Lactyllysine Esterification Enables Efficient Lactylprotein Expression via Genetic Code Expansion and Supports Functional Proteomics Studies.

Lysine lactylation has recently been discovered and demonstrated to be an essential player in immunity, cancer and neurodegenerative diseases. Genetic code expansion (GCE) technique is powerful in uncovering lactylation functions, since it allows site-specific incorporation of lactyllysine (Klac) into proteins of interest (POIs) in living cells. However, the inefficient uptake of Klac into cells, due to its high hydrophilicity, results in limited expression of lactylated POIs. To address this challenge, here we designed esterified Klac derivatives, exemplified by ethylated Klac (KlacOEt), to enhance Klac's lipophilicity and improve its cellular uptake. The expression level of site-specifically lactylated POIs was doubled using KlacOEt in both Escherichia coli and HEK293T cells. Immunoprecipitation mass spectrometry analysis verified the significantly increased yield of the precisely lactylated fructose-bisphosphate aldolase A using KlacOEt. Furthermore, in conjunction with the Target Responsive Accessibility Profiling approach, we found that lactylation at ALDOA-K147 altered the protein's conformation, which may explain the lactylation-induced reduction in enzyme activity. Together, we demonstrate that, through enhancing the yield of lactylated proteins with Klac esters via GCE, we are able to site-specifically reveal the effects of lactylation on POIs' interactions, conformations and activities using a suite of functional proteomics and biochemical tools.

HBeeID: a molecular tool that identifies honey bee subspecies from different geographic populations.

BACKGROUND: Honey bees are the principal commercial pollinators. Along with other arthropods, they are increasingly under threat from anthropogenic factors such as the incursion of invasive honey bee subspecies, pathogens and parasites. Better tools are needed to identify bee subspecies. Genomic data for economic and ecologically important organisms is increasing, but in its basic form its practical application to address ecological problems is limited. RESULTS: We introduce HBeeID a means to identify honey bees. The tool utilizes a knowledge-based network and diagnostic SNPs identified by discriminant analysis of principle components and hierarchical agglomerative clustering. Tests of HBeeID showed that it identifies African, Americas-Africanized, Asian, and European honey bees with a high degree of certainty even when samples lack the full 272 SNPs of HBeeID. Its prediction capacity decreases with highly admixed samples. CONCLUSION: HBeeID is a high-resolution genomic, SNP based tool, that can be used to identify honey bees and screen species that are invasive. Its flexible design allows for future improvements via sample data additions from other localities.

Emerging mechanisms of non-alcoholic steatohepatitis and novel drug therapies.

Non-alcoholic fatty liver disease (NAFLD) has become a leading cause of chronic liver disease globally. It initiates with simple steatosis (NAFL) and can progress to the more severe condition of non-alcoholic steatohepatitis (NASH). NASH often advances to end-stage liver diseases such as liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Notably, the transition from NASH to end-stage liver diseases is irreversible, and the precise mechanisms driving this progression are not yet fully understood. Consequently, there is a critical need for the development of effective therapies to arrest or reverse this progression. This review provides a comprehensive overview of the pathogenesis of NASH, examines the current therapeutic targets and pharmacological treatments, and offers insights for future drug discovery and development strategies for NASH therapy.

Reciprocal relations between future time perspective and academic achievement among adolescents: A four-wave longitudinal study.

INTRODUCTION: Adolescents' academic achievement is closely associated with their future time perspective. However, the reciprocal nature of this relationship remains ambiguous due to a lack of longitudinal studies. This study investigated the developmental trajectories of future time perspective and academic achievement among adolescents, as well as reciprocal relations between future time perspective and academic achievement. METHODS: Between 2017 and 2019, we collected 373 adolescents' (baseline Mage = 14.48, SD = 1.90; 49% girls) future time perspective and academic achievement four times from Henan and Hunan Province, China. Each is separated by a 6-month interval. RESULTS: Chinese adolescents' future time perspective was relatively stable. Regarding academic achievement, two distinct developmental trajectories of academic achievement were identified (i.e., high positive growth class and low negative growth class). Those who excel tended to experience an upward trajectory, while those with poorer grades continued to experience a downward trajectory. In the high positive growth class, the intercept of future time perspective was positively correlated with the rate of academic achievement growth, whereas, in the low negative growth class, it negatively predicted the rate of academic achievement decline. More importantly, reciprocal relations existed between future time perspective and academic achievement. CONCLUSIONS: Adolescents' future time perspective may serve as a protective factor for academic achievement, while high academic achievement may also benefit future time perspective. Interventions to enhance academic achievement should prioritize cultivating adolescents' future perspectives. Additionally, preventing the adverse consequences of subpar academic achievement on future time perspective is imperative.

Avian Influenza A(H5N1) Virus among Dairy Cattle, Texas, USA.

During March and April 2024, we studied dairy cattle specimens from a single farm in Texas, USA, using multiple molecular, cell culture, and next-generation sequencing pathogen detection techniques. Here, we report evidence that highly pathogenic avian influenza A(H5N1) virus strains of clade 2.3.4.4b were the sole cause of this epizootic.

Blocking cGAS-STING pathway promotes post-stroke functional recovery in an extended treatment window via facilitating remyelination.

BACKGROUND: Ischemic stroke is a major cause of worldwide death and disability, with recombinant tissue plasminogen activator being the sole effective treatment, albeit with a limited treatment window. The cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) pathway is emerging as the major DNA-sensing pathway to invoke immune responses in neuroinflammatory disorders. METHODS: By performing a series of neurobehavioral assessments, electrophysiological analysis, high-throughput sequencing, and cell-based assays based on the transient middle cerebral artery occlusion (tMCAO) mouse stroke model, we examined the effects and underlying mechanisms of genetic and pharmacological inhibition of the cGAS-STING pathway on long-term post-stroke neurological functional outcomes. FINDINGS: Blocking the cGAS-STING pathway, even 3 days after tMCAO, significantly promoted functional recovery in terms of white matter structural and functional integrity as well as sensorimotor and cognitive functions. Mechanistically, the neuroprotective effects via inhibiting the cGAS-STING pathway were contributed not only by inflammation repression at the early stage of tMCAO but also by modifying the cell state of phagocytes to facilitate remyelination at the sub-acute phase. The activation of the cGAS-STING pathway significantly impeded post-stroke remyelination through restraining myelin debris uptake and degradation and hindering oligodendrocyte differentiation and maturation. CONCLUSIONS: Manipulating the cGAS-STING pathway has an extended treatment window in promoting long-term post-stroke functional recovery via facilitating remyelination in a mouse stroke model. Our results highlight the roles of the cGAS-STING pathway in aggregating stroke pathology and propose a new way for improving functional recovery after ischemic stroke. FUNDING: This work was primarily funded by the National Key R&D Program of China.

Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Hepatocyte-specific CCAAT/enhancer binding protein α restricts liver fibrosis progression.

Metabolic dysfunction-associated steatohepatitis (MASH) - previously described as nonalcoholic steatohepatitis (NASH) - is a major driver of liver fibrosis in humans, while liver fibrosis is a key determinant of all-cause mortality in liver disease independent of MASH occurrence. CCAAT/enhancer binding protein α (CEBPA), as a versatile ligand-independent transcriptional factor, has an important function in myeloid cells, and is under clinical evaluation for cancer therapy. CEBPA is also expressed in hepatocytes and regulates glucolipid homeostasis; however, the role of hepatocyte-specific CEBPA in modulating liver fibrosis progression is largely unknown. Here, hepatic CEBPA expression was found to be decreased during MASH progression both in humans and mice, and hepatic CEBPA mRNA was negatively correlated with MASH fibrosis in the human liver. CebpaΔHep mice had markedly enhanced liver fibrosis induced by a high-fat, high-cholesterol, high-fructose diet or carbon tetrachloride. Temporal and spatial hepatocyte-specific CEBPA loss at the progressive stage of MASH in CebpaΔHep,ERT2 mice functionally promoted liver fibrosis. Mechanistically, hepatocyte CEBPA directly repressed Spp1 transactivation to reduce the secretion of osteopontin, a fibrogenesis inducer of hepatic stellate cells. Forced hepatocyte-specific CEBPA expression reduced MASH-associated liver fibrosis. These results demonstrate an important role for hepatocyte-specific CEBPA in liver fibrosis progression, and may help guide the therapeutic discoveries targeting hepatocyte CEBPA for the treatment of liver fibrosis.

Temporal Asymmetry of Pleasant and Unpleasant Feelings Among Chinese Adolescents.

Previous studies have shown that anticipation induces more emotions than retrospection, known as temporal emotion asymmetry. However, the majority of previous studies have been confined to Western contexts. Eastern populations tend to emphasize the past more than their Western counterparts and may exhibit distinct forms of temporal emotion asymmetry. Therefore, we conducted an investigation involving Chinese adolescents. Our research encompassed two experiments, investigating Chinese adolescents' temporal emotion asymmetry from a self-perspective (Experiment 1; N = 124) and an other-perspective (Experiment 2; N = 162). Participants were prompted to retrospect and anticipate events that elicited pleasant or unpleasant feelings. The results revealed that, whether from a self-perspective or an other-perspective, retrospection of past positive events elicited greater pleasure than the anticipation of future positive events. However, concerning adverse events, under a self-perspective, anticipation induced more displeasure than retrospection (Experiment 1); under an other-perspective, retrospection induced more displeasure than anticipation (Experiment 2). Our findings provide some support for the construal level theory, fading affect bias, and mobilization-minimization hypothesis of event cognition. Based on these results, retrospection seems to be a potential means for regulating the emotions of Chinese adolescents.

Ginsenosides retard atherogenesis via remodelling host-microbiome metabolic homeostasis.

BACKGROUND AND PURPOSE: Panax ginseng is widely applied in the adjuvant treatment of cardiometabolic diseases in clinical practice without clear mechanisms. This study aims to clearly define the efficacy and underlying mechanism of P. ginseng and its active components in protecting against atherosclerosis. EXPERIMENTAL APPROACH: The anti-atherogenic efficacy of total ginseng saponin extract (TGS) and its components was evaluated on Ldlr-/- mice. Gut microbial structure was analysed by 16S rRNA sequencing and PCR. Bile acid profiles were revealed using targeted metabolomics with LC-MS/MS analysis. The contribution of gut microbiota to atherosclerosis was assessed by co-housing experiments. KEY RESULTS: Ginsenoside Rb1, representing protopanaxadiol (PPD)-type saponins, increased intestinal Lactobacillus abundance, resulting in enhanced bile salt hydrolase (BSH) activity to promote intestinal conjugated bile acid hydrolysis and excretion, followed by suppression of enterohepatic farnesoid X receptor (FXR)-fibroblast growth factor 15 (FGF15) signal, and thereby increased cholesterol 7α-hydroxylase (CYP7A1) transcriptional expression and facilitated metabolic elimination of cholesterol. Synergistically, protopanaxatriol (PPT)-type saponins, represented by ginsenoside Rg1, protected against atherogenesis-triggered gut leak and metabolic endotoxaemia. Ginsenoside Rg1 directly induced mucin production to nutritionally maintain Akkermansia muciniphila, which reciprocally inhibited gut permeation. Rb1/Rg1 combination, rather than a single compound, can largely mimic the holistic efficacy of TGS in protecting Ldlr-/- mice from atherogenesis. CONCLUSION AND IMPLICATIONS: Our study provides strong evidence supporting TGS and ginsenoside Rb1/Rg1 combinations as effective therapies against atherogenesis, via targeting different signal nodes by different components and may provide some elucidation of the holistic mode of herbal medicines.

HERC5-catalyzed ISGylation potentiates cGAS-mediated innate immunity.

The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) is essential to elicit type I interferon cascade response; thus, the activity of cGAS must be strictly regulated to boost the antiviral innate immunity. Here, we report that cGAS is responsible for the DNA-induced ISG15 conjugation system. The E3 HERC5 catalyzes the ISGylation of cytoplasmic cGAS at lysine 21, 187, 219, and 458, whereas Ubl carboxy-terminal hydrolase 18 removes the ISGylation of cGAS. The interaction of cGAS and HERC5 depends on the cGAS C-terminal domain and the RRC1-4 and RRC1-5 domains of HERC5. Mechanically, HERC5-catalyzed ISGylation promotes DNA-induced cGAS oligomerization and enhances cGAS enzymatic activity. Deficiency of ISGylation attenuates the downstream inflammatory gene expression induced by the cGAS-STING axis and the antiviral ability in mouse and human cells. Mice deficient in Isg15 or Herc6 are more vulnerable to herpes simplex virus 1 infection. Collectively, our study shows a positive feedback regulation of the cGAS-mediated innate immune pathway by ISGylation.

Discovery of proqodine A derivatives with antitumor activity targeting NAD(P)H: quinone oxidoreductase 1 and nicotinamide phosphoribosyltransferase.

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavin protease highly expressed in various cancer cells. NQO1 catalyzes a futile redox cycle in substrates, leading to substantial reactive oxygen species (ROS) production. This ROS generation results in extensive DNA damage and elevated poly (ADP-ribose) polymerase 1 (PARP1)-mediated consumption of nicotinamide adenine dinucleotide (NAD+), ultimately causing cell death. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage synthesis pathway, emerges as a critical target in cancer therapy. The concurrent inhibition of NQO1 and NAMPT triggers hyperactivation of PARP1 and intensive NAD+ depletion. In this study, we designed, synthesized, and assessed a novel series of proqodine A derivatives targeting both NQO1 and NAMPT. Among these, compound T8 demonstrated potent antitumor properties. Specifically, T8 selectively inhibited the proliferation of MCF-7 cells and induced apoptosis through mechanisms dependent on both NQO1 and NAMPT. This discovery offers a promising new molecular entity for advancing anticancer research.

Psychological stress-induced microbial metabolite indole-3-acetate disrupts intestinal cell lineage commitment.

The brain and gut are intricately connected and respond to various stimuli. Stress-induced brain-gut communication is implicated in the pathogenesis and relapse of gut disorders. The mechanism that relays psychological stress to the intestinal epithelium, resulting in maladaptation, remains poorly understood. Here, we describe a stress-responsive brain-to-gut metabolic axis that impairs intestinal stem cell (ISC) lineage commitment. Psychological stress-triggered sympathetic output enriches gut commensal Lactobacillus murinus, increasing the production of indole-3-acetate (IAA), which contributes to a transferrable loss of intestinal secretory cells. Bacterial IAA disrupts ISC mitochondrial bioenergetics and thereby prevents secretory lineage commitment in a cell-intrinsic manner. Oral α-ketoglutarate supplementation bolsters ISC differentiation and confers resilience to stress-triggered intestinal epithelial injury. We confirm that fecal IAA is higher in patients with mental distress and is correlated with gut dysfunction. These findings uncover a microbe-mediated brain-gut pathway that could be therapeutically targeted for stress-driven gut-brain comorbidities.

A Gpr35-tuned gut microbe-brain metabolic axis regulates depressive-like behavior.

Gene-environment interactions shape behavior and susceptibility to depression. However, little is known about the signaling pathways integrating genetic and environmental inputs to impact neurobehavioral outcomes. We report that gut G-protein-coupled receptor, Gpr35, engages a microbe-to-brain metabolic pathway to modulate neuronal plasticity and depressive behavior in mice. Psychological stress decreases intestinal epithelial Gpr35, genetic deletion of which induces depressive-like behavior in a microbiome-dependent manner. Gpr35-/- mice and individuals with depression have increased Parabacteroides distasonis, and its colonization to wild-type mice induces depression. Gpr35-/- and Parabacteroides distasonis-colonized mice show reduced indole-3-carboxaldehyde (IAld) and increased indole-3-lactate (ILA), which are produced from opposing branches along the bacterial catabolic pathway of tryptophan. IAld and ILA counteractively modulate neuroplasticity in the nucleus accumbens, a brain region linked to depression. IAld supplementation produces anti-depressant effects in mice with stress or gut epithelial Gpr35 deficiency. Together, these findings elucidate a gut microbe-brain signaling mechanism that underlies susceptibility to depression.

STING signaling in islet macrophages impairs insulin secretion in obesity.

The innate immune regulator stimulator of interferon genes (STING) mediates self-DNA sensing and leads to the induction of type I interferons and inflammatory cytokines, which promotes the progression of various inflammatory and autoimmune diseases. Innate immune system plays a critical role in regulating obesity-induced islet dysfunction, whereas the potential effect of STING signaling is not fully understood. Here, we demonstrate that STING is mainly expressed and activated in islet macrophages upon high-fat diet (HFD) feeding. Sting-/- alleviates HFD-induced islet inflammation by inhibiting the expression of pro-inflammatory cytokines and the infiltration of macrophages. Mechanically, palmitic acid incubation promotes mitochondrial DNA leakage into the cytosol and subsequently activates STING pathway in macrophages. Additionally, STING activation in macrophages impairs glucose-stimulated insulin secretion by mediating the engulfment of ß cell insulin secretory granules. Pharmacologically inhibiting STING activation enhances insulin secretion to control hyperglycemia. Together, our results reveal a regulatory mechanism in controlling the islet inflammation and insulin secretion in diet--induced obesity and suggest that selective blocking of the STING activation may be a promising strategy for treating type 2 diabetes.

FXR agonists for MASH therapy: Lessons and perspectives from obeticholic acid.

Nonalcoholic fatty liver disease, also called metabolic dysfunction-associated steatotic liver disease, is the most common liver disease worldwide and has no approved pharmacotherapy. Due to its beneficial effects on metabolic regulation, inflammation suppression, cell death prevention, and fibrogenesis inhibition, farnesoid X receptor (FXR) is widely accepted as a promising therapeutic target for nonalcoholic steatosis (NASH) or called metabolic dysfunction-associated steatohepatitis (MASH). Many FXR agonists have been developed for NASH/MASH therapy. Obeticholic acid (OCA) is the pioneering frontrunner FXR agonist and the first demonstrating success in clinical trials. Unfortunately, OCA did not receive regulatory approval as a NASH pharmacotherapy because its moderate benefits did not outweigh its safety risks, which may cast a shadow over FXR-based drug development for NASH/MASH. This review summarizes the milestones in the development of OCA for NASH/MASH and discuss its limitations, including moderate hepatoprotection and the undesirable side effects of dyslipidemia, pruritus, cholelithiasis, and liver toxicity risk, in depth. More importantly, we provide perspectives on FXR-based therapy for NASH/MASH, hoping to support a successful bench-to-clinic transition.

Exhaustively Exploring the Prevalent Interaction Pathways of Ligands Targeting the Ligand-Binding Pocket of Farnesoid X Receptor via Combined Enhanced Sampling.

It is well-known that the potency of a drug is heavily associated with its kinetic and thermodynamic properties with the target. Nuclear receptors (NRs), as an important target family, play important roles in regulating a variety of physiological processes in vivo. However, it is hard to understand the drug-NR interaction process because of the closed structure of the ligand-binding domain (LBD) of the NR proteins, which apparently hinders the rational design of drugs with controllable kinetic properties. Therefore, understanding the underlying mechanism of the ligand-NR interaction process seems necessary to help NR drug design. However, it is usually difficult for experimental approaches to interpret the kinetic process of drug-target interactions. Therefore, in silico methods were utilized to explore the optimal binding/dissociation pathways of the NR ligands. Specifically, farnesoid X receptor (FXR) is considered here as the target system since it has been an important target for the treatment of bile acid metabolism-associated diseases, and a series of structures cocrystallized with diverse scaffold ligands were resolved. By using random acceleration molecular dynamics (RAMD) simulation and umbrella sampling (US), 5 main dissociation pathways (pathways I-V) were identified in 11 representative FXR ligands, with most of them (9/11) preferring to go through Pathway III and the remaining two favoring escaping from Pathway I and IV. Furthermore, key residues functioning in the three main dissociation pathways were revealed by the kinetic residue energy analysis (KREA) based on the US trajectories, which may serve as road-marker residues for rapid identification of the (un)binding pathways of FXR ligands. Moreover, the preferred pathways explored by RAMD simulations are in good agreement with the minimum free energy path identified by the US simulations with the Pearson R = 0.76 between the predicted binding affinity and the experimental data, suggesting that RAMD is suitable for applying in large-scale (un)binding-pathway exploration in the case of ligands with obscure binding tunnels to the target.

Repressed Blautia-acetate immunological axis underlies breast cancer progression promoted by chronic stress.

Chronic stress is a known risk factor for breast cancer, yet the underlying mechanisms are unclear. This study explores the potential involvement of microbial and metabolic signals in chronic stress-promoted breast cancer progression, revealing that reduced abundances of Blautia and its metabolite acetate may contribute to this process. Treatment with Blautia and acetate increases antitumor responses of CD8+ T cells and reverses stress-promoted breast cancer progression in female mice. Patients with depression exhibit lower abundances of Blautia and acetate, and breast cancer female patients with depression display lower abundances of acetate, decreased numbers of tumor-infiltrating CD8+ T cells, and an increased risk of metastasis. These results suggest that Blautia-derived acetate plays a crucial role in modulating the immune response to breast cancer, and its reduction may contribute to chronic stress-promoted cancer progression. Our findings advance the understanding of microbial and metabolic signals implicated in cancer in patients with depression and may provide therapeutic options for female patients with breast cancer and depression.

X-ray crystallography study and optimization of novel benzothiophene analogs as potent selective estrogen receptor covalent antagonists (SERCAs) with improved potency and safety profiles.

Endocrine therapy (ET) is a well-validated strategy for estrogen receptor α positive (ERα + ) breast cancer therapy. Despite the clinical success of current standard of care (SoC), endocrine-resistance inevitably emerges and remains a significant medical challenge. Herein, we describe the structural optimization and evaluation of a new series of selective estrogen receptor covalent antagonists (SERCAs) based on benzothiophene scaffold. Among them, compounds 15b and 39d were identified as two highly potent covalent antagonists, which exhibits superior antiproliferation activity than positive controls against MCF-7 cells and shows high selectivity over ERα negative (ERα-) cells. More importantly, their mode of covalent engagement at Cys530 residue was accurately illustrated by a cocrystal structure of 15b-bound ERαY537S (PDB ID: 7WNV) and intact mass spectrometry, respectively. Further in vivo studies demonstrated potent antitumor activity in MCF-7 xenograft mouse model and an improved safety profile. Collectively, these compounds could be promising candidates for future development of the next generation SERCAs for endocrine-resistant ERα + breast cancer.