Structures of AT8 and PHF1 phosphomimetic tau: Insights into the posttranslational modification code of tau aggregation.

The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphosphorylated, suggesting that this posttranslational modification (PTM) may induce tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phosphomimetic full-length tau fibrils assembled without anionic cofactors. We mutated key Ser and Thr residues to Glu in two regions of the protein. One construct contains three Glu mutations at the epitope of the anti-phospho-tau antibody AT8 (AT8-3E tau), whereas the other construct contains four Glu mutations at the epitope of the antibody PHF1 (PHF1-4E tau). Solid-state NMR data show that both phosphomimetic tau mutants form homogeneous fibrils with a single set of chemical shifts. The AT8-3E tau rigid core extends from the R3 repeat to the C terminus, whereas the PHF1-4E tau rigid core spans R2, R3, and R4 repeats. Cryoelectron microscopy data show that AT8-3E tau forms a triangular multi-layered core, whereas PHF1-4E tau forms a triple-stranded core. Interestingly, a construct combining all seven Glu mutations exhibits the same conformation as PHF1-4E tau. Scalar-coupled NMR data additionally reveal the dynamics and shape of the fuzzy coat surrounding the rigid cores. These results demonstrate that specific PTMs induce structurally specific tau aggregates, and the phosphorylation code of tau contains redundancy.

Structural and functional reorganization of inhibitory synapses by activity-dependent cleavage of neuroligin-2.

Recent evidence has demonstrated that the transsynaptic nanoscale organization of synaptic proteins plays a crucial role in regulating synaptic strength in excitatory synapses. However, the molecular mechanism underlying this transsynaptic nanostructure in inhibitory synapses still remains unclear and its impact on synapse function in physiological or pathological contexts has not been demonstrated. In this study, we utilized an engineered proteolysis technique to investigate the effects of acute cleavage of neuroligin-2 (NL2) on synaptic transmission. Our results show that the rapid cleavage of NL2 led to impaired synaptic transmission by reducing both neurotransmitter release probability and quantum size. These changes were attributed to the dispersion of RIM1/2 and GABAA receptors and a weakened spatial alignment between them at the subsynaptic scale, as observed through superresolution imaging and model simulations. Importantly, we found that endogenous NL2 undergoes rapid MMP9-dependent cleavage during epileptic activities, which further exacerbates the decrease in inhibitory transmission. Overall, our study demonstrates the significant impact of nanoscale structural reorganization on inhibitory transmission and unveils ongoing modulation of mature GABAergic synapses through active cleavage of NL2 in response to hyperactivity.

Predicting TCR sequences for unseen antigen epitopes using structural and sequence features.

T-cell receptor (TCR) recognition of antigens is fundamental to the adaptive immune response. With the expansion of experimental techniques, a substantial database of matched TCR-antigen pairs has emerged, presenting opportunities for computational prediction models. However, accurately forecasting the binding affinities of unseen antigen-TCR pairs remains a major challenge. Here, we present convolutional-self-attention TCR (CATCR), a novel framework tailored to enhance the prediction of epitope and TCR interactions. Our approach utilizes convolutional neural networks to extract peptide features from residue contact matrices, as generated by OpenFold, and a transformer to encode segment-based coded sequences. We introduce CATCR-D, a discriminator that can assess binding by analyzing the structural and sequence features of epitopes and CDR3-ß regions. Additionally, the framework comprises CATCR-G, a generative module designed for CDR3-ß sequences, which applies the pretrained encoder to deduce epitope characteristics and a transformer decoder for predicting matching CDR3-ß sequences. CATCR-D achieved an AUROC of 0.89 on previously unseen epitope-TCR pairs and outperformed four benchmark models by a margin of 17.4%. CATCR-G has demonstrated high precision, recall and F1 scores, surpassing 95% in bidirectional encoder representations from transformers score assessments. Our results indicate that CATCR is an effective tool for predicting unseen epitope-TCR interactions. Incorporating structural insights enhances our understanding of the general rules governing TCR-epitope recognition significantly. The ability to predict TCRs for novel epitopes using structural and sequence information is promising, and broadening the repository of experimental TCR-epitope data could further improve the precision of epitope-TCR binding predictions.

Structure of the nonhelical filament of the Alzheimer's disease tau core.

The microtubule-associated protein tau aggregates into neurofibrillary tangles in Alzheimer's disease (AD). The main type of aggregates, the paired helical filaments (PHF), incorporate about 20% of the full-length protein into the rigid core. Recently, cryo-electron microscopy data showed that a protease-resistant fragment of tau (residues 297-391) self-assembles in vitro in the presence of divalent cations to form twisted filaments whose molecular structure resembles that of AD PHF tau [S. Lövestam et al., Elife 11, e76494 (2022)]. To investigate whether this tau construct is uniquely predisposed to this morphology and structure, we fibrillized tau (297-391) under the reported conditions and determined its structure using solid-state NMR spectroscopy. Unexpectedly, the protein assembled predominantly into nontwisting ribbons whose rigid core spans residues 305-357. This rigid core forms a ß-arch that turns at residues 322CGS324. Two protofilaments stack together via a long interface that stretches from G323 to I354. Together, these two protofilaments form a four-layered ß-sheet core whose sidechains are stabilized by numerous polar and hydrophobic interactions. This structure gives insight into the fibril morphologies and molecular conformations that can be adopted by this protease-resistant core of AD tau under different pH and ionic conditions.

Milligram-scale assembly and NMR fingerprint of tau fibrils adopting the Alzheimer's disease fold.

In the Alzheimer's disease (AD) brain, the microtubule-associated protein tau aggregates into paired helical filaments in which each protofilament has a C-shaped conformation. In vitro assembly of tau fibrils adopting this fold is highly valuable for both fundamental and applied studies of AD without requiring patient-brain extracted fibrils. To date, reported methods for forming AD-fold tau fibrils have been irreproducible and sensitive to subtle variations in fibrillization conditions. Here, we describe a route to reproducibly assemble tau fibrils adopting the AD fold on the multi-milligram scale. We investigated the fibrillization conditions of two constructs and found that a tau (297-407) construct that contains four AD phospho-mimetic glutamate mutations robustly formed the C-shaped conformation. 2D and 3D correlation solid-state NMR spectra show a single predominant set of chemical shifts, indicating a single molecular conformation. Negative-stain electron microscopy and cryo-EM data confirm that the protofilament formed by 4E-tau (297-407) adopts the C-shaped conformation, which associates into paired, triple, and quadruple helical filaments. In comparison, NMR spectra indicate that a previously reported construct, tau (297-391), forms a mixture of a four-layered dimer structure and the C-shaped structure, whose populations are sensitive to the environmental conditions. The determination of the NMR chemical shifts of the AD-fold tau opens the possibility for future studies of tau fibril conformations and ligand binding by NMR. The quantitative assembly of tau fibrils adopting the AD fold should facilitate the development of diagnostic and therapeutic compounds that target AD tau.

Hippocampal excitation-inhibition balance underlies the 5-HT2C receptor in modulating depressive behaviours.

The implication of 5-hydroxytryptamine 2C receptor (5-HT2CR) in depression is a topic of debate, and the underlying mechanisms remain largely unclear. We now elucidate hippocampal excitation-inhibition (E/I) balance underlies the regulatory effects of 5-HT2CR in depression. Molecular biological analyses showed that chronic mild stress (CMS) reduced the expression of 5-HT2CR in hippocampus. We revealed that inhibition of 5-HT2CR induced depressive-like behaviors, reduced GABA release and shifted the E/I balance towards excitation in CA3 pyramidal neurons by using behavioral analyses, microdialysis coupled with mass spectrum, and electrophysiological recording. Moreover, 5-HT2CR modulated neuronal nitric oxide synthase (nNOS)-carboxy-terminal PDZ ligand of nNOS (CAPON) interaction through influencing intracellular Ca2+ release, as determined by fiber photometry and coimmunoprecipitation. Notably, disruption of nNOS-CAPON by specific small molecule compound ZLc-002 or AAV-CMV-CAPON-125C-GFP, abolished 5-HT2CR inhibition-induced depressive-like behaviors, as well as the impairment in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly-mediated GABA vesicle release and a consequent E/I imbalance. Importantly, optogenetic inhibition of CA3 GABAergic neurons prevented the effects of AAV-CMV-CAPON-125C-GFP on depressive behaviors in the presence of 5-HT2CR antagonist. Conclusively, our findings disclose the regulatory role of 5-HT2CR in depressive-like behaviors and highlight the hippocampal nNOS-CAPON coupling-triggered E/I imbalance as a pivotal cellular event underpinning the behavioral consequences of 5-HT2CR inhibition.

Distinguishing Different Hydrogen-Bonded Helices in Proteins by Efficient 1H-Detected Three-Dimensional Solid-State NMR.

Helical structures in proteins include not only α-helices but also 310 and π helices. These secondary structures differ in the registry of the C═O···H-N hydrogen bonds, which are i to i + 4 for α-helices, i to i + 3 for 310 helices, and i to i + 5 for π-helices. The standard NMR observable of protein secondary structures are chemical shifts, which are, however, insensitive to the precise type of helices. Here, we introduce a three-dimensional (3D) 1H-detected experiment that measures and assigns CO-HN cross-peaks to distinguish the different types of hydrogen-bonded helices. This hCOhNH experiment combines efficient cross-polarization from CO to HN with 13C, 15N, and 1H chemical shift correlation to detect the relative proximities of the COi-Hi+jN spin pairs. We demonstrate this experiment on the membrane-bound transmembrane domain of the SARS-CoV-2 envelope (E) protein (ETM). We show that the C-terminal five residues of ETM form a 310-helix, whereas the rest of the transmembrane domain have COi-Hi+4N hydrogen bonds that are characteristic of α-helices. This result confirms the recent high-resolution solid-state NMR structure of the open state of ETM, which was solved in the absence of explicit hydrogen-bonding restraints. This C-terminal 310 helix may facilitate proton and calcium conduction across the hydrophobic gate of the channel. This hCOhNH experiment is generally applicable and can be used to distinguish not only different types of helices but also different types of ß-strands and other hydrogen-bonded conformations in proteins.

Is Short-Course Antibiotic Therapy Suitable for Pseudomonas aeruginosa Bloodstream Infections in Onco-hematology Patients With Febrile Neutropenia? Results of a Multi-institutional Analysis.

BACKGROUND: Several studies have suggested that short-course antibiotic therapy was effective in Pseudomonas aeruginosa (PA) bloodstream infections (BSI) in immunocompetent patients. But similar studies in patients with hematological malignancies were rare. METHODS: This cohort study included onco-hematology patients at 2 hematology centers in China. Inverse probability of treatment weighting was used to balance the confounding factors. Multivariate regression model was used to evaluate the effect of short-course antibiotic therapy on clinical outcomes. RESULTS: In total, 434 patients met eligibility criteria (short-course, 7-11 days, n = 229; prolonged, 12-21 days, n = 205). In the weighted cohort, the univariate and multivariate analysis indicated that short course antibiotic therapy had similar outcomes to the prolonged course. The recurrent PA infection at any site or mortality within 30 days of completing therapy occurred in 8 (3.9%) patients in the short-course group and in 10 (4.9%) in the prolonged-course group (P = .979). The recurrent infection within 90 days occurred in 20 (9.8%) patients in the short-course group and in 13 (6.3%) patients in the prolonged-course group (P = .139), and the recurrent fever within 7 days occurred in 17 (8.3%) patients in the short-course group and in 15 (7.4%) in the prolonged-course group (P = .957). On average, patients who received short-course antibiotic therapy spent 3.3 fewer days in the hospital (P < .001). CONCLUSIONS: In the study, short-course therapy was non-inferior to prolonged-course therapy in terms of clinical outcomes. However, due to its biases and limitations, further prospective randomized controlled trials are needed to generalize our findings.

Oncogenic and immunological roles of RACGAP1 in pan-cancer and its potential value in nasopharyngeal carcinoma.

A particular GTPase-activating protein called RACGAP1 is involved in apoptosis, proliferation, invasion, metastasis, and drug resistance in a variety of malignancies. Nevertheless, the role of RACGAP1 in pan-cancer was less studied, and its value of the expression and prognostic of nasopharyngeal carcinoma (NPC) has not been explored. Hence, the goal of this study was to investigate the oncogenic and immunological roles of RACGAP1 in various cancers and its potential value in NPC. We comprehensively analyzed RACGAP1 expression, prognostic value, function, methylation levels, relationship with immune cells, immune infiltration, and immunotherapy response in pan-cancer utilizing multiple databases. The results discovered that RACGAP1 expression was elevated in most cancers and suggested poor prognosis, which could be related to the involvement of RACGAP1 in various cancer-related pathways such as the cell cycle and correlated with RACGAP1 methylation levels, immune cell infiltration and reaction to immunotherapy, and chemoresistance. RACGAP1 could inhibit anti-tumor immunity and immunotherapy responses by fostering immune cell infiltration and cytotoxic T lymphocyte dysfunction. Significantly, we validated that RACGAP1 mRNA and protein were highly expressed in NPC. The Gene Expression Omnibus database revealed that elevated RACGAP1 expression was associated with shorter PFS in patients with NPC, and RACGAP1 potentially influenced cell cycle progression, DNA replication, metabolism, and immune-related pathways, resulting in the recurrence and metastasis of NPC. This study indicated that RACGAP1 could be a potential biomarker in pan-cancer and NPC.

Altering the substitution and cross-linking of glucuronoarabinoxylans affects cell wall architecture in Brachypodium distachyon.

The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both BdXAX1A and GUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials.

Identification and genetic characterization of a recently identified enterovirus C116 in China.

Enterovirus C116 (EV-C116) is a new member of the enterovirus C group which is closely associated with several infectious diseases. Although sporadic studies have detected EV-C116 in clinical samples worldwide, there is currently limited information available. In this study, two EV-C-positive fecal specimens were detected in apparently healthy children, which harbored low abundance, through meta-transcriptome sequencing. Based on the prototypes of several EV-Cs, two lineages were observed. Lineage 1 included many types that could not cause EV-like cytopathic effect in cell culture. Three genogroups of EV-C116 were divided in the maximum likelihood tree, and the two strains in this study (XZ2 and XZ113) formed two different lineages, suggesting that EV-C116 still diffuses worldwide. Obvious inter-type recombination events were observed in the XZ2 strain, with CVA22 identified as a minor donor. However, another strain (XZ113) underwent different recombination situations, highlighting the importance of recombination in the formation of EV-Cs biodiversity. The EV-C116 strains could propagate in rhabdomyosarcoma cell cultures at low titer; however, EV-like cytopathic effects were not observed. HEp-2, L20B, VERO, and 293T cell lines did not provide an appropriate environment for EV-C116 growth. These results challenge the traditional recognition of the uncultured nature of EV-C116 strains and explain the difficulty of clinical detection.

miR-30e-5p regulates leukemia stem cell self-renewal through the Cyb561/ROS signaling pathway.

Leukemia stem cells (LSC) represent a crucial and rare subset of cells present in acute myeloid leukemia (AML); they play a pivotal role in the initiation, maintenance, and relapse of this disease. Targeting LSC holds great promise for preventing AML relapse and improving long-term outcomes. However the precise molecular mechanisms governing LSC self-renewal are still poorly understood. Here, we present compelling evidence that the expression of miR-30e-5p, a potential tumor-suppressive microRNA, is significantly lower in AML samples than in healthy bone marrow samples. Forced expression of miR- 30e effectively inhibits leukemogenesis, impairs LSC self-renewal, and delays leukemia progression. Mechanistically, Cyb561 acts as a direct target of miR-30e-5p in LSC, and its deficiency restricts the self-renewal of LSC by activating reactive oxygen series signaling and markedly prolongs recipients' survival. Moreover, genetic or pharmacological overexpression of miR-30e-5p or knockdown of Cyb561 suppresses the growth of human AML cells. In conclusion, our findings establish the crucial role of the miR-30e-5p/Cyb561/ROS axis in finely regulating LSC self-renewal, highlighting Cyb561 as a potential therapeutic target for LSC-directed therapies.

Genetic and prognostic analysis of blastoid and pleomorphic mantle cell lymphoma: a multicenter analysis in China.

Blastoid or pleomorphic mantle cell lymphoma (B/P-MCL) is characterized by high invasiveness and unfavorable outcomes, which is still a challenge for treating MCL. This retrospective study was performed to comprehensively analyze the clinical, genomic characteristics and treatment options of patients with B/PMCL from multicenter in China. Data were obtained from 693 patients with B/PMCL from three centers in China between April 1999 and December 2019. Seventy-four patients with BMCL (n = 43) or PMCL (n = 31) were included in the analysis. The median age of the cohort was 60.0 years with a male-to-female ratio of 2.89:1. The 3-year progression-free survival (PFS) and overall survival (OS) rates were 44.1% and 46.0%, respectively. Mutations of TP53, ATM, NOTCH1, NOTCH2, NSD2, SMARCA4, CREBBP, KMT2D, FAT1, and TRAF2 genes were the most common genetic changes in B/P-MCL. Progression of disease within 12 months (POD12) could independently predict the poor prognosis of patients with blastoid and pleomorphic variants. Patients with POD12 carried a distinct mutation profile (TP53, SMARCA4, NSD2, NOTCH2, KMT2D, PTPRD, CREBBP, and CDKN2A mutations) compared to patients with non-POD12. First-line high-dose cytosine arabinoside exposure obtained survival benefits in these populations, and BTKi combination therapy as the front-line treatment had somewhat improvement in survival with no significant difference in the statistic. In conclusion, B/P-MCL had inferior outcomes and a distinct genomic profile. Patients with POD12 displayed a distinct mutation profile and a poor prognosis. New therapeutic drugs and clinical trials for B/P-MCL need to be further explored.

Efficacy of chidamide maintenance therapy versus autologous stem cell transplantation versus observation as a post-remission choice in the survival of adult patients with peripheral T-cell lymphoma: Post hoc analysis of a prospective, multicenter, phase 2 study in China.

In this prospective, multicenter, Phase 2 clinical trial (NCT02987244), patients with peripheral T-cell lymphomas (PTCLs) who had responded to first-line chemotherapy with cyclophosphamide, doxorubicin or epirubicin, vincristine or vindesine, etoposide, and prednisone (Chi-CHOEP) were treated by autologous stem cell transplantation (ASCT) or with chidamide maintenance or observation. A total of 85 patients received one of the following interventions: ASCT (n = 15), chidamide maintenance (n = 44), and observation (n = 26). estimated 3 PFS and OS rates were 85.6%, 80.8%, and 49.4% (P = 0.001). The two-year OS rates were 85.6%, 80.8%, and 69.0% (P = 0.075).The ASCT and chidamide maintenance groups had significantly better progression-free survival (PFS) than the observation group (P = 0.001, and P = 0.01, respectively). The overall survival (OS) differed significantly between the chidamide maintenance group and the observation group ( P = 0.041). The multivariate and propensity score matching analyses for PFS revealed better outcomes in the subjects in the chidamide maintenance than observation groups (P = 0.02). The ASCT and chidamide maintenance groups had significant survival advantages over the observation group. In the post-remission stage of the untreated PTCL patients, single-agent chidamide maintenance demonstrated superior PFS and better OS than observation. Our findings highlight the potential benefit of chidamide in this patient subset, warranting further investigation through larger prospective trials. Clinical trial registration: clinicaltrial.gov, NCT02987244. Registered 8 December 2016, http://www.clinicaltrials.gov/ct2/show/NCT02987244 .

Children with severe neurological symptoms associated with SARS-CoV-2 infection during Omicron pandemic in China.

BACKGROUND: To analyze the clinical characteristics and outcomes of children with severe neurological symptoms associated with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection during the Omicron pandemic in China. METHODS: This study used a questionnaire to obtain data from pediatric intensive care unit (PICU) centers in seven tertiary hospitals in Northeast China from December 1, 2022, to January 31, 2023. RESULTS: A total of 255 patients were confirmed to have SARS-CoV-2 infection, and 45 patients (17.65 %) were included in this study. Of these, seven (15.6%) patients died, and the median time from admission to death was 35 h (IQR, 14-120 h). Twenty (52.6%) survivors experienced neurological sequelae. Patients with platelet counts lower than 100 × 109/L had a higher incidence of complications such as multiple organ dysfunction, mechanical ventilation rate, and mortality. Cranial magnetic resonance imaging (MRI) always reveals cerebral tissue edema, with some severe lesions forming a softening site. CONCLUSION: Children infected with SARS-CoV-2 often exhibit severe neurological symptoms, and in some cases, they may rapidly develop malignant cerebral edema or herniation, leading to a fatal outcome. An early decrease in platelet count may associated with an unfavorable prognosis. IMPACT: Since early December 2022, China has gradually adjusted its prevention and control policy of SARS-CoV-2; Omicron outbreaks have occurred in some areas for a relatively short period. Due to the differences in ethnicity, endemic strains and vaccination status, there was a little difference from what has been reported about children with SARS-CoV-2 infection with severe neurological symptoms in abroad. This is the first multicenter clinical study in children with nervous system involvement after acute SARS-CoV-2 infection in China, and helpful for pediatricians to have a more comprehensive understanding of the clinical symptoms and prognosis of such disease.

Blockade of endothelial adenosine receptor 2 A suppresses atherosclerosis in vivo through inhibiting CREB-ALK5-mediated endothelial to mesenchymal transition.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and morbidity and mortality rates continue to rise. Atherosclerosis constitutes the principal etiology of CVDs. Endothelial injury, inflammation, and dysfunction are the initiating factors of atherosclerosis. Recently, we reported that endothelial adenosine receptor 2 A (ADORA2A), a G protein-coupled receptor (GPCR), plays critical roles in neovascularization disease and cerebrovascular disease. However, the precise role of endothelial ADORA2A in atherosclerosis is still not fully understood. Here, we showed that ADORA2A expression was markedly increased in the aortic endothelium of humans with atherosclerosis or Apoe-/- mice fed a high-cholesterol diet. In vivo studies unraveled that endothelial-specific Adora2a deficiency alleviated endothelial-to-mesenchymal transition (EndMT) and prevented the formation and instability of atherosclerotic plaque in Apoe-/- mice. Moreover, pharmacologic inhibition of ADORA2A with KW6002 recapitulated the anti-atherogenic phenotypes observed in genetically Adora2a-deficient mice. In cultured human aortic endothelial cells (HAECs), siRNA knockdown of ADORA2A or KW6002 inhibition of ADORA2A decreased EndMT, whereas adenoviral overexpression of ADORA2A induced EndMT. Mechanistically, ADORA2A upregulated ALK5 expression via a cAMP/PKA/CREB axis, leading to TGFß-Smad2/3 signaling activation, thereby promoting EndMT. In conclusion, these findings, for the first time, demonstrate that blockade of ADORA2A attenuated atherosclerosis via inhibition of EndMT induced by the CREB1-ALK5 axis. This study discloses a new link between endothelial ADORA2A and EndMT and indicates that inhibiting endothelial ADORA2A could be an effective novel strategy for the prevention and treatment of atherosclerotic CVDs.

Safety, tolerability, pharmacokinetic, pharmacodynamic and immunogenicity profiles of Exendin-4-IgG4-Fc in healthy subjects: A phase 1, single-centre, randomized, double-blind, dose escalation study.

AIM: Novel long-acting drugs for type 2 diabetes mellitus may optimize patient compliance and glycaemic control. Exendin-4-IgG4-Fc (E4F4) is a long-acting glucagon-like peptide-1 receptor agonist. This first-in-human study investigated the safety, tolerability, pharmacokinetic, pharmacodynamic and immunogenicity profiles of a single subcutaneous injection of E4F4 in healthy subjects. METHODS: This single-centre, randomized, double-blind, placebo-controlled phase 1 clinical trial included 96 subjects in 10 sequential cohorts that were provided successively higher doses of E4F4 (0.45, 0.9, 1.8, 3.15, 4.5, 6.3, 8.1, 10.35, 12.6 and 14.85 mg) or placebo (ChinaDrugTrials.org.cn: ChiCTR2100049732). The primary endpoint was safety and tolerability of E4F4. Secondary endpoints were pharmacokinetic, pharmacodynamic and immunogenicity profiles of E4F4. Safety data to day 15 after the final subject in a cohort had been dosed were reviewed before commencing the next dose level. RESULTS: E4F4 was safe and well tolerated among healthy Chinese participants in this study. There was no obvious dose-dependent relationship between frequency, severity or causality of treatment-emergent adverse events. Cmax and area under the curve of E4F4 were dose proportional over the 0.45-14.85 mg dose range. Median Tmax and t1/2 ranged from 146 to 210 h and 199 to 252 h, respectively, across E4F4 doses, with no dose-dependent trends. For the intravenous glucose tolerance test, area under the curve of glucose in plasma from time 0 to 180 min showed a dose-response relationship in the 1.8-10.35 mg dose range, with an increased response at the higher doses. CONCLUSION: E4F4 exhibited an acceptable safety profile and linear pharmacokinetics in healthy subjects. The recommended phase 2 dose is 4.5-10.35 mg once every 2 weeks.

Surf4 (Surfeit Locus Protein 4) Deficiency Reduces Intestinal Lipid Absorption and Secretion and Decreases Metabolism in Mice.

BACKGROUND: Postprandial dyslipidemia is a causative risk factor for cardiovascular disease. The majority of absorbed dietary lipids are packaged into chylomicron and then delivered to circulation. Previous studies showed that Surf4 (surfeit locus protein 4) mediates very low-density lipoprotein secretion from hepatocytes. Silencing hepatic Surf4 markedly reduces the development of atherosclerosis in different mouse models of atherosclerosis without causing hepatic steatosis. However, the role of Surf4 in chylomicron secretion is unknown. METHODS: We developed inducible intestinal-specific Surf4 knockdown mice (Surf4IKO) using Vil1Cre-ERT2 and Surf4flox mice. Metabolic cages were used to monitor mouse metabolism. Enzymatic kits were employed to measure serum and tissue lipid levels. The expression of target genes was detected by qRT-PCR and Western Blot. Transmission electron microscopy and radiolabeled oleic acid were used to assess the structure of enterocytes and intestinal lipid absorption and secretion, respectively. Proteomics was performed to determine changes in protein expression in serum and jejunum. RESULTS: Surf4IKO mice, especially male Surf4IKO mice, displayed significant body weight loss, increased mortality, and reduced metabolism. Surf4IKO mice exhibited lipid accumulation in enterocytes and impaired fat absorption and secretion. Lipid droplets and small lipid vacuoles were accumulated in the cytosol and the endoplasmic reticulum lumen of the enterocytes of Surf4IKO mice, respectively. Surf4 colocalized with apoB and co-immunoprecipitated with apoB48 in differentiated Caco-2 cells. Intestinal Surf4 deficiency also significantly reduced serum triglyceride, cholesterol, and free fatty acid levels in mice. Proteomics data revealed that diverse pathways were altered in Surf4IKO mice. In addition, Surf4IKO mice had mild liver damage, decreased liver size and weight, and reduced hepatic triglyceride levels. CONCLUSIONS: Our findings demonstrate that intestinal Surf4 plays an essential role in lipid absorption and chylomicron secretion and suggest that the therapeutic use of Surf4 inhibition requires highly cell/tissue-specific targeting.

Clinicopathological characteristics and prognosis of Epstein-Barr virus-associated gastric cancer.

OBJECTIVE: Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct molecular subtype of gastric cancer (GC). At present, the clinical characteristics and prognostic implications of EBV infection and the potential clinical benefits of immune checkpoint blockade in GC remain to be clarified. Hence, this study was designed to analyze the clinical and pathological characteristics of GC patients with varying EBV infection states and compare their overall survival (OS). METHODS: A retrospective study was performed on 1031 consecutive GC patients who underwent gastrectomy at the Affiliated Hospital of Xuzhou Medical University from February 2018 to November 2022. EBV-encoded RNA (EBER) in situ hybridization (ISH) was used for EBV assessment, and immunohistochemical staining was used for evaluation of human epidermal growth factor receptor 2 (HER2), programmed death ligand 1 (PD-L1), and Ki67 expression. EBVaGC was defined as tumors with EBV positivity. In addition, EBV-negative GC (EBVnGC) patients were matched with EBVaGC patients based on seven clinicopathological parameters (age, gender, anatomic subsite, tumor size, Lauren classification, degree of differentiation, and tumor-node-metastasis [TNM] stage). The correlations of clinical features with HER2, PD-L1, and Ki67 expression were evaluated statistically. The survival of patients was assessed through medical records, telephone, or WeChat communication, and prognostic analysis was performed using the logrank test as well as univariable and multivariable regression analysis. RESULTS: Out of 1031 GC patients tested, 35 (3.4%) were diagnosed with EBVaGC. Notably, the EBVaGC group exhibited a distinct predominance of males and younger patients, significantly higher Ki67 and PD-L1 expression levels, and a lower prevalence of pericancerous nerve invasion than the EBVnGC group (P < 0.01). In the 35 EBVaGC cases, Ki67 expression was negatively correlated with age (P < 0.05), suggesting that a younger onset age was associated with higher Ki67 expression. In addition, PD-L1 expression was correlated with the degree of differentiation, T-stage, and clinical stage of the patient. Furthermore, PD-L1 expression was elevated in tumors with lower differentiation or at later stages (P < 0.05). Using univariate analysis, Ki67, PD-L1, and clinical stage were identified as significant factors influencing the overall survival (OS) of EBVaGC patients (P < 0.05). Moreover, multivariate survival analysis revealed that clinical stage and Ki67 expression were independent risk factors for the OS of the patients (P < 0.05), and the three-year OS rate of EBVaGC patients was 64.2%. CONCLUSION: EBV-ISH is a practical and valuable method to identify EBVaGC. Owing to its unique etiological, pathological, and clinical characteristics, patients with EBVaGC might benefit from immune checkpoint blockade therapy.

From Angstroms to Nanometers: Measuring Interatomic Distances by Solid-State NMR.

Internuclear distances represent one of the main structural constraints in molecular structure determination using solid-state NMR spectroscopy, complementing chemical shifts and orientational restraints. Although a large number of magic-angle-spinning (MAS) NMR techniques have been available for distance measurements, traditional 13C and 15N NMR experiments are inherently limited to distances of a few angstroms due to the low gyromagnetic ratios of these nuclei. Recent development of fast MAS triple-resonance 19F and 1H NMR probes has stimulated the design of MAS NMR experiments that measure distances in the 1-2 nm range with high sensitivity. This review describes the principles and applications of these multiplexed multidimensional correlation distance NMR experiments, with an emphasis on 19F- and 1H-based distance experiments. Representative applications of these long-distance NMR methods to biological macromolecules as well as small molecules are reviewed.