Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation.

BACKGROUND: Phosphatase and tensin homolog deleted on chromosome ten (PTEN) serves as a powerful tumor suppressor, and has been found to be downregulated in human bladder cancer (BC) tissues. Despite this observation, the mechanisms contributing to PTEN's downregulation have remained elusive. METHODS: We established targeted genes' knockdown or overexpressed cell lines to explore the mechanism how it drove the malignant transformation of urothelial cells or promoted anchorageindependent growth of human basal muscle invasive BC (BMIBC) cells. The mice model was used to validate the conclusion in vivo. The important findings were also extended to human studies. RESULTS: In this study, we discovered that mice exposed to N-butyl-N-(4-hydroxybu-tyl)nitrosamine (BBN), a specific bladder chemical carcinogen, exhibited primary BMIBC accompanied by a pronounced reduction in PTEN protein expression in vivo. Utilizing a lncRNA deep sequencing high-throughput platform, along with gain- and loss-of-function analyses, we identified small nucleolar RNA host gene 1 (SNHG1) as a critical lncRNA that might drive the formation of primary BMIBCs in BBN-treated mice. Cell culture results further demonstrated that BBN exposure significantly induced SNHG1 in normal human bladder urothelial cell UROtsa. Notably, the ectopic expression of SNHG1 alone was sufficient to induce malignant transformation in human urothelial cells, while SNHG1 knockdown effectively inhibited anchorage-independent growth of human BMIBCs. Our detailed investigation revealed that SNHG1 overexpression led to PTEN protein degradation through its direct interaction with HUR. This interaction reduced HUR binding to ubiquitin-specific peptidase 8 (USP8) mRNA, causing degradation of USP8 mRNA and a subsequent decrease in USP8 protein expression. The downregulation of USP8, in turn, increased PTEN polyubiquitination and degradation, culminating in cell malignant transformation and BMIBC anchorageindependent growth. In vivo studies confirmed the downregulation of PTEN and USP8, as well as their positive correlations in both BBN-treated mouse bladder urothelium and tumor tissues of bladder cancer in nude mice. CONCLUSIONS: Our findings, for the first time, demonstrate that overexpressed SNHG1 competes with USP8 for binding to HUR. This competition attenuates USP8 mRNA stability and protein expression, leading to PTEN protein degradation, consequently, this process drives urothelial cell malignant transformation and fosters BMIBC growth and primary BMIBC formation.

Genome scale CRISPR screens identify actin capping proteins as key modulators of therapeutic responses to radiation and immunotherapy.

Radiotherapy (RT), is a fundamental treatment for malignant tumors and is used in over half of cancer patients. As radiation can promote anti-tumor immune effects, a promising therapeutic strategy is to combine radiation with immune checkpoint inhibitors (ICIs). However, the genetic determinants that impact therapeutic response in the context of combination therapy with radiation and ICI have not been systematically investigated. To unbiasedly identify the tumor intrinsic genetic factors governing such responses, we perform a set of genome-scale CRISPR screens in melanoma cells for cancer survival in response to low-dose genotoxic radiation treatment, in the context of CD8 T cell co-culture and with anti-PD1 checkpoint blockade antibody. Two actin capping proteins, Capza3 and Capg, emerge as top hits that upon inactivation promote the survival of melanoma cells in such settings. Capza3 and Capg knockouts (KOs) in mouse and human cancer cells display persistent DNA damage due to impaired homology directed repair (HDR); along with increased radiation, chemotherapy, and DNA repair inhibitor sensitivity. However, when cancer cells with these genes inactivated were exposed to sublethal radiation, inactivation of such actin capping protein promotes activation of the STING pathway, induction of inhibitory CEACAM1 ligand expression and resistance to CD8 T cell killing. Patient cancer genomics analysis reveals an increased mutational burden in patients with inactivating mutations in CAPG and/or CAPZA3, at levels comparable to other HDR associated genes. There is also a positive correlation between CAPG expression and activation of immune related pathways and CD8 T cell tumor infiltration. Our results unveil the critical roles of actin binding proteins for efficient HDR within cancer cells and demonstrate a previously unrecognized regulatory mechanism of therapeutic response to radiation and immunotherapy.

AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells.

Engineering cells for adoptive therapy requires overcoming limitations in cell viability and, in the efficiency of transgene delivery, the duration of transgene expression and the stability of genomic integration. Here we report a gene-delivery system consisting of a Sleeping Beauty (SB) transposase encoded into a messenger RNA delivered by an adeno-associated virus (AAV) encoding an SB transposon that includes the desired transgene, for mediating the permanent integration of the transgene. Compared with lentiviral vectors and with the electroporation of plasmids of transposon DNA or minicircle DNA, the gene-delivery system, which we named MAJESTIC (for 'mRNA AAV-SB joint engineering of stable therapeutic immune cells'), offers prolonged transgene expression, as well as higher transgene expression, therapeutic-cell yield and cell viability. MAJESTIC can deliver chimeric antigen receptors (CARs) into T cells (which we show lead to strong anti-tumour activity in vivo) and also transduce natural killer cells, myeloid cells and induced pluripotent stem cells with bi-specific CARs, kill-switch CARs and synthetic T-cell receptors.

Perturbomics of tumor-infiltrating NK cells.

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly reveal the functional genetic landscape underlying critical NK cell characteristics against cancer, we perform perturbomics mapping of tumor infiltrating NK cells by joint in vivo AAV-CRISPR screens and single cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)- CRISPR screening leveraging a custom high-density sgRNA library targeting cell surface genes, and perform four independent in vivo tumor infiltration screens in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator that emerges from both screen and single cell analyses, shows both in vitro and in vivo efficacy enhancement when perturbed in chimeric antigen receptor (CAR)-NK cells. Differential gene expression analysis reveals that CALHM2 knockout reshapes cytokine production, cell adhesion, and signaling pathways in CAR- NKs. These data directly and systematically map out endogenous factors that naturally limit NK cell function in the TME to offer a broad range of cellular genetic checkpoints as candidates for future engineering to enhance NK cell-based immunotherapies.

Therapeutic immune cell engineering with an mRNA : AAV- Sleeping Beauty composite system.

Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC ( m RNA A AV-Sleeping-Beauty J oint E ngineering of S table T herapeutic I mmune C ells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo . This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.

Improved Carrier Management via a Multifunctional Modifier for High-Quality Low-Bandgap Sn-Pb Perovskites and Efficient All-Perovskite Tandem Solar Cells.

All-perovskite tandem solar cells (TSCs) hold great promise in terms of ultrahigh efficiency, low manufacturing cost, and flexibility, stepping forward to the next-generation photovoltaics. However, their further development is hampered by the relatively low performance of low-bandgap (LBG) tin (Sn)-lead (Pb) perovskite solar cells (PSCs). Improving the carrier management, including suppressing trap-assisted non-radiative recombination and promoting carrier transfer, is of great significance to enhance the performance of Sn-Pb PSCs. Herein, a carrier management strategy is reported for using cysteine hydrochloride (CysHCl) simultaneously as a bulky passivator and a surface anchoring agent for Sn-Pb perovskite. CysHCl processing effectively reduces trap density and suppresses non-radiative recombination, enabling the growth of high-quality Sn-Pb perovskite with greatly improved carrier diffusion length of >8 µm. Furthermore, the electron transfer at the perovskite/C60 interface is accelerated due to the formation of surface dipoles and favorable energy band bending. As a result, these advances enable the demonstration of champion efficiency of 22.15% for CysHCl-processed LBG Sn-Pb PSCs with remarkable enhancement in both open-circuit voltage and fill factor. When paired with a wide-bandgap (WBG) perovskite subcell, a certified 25.7%-efficient all-perovskite monolithic tandem device is further demonstrated.

RAMIHM generates fully human monoclonal antibodies by rapid mRNA immunization of humanized mice and BCR-seq.

As a clinical vaccine, lipid nanoparticle (LNP) mRNA has demonstrated potent and broad antibody responses, leading to speculation about its potential for antibody discovery. Here, we developed RAMIHM, a highly efficient strategy for developing fully human monoclonal antibodies that employs rapid mRNA immunization of humanized mice followed by single B cell sequencing (scBCR-seq). We immunized humanized transgenic mice with RAMIHM and generated 15 top-ranked clones from peripheral blood, plasma B, and memory B cell populations, demonstrating a high rate of antigen-specificity (93.3%). Two Omicron-specific neutralizing antibodies with high potency and one broad-spectrum neutralizing antibody were discovered. Furthermore, we extended the application of RAMIHM to cancer immunotherapy targets, including a single transmembrane protein CD22 and a multi-transmembrane G protein-coupled receptor target, GPRC5D, which is difficult for traditional protein immunization methods. RAMIHM-scBCR-seq is a broadly applicable platform for the rapid and efficient development of fully human monoclonal antibodies against an assortment of targets.

Surfactant-Free Synthesis of Ultrafine Pt Nanoparticles on MoS2 Nanosheets as Bifunctional Catalysts for the Hydrodeoxygenation of Bio-Oil.

Hydrodeoxygenation (HDO) of bio-oil is a crucial step for improving the bio-fuel quality, but developing highly dispersed Pt-based catalysts with high selectivity for target alkanes remains a great challenge. This study presents a fast surfactant-free method to prepare the MoS2-supported Pt catalyst for HDO. Ultrafine Pt nanoparticles with sizes of <5 nm can be readily grown on chemically exfoliated MoS2 nanosheets (NSs) via the direct microwave-assisted thermal reduction. The obtained Pt NPs/MoS2 composites show excellent catalytic performance in the conversion of palmitic acid, and the best selectivity (also the yield) of hexadecane and pentadecane is 80.56 and 19.43%, respectively.

A novel approach toward Snowman-like polymer/SiO2 hybrid nanoparticles via gas-driving.

For the first time, we report an interesting transition from conventional core-shell polymer/SiO2 particles to self-stable snowman-like particles, which can be achieved by adding a low-boiling point oil-soluble monomer because the volatile monomer not only plays a lubrication role, but also acts as a gas "motor" to drive the silica precursor polycondensate migration.

[Effect of bladder training on bladder function recovery in the male patients after mid-low rectal cancer surgery: a prospective, open, randomized controlled study].

OBJECTIVE: To investigate the efficacy and safety of the bladder training in male patients before urinary catheter removal after mid-low rectal cancer surgery. METHODS: This was a prospective, open, randomized controlled study. INCLUSION CRITERIA: male patients; pathologically diagnosed as mid-low rectal adenocarcinoma; distance from tumor lower edge to anal margin ≤10 cm; standard radical surgery for rectal cancer, including intestinal resection and regional lymph node dissection. EXCLUSION CRITERIA: previous history of benign prostatic hyperplasia or history of prostate surgery; bladder dysfunction such as dysuria and urinary retention before surgery; local resection of rectal tumor or extended resection. According to the above criteria, 92 patients who underwent colorectal surgery at the Union Hospital of Fujian Medical University from June to December 2016 were prospectively included. The patients were randomly divided into bladder training group (n=43) and bladder non-training group (n=49) according to the random number table method. The study was approved by the Ethics Committee of the Union Hospital of Fujian Medical University (ethical approval number: 2016KY005) and registered with the China Clinical Trial Registration Center (ChiCTR) (registration No.ChiCTR-IOR-16007995). The implementation of patient's treatment measures, the data collection and analysis were based on the three-blind principle, using envelopes for distribution concealment. In the bladder training group, bladder training was routinely performed from the first day after operation to catheter removal, and in bladder non-training group the catheter was kept open till its removal. The catheter was removed in the early morning at the 5th day after surgery, and the spontaneous urine output was recorded and the residual urine volume of the bladder was measured after the first urination. The international prostate symptom score (IPSS) was applied to evaluate the patient's urinary function before and after surgery. RESULTS: The age of whole group was (58.6±10.9) years old, the body mass index was (22.4±2.7) kg/m 2, and the distance from tumor lower edge to anal margin was (6.5±1.9) cm. The baseline data, such as age, body mass index, distance from tumor lower edge to anal margin, preoperative IPSS score, preoperative bladder residual urine volume, neoadjuvant radiotherapy and chemotherapy, preventive ileostomy and surgical procedure were not significantly different between two groups (all P>0.05). There was no significant difference in IPSS scores evaluated at the second day (3.6±4.0 vs. 3.5±3.4, t=0.128, P=0.899) and one month (3.7±2.9 vs. 3.0±3.1, t=1.113, P=0.269) after catheter removal between the bladder training group and bladder non-training group. No significant difference in the postoperative residual urine volume of bladder (media 44 ml vs. 24 ml, Z=-1.466, P=0.143), the first spontaneous urination volume (median 200 ml vs. 150 ml, Z=-1.228, P=0.219) after catheter removal, and postoperative hospital stay [(8.2±4.5) days vs. (9.1±5.5) days, t=-0.805, P=0.423] was found. Urinary infection rate was 20.9%(9/43) in the training group, which was even higher than 8.2%(4/49) in the non-training group, but the difference was not significant(χ²=3.077, P=0.079). No patient needed re-catheterization in either group. CONCLUSIONS: The routine bladder training after mid-low rectal cancer surgery does not improve the urinary function, and can not reduce the residual urine volume of bladder after catheter removal. This routine clinical practice is not helpful for the bladder function recovery after rectal cancer surgery.

Cloning, expression and molecular characterization of a 14-3-3 gene from a parasitic ciliate, Cryptocaryon irritans.

Cryptocaryon irritans is a parasitic ciliate and responsible for cryptocaryosis of ocean teleostean. In this paper, one gene homologous to 14-3-3 was isolated from cDNA library of C. irritans trophont/protomont stage and designated as Ci14-3-3. The full-length cDNA of the gene was 892bp with an open reading frame of 744bp, which encoded a polypeptide of 247 amino acids with a predicted molecular weight of 28.4kDa. After modification of the non-universal genetic codes, the open reading frame of Ci14-3-3 was inserted into plasmid pGEX-4T-1, transformed into Escherichia coli DH5α strain and then expressed as a glutathione S transferase fusion protein (rCi14-3-3). The result of western blot analysis showed that the rCi14-3-3 had antigenicity and the Ci14-3-3 gene in C. irritans was expressed at all stages of life cycle. The endogenous Ci14-3-3 not only distributed in cytoplasm, but also presented on the plasma membrane and the front end of cytostome in newly hatched theronts. However, when theronts were dying the protein appeared as dot-like aggregates around the nucleuses. The murine anti-rCi14-3-3 sera were capable of causing agglutination/immobilization of theronts, suggesting its potential for vaccine development.

Molecular characterization of an actin depolymerizing factor from Cryptocaryon irritans.

Actin depolymerizing factors regulate actin dynamics involved in cellular processes such as morphogenesis, motility, development and infection. Here, a novel actin depolymerizing factor gene (CiADF 2 ) was cloned from the cDNA library of Cryptocaryon irritans, a parasitic ciliate causing cryptocaryonosis. The full-length cDNA of CiADF 2 was 531 bp. Its open reading frame (ORF) was 417 bp, encoding a polypeptide of 138 aa with typical features of the ADF/cofilin family. Reverse transcription-PCR suggested that CiADF 2 is expressed in all stages of the life cycle. After site-directed mutagenesis of a non-universal genetic code, the ORF was subcloned in Escherichia coli. The bacteria were induced with the addition of isopropylthio-ß-D-galactoside to express a fusion protein of recombinant CiADF2 (rCiADF2) with glutathione S transferase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed the predicted molecular mass of rCiADF2 of 16·2 kDa. A mouse antibody against rCiADF2 recognized native CiADF2, and rCiADF2 reacted with mouse antisera against C. irritans trophonts. CiADF2 was abundant in the plasma around cytostomes, suggesting that CiADF2 is involved in ciliate movement. Moreover, rCiADF2 showed F-actin binding and depolymerizing activity. This study will help to clarify the pathogenic biology of the parasite and develop effective control measures for cryptocaryonosis.