Bioorthogonal Quinone Methide Decaging Enables Live-Cell Quantification of Tumor-Specific Immune Interactions.

Effective antitumor immunity hinges on the specific engagement between tumor and cytotoxic immune cells, especially cytotoxic T cells. Although investigating these intercellular interactions is crucial for characterizing immune responses and guiding immunotherapeutic applications, direct and quantitative detection of tumor-T cell interactions within a live-cell context remains challenging. We herein report a photocatalytic live-cell interaction labeling strategy (CAT-Cell) relying on the bioorthogonal decaging of quinone methide moieties for sensitive and selective investigation and quantification of tumor-T cell interactions. By developing quinone methide-derived probes optimized for capturing cell-cell interactions (CCIs), we demonstrated the capacity of CAT-Cell for detecting CCIs directed by various types of receptor-ligand pairs (e.g., CD40-CD40L, TCR-pMHC) and further quantified the strengths of tumor-T cell interactions that are crucial for evaluating the antitumor immune responses. We further applied CAT-Cell for ex vivo quantification of tumor-specific T cell interactions on splenocyte and solid tumor samples from mouse models. Finally, the broad compatibility and utility of CAT-Cell were demonstrated by integrating it with the antigen-specific targeting system as well as for tumor-natural killer cell interaction detection. By leveraging the bioorthogonal photocatalytic decaging chemistry on quinone methide, CAT-Cell provides a sensitive, tunable, universal, and noninvasive toolbox for unraveling and quantifying the crucial but delicate tumor-immune interactions under live-cell settings.

Effects of 3-nitrooxypropanol (3-NOP, Bovaer®10) and whole cottonseed on milk production and enteric methane emissions from dairy cows under Swiss management conditions.

The objective of this study was to determine the potential effect and interaction of 3- nitrooxypropanol (3-NOP; Bovaer®) and whole cottonseed (WCS) on lactational performance, and enteric methane (CH4) emission of dairy cows. A total of 16 multiparous cows, including 8 Holstein Friesian (HF) and 8 Brown Swiss (BS) [224 ± 36 d in milk, 26 ± 3.7 kg milk yield], were used in a split-plot design, where the main plot was the breed of cows. Within each subplot, cows were randomly assigned to a treatment sequence in a replicated 4 × 4 Latin Square design with 2 × 2 factorial arrangements of treatments with 4, 24-d periods. The experimental treatments were: 1) Control (basal TMR), 2) 3-NOP (60 mg/kg TMR DM), 3) WCS (5% TMR DM), and 4) 3-NOP + WCS. The treatment diets were balanced for ether extract, crude protein, and NDF contents (4%, 16%, and 43% of TMR DM, respectively). The basal diets were fed twice daily at 0800 and 1800 h. Dry matter intake (DMI) and milk yield were measured daily, and enteric gas emissions were measured (using the GreenFeed system) during the last 3 d of each 24-d experimental period when animals were housed in tie stalls. There was no difference in DMI on treatment level, whereas the WCS treatment increased ECM yield and milk fat yield. There was no interaction of 3-NOP and WCS for any of the enteric gas emission parameters, but 3-NOP decreased CH4 production (g/d), CH4 yield (g/kg DMI), and CH4 intensity (g/kg ECM) by 13, 14 and 13%, respectively. Further, an unexpected interaction of breed by 3-NOP was observed for different enteric CH4 emission metrics: HF cows had a greater CH4 mitigation effect compared with BS cows for CH4 production (g/d; 18 vs. 8%), CH4 intensity (g/kg MY; 19% vs. 3%) and CH4 intensity (g/kg ECM; 19 vs. 4%). Hydrogen production was increased by 2.85 folds in HF and 1.53 folds in BS cows receiving 3-NOP. Further, there was a 3-NOP ' Time interaction for both breeds. In BS cows, 3-NOP tended to reduce CH4 production by 18% at around 4 h after morning feeding but no effect was observed at other time points. In HF cows, the greatest mitigation effect of 3-NOP (29.6%) was observed immediately after morning feeding and it persisted at around 23% to 26% for 10 h until the second feed provision, and 3 h thereafter, in the evening. In conclusion, supplementing 3-NOP at 60 mg/kg DM to a high fiber diet resulted in 18 to 19% reduction in enteric CH4 emission in Swiss Holstein Friesian cows. The lower response to 3-NOP by BS cows was unexpected and has not been observed in other studies. These results should be interpreted with caution due to low number of cows per breed. Lastly, supplementing WCS at 5% of DM improved ECM and milk fat yield but did not enhance CH4 inhibition effect of 3-NOP of dairy cows.

Bioorthogonal photocatalytic proximity labeling in primary living samples.

In situ profiling of subcellular proteomics in primary living systems, such as native tissues or clinic samples, is crucial for understanding life processes and diseases, yet challenging due to methodological obstacles. Here we report CAT-S, a bioorthogonal photocatalytic chemistry-enabled proximity labeling method, that expands proximity labeling to a wide range of primary living samples for in situ profiling of mitochondrial proteomes. Powered by our thioQM labeling warhead development and targeted bioorthogonal photocatalytic chemistry, CAT-S enables the labeling of mitochondrial proteins in living cells with high efficiency and specificity. We apply CAT-S to diverse cell cultures, dissociated mouse tissues as well as primary T cells from human blood, portraying the native-state mitochondrial proteomic characteristics, and unveiled hidden mitochondrial proteins (PTPN1, SLC35A4 uORF, and TRABD). Furthermore, CAT-S allows quantification of proteomic perturbations on dysfunctional tissues, exampled by diabetic mouse kidneys, revealing the alterations of lipid metabolism that may drive disease progression. Given the advantages of non-genetic operation, generality, and spatiotemporal resolution, CAT-S may open exciting avenues for subcellular proteomic investigations of primary samples that are otherwise inaccessible.

Experience with a hybrid recruitment approach of patient-facing web portal screening and subsequent phone and medical record review for a neurosurgical intervention trial for chronic ischemic stroke disability (PISCES III).

BACKGROUND: Recruitment of participants is the greatest risk to completion of most clinical trials, with 20-40% of trials failing to reach the targeted enrollment. This is particularly true of trials of central nervous system (CNS) therapies such as intervention for chronic stroke. The PISCES III trial was an invasive trial of stereotactically guided intracerebral injection of CTX0E03, a fetal derived neural stem cell line, in patients with chronic disability due to ischemic stroke. We report on the experience using a novel hybrid recruitment approach of a patient-facing portal to self-identify and perform an initial screen for general trial eligibility (tier 1), followed by phone screening and medical records review (tier 2) prior to a final in-person visit to confirm eligibility and consent. METHODS: Two tiers of screening were established: an initial screen of general eligibility using a patient-facing web portal (tier 1), followed by a more detailed screen that included phone survey and medical record review (tier 2). If potential participants passed the tier 2 screen, they were referred directly to visit 1 at a study site, where final in-person screening and consent were performed. Rates of screening were tracked during the period of trial recruitment and sources of referrals were noted. RESULTS: The approach to screening and recruitment resulted in 6125 tier 1 screens, leading to 1121 referrals to tier 2. The tier 2 screening resulted in 224 medical record requests and identification of 86 qualifying participants for referral to sites. The study attained a viable recruitment rate of 6 enrolled per month prior to being disrupted by COVID 19. CONCLUSIONS: A tiered approach to eligibility screening using a hybrid of web-based portals to self-identify and screen for general eligibility followed by a more detailed phone and medical record review allowed the study to use fewer sites and reduce cost. Despite the difficult and narrow population of patients suffering moderate chronic disability from stroke, this strategy produced a viable recruitment rate for this invasive study of intracranially injected neural stem cells. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03629275.

Genetically encoded bioorthogonal tryptophan decaging in living cells.

Tryptophan (Trp) plays a critical role in the regulation of protein structure, interactions and functions through its π system and indole N-H group. A generalizable method for blocking and rescuing Trp interactions would enable the gain-of-function manipulation of various Trp-containing proteins in vivo, but generating such a platform remains challenging. Here we develop a genetically encoded N1-vinyl-caged Trp capable of rapid and bioorthogonal decaging through an optimized inverse electron-demand Diels-Alder reaction, allowing site-specific activation of Trp on a protein of interest in living cells. This chemical activation of a genetically encoded caged-tryptophan (Trp-CAGE) strategy enables precise activation of the Trp of interest underlying diverse important molecular interactions. We demonstrate the utility of Trp-CAGE across various protein families, such as catalase-peroxidases and kinases, as translation initiators and posttranslational modification readers, allowing the modulation of epigenetic signalling in a temporally controlled manner. Coupled with computer-aided prediction, our strategy paves the way for bioorthogonal Trp activation on more than 28,000 candidate proteins within their native cellular settings.

DeKinomics pulse-chases kinase functions in living cells.

Cellular context is crucial for understanding the complex and dynamic kinase functions in health and disease. Systematic dissection of kinase-mediated cellular processes requires rapid and precise stimulation ('pulse') of a kinase of interest, as well as global and in-depth characterization ('chase') of the perturbed proteome under living conditions. Here we developed an optogenetic 'pulse-chase' strategy, termed decaging kinase coupled proteomics (DeKinomics), for proteome-wide profiling of kinase-driven phosphorylation at second-timescale in living cells. We took advantage of the 'gain-of-function' feature of DeKinomics to identify direct kinase substrates and further portrayed the global phosphorylation of understudied receptor tyrosine kinases under native cellular settings. DeKinomics offered a general activation-based strategy to study kinase functions with high specificity and temporal resolution under living conditions.

Decaging-to-labeling: Development and investigation of quinone methide warhead for protein labeling.

Biomolecule labeling in living systems is crucial for understanding biological processes and discovering therapeutic targets. A variety of labeling warheads have been developed for multiple biological applications, including proteomics, bioimaging, sequencing, and drug development. Quinone methides (QMs), a class of highly reactive Michael receptors, have recently emerged as prominent warheads for on-demand biomolecule labeling. Their highly flexible functionality and tunability allow for diverse biological applications, but remain poorly explored at present. In this regard, we designed, synthesized, and evaluated a series of new QM probes with a trifluoromethyl group at the benzyl position and substituents on the aromatic ring to manipulate their chemical properties for biomolecule labeling. The engineered QM warhead efficiently labeled proteins both in vitro and under living cell conditions, with significantly enhanced activity compared to previous QM warheads. We further analyzed the labeling efficacy with the assistance of density functional theory (DFT) calculations, which revealed that the QM generation process, rather than the reactivity of QM, contributes more predominantly to the labeling efficacy. Noteworthy, twelve nucleophilic residues on the BSA were labeled by the probe, including Cys, Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr, Trp and Tyr. Given their high efficiency and tunability, these new QM warheads may hold great promise for a broad range of applications, especially spatiotemporal proteomic profiling for in-depth biological studies.

[Risk factors of cytomegalovirus infection in patients with failed corneal grafts].

Objective: To investigate the levels of cytomegalovirus (CMV) infection and associated risk factors in corneal transplant recipients who experienced transplant failure. Methods: This was a case-control study. Clinical data from 576 cases (576 eyes) of patients who underwent repeat corneal transplant surgery at the Department of Ophthalmology, Peking University Third Hospital, due to corneal transplant failure from January 2016 to May 2022 were collected. Of these, 305 were male and 271 were female, with a median age of 44.0 (0.7, 91.0) years. The CMV infection rate was analyzed based on the detection of CMV DNA in aqueous humor or corneal tissue during corneal transplant surgery. Patients were divided into the CMV group (CMV DNA positive) and the control group (herpes virus DNA negative). The main research indicators included the CMV infection rate, clinical characteristics, and risk factors in corneal transplant recipients. Chi-square tests and binary logistic analysis were used to compare differences between the two groups in general information, systemic diseases, ocular lesions, ocular surgical history, and local and systemic medications. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for each CMV infection risk factor. Results: The overall CMV infection rate was 21.9%(126/576), with annual rates ranging from 10.9% to 37.7% from 2016 to 2021. After applying inclusion and exclusion criteria, 378 patients were included in the control trial, with 126 in the CMV group and 252 in the control group. Statistically significant differences between the two groups were observed in systemic immune-related corneal lesions [CMV group: 38 (30.2%), control group: 26 (10.3%)], local immune and inflammatory corneal lesions [CMV group: 46 (36.5%), control group: 40 (15.9%)], congenital corneal opacity [CMV group: 46 (36.5%), control group: 48 (19.0%)] total number of corneal transplants (CMV group: 178 times, control group: 276 times), corneal deep neovascularization crossing the graft [CMV group: 104 (82.5%), control group: 68 (27.0%)] and severe opacity [CMV group: 44 (34.9%), control group: 30 (11.0%)]. Binary logistic regression analysis showed that systemic immune-related corneal lesions (OR=4.044, 95%CI 1.810-9.033, P<0.001), local immune and inflammatory corneal lesions (OR=3.554, 95%CI 1.569-8.052, P=0.002), congenital corneal opacity (OR=2.606, 95%CI 1.216-5.589, P=0.014), total number of corneal transplants (OR=3.206, 95%CI 1.753-5.864, P<0.001), corneal deep neovascularization crossing the graft (OR=8.347, 95%CI 3.967-17.559, P<0.001), and severe opacity (OR=3.063, 95%CI 1.221-7.682, P=0.017) were independent risk factors for CMV infection after corneal transplant. Conclusions: CMV infection was present in more than 1/5 of corneal transplant recipients who experienced transplant failure. CMV infection after corneal transplant may be related to immune rejection reactions and ocular inflammatory responses. Inflammatory corneal lesions associated with systemic or local immune abnormalities, congenital corneal opacity, and multiple corneal transplants may exacerbate the levels of inflammatory factors during the perioperative period of corneal transplant, increasing the risk of post-transplant CMV infection, leading to the infiltration of deep neovascularization and severe opacity in the cornea.

Cerebrovascular Complications Associated With Iatrogenic Fungal Meningitis Following Surgical Procedures in Mexico.

BACKGROUND: The current fungal meningitis outbreak caused by contaminated epidural anesthesia with Fusarium solani among patients who underwent surgical procedures in Matamoros, Mexico remains a cause of concern. Its association with an increased susceptibility for cerebrovascular complications (CVC) has not been reported. This single-center study describes 3 patients with a unique pattern of CVC attributed to fungal meningitis. METHODS: A retrospective case series of patients diagnosed with fungal meningitis following surgical procedures under contaminated epidural anesthesia who developed a unique pattern of CVC during their hospitalization. RESULTS: Three female patients (mean age, 35 years) with CVC due to iatrogenic fungal meningitis were included. Positive Fungitell ß-D-glucan assay in cerebrospinal fluid was documented in all cases, and F. solani was confirmed by polymerase chain reaction in case 3. All cases were complicated by severe vertebrobasilar circulation vasculopathy and arterial dissections with resultant subarachnoid hemorrhage and intraventricular hemorrhage, ultimately leading to patients' death. CONCLUSIONS: The death toll from the ongoing fungal meningitis outbreak keeps rising, underscoring the need for early recognition and aggressive treatment. We highlight the risk for vertebrobasilar circulation CVC among these patients. The angioinvasive nature of F. solani is yet to be clarified; however, a clear pattern has been observed. Public health awareness should be raised and a strong response should be pursued.

Global burden and inequality of maternal and neonatal disorders: based on data from the 2019 Global Burden of Disease study.

BACKGROUND: Maternal and neonatal disorders account for substantial health loss across the lifespan from early childhood. These problems may be related to health inequality. AIM: To provide evidence for improvement in health policies regarding maternal and neonatal disorder inequity. DESIGN: This was a population-based cross-sectional study based on 2019 Global Burden of Disease data. METHODS: Annual cases and age-standardized rates (ASRs) of incidence, prevalence, death, and disability-adjusted life-years (DALYs) in maternal and neonatal disorders between 1990 and 2019 were collected from the 2019 Global Burden of Disease study. Concentration curves and concentration indices were used to summarize the degree of socioeconomic-related inequality. RESULTS: For maternal disorders, the global ASRs of incidence, prevalence, death and DALYs were 2889.4 (95% uncertainty interval (UI), 2562.9-3251.9), 502.9 (95% UI 418.7-598.0), 5.0 (95% UI 4.4-5.8) and 324.9 (95% UI 284.0-369.1) per 100 000 women in 2019, respectively. The ASRs of maternal disorders were all obviously reduced and remained pro-poor from 1990 to 2019. In neonatal disorders, the global ASRs of incidence, prevalence, death and DALYs were 363.3 (95% UI 334.6-396.8), 1239.8 (95% UI 1142.1-1356.7), 29.1 (95% UI 24.8-34.5) and 2828.3 (95% UI 2441.6-3329.6) per 100 000 people in 2019, respectively. The global ASRs of incidence, death and DALYs in neonatal disorders have remained pro-poor. However, the socioeconomic-related fairness in the ASR of neonatal disorder prevalence is being levelled. CONCLUSIONS: The global burden of maternal and neonatal disorders has remained high, and socioeconomic-related inequality (pro-poor) tended not to change between 1990 and 2019.

A Dual-Mechanism Targeted Bioorthogonal Prodrug Therapy.

Bioorthogonal prodrug therapies offer an intriguing two-component system that features enhanced circulating stability and controlled activation on demand. Current strategies often deliver either the prodrug or its complementary activator to the tumor with a monomechanism targeted mechanism, which cannot achieve the desired antitumor efficacy and safety profile. The orchestration of two distinct and orthogonal mechanisms should overcome the hierarchical heterogeneity of solid tumors to improve the delivery efficiency of both components simultaneously for bio-orthogonal prodrug therapies. We herein developed a dual-mechanism targeted bioorthogonal prodrug therapy by integrating two orthogonal, receptor-independent tumor-targeting strategies. We first employed the endogenous albumin transport system to generate the in situ albumin-bound, bioorthogonal-caged doxorubicin prodrug with extended plasma circulation and selective accumulation at the tumor site. We then employed enzyme-instructed self-assembly (EISA) to specifically enrich the bioorthogonal activators within tumor cells. As each targeted delivery mode induced an intrinsic pharmacokinetic profile, further optimization of the administration sequence according to their pharmacokinetics allowed the spatiotemporally controlled prodrug activation on-target and on-demand. Taken together, by orchestrating two discrete and receptor-independent targeting strategies, we developed an all-small-molecule based bioorthogonal prodrug system for dual-mechanism targeted anticancer therapies to maximize therapeutic efficacy and minimize adverse drug reactions for chemotherapeutic agents.

[Efficacy evaluation of 0.05% cyclosporine A and 0.1% tacrolimus eye drops in the treatment of severe dry eye associated with chronic graft-versus-host disease].

Objective: To evaluate the effectiveness and safety of 0.05% cyclosporine A and 0.1% tacrolimus eye drops in treating severe dry eye associated with chronic graft-versus-host disease (cGVHD). Methods: This non-randomized concurrent control trial enrolled 83 eyes from 83 patients with cGVHD-associated severe dry eye. The treatment had two phases. During the initial shock treatment period (0-3 months), 44 patients received 0.05% cyclosporine A eye drops (4 times/day; group A) and 39 patients received 0.1% tacrolimus eye drops (twice/day; group B) alongside basic treatment. In the maintenance treatment period (3-6 months), both groups used 0.05% cyclosporine A eye drops (twice/day) and sodium hyaluronate. Examinations were conducted at 1, 3, and 6 months after treatment initiation, assessing the Ocular Surface Disease Index (OSDI), corneal fluorescein staining (CFS) score, and fluorescein tear break-up time (BUT) for efficacy. visual acuity and intraocular pressure (IOP) were evaluated for safety, and patients' post-medication irritation symptoms were recorded. Results: The study included 52 males and 31 females, aged (28.57±15.67) years. After 1 month of treatment, the CFS score in group A significantly decreased from 10.0 (6.0, 14.0) to 5.0 (3.0, 8.5) (P<0.001). in group B, the CFS score also significantly decreased from 10.0 (6.0, 15.0) to 6.0 (2.0, 10.0), and the BUT increased from 2.0 (1.0, 2.0) s to 2.0 (1.8, 3.3) s (P<0.001). No significant OSDI decrease was observed in either group. No significant differences were found in OSDI, CFS score, and BUT between the two groups. After 3 months, group A showed significant improvement in OSDI, CFS score, and BUT (P<0.05), while group B only demonstrated significant CFS score decrease (P<0.05). OSDI was significantly lower in group A than group B (P<0.05). No significant differences were noted in CFS score and BUT between groups. After 6 months, OSDI, CFS score, and BUT were 18.9 (9.3, 34.2), 7.0 (3.0, 8.5), and 2.0 (1.0, 3.0) s in group A, and 10.9 (3.6, 35.4), 5.5 (2.8, 10.0), and 2.0 (1.0, 10.0) s in group B. In both groups, CFS scores significantly decreased and BUT increased (P<0.05). Visual acuity improved significantly in group A at 1, 3, and 6 months (P<0.05), while no significant changes were seen in group B. Irritation symptoms were transient and self-resolving in both groups. Conclusions: Both 0.05% cyclosporine A and 0.1% tacrolimus eye drops, when combined with local glucocorticoids, exhibited significant anti-inflammatory effects, effectively and safely treating severe dry eye in cGVHD patients. Although the onset of 0.05% cyclosporine A was slower than 0.1% tacrolimus, it offered more stable long-term effects and better symptom improvement.

Near Infrared Light-Triggered Photocatalytic Decaging for Remote-Controlled Spatiotemporal Activation in Living Mice.

Spatiotemporal manipulation of biological processes in living animals using noninvasive, remote-controlled stimuli is a captivating but challenging endeavor. Herein, we present the development of a biocompatible photocatalytic technology termed CAT-NIR, which uses external near infrared light (NIR, 740 nm) to trigger decaging reactions in living mice. The Os(II) terpyridine complex was identified as an efficient NIR photocatalyst for promoting deboronative hydroxylation reactions via superoxide generation in the presence of NIR light, resulting in the deprotection of phenol groups and the release of bioactive molecules under living conditions. The validation of the CAT-NIR system was demonstrated through the NIR-triggered rescue of fluorophores, prodrugs as well as biomolecules ranging from amino acids, peptides to proteins. Furthermore, by combining genetic code expansion and computer-aided screening, CAT-NIR could regulate affibody binding to the cell surface receptor HER2, providing a selective cell tagging technology through external NIR light. In particular, the tissue-penetrating ability of NIR light allowed for facile prodrug activation in living mice, enabling noninvasive, remote-controlled rescue of drug molecules. Given its broad adaptability, this CAT-NIR system may open new opportunities for manipulating the functions of bioactive molecules in living animals using external NIR light with spatiotemporal resolution.

Opening New Avenues in Bioorganic Chemistry: Prof. Chuan He Receives the Tetrahedron Prize.

Prof. Chuan He was awarded the Tetrahedron Prize this year, one of the world's most prestigious prizes in organic chemistry. This In Focus briefly delves into the remarkable work of Prof. Chuan He and explores how his recent accolades underscore his impact on the world of science. His seminal contributions have paved the way for new directions at the interface of organic chemistry and life sciences.

Optical Control of Protein Functions via Genetically Encoded Photocaged Aspartic Acids.

Site-specific protein decaging by light has become an effective approach for in situ manipulation of protein activities in a gain-of-function fashion. Although successful decaging of amino acid side chains of Lys, Tyr, Cys, and Glu has been demonstrated, this strategy has not been extended to aspartic acid (Asp), an essential amino acid residue with a range of protein functions and protein-protein interactions. We herein reported a genetically encoded photocaged Asp and applied it to the photocontrolled manipulation of a panel of proteins including firefly luciferase, kinases (e.g., BRAF), and GTPase (e.g., KRAS) as well as mimicking the in situ phosphorylation event on kinases. As a new member of the increasingly expanded amino acid-decaging toolbox, photocaged Asp may find broad applications for gain-of-function study of diverse proteins as well as biological processes in living cells.

Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes.

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI-COT-Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ -30% ΔF/F0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI-COT-Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI-COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore.

Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site-specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine-based aminoacyl-tRNA synthetase-tRNA pair for a high yield of pore-forming protein. Both molecular dynamics (MD) simulations and single-molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches.

Linking chromatin acylation mark-defined proteome and genome in living cells.

A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and ß-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr's gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the "metabolites-modification-regulation" axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.

[Single-center study of different treatment for advanced or unresectable angiosarcoma patients].

Objective: To evaluate the efficacy and safety of different medical treatment in advanced or unresectable angiosarcoma. Methods: This study was a single-center retrospective clinical study. Fifty-five advanced or unresectable angiosarcoma patients treated in Sun-Yat Sen University Cancer Center from January 2005 to August 2020 were enrolled. There were 34 patients who received first-line doxorubicin-based chemotherapy (doxorubicin group), 12 patients received first-line doxorubicin or liposome doxorubicin plus paclitaxel or albumin bound paclitaxel chemotherapy (combination therapy group), and 4 patients received first-line paclitaxel-based treatment (paclitaxel group). There were 6 patients who received anti-angiogenesis targeted therapy, another 2 patients received anti-PD-1 antibody plus anti-angiogenesis targeted therapy. Targeted therapy and immunotherapy plus targeted therapy included 5 cases of first-line therapy and 3 cases of second-line therapy. The therapeutic effect was evaluated by RECIST 1.1 standard. The adverse reactions were evaluated by CTCAE4.0 standard. Kaplan-Meier survival analysis was evaluated with Log rank test. Cox proportional hazard model was used to analyze the influencing factors. Results: There were 18 patients achieved partial response (PR) in 34 patients in the doxorubicin group, median progression-free survival (mPFS) was 4.5 months, and median overall survival (mOS) was 15 months. Four patients achieved PR in 12 patients in the combination therapy group, mPFS and mOS were 4 months and 19 months. Two patients achieved PR in 4 patients in the paclitaxel group, mPFS and mOS were 3 months and 9 months. However, only 1 in 6 patients achieved PR for anti-angiogenesis targeted therapy, mPFS and mOS were 3 months and 16 months. Two patients who received anti-PD-1 immunotherapy combined with anti-angiogenesis targeted therapy acquired PR for 17 months and more than 16 months. Median PFS (7.5 months) were longer in those with primary liver, lung and spleen angiosarcoma than in those with other primary site (3.0 months, P=0.028). The mOS (20 months) was longer in females than that in males (12 months, P=0.045). Primary tumor site, sex, age and treatment were not independent prognostic factors for angiosarcoma patients (P>0.05). Grade 3-4 cardiac toxicity was found in 2 patients in the combination therapy group. Conclusions: Doxorubicin-based and paclitaxel-based chemotherapy are the most important treatment for advanced angiosarcoma. Potential efficacy for targeted therapy combined with anti-PD-1 immunotherapy are showed in some patients with long duration of response and moderate adverse event.