1 and K2 were strongly associated with hypervirulence (P < 0.05). Nineteen sequence types were scattered in the 29 hvKP strains, and the most common was ST23 (24.1%). None of the hvKP strains were carbapenem resistant. Compared with cKP, hvKP was more capable of developing a liver abscess. However, the 30-day mortality rate was lower (13.8% vs 21.6%) in the hvKP subgroup than in the cKP subgroup. Conclusion: This study demonstrated a high proportion of hvKP in BSI K. pneumoniae, most of which were RmpA and siderophore producing, and of multiclonal origin.

Identification of STAU1 as a regulator of HBV replication by TurboID-based proximity labeling.

The core promoter (CP) of hepatitis B virus (HBV) is critical for HBV replication by controlling the transcription of pregenomic RNA (pgRNA). Host factors regulating the activity of the CP can be identified by different methods. Biotin-based proximity labeling, a powerful method with the capability to capture weak or dynamic interactions, has not yet been used to map proteins interacting with the CP. Here, we established a strategy, based on the newly evolved promiscuous enzyme TurboID, for interrogating host factors regulating the activity of HBV CP. Using this strategy, we identified STAU1 as an important factor involved in the regulation of HBV CP. Mechanistically, STAU1 indirectly binds to CP mediated by TARDBP, and recruits the SAGA transcription coactivator complex to the CP to upregulate its activity. Moreover, STAU1 binds to HBx and enhances the level of HBx by stabilizing it in a ubiquitin-independent manner.

Comprehensive Characterization of Transforming Growth Factor Beta Receptor 1 in Stomach Adenocarcinoma Identifies a Prognostic Signature for Predicting Clinical Outcomes and Immune Infiltrates.

Background: Stomach adenocarcinoma (STAD) ranks as the third leading cause of cancer death worldwide. TGF­ß receptor 1 (TGFBR1), serving important roles in the TGF­ß family, the mechanisms whereby TGFBR1 governs tumor progression, immune cell infiltration in STAD remains unintelligible. Methods: We used the TCGA, GEPIA, and HPA databases to explore TGFBR1 expression in STAD, the correlation between TGFBR1 expression and the clinical features. A receiver operating characteristic (ROC) curve and nomogram were constructed, and LASSO (the Least Absolute Shrinkage and Selection Operator)-selected features were used to build the TGFBR1 prognostic signature. GSEA is used to find the potential mechanism of TGFBR1 to promote the malignant process of STAD. We explored the influence of the TGFBR1 on the immune microenvironment of STAD through the TIMER2.0 and GEPIA database. Results: In our study, TGFBR1 expression was significantly elevated in STAD and positively co-expression with pathologic stage, lymph node metastases (LNM) stage and histopathological grade. Nine factors with non-zero coefficients were identified by LASSO-selected features. Survival analysis revealed that patients with high TGFBR1 had shorter OS, FP, and PPS. Multivariate Cox analysis revealed that TGFBR1 was an independent prognostic factor for OS in STAD. The ROC analysis suggested that high diagnostic value with the AUC of TGFBR1 was 0.739. GSEA revealed that high TGFBR1 expression was correlated with pathway in cancer, MAPK signaling pathway, NOTCH signaling pathway, and VEGF-C production. ssGSEA showed that TGFBR1 is correlated with NK cells, Tem and Th17 cells. Furthermore, elevated TGFBR1 expression was found to be significantly correlated with several immune checkpoint and immune markers associated with immune cell subsets. Conclusion: In summary, TGFBR1 could be a prognostic biomarker and an important regulator of immune cell infiltration in STAD. The present study revealed the probable underlying molecular mechanisms of TGFBR1 in STAD and provided a potential target for improving the prognosis.

Site-Specific Integration of TRAIL in iPSC-Derived Mesenchymal Stem Cells for Targeted Cancer Therapy.

Mesenchymal stem cells (MSCs) are a promising cellular vehicle for transferring anti-cancer factors to malignant tumors. Currently, a variety of anti-cancer agents, including the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been loaded into MSCs derived from a range of sources through different engineering methods. These engineered MSCs exhibit enormous therapeutic potential for various cancers. To avoid the intrinsic defects of MSCs derived from tissues and the potential risk of viral vectors, TRAIL was site-specifically integrated into the ribosomal DNA (rDNA) locus of human-induced pluripotent stem cells (iPSCs) using a non-viral rDNA-targeting vector and transcription activator-like effector nickases (TALENickases). These genetically modified human iPSCs were differentiated into an unlimited number of homogeneous induced MSCs (TRAIL-iMSCs) that overexpressed TRAIL in both culture supernatants and cell lysates while maintaining MSC-like characteristics over continuous passages. We found that TRAIL-iMSCs significantly induced apoptosis in A375, A549, HepG2, and MCF-7 cells in vitro. After intravenous infusion, TRAIL-iMSCs had a prominent tissue tropism for A549 or MCF-7 xenografts and significantly inhibited tumor growth through the activation of apoptotic signaling pathways without obvious side effects in tumor-bearing mice models. Altogether, our results showed that TRAIL-iMSCs have strong anti-tumor effects in vitro and in vivo on a range of cancers. This study allows for the development of an unlimited number of therapeutic gene-targeted MSCs with stable quality and high homogeneity for cancer therapy, thus highlighting a universal and safe strategy for stem cell-based gene therapy with high potential for clinical applications.

Exosome-delivered miR-221/222 exacerbates tumor liver metastasis by targeting SPINT1 in colorectal cancer.

MicroRNAs (miRNAs) are involved in the progression of many cancers through largely unelucidated mechanisms. The results of our present study identified a gene cluster, miR-221/222, that is constitutively upregulated in serum exosome samples of patients with colorectal carcinoma (CRC) with liver metastasis (LM); this upregulation predicts a poor overall survival rate. Using an in vitro cell coculture model, we demonstrated that CRC exosomes harboring miR-221/222 activate liver hepatocyte growth factor (HGF) by suppressing SPINT1 expression. Importantly, miR-221/222 plays a key role in forming a favorable premetastatic niche (PMN) that leads to the aggressive nature of CRC, which was further shown through in vivo studies. Overall, our results show that exosomal miR-221/222 promotes CRC progression and may serve as a novel prognostic marker and therapeutic target for CRC with LM.

Comparison of the clinical outcomes of skin bridge loop ileostomy and traditional loop ileostomy in patients with low rectal cancer.

To compare the clinical results of patients with low rectal cancer who underwent skin bridge loop ileostomy and traditional loop ileostomy, and provide clinical evidence for choosing a better ostomy method. We retrospectively collected data of 118 patients with rectal cancer who underwent low anterior resection and loop ileostomy. To investigate the patients characteristics, postoperative stoma-related complications and the frequency of exchanged ostomy bags. The differences of these indicators between the two groups of patients who underwent skin bridge loop ileostomy and traditional loop ileostomy were compared. The Visual Analog Scale (VAS) score of the skin bridge loop ileostomy group was lower than that of the traditional ileostomy loop group (P < 0.05). The skin bridge group had a lower Discoloration, Erosion, Tissue overgrowth (DET) score and incidence of mucocutaneous separation than the traditional group at the 1st and 2nd weeks after operation (P < 0.05). The average number of weekly exchanged ostomy bags was significantly less in the skin bridge group than in the traditional group within 4 weeks after surgery (P < 0.05). Our experience demonstrates that the skin bridge loop ileostomy may significantly reduce early postoperative stoma-related complications, the frequency of exchanged ostomy bags and patients' medical costs after discharge.

Laboratory-based Surveillance and Clinical Profile of Sporadic HEV Infection in Shanghai, China.

The study aimed to describe the epidemiological, virological and clinical features of sporadic HEV infection in eastern China. A total of 6112 patient sera were tested for anti-HEV IgG or anti-HEV IgM during one consecutive year (between August 2018 and July 2019). HEV RNA presence was evaluated by RT-PCR and HEV sequences were phylogenetically analyzed. Clinical features of confirmed HEV-infected patients were delineated. The sero-positivity rate of anti-HEV IgG maintained stable around 40%, while an obvious winter spike of anti-HEV IgM prevalence was observed. A total of 111 patients were confirmed of HEV viremia by molecular diagnosis. Subtype 4d was predominant. Phylogenetic analyses suggest that certain strains circulate across species and around the country. Subjects with confirmed current HEV infection had a high median age (58 years) and males were predominant (62.2%). Most patients presented with jaundice (75.7%) and anorexia (68.0%). Significantly elevated levels of liver enzymes and bilirubin were observed. Remarkably, the baseline bilirubin level was positively correlated with illness severity. Pre-existing HBV carriage may deteriorate illness. The clinical burden caused by locally acquired HEV infection is increasing. Surveillance should be enforced especially during the transition period from winter to spring. Patients with higher level of bilirubin at disease onset had slower recovery from HEV infection.

Selective Laser Trabeculoplasty: Outcomes of Multiple Repeat Treatments.

PURPOSE: To characterize intraocular pressure (IOP) reduction, effect duration, and side effect profile of repeat selective laser trabeculoplasty (SLT) used as primary stand-alone treatment for open-angle glaucoma (OAG). The secondary aim was to investigate covariates associated with treatment response to SLT. DESIGN: Retrospective chart review. PARTICIPANTS: A total of 52 patients with treatment-naïve OAG who received 3 installments of 3600 SLT as stand-alone glaucoma therapy. When both eyes met the inclusion criteria, only right eye data were used for analysis. METHODS: The study was conducted in a single specialist practice. First, second, and third SLT (SLT1, SLT2, SLT3, respectively) treatments were compared for IOP reduction and effect duration. Eyes were classified as "treatment responders" if they had ≥20% IOP reduction 4 to 8 weeks post-SLT compared with baseline. Effect duration was the interval between SLT and the time point at which the surgeon decided inadequate IOP control necessitated repeat SLT. Individuals were excluded if they underwent intraocular surgery during the study period or received treatment with adjunctive ocular hypotensive medications. MAIN OUTCOME MEASURES: Reduction in IOP post-SLT and effect duration between treatments. RESULTS: Mean age at SLT1 was 58 years; 50% were male, and 92% were phakic. The SLT1 and SLT3 both resulted in mean 27% IOP reduction at 4 to 8 weeks, whereas SLT2 led to 26% IOP reduction. Response rate (≥20% IOP reduction at 4-8 weeks) was 79% for SLT1, 73% for SLT2, and 81% for SLT3, but the difference was not statistically significant. Response to repeat SLT was not significantly associated with previous SLT outcome. Effect duration was 22.2 months, 33.8 months, and 28.9 months after SLT1, SLT2, and SLT3, respectively. Effect duration was significantly longer after SLT2 (P = 0.0006) and SLT3 (P = 0.0444) compared with SLT1. There was no significant association between SLT response and gender, lens status, or OAG subtype. CONCLUSIONS: For primary stand-alone treatment in OAG, initial and repeat SLTs produced comparable percentage IOP reduction, but repeat SLTs had longer effect duration. Intraocular pressure response to SLT was not predictive of the IOP response to subsequent, repeat SLT treatment.