Algorithm for optimized mRNA design improves stability and immunogenicity.

Messenger RNA (mRNA) vaccines are being used to combat the spread of COVID-19 (refs. 1-3), but they still exhibit critical limitations caused by mRNA instability and degradation, which are major obstacles for the storage, distribution and efficacy of the vaccine products4. Increasing secondary structure lengthens mRNA half-life, which, together with optimal codons, improves protein expression5. Therefore, a principled mRNA design algorithm must optimize both structural stability and codon usage. However, owing to synonymous codons, the mRNA design space is prohibitively large-for example, there are around 2.4 × 10632 candidate mRNA sequences for the SARS-CoV-2 spike protein. This poses insurmountable computational challenges. Here we provide a simple and unexpected solution using the classical concept of lattice parsing in computational linguistics, where finding the optimal mRNA sequence is analogous to identifying the most likely sentence among similar-sounding alternatives6. Our algorithm LinearDesign finds an optimal mRNA design for the spike protein in just 11 minutes, and can concurrently optimize stability and codon usage. LinearDesign substantially improves mRNA half-life and protein expression, and profoundly increases antibody titre by up to 128 times in mice compared to the codon-optimization benchmark on mRNA vaccines for COVID-19 and varicella-zoster virus. This result reveals the great potential of principled mRNA design and enables the exploration of previously unreachable but highly stable and efficient designs. Our work is a timely tool for vaccines and other mRNA-based medicines encoding therapeutic proteins such as monoclonal antibodies and anti-cancer drugs7,8.

Activation of ERß hijacks the splicing machinery to trigger R-loop formation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with aggressive behavior and poor prognosis. Current therapeutic options available for TNBC patients are primarily chemotherapy. With our evolving understanding of this disease, novel targeted therapies, including poly ADP-ribose polymerase (PARP) inhibitors, antibody-drug conjugates, and immune-checkpoint inhibitors, have been developed for clinical use. Previous reports have demonstrated the essential role of estrogen receptor ß (ERß) in TNBC, but the detailed molecular mechanisms downstream ERß activation in TNBC are still far from elucidated. In this study, we demonstrated that a specific ERß agonist, LY500307, potently induces R-loop formation and DNA damage in TNBC cells. Subsequent interactome experiments indicated that the residues 151 to 165 of U2 small nuclear RNA auxiliary factor 1 (U2AF1) and the Trp439 and Lys443 of ERß were critical for the binding between U2AF1 and ERß. Combined RNA sequencing and ribosome sequencing analysis demonstrated that U2AF1-regulated downstream RNA splicing of 5-oxoprolinase (OPLAH) could affect its enzymatic activity and is essential for ERß-induced R-loop formation and DNA damage. In clinical samples including 115 patients from The Cancer Genome Atlas (TCGA) and 32 patients from an in-house cohort, we found a close correlation in the expression of ESR2 and U2AF1 in TNBC patients. Collectively, our study has unraveled the molecular mechanisms that explain the therapeutic effects of ERß activation in TNBC, which provides rationale for ERß activation-based single or combined therapy for patients with TNBC.

SP1-stimulated miR-208a-5p aggravates sepsis-induced myocardial injury via targeting XIAP.

The development of sepsis can lead to many organ dysfunction and even death. Myocardial injury is one of the serious complications of sepsis leading to death. New evidence suggests that microRNAs (miRNAs) play a critical role in infection myocardial injury. However, the mechanism which miR-208a-5p regulates sepsis-induced myocardial injury remains unclear. To mimic sepsis-induced myocardial injury in vitro, rat primary cardiomyocytes were treated with LPS. Cell viability and apoptosis were tested by CCK-8 and flow cytometry, respectively. The secretion of inflammatory factors was analyzed by ELISA. mRNA and protein levels were detected by RT-qPCR and Western blotting. The interaction among SP1, XIAP and miR-208a-5p was detected using dual luciferase report assay. Ultrasonic analysis and HE staining was performed to observe the effect of miR-208a-5p in sepsis-induced rats. Our findings indicated that miR-208a-5p expression in primary rat cardiomyocytes was increased by LPS. MiR-208a-5p inhibitor reversed LPS-induced cardiomyocytes injury through inhibiting the apoptosis. Furthermore, the inflammatory injury in cardiomyocytes was induced by LPS, which was rescued by miR-208a-5p inhibitor. In addition, downregulation of miR-208a-5p improved LPS-induced sepsis myocardial injury in vivo. Mechanistically, XIAP might be a target gene of miR-208a-5p. SP1 promoted transcription of miR-208a by binding to the miR-208a promoter region. Moreover, silencing of XIAP reversed the regulatory of miR-208a-5p inhibitor on cardiomyocytes injury. To sum up, those findings revealed silencing of miR-208a-5p could alleviate sepsis-induced myocardial injury, which would grant a new process for the treatment of sepsis.

LinearCoFold and LinearCoPartition: linear-time algorithms for secondary structure prediction of interacting RNA molecules.

Many RNAs function through RNA-RNA interactions. Fast and reliable RNA structure prediction with consideration of RNA-RNA interaction is useful, however, existing tools are either too simplistic or too slow. To address this issue, we present LinearCoFold, which approximates the complete minimum free energy structure of two strands in linear time, and LinearCoPartition, which approximates the cofolding partition function and base pairing probabilities in linear time. LinearCoFold and LinearCoPartition are orders of magnitude faster than RNAcofold. For example, on a sequence pair with combined length of 26,190 nt, LinearCoFold is 86.8× faster than RNAcofold MFE mode, and LinearCoPartition is 642.3× faster than RNAcofold partition function mode. Surprisingly, LinearCoFold and LinearCoPartition's predictions have higher PPV and sensitivity of intermolecular base pairs. Furthermore, we apply LinearCoFold to predict the RNA-RNA interaction between SARS-CoV-2 genomic RNA (gRNA) and human U4 small nuclear RNA (snRNA), which has been experimentally studied, and observe that LinearCoFold's prediction correlates better with the wet lab results than RNAcofold's.

LazySampling and LinearSampling: fast stochastic sampling of RNA secondary structure with applications to SARS-CoV-2.

Many RNAs fold into multiple structures at equilibrium, and there is a need to sample these structures according to their probabilities in the ensemble. The conventional sampling algorithm suffers from two limitations: (i) the sampling phase is slow due to many repeated calculations; and (ii) the end-to-end runtime scales cubically with the sequence length. These issues make it difficult to be applied to long RNAs, such as the full genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address these problems, we devise a new sampling algorithm, LazySampling, which eliminates redundant work via on-demand caching. Based on LazySampling, we further derive LinearSampling, an end-to-end linear time sampling algorithm. Benchmarking on nine diverse RNA families, the sampled structures from LinearSampling correlate better with the well-established secondary structures than Vienna RNAsubopt and RNAplfold. More importantly, LinearSampling is orders of magnitude faster than standard tools, being 428× faster (72 s versus 8.6 h) than RNAsubopt on the full genome of SARS-CoV-2 (29 903 nt). The resulting sample landscape correlates well with the experimentally guided secondary structure models, and is closer to the alternative conformations revealed by experimentally driven analysis. Finally, LinearSampling finds 23 regions of 15 nt with high accessibilities in the SARS-CoV-2 genome, which are potential targets for COVID-19 diagnostics and therapeutics.

An antagonistic pleiotropic gene regulates the reproduction and longevity tradeoff.

The antagonistic pleiotropy theory of aging proposes that genes enhancing fitness in early life limit the lifespan, but the molecular evidence remains underexplored. By profiling translatome changes in Caenorhabditis elegans during starvation recovery, we find that an open reading frame (ORF) trl-1 "hidden" within an annotated pseudogene significantly translates upon refeeding. trl-1 mutant animals increase brood sizes but shorten the lifespan and specifically impair germline deficiency­induced longevity. The loss of trl-1 abnormally up-regulates the translation of vitellogenin that produces copious yolk to provision eggs, whereas vitellogenin overexpression is known to reduce the lifespan. We show that the TRL-1 protein undergoes liquid­liquid phase separation (LLPS), through which TRL-1 granules recruit vitellogenin messenger RNA and inhibit its translation. These results indicate that trl-1 functions as an antagonistic pleiotropic gene to regulate the reproduction­longevity tradeoff by optimizing nutrient production for the next generation.

Amyloid pathology mediates the associations between plasma fibrinogen and cognition in non-demented adults.

Though previous studies revealed the potential associations of elevated levels of plasma fibrinogen with dementia, there is still limited understanding regarding the influence of Alzheimer's disease (AD) biomarkers on these associations. We sought to investigate the interrelationships among fibrinogen, cerebrospinal fluid (CSF) AD biomarkers, and cognition in non-demented adults. We included 1996 non-demented adults from the Chinese Alzheimer's Biomarker and LifestylE (CABLE) study and 337 from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. The associations of fibrinogen with AD biomarkers and cognition were explored using multiple linear regression models. The mediation analyses with 10 000 bootstrapped iterations were conducted to explore the mediating effects of AD biomarkers on cognition. In addition, interaction analyses and subgroup analyses were conducted to assess the influence of covariates on the relationships between fibrinogen and AD biomarkers. Participants exhibiting low Aß42 were designated as A+, while those demonstrating high phosphorylated tau (P-tau) and total tau (Tau) were labeled as T+ and N+, respectively. Individuals with normal measures of Aß42 and P-tau were categorized as the A-T- group, and those with abnormal levels of both Aß42 and P-tau were grouped under A+T+. Fibrinogen was higher in the A+ subgroup compared to that in the A- subgroup (p = 0.026). Fibrinogen was higher in the A+T+ subgroup compared to that in the A-T- subgroup (p = 0.011). Higher fibrinogen was associated with worse cognition and Aß pathology (all p < 0.05). Additionally, the associations between fibrinogen and cognition were partially mediated by Aß pathology (mediation proportion range 8%-28%). Interaction analyses and subgroup analyses showed that age and ApoE ε4 affect the relationships between fibrinogen and Aß pathology. Fibrinogen was associated with both cognition and Aß pathology. Aß pathology may be a critical mediator for impacts of fibrinogen on cognition.

Multiomics-based molecular subtyping based on the commensal microbiome predicts molecular characteristics and the therapeutic response in breast cancer.

The gut microbiota has been demonstrated to be correlated with the clinical phenotypes of diseases, including cancers. However, there are few studies on clinical subtyping based on the gut microbiota, especially in breast cancer (BC) patients. Here, using machine learning methods, we analysed the gut microbiota of BC, colorectal cancer (CRC), and gastric cancer (GC) patients to identify their shared metabolic pathways and the importance of these pathways in cancer development. Based on the gut microbiota-related metabolic pathways, human gene expression profile and patient prognosis, we established a novel BC subtyping system and identified a subtype called "challenging BC". Tumours with this subtype have more genetic mutations and a more complex immune environment than those of other subtypes. A score index was proposed for in-depth analysis and showed a significant negative correlation with patient prognosis. Notably, activation of the TPK1-FOXP3-mediated Hedgehog signalling pathway and TPK1-ITGAE-mediated mTOR signalling pathway was linked to poor prognosis in "challenging BC" patients with high scores, as validated in a patient-derived xenograft (PDX) model. Furthermore, our subtyping system and score index are effective predictors of the response to current neoadjuvant therapy regimens, with the score index significantly negatively correlated with both treatment efficacy and the number of immune cells. Therefore, our findings provide valuable insights into predicting molecular characteristics and treatment responses in "challenging BC" patients.

Mitochondrial DNA copy number plays opposing roles in T-lymphocyte infiltration of colorectal cancer based on mismatch repair status: new directions for immunotherapy?

BACKGROUND: Researchers have previously reported that mitochondrial DNA copy number (mtDNA-CN) can play different roles in microsatellite instable/mismatch repair-deficient (MSI/dMMR) and microsatellite stable/mismatch repair-proficient (MSS/pMMR) colorectal cancer (CRC). To support malignancy, dMMR CRC relies on glycolysis, while pMMR CRC favors oxidative phosphorylation. However, it is unclear whether mtDNA-CN changes are related to T cell infiltration in CRC. METHODS: The mtDNA-CN was detected by qRT-PCR in 532 patients, and the expression of CD3 and CD8 in 485 patients was detected by immunohistochemistry. The correlation between mtDNA-CN and the prognosis of CRC patients was further analyzed, and the correlation between mtDNA-CN and T lymphocyte infiltration was also analyzed. Biopsy specimens from the immune checkpoint inhibitors (ICIs) treatment cohort were obtained to verify the correlation between mtDNA-CN and the efficacy of ICIs. The effects of mtDNA-CN and MMR status on gene expression were analyzed by RNA-seq. RESULTS: Our results show that mtDNA-CN has inverse relationships to CRC prognosis in cases with different MMR statuses, potentially inducing the U-shaped association in CRC. The opposing correlations between mtDNA-CN and T lymphocyte infiltration in cases of dMMR CRC and pMMR CRC further suggest that mtDNA-CN might play an important role in CRC development. More importantly, cases of pMMR CRC with lower mtDNA-CN and of dMMR CRC with higher mtDNA-CN can benefit more dramatically from ICIs. Furthermore, RNA-seq revealed a link between the level of mtDNA-CN and T lymphocyte infiltration in CRC cases with different MMR statuses. CONCLUSION: Our study found a potential relationship between mtDNA-CN and CRC development that differs by MMR status, potentially providing a rationale for the use of mtDNA-CN as both a predictive biomarker and a therapeutic target for ICIs.

Up-front autologous stem cell transplant in peripheral T-cell lymphoma patients achieving complete response after first-line treatment: A multicentre real-world analysis.

We conducted a retrospective, multicentre study to compare consolidation therapy with or without first-line autologous stem cell transplant (ASCT) for peripheral T-cell lymphoma (PTCL) patients in a real-world setting. We enrolled 347 PTCL patients who achieved complete response after first-line treatment. Of these, 257 received consolidation chemotherapy (non-ASCT group) and 90 received ASCT (ASCT group). Clinical outcomes were comparable between ASCT and non-ASCT groups. After propensity score matching, the 2-year cumulative incidence of treatment-related mortality and relapse remained similar between groups (1.9% vs. 2.0%, p = 0.985; 24.7% vs. 47.1%, p = 0.021). However, significant differences emerged in progression-free survival and overall survival probabilities. Within the T-cell lymphoma subgroup, ASCT patients exhibited favourable outcomes compared to non-ASCT patients: 2-year progression-free survival (73.4% vs. 50.8%, p = 0.024) and overall survival (92.1% vs. 73.5%, p = 0.021). Notably, no significant differences were observed for patients with NK/T-cell lymphoma. These real-world data suggest that up-front ASCT is a safe and effective consolidation option for PTCL patients in remission, particularly those with T-cell lymphoma.

CRISPR-Cas12a-Mediated Hue-Recognition Lateral Flow Assay for Point-of-Need Detection of Salmonella.

Nucleic acid detection of pathogens in a point-of-need (PON) manner is of great significance yet remains challenging for sensitive and accurate visual discrimination. Here, we report a CRISPR-Cas12a-mediated lateral flow assay for PON detection of Salmonella typhimurium (S.ty) that is a prevailing pathogen disseminated through tainted food. The variation of the fluorescence color of the test line is exploited to interpret the results, enabling the discrimination between positive and negative samples on the basis of a hue-recognition mechanism. By leveraging the cleavage activity of Cas12a and hue-recognition readout, the assay facilitated by recombinase polymerase amplification can yield a visual detection limit of 1 copy µL-1 for S.ty genomic DNA within 1 h. The assay also displays a high specificity toward S.ty in fresh chicken samples, as well as a sensitivity 10-fold better than that of the commercial test strip. Moreover, a semiquantitative detection of S.ty ranging from 0 to 4 × 103 CFU/mL by the naked eye is made possible, thanks to the easily discernible color change of the test line. This approach provides an easy, rapid, accurate, and user-friendly solution for the PON detection of Salmonella and other pathogens.

CAR T Cell Membrane Camouflaged Nanocatalyst Augments CAR T Cell Therapy Efficacy Against Solid Tumor.

The immunosuppressive tumor microenvironment (TME) reduces the chimeric antigen receptor (CAR) T-cell therapy against solid tumors. Here, a CAR T cell membrane-camouflaged nanocatalyst (ACSP@TCM) is prepared to augment CAR T cell therapy efficacy against solid tumors. ACSP@TCM is prepared by encapsulating core/shell Au/Cu2- xSe and 3-bromopyruvate with a CAR T cell membrane. It is demonstrated that the CAR T cell membrane camouflaging has much better-targeting effect than the homologous tumors cell membrane camouflaging. ACSP@TCM has an appealing synergistic chemodynamic/photothermal therapy (CDT/PTT) effect that can induce the immunogenic cell death (ICD) of NALM 6 cells. Moreover, 3-bromopyruvate can inhibit the efflux of lactic acid by inhibiting the glycolysis process, regulating the acidity of TME, and providing a more favorable environment for the survival of CAR T cells. In addition, the photoacoustic (PA) imaging and computed tomography (CT) imaging performance can guide the ACSP@TCM-mediated tumor therapy. The results demonstrated that the ACSP@TCM significantly enhanced the CAR T cell therapy efficacy against NALM 6 solid tumor mass, and completely eliminated tumors. This work provides an effective tumor strategy for CAR T cell therapy in solid tumors.

Kiwi-Inspired Rational Nanoarchitecture with Intensified and Discrete Magneto-Fluorescent Functionalities for Ultrasensitive Point-of-Care Immunoassay.

Fluorescent lateral flow immunoassays (FLFIA) is a well-established rapid detection technique for quantitative analysis. However, achieving accurate analysis of biomarkers at the pg mL-1 level using FLFIA still poses challenges. Herein, an ultrasensitive FLFIA platform is reported utilizing a kiwi-type magneto-fluorescent silica nanohybrid (designated as MFS) that serves as both a target-enrichment substrate and an optical signal enhancement label. The spatially-layered architecture comprises a Fe3O4 core, an endocarp-fibers like dendritic mesoporous silica, seed-like quantum dots, and a kiwi-flesh like silica matrix. The MFS demonstrates heightened fluorescence brightness, swift magnetic response, excellent size uniformity, and dispersibility in water. Through liquid-phase capturing and fluorescence-enhanced signal amplification, as well as magnetic-enrichment sample amplification and magnetic-separation noise reduction, the MFS-based FLFIA is successfully applied to the detection of cardiac troponin I that achieved a limit of detection at 8.4 pg mL-1, tens of times lower than those of previously published fluorescent and colorimetric lateral flow immunoassays. This work offers insights into the strategic design of magneto-fluorescent synergetic signal amplification on LFIA platform and underscores their prospects in high-sensitive rapid and on-site diagnosis of biomarkers.

Accelerating the discovery of anticancer peptides targeting lung and breast cancers with the Wasserstein autoencoder model and PSO algorithm.

In the development of targeted drugs, anticancer peptides (ACPs) have attracted great attention because of their high selectivity, low toxicity and minimal non-specificity. In this work, we report a framework of ACPs generation, which combines Wasserstein autoencoder (WAE) generative model and Particle Swarm Optimization (PSO) forward search algorithm guided by attribute predictive model to generate ACPs with desired properties. It is well known that generative models based on Variational AutoEncoder (VAE) and Generative Adversarial Networks (GAN) are difficult to be used for de novo design due to the problems of posterior collapse and difficult convergence of training. Our WAE-based generative model trains more successfully (lower perplexity and reconstruction loss) than both VAE and GAN-based generative models, and the semantic connections in the latent space of WAE accelerate the process of forward controlled generation of PSO, while VAE fails to capture this feature. Finally, we validated our pipeline on breast cancer targets (HIF-1) and lung cancer targets (VEGR, ErbB2), respectively. By peptide-protein docking, we found candidate compounds with the same binding sites as the peptides carried in the crystal structure but with higher binding affinity and novel structures, which may be potent antagonists that interfere with these target-mediated signaling.

RNA design via structure-aware multifrontier ensemble optimization.

MOTIVATION: RNA design is the search for a sequence or set of sequences that will fold to desired structure, also known as the inverse problem of RNA folding. However, the sequences designed by existing algorithms often suffer from low ensemble stability, which worsens for long sequence design. Additionally, for many methods only a small number of sequences satisfying the MFE criterion can be found by each run of design. These drawbacks limit their use cases. RESULTS: We propose an innovative optimization paradigm, SAMFEO, which optimizes ensemble objectives (equilibrium probability or ensemble defect) by iterative search and yields a very large number of successfully designed RNA sequences as byproducts. We develop a search method which leverages structure level and ensemble level information at different stages of the optimization: initialization, sampling, mutation, and updating. Our work, while being less complicated than others, is the first algorithm that is able to design thousands of RNA sequences for the puzzles from the Eterna100 benchmark. In addition, our algorithm solves the most Eterna100 puzzles among all the general optimization based methods in our study. The only baseline solving more puzzles than our work is dependent on handcrafted heuristics designed for a specific folding model. Surprisingly, our approach shows superiority on designing long sequences for structures adapted from the database of 16S Ribosomal RNAs. AVAILABILITY AND IMPLEMENTATION: Our source code and data used in this article is available at https://github.com/shanry/SAMFEO.

KSHV RTA antagonizes SMC5/6 complex-induced viral chromatin compaction by hijacking the ubiquitin-proteasome system.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus with the capacity to establish life-long latent infection. During latent infection, the viral genome persists as a circular episome that associates with cellular histones and exists as a nonintegrated minichromosome in the nucleus of infected cells. Chromatin structure and epigenetic programming are required for the proper control of viral gene expression and stable maintenance of viral DNA. However, there is still limited knowledge regarding how the host regulates the chromatin structure and maintenance of episomal DNA. Here, we found that the cellular protein structural maintenance of chromosome (SMC) complex SMC5/6 recognizes and associates with the KSHV genome to inhibit its replication. The SMC5/6 complex can bind to the KSHV genome and suppress KSHV gene transcription by condensing the viral chromatin and creating a repressive chromatin structure. Correspondingly, KSHV employs an antagonistic strategy by utilizing the viral protein RTA to degrade the SMC5/6 complex and antagonize the inhibitory effect of this complex on viral gene transcription. Interestingly, this antagonistic mechanism of RTA is evolutionarily conserved among γ-herpesviruses. Our work suggests that the SMC5/6 complex is a new host factor that restricts KSHV replication.

Ultra-broadband TM-pass polarizer based on anisotropic metamaterials in lithium niobate on an insulator.

An ultra-broadband TM-pass polarizer is designed, fabricated, and experimentally demonstrated based on subwavelength grating (SWG) metamaterials in a lithium niobate on an insulator (LNOI) platform. According to our simulation, the designed device is predicted to work at a 220 nm wavelength range from 1460 to 1680 nm, covering the S-, C-, L-, U-bands of optical fiber communication. By depositing and subsequently etching a silicon nitride thin film atop the LNOI chip, the SWG structures are formed successfully by using complementary metal-oxide semiconductor (CMOS)-compatible fabrication processes. The measured results show a high polarization extinction ratio larger than 20 dB and a relatively low insertion loss below 2.5 dB over a 130 nm wavelength range from 1500 to 1630 nm, mainly limited by the operation bandwidth of our laser source.

USP5 facilitates bladder cancer progression by stabilizing the c-Jun protein.

BACKGROUND: Bladder cancer is the second most common genitourinary malignancy worldwide. The death rate of bladder cancer has increased every year. However, the molecular mechanism of bladder cancer is not sufficiently studied. Deubiquitinating enzymes (DUBs) play an important role in carcinogenesis. Several studies have demonstrated that USP5 associated with malignancy and pathological progression in hepatocellular carcinoma, colorectal and non-small cell lung cancer. However, the role of USP5 in bladder cancer need to be explored. METHODS: The USP5 expression was analysed using the web server GEPIA. To explore USP5 function in bladder cancer, we constructed USP5-knockout cell lines in T24 cells. A FLAG-USP5 (WT USP5) plasmid and a plasmid FLAG-USP5 C335A (catalytic-inactive mutant) used to overexpress USP5 in EJ cells. CCK8, colony formation, transwell and scratch assays were used to assess cell viability, proliferation and migration. RNA sequencing (RNA-seq) and dual-luciferase reporter assays were performed to screen the pathway. Coimmunoprecipitation and immunofluorescence were used to explore the interaction between USP5 and c-Jun. Cycloheximide (CHX) chase assays were performed to establish the effect of USP5 on c-Jun stability. Xenograft mouse model was used to study the role of USP5 in bladder cancer. RESULTS: USP5 expression is increased in bladder cancer patients. Genetic ablation of USP5 markedly inhibited bladder cancer cell proliferation, viability, and migration both in vitro and in vivo. RNA-seq and luciferase pathway screening showed that USP5 activated JNK signalling, and we identified the interaction between USP5 and c-Jun. USP5 was found to activate c-Jun by inhibiting its ubiquitination. CONCLUSIONS: Our results show that high USP5 expression promotes bladder cancer progression by stabilizing c-Jun and that USP5 is a potential therapeutic target in bladder cancer.

Prognostic differences between carmustine, etoposide, cytarabine and melphalan (BEAM) and carmustine, etoposide, cytarabine, melphalan and fludarabine (BEAMF) regimens before autologous stem cell transplantation plus chimeric antigen receptor T therapy in patients with refractory/relapsed B-cell non-Hodgkin-lymphoma.

BACKGROUND AIMS: The combination therapy of autologous hematopoietic stem cell transplantation (ASCT) and chimeric antigen receptor T-cell (CART) therapy has been employed to improve outcomes for relapsed or refractory (R/R) B-cell non-Hodgkin-lymphoma (B-NHL). The widely used conditioning regimen before ASCT plus CART therapy reported in the literature was carmustine, etoposide, cytarabine and melphalan (BEAM). However, whether adding fludarabine to the BEAM regimen (BEAMF) can improve the survival of patients with R/R B-NHL remains unknown. METHODS: In total, 39 and 19 patients with R/R B-NHL were enrolled to compare clinical outcomes in the BEAM and BEAMF regimens before ASCT plus CD19/22 CART therapy, respectively. RESULTS: The objective response (OR) rates at 3 months to BEAM and BEAMF regimens before ASCT plus CD19/22 CART therapy were 71.8% and 94.7%, respectively (P = 0.093). The BEAMF regimen showed a trend towards a superior duration of response compared with the BEAM regimen (P = 0.09). After a median follow-up of 28 months (range: 0.93-51.9 months), the BEAMF regimen demonstrated superior 2-year progression-free survival (PFS) (89.5% versus 63.9%; P = 0.048) and 2-year overall survival (OS) (100% vs 77.3%; P = 0.035) compared with the BEAM regimen. In the multivariable Cox regression analysis, OR at month 3 (responders) was remarkably correlated with better OS (hazard ratio: 0.112, P = 0.005) compared with OR (non-responders). CONCLUSIONS: For patients with R/R B-NHL, the BEAMF regimen before ASCT plus CD19/22 CART therapy was correlated with superior PFS and OS than the BEAM regimen, and the BEAMF regimen is a promising alternative conditioning regimen for ASCT plus CAR-T therapy.

Evidence from an Avian Embryo Model that Zinc-Inducible MT4 Expression Protects Mitochondrial Function Against Oxidative Stress.

BACKGROUND: Metallothioneins (MTs) have a strong affinity for zinc (Zn) and remain at a sufficiently high level in mitochondria. As the avian embryo is highly susceptible to oxidative damage and relatively easy to manipulate in a naturally closed chamber, it is an ideal model of the effects of oxidative stress on mitochondrial function. However, the protective roles and molecular mechanisms of Zn-inducible protein expression on mitochondrial function in response to various stressors are poorly understood. OBJECTIVES: The study aimed to investigate the mechanisms by which Zn-induced MT4 expression protects mitochondrial function and energy metabolism subjected to oxidative stress using the avian embryo and embryonic primary hepatocyte models. METHODS: First, we investigated whether MT4 expression alters mitochondrial function. Then, we examined the effects of Zn-induced MT4 overexpression and MT4 silencing on embryonic primary hepatocytes from breeder hens fed a normal Zn diet subjected to a tert-butyl hydroperoxide (BHP) oxidative stress challenge during incubation. In vivo, the avian embryos from hens fed the Zn-deficient and Zn-adequate diets were used to determine the protective roles of Zn-induced MT4 expression on the function of mitochondria exposed to oxidative stress induced by in ovo BHP injection. RESULTS: An in vitro study revealed that Zn-induced MT4 expression reduced reactive oxygen species accumulation in primary hepatocytes. MT4 silencing exacerbated BHP-mediated mitochondrial dysfunction whereas Zn-inducible MT4 overexpression mitigated it. Another in vivo study disclosed that maternal Zn-induced MT4 expression protected mitochondrial function in chick embryo hepatocytes against oxidative stress by inhibiting the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α)/peroxisome proliferators-activated receptor-γ (PPAR-γ) pathway. CONCLUSION: This study underscores the potential protective roles of Zn-induced MT4 expression via the downregulation of the PGC-1α/PPAR-γ pathway on mitochondrial function stimulated by the stress challenge in the primary hepatocytes in an avian embryo model. Our findings suggested that Zn-induced MT4 expression could provide a new therapeutic target and preventive strategy for repairing mitochondrial dysfunction in disease.