The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins.

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

RGD-p21Ras-scFv expressed prokaryotically on a pilot scale inhibits ras-driven colorectal cancer growth by blocking p21Ras-GTP.

BACKGROUND: Ras gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling. METHODS: The E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models. RESULTS: The technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines. CONCLUSION: RGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.

Consensus statement on extracellular vesicles in liquid biopsy for advancing laboratory medicine.

Extracellular vesicles (EVs) represent a diverse class of nanoscale membrane vesicles actively released by cells. These EVs can be further subdivided into categories like exosomes and microvesicles, based on their origins, sizes, and physical attributes. Significantly, disease-derived EVs have been detected in virtually all types of body fluids, providing a comprehensive molecular profile of their cellular origins. As a result, EVs are emerging as a valuable addition to liquid biopsy techniques. In this collective statement, the authors share their current perspectives on EV-related research and product development, with a shared commitment to translating this newfound knowledge into clinical applications for cancer and other diseases, particularly as disease biomarkers. The consensus within this document revolves around the overarching recognition of the merits, unresolved questions, and existing challenges surrounding EVs. This consensus manuscript is a collaborative effort led by the Committee of Exosomes, Society of Tumor Markers, Chinese anti-Cancer Association, aimed at expediting the cultivation of robust scientific and clinically applicable breakthroughs and propelling the field forward with greater swiftness and efficacy.

Human peri-gastruloids: a significant advancement in embryology research.

The peri-gastruloids comprise both embryonic (epiblast) and extraembryonic (hypoblast) tissues, faithfully mirroring crucial developmental events spanning from the immediate post-implantation phase to early organogenesis, encompassing the emergence of amniotic and yolk sac cavities, as well as the progression from bilaminar to trilaminar embryonic discs.

TRIM25 predominately associates with anti-viral stress granules.

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.

Deficiency in Prader-Willi syndrome gene necdin leads to attenuated cardiac contractility.

Prader-Willi syndrome (PWS) is a genetic disorder characterized by behavioral disturbances, hyperphagia, and intellectual disability. Several surveys indicate that PWS is also associated with cardiac abnormalities, possibly contributing to a high incidence of sudden death. However, the pathological mechanisms underlying cardiac dysfunction in PWS remain unclear. In this study, we found that deficiency in necdin, an intronless gene within PWS region, led to heart systolic and diastolic dysfunction in mice. Through yeast two-hybrid screening, we identified an interaction between necdin and non-muscle myosin regulatory light chain 12a/b (MYL12 A/B). We further showed that necdin stabilized MYL12 A/B via SGT1-heat shock protein 90 (HSP90) chaperone machinery. The zebrafish lacking the MYL12 A/B analog, MYL12.1, exhibited impaired heart function, while cardiac-specific overexpression of MYL12A normalized the heart dysfunction in necdin-deficient mice. Our findings revealed necdin dysfunction as a contributing factor to cardiomyopathy in PWS patients and emphasized the importance of HSP90 chaperone machinery and non-muscle myosin in heart fitness.

TBC1D23 mediates Golgi-specific LKB1 signaling.

Liver kinase B1 (LKB1), an evolutionarily conserved serine/threonine kinase, is a master regulator of the AMPK subfamily and controls cellular events such as polarity, proliferation, and energy homeostasis. Functions and mechanisms of the LKB1-AMPK axis at specific subcellular compartments, such as lysosome and mitochondria, have been established. AMPK is known to be activated at the Golgi; however, functions and regulatory mechanisms of the LKB1-AMPK axis at the Golgi apparatus remain elusive. Here, we show that TBC1D23, a Golgi-localized protein that is frequently mutated in the neurodevelopment disorder pontocerebellar hypoplasia (PCH), is specifically required for the LKB1 signaling at the Golgi. TBC1D23 directly interacts with LKB1 and recruits LKB1 to Golgi, promoting Golgi-specific activation of AMPK upon energy stress. Notably, Golgi-targeted expression of LKB1 rescues TBC1D23 deficiency in zebrafish models. Furthermore, the loss of LKB1 causes neurodevelopmental abnormalities in zebrafish, which partially recapitulates defects in TBC1D23-deficient zebrafish, and LKB1 sustains normal neuronal development via TBC1D23 interaction. Our study uncovers a regulatory mechanism of the LKB1 signaling, and reveals that a disrupted Golgi-LKB1 signaling underlies the pathogenesis of PCH.

Prenatal low-dose Bisphenol A exposure impacts cortical development via cAMP-PKA-CREB pathway in offspring.

Bisphenol A (BPA) is a widely used plasticizer known to cause various disorders. Despite a global reduction in the use of BPA-containing products, prenatal exposure to low-dose BPA, even those below established safety limits, has been linked to neurological and behavioral deficits in childhood. The precise mechanisms underlying these effects remain unclear. In the present study, we observed a significant increase in the number of cortical neurons in offspring born to dams exposed to low-dose BPA during pregnancy. We also found that this prenatal exposure to low-dose BPA led to increased proliferation but reduced migration of cortical neurons. Transcriptomic analysis via RNA sequencing revealed an aberrant activation of the cAMP-PKA-CREB pathway in offspring exposed to BPA. The use of H89, a selective PKA inhibitor, effectively rescued the deficits in both proliferation and migration of cortical neurons. Furthermore, offspring from dams exposed to low-dose BPA exhibited manic-like behaviors, including hyperactivity, anti-depressant-like responses, and reduced anxiety. While H89 normalized hyperactivity, it didn't affect the other behavioral changes. These results suggest that the overactivation of PKA plays a causative role in BPA-induced changes in neuronal development. Our data also indicate that manic-like behaviors induced by prenatal low-dose BPA exposure may be influenced by both altered neuronal development and abnormal PKA signaling in adulthood.

Targeting BMAL1 reverses drug resistance of acute myeloid leukemia cells and promotes ferroptosis through HMGB1-GPX4 signaling pathway.

PURPOSE: Acute myeloid leukemia (AML) is a refractory hematologic malignancy that poses a serious threat to human health. Exploring alternative therapeutic strategies capable of inducing alternative modes of cell death, such as ferroptosis, holds great promise as a viable and effective intervention. METHODS: We analyzed online database data and collected clinical samples to verify the expression and function of BMAL1 in AML. We conducted experiments on AML cell proliferation, cell cycle, ferroptosis, and chemotherapy resistance by overexpressing/knocking down BMAL1 and using assays such as MDA detection and BODIPY 581/591 C11 staining. We validated the transcriptional regulation of HMGB1 by BMAL1 through ChIP assay, luciferase assay, RNA level detection, and western blotting. Finally, we confirmed the results of our cell experiments at the animal level. RESULTS: BMAL1 up-regulation is an observed phenomenon in AML patients. Furthermore, there existed a strong correlation between elevated levels of BMAL1 expression and inferior prognosis in individuals with AML. We found that knocking down BMAL1 inhibited AML cell growth by blocking the cell cycle. Conversely, overexpressing BMAL1 promoted AML cell proliferation. Moreover, our research results revealed that BMAL1 inhibited ferroptosis in AML cells through BMAL1-HMGB1-GPX4 pathway. Finally, knocking down BMAL1 can enhance the efficacy of certain first-line cancer therapeutic drugs, including venetoclax, dasatinib, and sorafenib. CONCLUSION: Our research results suggest that BMAL1 plays a crucial regulatory role in AML cell proliferation, drug resistance, and ferroptosis. BMAL1 could be a potential important therapeutic target for AML.

Ketogenic diet reshapes cancer metabolism through lysine ß-hydroxybutyrylation.

Lysine ß-hydroxybutyrylation (Kbhb) is a post-translational modification induced by the ketogenic diet (KD), a diet showing therapeutic effects on multiple human diseases. Little is known how cellular processes are regulated by Kbhb. Here we show that protein Kbhb is strongly affected by the KD through a multi-omics analysis of mouse livers. Using a small training dataset with known functions, we developed a bioinformatics method for the prediction of functionally important lysine modification sites (pFunK), which revealed functionally relevant Kbhb sites on various proteins, including aldolase B (ALDOB) Lys108. KD consumption or ß-hydroxybutyrate supplementation in hepatocellular carcinoma cells increases ALDOB Lys108bhb and inhibits the enzymatic activity of ALDOB. A Kbhb-mimicking mutation (p.Lys108Gln) attenuates ALDOB activity and its binding to substrate fructose-1,6-bisphosphate, inhibits mammalian target of rapamycin signalling and glycolysis, and markedly suppresses cancer cell proliferation. Our study reveals a critical role of Kbhb in regulating cancer cell metabolism and provides a generally applicable algorithm for predicting functionally important lysine modification sites.

Interaction between the gut microbiota and colonic enteroendocrine cells regulates host metabolism.

Nutrient handling is an essential function of the gastrointestinal tract. Hormonal responses of small intestinal enteroendocrine cells (EECs) have been extensively studied but much less is known about the role of colonic EECs in metabolic regulation. To address this core question, we investigated a mouse model deficient in colonic EECs. Here we show that colonic EEC deficiency leads to hyperphagia and obesity. Furthermore, colonic EEC deficiency results in altered microbiota composition and metabolism, which we found through antibiotic treatment, germ-free rederivation and transfer to germ-free recipients, to be both necessary and sufficient for the development of obesity. Moreover, studying stool and blood metabolomes, we show that differential glutamate production by intestinal microbiota corresponds to increased appetite and that colonic glutamate administration can directly increase food intake. These observations shed light on an unanticipated host-microbiota axis in the colon, part of a larger gut-brain axis, that regulates host metabolism and body weight.

Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes.

Mutations in the Contactin-associated protein-like 2 (CNTNAP2) gene are associated with autism spectrum disorder (ASD), and ectodomain shedding of the CNTNAP2 protein plays a role in its function. However, key enzymes involved in the C-terminal cleavage of CNTNAP2 remain largely unknown, and the effect of ASD-associated mutations on this process and its role in ASD pathogenesis remain elusive. In this report we showed that CNTNAP2 undergoes sequential cleavages by furin, ADAM10/17-dependent α-secretase and presenilin-dependent γ-secretase. We identified that the cleavage sites of ADAM10 and ADAM17 in CNTNAP2 locate at its C-terminal residue I79 and L96, and the main α-cleavage product C79 by ADAM10 is required for the subsequent γ-secretase cleavage to generate CNTNAP2 intracellular domain (CICD). ASD-associated CNTNAP2 mutations impair the α-cleavage to generate C79, and the inhibition leads to ASD-like repetitive and social behavior abnormalities in the Cntnap2-I1254T knock-in mice. Finally, exogenous expression of C79 improves autism-like phenotypes in the Cntnap2-I1254T knock-in and Cntnap2-/- knockout mice. This data demonstrates that the α-secretase is essential for CNTNAP2 processing and its function. Our study indicates that inhibition of the cleavage by pathogenic mutations underlies ASD pathogenesis, and upregulation of its C-terminal fragments could have therapeutical potentials for ASD treatment.

PCDH17 restricts dendritic spine morphogenesis by regulating ROCK2-dependent control of the actin cytoskeleton, modulating emotional behavior.

Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function. Synaptic abnormalities, such as defects in the density and morphology of postsynaptic dendritic spines, underlie the pathology of various neuropsychiatric disorders. Protocadherin 17 (PCDH17) is associated with major mood disorders, including bipolar disorder and depression. However, the molecular mechanisms by which PCDH17 regulates spine number, morphology, and behavior remain elusive. In this study, we found that PCDH17 functions at postsynaptic sites, restricting the number and size of dendritic spines in excitatory neurons. Selective overexpression of PCDH17 in the ventral hippocampal CA1 results in spine loss and anxiety- and depression-like behaviors in mice. Mechanistically, PCDH17 interacts with actin-relevant proteins and regulates actin filament (F-actin) organization. Specifically, PCDH17 binds to ROCK2, increasing its expression and subsequently enhancing the activity of downstream targets such as LIMK1 and the phosphorylation of cofilin serine-3 (Ser3). Inhibition of ROCK2 activity with belumosudil (KD025) ameliorates the defective F-actin organization and spine structure induced by PCDH17 overexpression, suggesting that ROCK2 mediates the effects of PCDH17 on F-actin content and spine development. Hence, these findings reveal a novel mechanism by which PCDH17 regulates synapse development and behavior, providing pathological insights into the neurobiological basis of mood disorders.

Application of Machine Learning to Ultrasonography in Identifying Anatomical Landmarks for Cricothyroidotomy Among Female Adults: A Multi-center Prospective Observational Study.

We aimed to develop machine learning (ML)-based algorithms to assist physicians in ultrasound-guided localization of cricoid cartilage (CC) and thyroid cartilage (TC) in cricothyroidotomy. Adult female volunteers were prospectively recruited from two hospitals between September and December, 2020. Ultrasonographic images were collected via a modified longitudinal technique. You Only Look Once (YOLOv5s), Faster Regions with Convolutional Neural Network features (Faster R-CNN), and Single Shot Detector (SSD) were selected as the model architectures. A total of 488 women (mean age: 36.0 years) participated in the study, contributing to a total of 292,053 frames of ultrasonographic images. The derived ML-based algorithms demonstrated excellent discriminative performance for the presence of CC (area under the receiver operating characteristic curve [AUC]: YOLOv5s, 0.989, 95% confidence interval [CI]: 0.982-0.994; Faster R-CNN, 0.986, 95% CI: 0.980-0.991; SSD, 0.968, 95% CI: 0.956-0.977) and TC (AUC: YOLOv5s, 0.989, 95% CI: 0.977-0.997; Faster R-CNN, 0.981, 95% CI: 0.965-0.991; SSD, 0.982, 95% CI: 0.973-0.990). Furthermore, in the frames where the model could correctly indicate the presence of CC or TC, it also accurately localized CC (intersection-over-union: YOLOv5s, 0.753, 95% CI: 0.739-0.765; Faster R-CNN, 0.720, 95% CI: 0.709-0.732; SSD, 0.739, 95% CI: 0.726-0.751) or TC (intersection-over-union: YOLOv5s, 0.739, 95% CI: 0.722-0.755; Faster R-CNN, 0.709, 95% CI: 0.687-0.730; SSD, 0.713, 95% CI: 0.695-0.730). The ML-based algorithms could identify anatomical landmarks for cricothyroidotomy in adult females with favorable discriminative and localization performance. Further studies are warranted to transfer this algorithm to hand-held portable ultrasound devices for clinical use.

Discovery of a Hidden Pocket beneath the NES Groove by Novel Noncovalent CRM1 Inhibitors.

Protein localization is frequently manipulated to favor tumor initiation and progression. In cancer cells, the nuclear export factor CRM1 is often overexpressed and aberrantly localizes many tumor suppressors via protein-protein interactions. Although targeting protein-protein interactions is usually challenging, covalent inhibitors, including the FDA-approved drug KPT-330 (selinexor), were successfully developed. The development of noncovalent CRM1 inhibitors remains scarce. Here, by shifting the side chain of two methionine residues and virtually screening against a large compound library, we successfully identified a series of noncovalent CRM1 inhibitors with a stable scaffold. Crystal structures of inhibitor-protein complexes revealed that one of the compounds, B28, utilized a deeply hidden protein interior cavity for binding. SAR analysis guided the development of several B28 derivatives with enhanced inhibition on nuclear export and growth of multiple cancer cell lines. This work may benefit the development of new CRM1-targeted therapies.