Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

Tautomeric mixture coordination enables efficient lead-free perovskite LEDs.

Lead halide perovskite light-emitting diodes (PeLEDs) have demonstrated remarkable optoelectronic performance1-3. However, there are potential toxicity issues with lead4,5 and removing lead from the best-performing PeLEDs-without compromising their high external quantum efficiencies-remains a challenge. Here we report a tautomeric-mixture-coordination-induced electron localization strategy to stabilize the lead-free tin perovskite TEA2SnI4 (TEAI is 2-thiopheneethylammonium iodide) by incorporating cyanuric acid. We demonstrate that a crucial function of the coordination is to amplify the electronic effects, even for those Sn atoms that aren't strongly bonded with cyanuric acid owing to the formation of hydrogen-bonded tautomeric dimer and trimer superstructures on the perovskite surface. This electron localization weakens adverse effects from Anderson localization and improves ordering in the crystal structure of TEA2SnI4. These factors result in a two-orders-of-magnitude reduction in the non-radiative recombination capture coefficient and an approximately twofold enhancement in the exciton binding energy. Our lead-free PeLED has an external quantum efficiency of up to 20.29%, representing a performance comparable to that of state-of-the-art lead-containing PeLEDs6-12. We anticipate that these findings will provide insights into the stabilization of Sn(II) perovskites and further the development of lead-free perovskite applications.

OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

WWTR1(TAZ)-CAMTA1 reprograms endothelial cells to drive epithelioid hemangioendothelioma.

Epithelioid hemangioendothelioma (EHE) is a poorly understood and devastating vascular cancer. Sequencing of EHE has revealed a unique gene fusion between the Hippo pathway nuclear effector TAZ (WWTR1) and the brain-enriched transcription factor CAMTA1 in ∼90% of cases. However, it remains unclear whether the TAZ-CAMTA1 gene fusion is a driver of EHE, and potential targeted therapies are unknown. Here, we show that TAZ-CAMTA1 expression in endothelial cells is sufficient to drive the formation of vascular tumors with the distinctive features of EHE, and inhibition of TAZ-CAMTA1 results in the regression of these vascular tumors. We further show that activated TAZ resembles TAZ-CAMTA1 in driving the formation of EHE-like vascular tumors, suggesting that constitutive activation of TAZ underlies the pathological features of EHE. We show that TAZ-CAMTA1 initiates an angiogenic and regenerative-like transcriptional program in endothelial cells, and disruption of the TAZ-CAMTA1-TEAD interaction or ectopic expression of a dominant negative TEAD in vivo inhibits TAZ-CAMTA1-mediated transformation. Our study provides the first genetic model of a TAZ fusion oncoprotein driving its associated human cancer, pinpointing TAZ-CAMTA1 as the key driver and a valid therapeutic target of EHE.

Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.

The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

DeepBAM: a high-accuracy single-molecule CpG methylation detection tool for Oxford nanopore sequencing.

Recent nanopore sequencing system (R10.4) has enhanced base calling accuracy and is being increasingly utilized for detecting CpG methylation state. However, the robustness and universality of the methylation calling model in officially supplied Dorado remains poorly tested. In this study, we obtained heterogeneous datasets from human and plant sources to carry out comprehensive evaluations, which showed that Dorado performed significantly different across datasets. We therefore developed deep neural networks and implemented several optimizations in training a new model called DeepBAM. DeepBAM achieved superior and more stable performances compared with Dorado, including higher area under the ROC curves (98.47% on average and up to 7.36% improvement) and F1 scores (94.97% on average and up to 16.24% improvement) across the datasets. DeepBAM-based whole genome methylation frequencies have achieved >0.95 correlations with BS-seq on four of five datasets, outperforming Dorado in all instances. It enables unraveling allele-specific methylation patterns, including regions of transposable elements. The enhanced performance of DeepBAM paves the way for broader applications of nanopore sequencing in CpG methylation studies.

Cathelicidin peptide analogues inhibit EV71 infection through blocking viral entry and uncoating.

Given the serious neurological complications and deaths associated with enterovirus 71 (EV71) infection, there is an urgent need to develop effective antivirals against this viral infection. In this study, we demonstrated that two Cathelicidin-derived peptides, LL-18 and FF-18 were more potent against EV71 infection than the parent peptide LL-37, which is the mature and processed form of Cathelicidin. These peptides could directly bind to the EV71 virus particles, but not to coxsackievirus, indicative of their high specificity. The binding of peptides with the virus surface occupied the viral canyon region in a way that could block virus-receptor interactions and inhibit viral uncoating. In addition, these peptide analogues could also relieve the deleterious effect of EV71 infection in vivo. Therefore, Cathelicidin-derived peptides might be excellent candidates for further development of antivirals to treat EV71 infection.

Targeting Smurf1 to block PDK1-Akt signaling in KRAS-mutated colorectal cancer.

The phosphoinositide 3-kinase (PI3K)-Akt axis is one of the most frequently activated pathways and is demonstrated as a therapeutic target in Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC). Targeting the PI3K-Akt pathway has been a challenging undertaking through the decades. Here we unveiled an essential role of E3 ligase SMAD ubiquitylation regulatory factor 1 (Smurf1)-mediated phosphoinositide-dependent protein kinase 1 (PDK1) neddylation in PI3K-Akt signaling and tumorigenesis. Upon growth factor stimulation, Smurf1 immediately triggers PDK1 neddylation and the poly-neural precursor cell expressed developmentally downregulated protein 8 (poly-Nedd8) chains recruit methyltransferase SET domain bifurcated histone lysine methyltransferase 1 (SETDB1). The cytoplasmic complex of PDK1 assembled with Smurf1 and SETDB1 (cCOMPASS) consisting of PDK1, Smurf1 and SETDB1 directs Akt membrane attachment and T308 phosphorylation. Smurf1 deficiency dramatically reduces CRC tumorigenesis in a genetic mouse model. Furthermore, we developed a highly selective Smurf1 degrader, Smurf1-antagonizing repressor of tumor 1, which exhibits efficient PDK1-Akt blockade and potent tumor suppression alone or combined with PDK1 inhibitor in KRAS-mutated CRC. The findings presented here unveil previously unrecognized roles of PDK1 neddylation and offer a potential strategy for targeting the PI3K-Akt pathway and KRAS mutant cancer therapy.

Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

The class III phosphatidylinositol 3-kinase VPS34 supports EV71 replication by promoting viral replication organelle formation.

Enterovirus 71 (EV71) belongs to the family of Picornaviridae; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virus-host interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34-PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development.IMPORTANCEEnterovirus 71 (EV71) is a major pathogen that causes hand-foot-and-mouth disease (HFMD) and other serious complications, which are big threats to children under 5 years old. Unravelling the interactions between virus and the host cells will open new avenues in antiviral research. Here, we found the class III phosphatidylinositol 3-kinase, VPS34, and its effector, double FYVE-containing protein 1 (DFCP1), were essential for EV71 infection, both of which could support EV71 viral replication by enhancing the biogenesis of viral replication organelles (ROs). As DFCP1 localizes to lipid droplets, hijacking of these host factors will enable viral utilization of lipids from LDs for the generation of membrane structures during RO biogenesis. In addition, the VPS34 kinase inhibitor was found to be potent against EV71 infection; therefore, this study also brings up a novel target for future anti-EV71 drug development.

N-terminal acetyltransferase 6 facilitates enterovirus 71 replication by regulating PI4KB expression and replication organelle biogenesis.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease in children under 5 years old, which can result in severe neurological complications and even death. Due to limited treatments for EV71 infection, the identification of novel host factors and elucidation of mechanisms involved will help to counter this viral infection. N-terminal acetyltransferase 6 (NAT6) was identified as an essential host factor for EV71 infection with genome-wide CRISPR/Cas9 screening. NAT6 facilitates EV71 viral replication depending on its acetyltransferase activity but has little effect on viral release. In addition, NAT6 is also required for Echovirus 7 and coxsackievirus B5 infection, suggesting it might be a pan-enterovirus host factor. We further demonstrated that NAT6 is required for Golgi integrity and viral replication organelle (RO) biogenesis. NAT6 knockout significantly inhibited phosphatidylinositol 4-kinase IIIß (PI4KB) expression and PI4P production, both of which are key host factors for enterovirus infection and RO biogenesis. Further mechanism studies confirmed that NAT6 formed a complex with its substrate actin and one of the PI4KB recruiters-acyl-coenzyme A binding domain containing 3 (ACBD3). Through modulating actin dynamics, NAT6 maintained the integrity of the Golgi and the stability of ACBD3, thereby enhancing EV71 infection. Collectively, these results uncovered a novel mechanism of N-acetyltransferase supporting EV71 infection.IMPORTANCEEnterovirus 71 (EV71) is an important pathogen for children under the age of five, and currently, no effective treatment is available. Elucidating the mechanism of novel host factors supporting viral infection will reveal potential antiviral targets and aid antiviral development. Here, we demonstrated that a novel N-acetyltransferase, NAT6, is an essential host factor for EV71 replication. NAT6 could promote viral replication organelle (RO) formation to enhance viral replication. The formation of enterovirus ROs requires numerous host factors, including acyl-coenzyme A binding domain containing 3 (ACBD3) and phosphatidylinositol 4-kinase IIIß (PI4KB). NAT6 could stabilize the PI4KB recruiter, ACBD3, by inhibiting the autophagy degradation pathway. This study provides a fresh insight into the relationship between N-acetyltransferase and viral infection.

RNA-Seq profiling of circular RNAs in mice with lipopolysaccharide-induced acute lung injury.

Acute lung injury (ALI) is a serious illness that develops suddenly, progresses rapidly, has a poor treatment response and a high mortality rate. Studies have found that circular RNAs (circRNA) play a critical role in several diseases, but their role in ALI remains unclear. The aim of this study was to identify circRNAs that are associated with ALI and investigate their potential molecular mechanisms. A comparison of lung circRNA and microRNA expression profiles in mice with ALI and controls was performed by RNA-sequencing. A bioinformatic analysis was conducted to identify differentially expressed (DE) RNAs, to construct competitive endogenous RNA (ceRNA) networks, and to analyze their function and pathways. Then, a protein-protein interaction (PPI) network was generated by the Search Tool for the Retrieval of Interacting Genes database, and hub genes were identified using Cytoscape. Furthermore, a key ceRNA subnetwork was constructed based on these hub genes. Overall, we found 239 DE circRNAs and 42 DE microRNAs in ALI mice compared to controls. Additionally, the molecular mechanism of ALI was further understood by building ceRNA networks based on these DE genes. ALI-induced circRNAs are mostly function in the inflammatory response and metabolic processes. Moreover, DE circRNAs are primarily involved in the nuclear factor (NF)-kappa B and PI3K-Akt signaling pathways. Seven hub genes were derived from the PPI network of 191 genes, followed by the construction of circRNA-miRNA-hub gene subnetworks. In this study, circRNA profiles are remarkably changed in mice with LPS-triggered ALI, and their potential contribution to the disease is revealed.

CO-Assisted Methane Oxidation into Oxygenates over Surface Platinum-Titanium Alloyed Layers.

Methane oxidation using molecular oxygen remains a grand challenge in which the obstacle is not only the activation of methane but also the reaction with oxygen, considering the mismatch of the ground spin states. Herein, we report TiO2-supported Pt nanocrystals (Pt/TiO2) with surface Pt-Ti alloyed layers that directly convert methane into oxygenates by using O2 as the oxidant with the assistance of CO. The oxygenate yield reached 749.8 mmol gPt-1 in a H2O aqueous solution over 0.1% Pt/TiO2 under 31 bar of mixed gas (20:5:6 CH4:CO:O2) at 150 °C for 3 h, while the CH3OH selectivity was 62.3%. On the basis of the control experiments and spectroscopic results, we identified the surface Pt-Ti alloy as the active sites. Moreover, CO promoted the dissociation of O2 on the surface of Pt-Ti alloyed layers and the subsequent activation of CH4 to form oxygenated products.

Glass Disorder Modulated Luminescence in Zero-Dimensional Antimony-Chloride Coplanar Dimers for Optical Anti-counterfeiting.

Zero-dimensional metal halides have received wide attention due to their structural diversity, strong quantum confinement, and associated excellent photoluminescence properties. A reversible and tunable luminescence would be desirable for applications such as anti-counterfeiting, information encryption, and artificial intelligence. Yet, these materials are underexplored, with little known about their luminescence tuning mechanisms. Here we report a pyramidal coplanar dimer, (TBA)Sb2Cl7 (TBA = tetrabutylammonium), showing broadband emission wavelength tuning (585-650 nm) by simple thermal treatment. We attribute the broad color change to structural disorder induced by varying the heat treatment temperatures. Increasing the heating temperature transitions the material from long-range ordered crystalline phase to highly disordered glassy phase. The latter exhibits stronger electron-phonon coupling, enhancing the self-trapped exciton emission efficiency. The work provides a new material platform for manifold optical anti-counterfeiting applications and sheds light on the emission color tuning mechanisms for further design of stimuli-responsive materials.

A Solid-State Electrolyte Facilitates Acidic CO2 Electrolysis without Alkali Metal Cations by Regulating Proton Transport.

Electrochemical CO2 reduction (CO2R) in acidic media provides a pathway to curtail CO2 losses by suppressing the formation of (bi)carbonates. In such systems, a high concentration of alkali metal cations is necessary for mitigating the proton-rich environment and suppressing the competing hydrogen evolution reaction. However, a high cation concentration also promotes salt precipitation within the gas diffusion layer, resulting in poor system durability. Here, we resolve this conundrum by replacing the liquid catholyte with a solid-state proton conductor to regulate H+ transport. This is postulated to allow for a locally alkaline environment at the cathode, enabling selective CO2R even without alkali metal cations. We show that this strategy is effective over a broad range of catalyst systems. For instance, we achieve an 87% CO faradaic efficiency (FE) at 300 mA/cm2 using a composite nanoporous Au and single-atom Ni catalyst, with 0.25 M H2SO4 as the anolyte. Stable operation over 110 h and a high single-pass carbon efficiency of 82.8% were also successfully demonstrated. Importantly, we find that this solid-state system is also particularly effective at converting dilute feedstock (5% CO2) with a CO FE of 47.7%, a factor of 16.4 times higher than a conventional system. Our results introduce a simple yet effective design approach for developing efficient acidic CO2R electrolyzers.

LAMTOR1 decreased exosomal PD-L1 to enhance immunotherapy efficacy in non-small cell lung cancer.

Great progress has been made in utilizing immune checkpoint blockade (ICB) for the treatment of non-small-cell lung cancer (NSCLC). Therapies targeting programmed cell death protein 1 (PD-1) and its ligand PD-L1, expressed on tumor cells, have demonstrated potential in improving patient survival rates. An unresolved issue involves immune suppression induced by exosomal PD-L1 within the tumor microenvironment (TME), particularly regarding CD8+ T cells. Our study unveiled the crucial involvement of LAMTOR1 in suppressing the exosomes of PD-L1 and promoting CD8+ T cell infiltration in NSCLC. Through its interaction with HRS, LAMTOR1 facilitates PD-L1 lysosomal degradation, thereby reducing exosomal PD-L1 release. Notably, the ability of LAMTOR1 to promote PD-L1 lysosomal degradation relies on a specific ubiquitination site and an HRS binding sequence. The findings suggest that employing LAMTOR1 to construct peptides could serve as a promising strategy for bolstering the efficacy of immunotherapy in NSCLC. The discovery and comprehension of how LAMTOR1 inhibits the release of exosomal PD-L1 offer insights into potential therapeutic strategies for improving immunotherapy. It is imperative to conduct further research and clinical trials to investigate the feasibility and efficacy of targeting LAMTOR1 in NSCLC treatment.

The third Intensive Care Bundle with Blood Pressure Reduction in Acute Cerebral Haemorrhage Trial (INTERACT3): an international, stepped wedge cluster randomised controlled trial.

BACKGROUND: Early control of elevated blood pressure is the most promising treatment for acute intracerebral haemorrhage. We aimed to establish whether implementing a goal-directed care bundle incorporating protocols for early intensive blood pressure lowering and management algorithms for hyperglycaemia, pyrexia, and abnormal anticoagulation, implemented in a hospital setting, could improve outcomes for patients with acute spontaneous intracerebral haemorrhage. METHODS: We performed a pragmatic, international, multicentre, blinded endpoint, stepped wedge cluster randomised controlled trial at hospitals in nine low-income and middle-income countries (Brazil, China, India, Mexico, Nigeria, Pakistan, Peru, Sri Lanka, and Viet Nam) and one high-income country (Chile). Hospitals were eligible if they had no or inconsistent relevant, disease-specific protocols, and were willing to implement the care bundle to consecutive patients (aged ≥18 years) with imaging-confirmed spontaneous intracerebral haemorrhage presenting within 6 h of the onset of symptoms, had a local champion, and could provide the required study data. Hospitals were centrally randomly allocated using permuted blocks to three sequences of implementation, stratified by country and the projected number of patients to be recruited over the 12 months of the study period. These sequences had four periods that dictated the order in which the hospitals were to switch from the control usual care procedure to the intervention implementation of the care bundle procedure to different clusters of patients in a stepped manner. To avoid contamination, details of the intervention, sequence, and allocation periods were concealed from sites until they had completed the usual care control periods. The care bundle protocol included the early intensive lowering of systolic blood pressure (target <140 mm Hg), strict glucose control (target 6·1-7·8 mmol/L in those without diabetes and 7·8-10·0 mmol/L in those with diabetes), antipyrexia treatment (target body temperature ≤37·5°C), and rapid reversal of warfarin-related anticoagulation (target international normalised ratio <1·5) within 1 h of treatment, in patients where these variables were abnormal. Analyses were performed according to a modified intention-to-treat population with available outcome data (ie, excluding sites that withdrew during the study). The primary outcome was functional recovery, measured with the modified Rankin scale (mRS; range 0 [no symptoms] to 6 [death]) at 6 months by masked research staff, analysed using proportional ordinal logistic regression to assess the distribution in scores on the mRS, with adjustments for cluster (hospital site), group assignment of cluster per period, and time (6-month periods from Dec 12, 2017). This trial is registered at Clinicaltrials.gov (NCT03209258) and the Chinese Clinical Trial Registry (ChiCTR-IOC-17011787) and is completed. FINDINGS: Between May 27, 2017, and July 8, 2021, 206 hospitals were assessed for eligibility, of which 144 hospitals in ten countries agreed to join and were randomly assigned in the trial, but 22 hospitals withdrew before starting to enrol patients and another hospital was withdrawn and their data on enrolled patients was deleted because regulatory approval was not obtained. Between Dec 12, 2017, and Dec 31, 2021, 10 857 patients were screened but 3821 were excluded. Overall, the modified intention-to-treat population included 7036 patients enrolled at 121 hospitals, with 3221 assigned to the care bundle group and 3815 to the usual care group, with primary outcome data available in 2892 patients in the care bundle group and 3363 patients in the usual care group. The likelihood of a poor functional outcome was lower in the care bundle group (common odds ratio 0·86; 95% CI 0·76-0·97; p=0·015). The favourable shift in mRS scores in the care bundle group was generally consistent across a range of sensitivity analyses that included additional adjustments for country and patient variables (0·84; 0·73-0·97; p=0·017), and with different approaches to the use of multiple imputations for missing data. Patients in the care bundle group had fewer serious adverse events than those in the usual care group (16·0% vs 20·1%; p=0·0098). INTERPRETATION: Implementation of a care bundle protocol for intensive blood pressure lowering and other management algorithms for physiological control within several hours of the onset of symptoms resulted in improved functional outcome for patients with acute intracerebral haemorrhage. Hospitals should incorporate this approach into clinical practice as part of active management for this serious condition. FUNDING: Joint Global Health Trials scheme from the Department of Health and Social Care, the Foreign, Commonwealth & Development Office, and the Medical Research Council and Wellcome Trust; West China Hospital; the National Health and Medical Research Council of Australia; Sichuan Credit Pharmaceutic and Takeda China.

Response mechanisms of different Saccharomyces cerevisiae strains to succinic acid.

BACKGROUND: The production of succinic acid (SA) from biomass has attracted worldwide interest. Saccharomyces cerevisiae is preferred for SA production due to its strong tolerance to low pH conditions, ease of genetic manipulation, and extensive application in industrial processes. However, when compared with bacterial producers, the SA titers and productivities achieved by engineered S. cerevisiae strains were relatively low. To develop efficient SA-producing strains, it's necessary to clearly understand how S. cerevisiae cells respond to SA. RESULTS: In this study, we cultivated five S. cerevisiae strains with different genetic backgrounds under different concentrations of SA. Among them, KF7 and NBRC1958 demonstrated high tolerance to SA, whereas NBRC2018 displayed the least tolerance. Therefore, these three strains were chosen to study how S. cerevisiae responds to SA. Under a concentration of 20 g/L SA, only a few differentially expressed genes were observed in three strains. At the higher concentration of 60 g/L SA, the response mechanisms of the three strains diverged notably. For KF7, genes involved in the glyoxylate cycle were significantly downregulated, whereas genes involved in gluconeogenesis, the pentose phosphate pathway, protein folding, and meiosis were significantly upregulated. For NBRC1958, genes related to the biosynthesis of vitamin B6, thiamin, and purine were significantly downregulated, whereas genes related to protein folding, toxin efflux, and cell wall remodeling were significantly upregulated. For NBRC2018, there was a significant upregulation of genes connected to the pentose phosphate pathway, gluconeogenesis, fatty acid utilization, and protein folding, except for the small heat shock protein gene HSP26. Overexpression of HSP26 and HSP42 notably enhanced the cell growth of NBRC1958 both in the presence and absence of SA. CONCLUSIONS: The inherent activities of small heat shock proteins, the levels of acetyl-CoA and the strains' potential capacity to consume SA all seem to affect the responses and tolerances of S. cerevisiae strains to SA. These factors should be taken into consideration when choosing host strains for SA production. This study provides a theoretical basis and identifies potential host strains for the development of robust and efficient SA-producing strains.

Biomimetic Asymmetric Reduction Based on the Regenerable Coenzyme NAD(P)H Models.

ConspectusIn nature, the coenzyme NAD(P)H is utilized for the transfer of hydrogen and electrons in biocatalytic reduction, which involves the process of recycling, coenzyme usage, and reduction. Inspired by the biological system, a series of nonregenerable achiral and chiral NAD(P)H models were synthesized and employed. However, this approach faced intractable limitations, such as the need for an equivalent amount of mimics, accompanied by the production of byproducts, which resulted in poor atom economy and difficult separation of products. Therefore, the development of new and efficient methodologies for synthesis, regeneration, and application of the NAD(P)H models in organic synthesis is greatly desired.To tackle these challenges, the regenerable achiral and chiral coenzyme NAD(P)H models were designed and synthesized based on the principles of biocatalytic reduction and applied them in biomimetic asymmetric reduction (BMAR) reactions. This Account summarizes our endeavors in rational design, synthesis, regeneration, and application of the NAD(P)H models. First, we will introduce the design and synthesis of regenerable and achiral coenzyme NAD(P)H models (dihydrophenanthridine and dihydropyrroloquinoxaline), which were successfully applied to BMAR of imines and heteroaromatics using homogeneous ruthenium complex as a regeneration catalyst, chiral phosphoric acid as a transfer catalyst, and hydrogen as the terminal reductant. Regenerable and achiral NAD(P)H models require the addition of chiral catalysts or chiral ligands for stereoselective control during the BMAR process. However, the screening of the chiral transfer catalysts is tedious. Narrow substrate scope further limited their application in organic synthesis. Therefore, we designed and synthesized regenerable and chiral NAD(P)H models (CYNAM and FENAM) with planar chirality, which were successfully applied in asymmetric reduction of imines and heteroaromatics using commercially available achiral Brønsted acids, Lewis acids, or organocatalysts as transfer catalysts and a homogeneous ruthenium complex as a regeneration catalyst. Notably, the original factor of enantioselective control is from the chiral NAD(P)H models. In addition, this strategy could also realize the asymmetric reduction of a myriad of electron-deficient tetrasubstituted alkenes, which are challenging substrates in transition metal catalyzed asymmetric hydrogenation. This methodology provides an efficient strategy for the synthesis of chiral building blocks and bioactive molecules. Finally, the detailed mechanism of BMAR, based on the regenerable NAD(P)H models, was elaborated through a combination of experiments and density functional theory calculations. In summary, we believe that the results presented in this Account hold significant implications beyond our work and have the potential for further applications in the field of biomimetic asymmetric catalysis and synthetic methodology.