Protein B1 Research Articles

Nonsedating anxiolytics.

Anxiety disorders are the most prevalent psychiatric pathology with substantial cost to society, but the existing treatments are often inadequate. This has rekindled the interest in the GABAA-receptor (GABAAR) positive allosteric modulator (PAM) compounds, which have a long history in treatment of anxiety beginning with diazepam, chlordiazepoxide, and alprazolam. While the GABAAR PAMs possess remarkable anxiolytic efficacy, they have fallen out of favor due to a host of adverse effects including sedation, motor impairment, addictive potential and tolerance development. A substantial effort was thus devoted to the design of GABAAR PAMs as anxiolytics with reduced sedative liabilities. Several non-benzodiazepine (BZD) GABAAPAMs progressed to clinical trials (bretazenil, abecarnil, alpidem, and ocinaplon) with alpidem obtaining regulatory approval as anxiolytic, but later withdrawn from market due to hepatotoxicity. Advances in molecular biology gave birth to a host of subtype selective GABAAR-PAMs which suffered from signs of sedation and motor impairment and only three compounds progressed to proof-of-concept studies (TPA-023, AZD7325 and PF-06372865). TPA-023 was terminated due to toxicity in preclinical species while AZD7325 and PF-06372865 did not achieve efficacy endpoints in patients. We highlight a new compound, KRM-II-81, that is an imidazodiazepine selective for GABAAR containing α2/3 and β3 proteins. In preclinical studies KRM-II-81 produced anxiolytic-like effects but with minimal sedation, respiratory depression, and abuse liability. Thus, KRM-II-81 is a newly discovered, non- BZD anxiolytic compound, which targets a selective population of GABAAR for improved therapeutic gain and reduced side effects.

Co-treatment with erythropoietin derived HBSP and caspase-3 siRNA: A promising approach to prevent fibrosis after acute kidney injury.

Acute kidney injury (AKI) is a risk factor of chronic kidney disease, without specific treatment. This study investigated the effect of co-treatment using erythropoietin-derived helix B surface peptide (HBSP) and caspase-3 small interfering RNA (CASP3siRNA) on preventing fibrosis post AKI in order to achieve better efficacy by different action mechanisms. Ischemia-reperfusion (IR) in mice was induced by clamping bilateral renal pedicles for 30 min followed by 2-week reperfusion, with HBSP and/or CASP3siRNA administered at the onset of IR. Serum creatinine, apoptosis, active caspase-3 and high mobility group protein B1 (HMGB1) in kidneys were decreased by HBSP, CASP3siRNA or both, with increased PCNA. α-SMA expression and collagen I deposition were also reduced by CASP3siRNA and both. Most interestingly, the co-treatment further reduced tubulointerstitial damage and fibrosis, but raised PCNA compared to CASP3siRNA. EPOR/βcR was reduced by HBSP, and positively correlated with Sirius red staining, whereas EPOR was unchanged. In TCMK-1 cells, H2O2 raised apoptosis and α-SMA were reduced by HBSP, while the same was occurred to HMGB1. However, HMGB1 was further increased by EPOR siRNA under H2O2 stimulation with/without HBSP treatment. In conclusion, this study demonstrated synergistic long-term renoprotection post IR-AKI by HBSP and CASP3siRNA, which may be due to co-inhibiting inflammation and stimulating repair at early stage, and subsequently preventing fibrosis.

Proteomic Analysis Identifies Multiple Mechanisms of 5-Fluorouracil-Induced Gut Mucositis in Mice

Background/Objectives. Damage of the gastrointestinal mucosa is a major side effect of the anticancer drug 5-fluorouracil (5-FU). Insight into the molecular pathogenesis of 5-FU-induced gut mucositis is expected to justify the strategies of prophylaxis. Methods. We analyzed intestinal specimens obtained from Balb/c mice treated with 70 mg/kg 5-FU daily for up to 6 days. Results. Manifestations of mucositis in the ileum and the colon included diarrhea, weight loss, and morphological lesions. The proteomic analysis revealed dozens of differentially expressed proteins governed by a set of master regulator proteins that regulated downstream pathways culminating in the complexes of specific transcription factors. Among the most important mechanisms of 5-FU-induced gut damage predicted by bioinformatics tools was stimulation of insulin-like growth factor 1 concomitant with inhibition of insulin receptor substrate 1, suggesting an involvement of the insulin pathway. Furthermore, the levels of 14-3-3γ protein and epinephrin B2 tyrosine kinase were interpreted as key inhibitory effects of 5-FU. These changes were detectable in the ileum as well as in the colon, pointing to the commonality of 5-FU responses across the gut. Conclusion. These results demonstrated a hierarchical network of gut injury mechanisms differentially regulated in the course of the emergence of 5-FU-induced mucositis.

The Correlation between BRAFV600E Expression and Integrin β3 in Melanoma Cells

This study investigated the relationship between ITGB3 and BRAFV600E expression in melanoma. It specifically focused on how the overexpression of BRAFV600E and Integrin $\beta $3 impacts wound healing and the TGF-β pathway. The findings provide valuable insights into melanoma progression and highlight potential therapeutic targets. Homozygous BRAFV600E-carrying A375 and heterozygous BRAFV600E-carrying M14 melanoma cell lines were utilized. ITGB3 mRNA levels were measured in both A375 and M14 cells, while Integrin β3 protein levels were analyzed in BRAFV600E-overexpressing A375 cells. The impact of increased BRAFV600E and Integrin β3 on cell migration was assessed using an in vitro wound healing assay. Additionally, the influence of BRAFV600E and Integrin β3 overexpression on the TGF-β pathway was evaluated through luciferase reporter assays and real-time PCR. ITGB3 levels were significantly higher in A375 cells compared to M14 cells. Overexpression of BRAFV600E in A375 cells resulted in a marked increase in Integrin β3 levels. Cells overexpressing both BRAFV600E and Integrin β3 demonstrated enhanced wound healing rates and elevated TGF-β activity. These findings suggest a strong correlation between BRAFV600E expression and Integrin β3 levels in melanoma cells. This study uncovers a significant relationship between BRAFV600E expression and Integrin β3 levels in melanoma cells. The overexpression of both BRAFV600E and Integrin β3 enhances wound healing and promotes TGF-β signalling. These findings suggest that Integrin β3 may contribute to melanoma progression and poor prognosis by influencing cell migration and the TGF-β pathway. Targeting integrins, the TGF-β pathway, and BRAFV600E present potential therapeutic strategies for melanoma treatment.

Synthesis, Biological Evaluation, and Mechanistic Insights of Rubrolide Analogues as Antitumor Agents.

Marine natural products and their analogues have as of now been acknowledged as an important source of bioactive molecules for the treatment of cancer. Rubrolides, a unique group of γ-butenolides derived from marine microorganisms, have shown strong cytotoxic activity against various tumor cells. In this study, we synthesized and characterized 21 rubrolide analogues (including 16 new compounds) and investigated their antitumor activities in order to screen more active molecules and elucidate their mechanism of action. Primary MTT assay showed that compounds 1 and 4-9 all exhibited excellent antiproliferative activities. In particular, compound 7 showed broad-spectrum cytotoxic activity against six tumor cell lines, with IC50 values mostly ranging from 2.5 to 0.2 μM. Further mechanistic studies revealed that compound 7 could penetrate HCT116 and Hela cells, localize in the endoplasmic reticulum, and upregulate the PERK-eIF2α-CHOP pathway, inducing ER stress and increasing intracellular reactive oxygen species (ROS) levels to ultimately trigger apoptosis in tumor cells. Additionally, compound 7 was found to upregulate Cyclin B1 protein expression, causing cell cycle reticulum at the G2/M phase. In vivo studies further demonstrated that liposomal delivery of compound 7 exhibited a potent antitumor efficacy against Hela xenograft tumors. Based on these results, marine-derived rubrolide analogues show significant potential as novel lead compounds for antitumor drug development.

Promoting Polarization and Differentiation of Primary Human Salivary Gland Stem/Progenitor Cells in Protease-Degradable Hydrogels via ROCK Inhibition.

Towards the goal of in vitro engineering of functional salivary gland tissues, we cultured primary human salivary stem/progenitor cells (hS/PCs) in hyaluronic acid-based matrices with varying percentages of proteolytically degradable crosslinks in the presence of Rho kinase (ROCK) inhibitor. Single cells encapsulated in the hydrogel grew into organized multicellular structures by day 15, and over 60% of the structures developed in the non-degradable and 50% degradable hydrogels contained a central lumen. Importantly, ROCK inhibition led to the establishment of multicellular structures that were correctly polarized, as evidenced by apical localization of a Golgi marker GM130, apical/lateral localization of tight junction protein zonula occludens-1 (ZO-1), and basal localization of integrin β1 and basement membrane proteins laminin α1 and collagen IV. Cultures maintained in 50% degradable gels with ROCK inhibition exhibited an increased expression of acinar markers AQP5 and SLC12A2 (at the transcript level) and AQP5 and NKCC1 (at the protein level) as compared to those without ROCK inhibition. Upon stimulation with isoproterenol, α-amylase secretion into the lumen was observed. Particle-tracking microrheology was employed to analyze the stiffness of cells using mitochondria as the passive tracer particles. Our results indicated that cells grown in 100% degradable gels were stiffer than those maintained in non-degradable gels, and cells cultured with the ROCK inhibitor were softer than those maintained without the inhibitor. We conclude that reducing cellular contractility via ROCK inhibition while retaining some degree of matrix confinement promotes the establishment of multicellular structures containing pro-acinar cells with correct apicobasal polarization.

Essential role of the interaction between classical swine fever virus core protein and cellular MYO1B in viral components transport to exosomes and titer maintenance

Classical swine fever (CSF) is a severe disease caused by the highly contagious CSFV. Our previous study demonstrated that exosomes from CSFV-infected cells contained significant amounts of viral genome and Core (C) protein and were infectious. To further elucidate the mechanisms underlying the formation of these infectious exosomes, we investigated the intracellular transport of the C protein in this study. We first identified the synchronized transport of the C protein and viral genome to exosomes, distinguishing it from other structural proteins. This suggests that the C protein likely binds to the viral genome and is transported to exosomes as a nucleocapsid. Subsequently, Co-IP and co-localization experiments confirmed the interaction between the host Myosin 1B (MYO1B) protein and the C protein. Key interaction sites were identified by generating and analyzing various C protein point mutations and truncation variants. The results indicate that specific sites at the N-terminus of the C protein significantly impact its interaction with MYO1B. Ultimately, by modulating MYO1B expression, we found that MYO1B knockdown significantly reduced the C protein and viral genome content in exosomes, leading to a decrease in CSFV titers. These findings underscore the critical role of MYO1B in facilitating the transport of the C protein and viral genome into exosomes during CSFV infection. Overall, this study explores the mechanism of infectious exosome formation during CSFV infection, revealing the critical role of the host MYO1B in this process. This is the first study to identify the involvement of MYO1B in viral infection, not only offering important insights into host-virus interactions but also identifying a new target for antiviral drug development.

The PvRBP2b-TfR1 interaction is not essential for reticulocytes invasion by Plasmodium vivax isolates from Cambodia

Plasmodium vivax is the most widespread of the different Plasmodium species able to infect humans and is responsible for most malaria cases outside Africa. An effective, strain-transcending vaccine that alleviates or suppresses erythrocyte invasion would be a game-changer in eliminating vivax malaria. Recently, the binding of P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) to human Transferrin receptor (TfR1) has been described as essential for reticulocyte invasion, making this parasite protein an appealing vaccine candidate. Here, using P. vivax Cambodian clinical isolates in robust ex vivo invasion assays, we show that anti-PvRBP2b polyclonal and monoclonal antibodies that inhibit binding of PvRBP2b to TfR1 do not block P. vivax invasion into reticulocytes even at high concentrations. Anti-TfR1 antibodies do not inhibit P. vivax invasion either. Combinations at high concentrations of human monoclonal antibodies targeting different PvRBP2b epitopes do not inhibit invasion. Combinations of anti-PvRBP2b with anti-PvDBP do not enhance invasion inhibition caused by anti-PvDBP alone. We also show that the invasion of Cambodian P. vivax is trypsin-resistant while TfR1 is trypsin-sensitive, and we demonstrate that TfR1 is not recycled following trypsin treatment. We determined the PvRBP2b sequence of all isolates used in the invasion assays and analyzed polymorphism within epitopes recognized by anti-PvRBP2b antibodies. We show that polymorphism does not explain the absence of neutralization. Anti-PvRBP2b polyclonal antibodies recognized all four isolates tested in immunofluorescence assays while not inhibiting P. vivax invasion. Overall, our results demonstrate that PvRBP2b binding to TfR1 is not essential for invasion into reticulocytes of P. vivax Cambodian strains questioning the relevance of PvRBP2b as vaccine candidate.

Kallikrein-Related Peptidase 6 Contributes to Murine Intestinal Tumorigenesis Driven by a Mutant Adenomatous polyposis coli Gene.

The objective of this study was to assess the role of a secreted serine protease, kallikrein-related peptidase 6 (KLK6), during colorectal tumorigenesis driven by a mutant Adenomatous polyposis coli (APC) tumor suppressor gene. A first analysis of KLK6 expression in the intestinal tract of Apc-mutant multiple intestinal neoplasia (ApcMin/+) mice revealed up to four-fold induction of Klk6 mRNA levels in adenomas relative to its level in the adjacent mucosa. The presence of KLK6 protein in the adenomatous areas was confirmed by immunohistochemistry and optical coherence tomography/laser-induced fluorescence (OCT/LIF) imaging. To assess the contribution of the KLK6 expression on the Apc-mutant intestinal and colon tumorigenesis, we engineered a mouse with floxed alleles of the Klk6 gene (Klk6lox/lox) and crossed it with a mouse expressing the truncated APC protein under control of the intestinal tract-specific human CDX2P9.5-NLS Cre transgene (CPC;Apcfl/fl;Klk6+/+). We found that CPC;Apcfl/fl mice with disrupted Klk6 gene expression (CPC;Apcfl/fl;Klk6fl/fl) had a significantly smaller average size of the small intestinal and colon crypts (p < 0.001 and p = 0.04, respectively) and developed a significantly fewer adenomas (p = 0.01). Moreover, a decrease in high-grade adenomas (p = 0.03) and adenomas with a diameter above 2 mm (p < 0.0001) was noted in CPC;Apcfl/fl;Klk6fl/fl mice. Further molecular analysis showed that Klk6 gene inactivation in the small intestine and colon tissues of CPC;Apcfl/fl;Klk6fl/fl mice resulted in a significant suppression of transforming growth factor β2 (TGF-β2) protein (p ≤ 0.02) and mitogen-activated protein kinase (MAPK) phosphorylation (p ≤ 0.01). These findings demonstrate the oncogenic role of KLK6 in the mutant Apc-mediated intestinal tumorigenesis and suggest the utility of KLK6 for early diagnosis of colorectal tumors.

Shear stress-stimulated AMPK couples endothelial cell mechanics, metabolism, and vasodilation.

Endothelial cells respond to mechanical force by stimulating cellular signaling, but how these pathways are linked to elevations in cell metabolism and whether metabolism supports the mechanical response remains poorly understood. Here, we show that the application of force to endothelial cells stimulates VE-cadherin to activate liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis. VE-cadherin stimulated AMPK increases eNOS activity and localization to the plasma membrane, reinforcement of the actin cytoskeleton and cadherin adhesion complex, and glucose uptake. We present evidence for the increase in metabolism being necessary to fortify the adhesion complex, actin cytoskeleton, and cellular alignment. Together these data extend the paradigm for how mechanotransduction and metabolism are linked to include a connection to vasodilation, thereby providing new insight into how diseases involving contractile, metabolic, and vasodilatory disturbances arise.

Autophagosome development and chloroplast segmentation occur synchronously for piecemeal degradation of chloroplasts.

Plants distribute many nutrients to chloroplasts during leaf development and maturation. When leaves senesce or experience sugar starvation, the autophagy machinery degrades chloroplast proteins to facilitate efficient nutrient reuse. Here, we report on the intracellular dynamics of an autophagy pathway responsible for piecemeal degradation of chloroplast components. Through live-cell monitoring of chloroplast morphology, we observed the formation of chloroplast budding structures in sugar-starved leaves. These buds were then released and incorporated into the vacuolar lumen as an autophagic cargo termed a Rubisco-containing body. The budding structures did not accumulate in mutants of core autophagy machinery, suggesting that autophagosome creation is required for forming chloroplast buds. Simultaneous tracking of chloroplast morphology and autophagosome development revealed that the isolation membranes of autophagosomes interact closely with part of the chloroplast surface before forming chloroplast buds. Chloroplasts then protrude at the site associated with the isolation membranes, which divide synchronously with autophagosome maturation. This autophagy-related division does not require DYNAMIN-RELATED PROTEIN 5B, which constitutes the division ring for chloroplast proliferation in growing leaves. An unidentified division machinery may thus fragment chloroplasts for degradation in coordination with the development of the chloroplast-associated isolation membrane.

Pathogenesis-related protein, thaumatin-like protein 1b is a transdermal sensitizing allergen of mango fruits in mouse models

ABSTRACT Food allergens can enter the skin and increase the susceptibility to food allergies by producing immunoglobulin (Ig) E. Mango is a popular fruit worldwide and known to be allergenic. Dermal sensitization may occur around the oral cavity during ingestion of mangoes. However, to date, no allergenic proteins in mangoes have been shown to cause dermal sensitization in humans or animal models. Therefore, we aimed to identify mango proteins that sensitize mice percutaneously. The occiput of BALB/c mice was shaved, and a crude mango extract containing sodium dodecyl sulfate was applied to the skin. Mango protein-specific IgE and IgG1 were produced in response to this treatment. Proteins in mangoes that bind to mouse IgG1 and IgE were purified and identified as thaumatin-like protein 1b (TLP1b). This is the first study to identify TLP1b, a pan-allergen belonging to a family of pathogenesis-related proteins, as a mango allergen causing transdermal sensitization in mice.

Aloe Emodin Alleviates Radiation-Induced Heart Disease via Blocking P4HB Lactylation and Mitigating Kynurenine Metabolic Disruption.

Aloe emodin is an anthraquinone of traditional Chinese medicine monomer, which plays a protective action in cardiovascular diseases. However, the regulatory mechanisms of aloe emodin in the protection of radiation-induced heart damage (RIHD) are unclear. As a novel post-translational modification, lactylation is considered as a critical mediator in inflammatory cascade and cardiac injury. Here, using a cross of differential omics and 4D label-free lactylation omics, protein disulfide-isomerase (P4HB) is identified as a novel target for lactylation, and aloe emodin inhibits the binding of lactate to the K311 site of P4HB. Aloe emodin stabilizes kynurenine metabolism through inhibition of aspartate aminotransferase (GOT2) accumulation on damaged mitochondria. Mechanistically, aloe emodin inhibits phosphorylated glycogen synthase kinase 3B (p-GSK3B) transcription in the nucleus to repress the interaction of prostaglandin G/H synthase 2 (PTGS2) with SH3 domain of SH3 domain-containing GRB2-like protein B1 (SH3GLB1), thereby disrupting the functions of mitochondrial complexes and reducing SH3GLB1-mediated mitoROS accumulation, eventually suppressing calcium-binding and coiled-coil domain-containing protein 2 (NDP52)-induced mitophagy. This study unveils the regulatory role of aloe emodin in RIHD alleviation through PTGS2/SH3GLB1/NDP52 axis, indicates aloe emodin stabilizes GOT2-mediated kynurenine metabolism through P4HB lactylation. Collectively, this study provides novel insights into the regulatory mechanisms underlying the protective role of aloe emodin in cardiac injury, and opens new avenues for therapeutic strategies of aloe emodin in preventing RIHD by regulating lactylation.

HMGB2 promotes smooth muscle cell proliferation through PPAR-γ/PGC-1α pathway-mediated glucose changes in aortic dissection

Background and aimsAortic dissection (AD) is a fatal condition with a complicated pathogenesis. High mobility group protein B2 (HMGB2) is a member of the high mobility group protein family; HMGB2 is involved in innate immunity and inflammatory diseases, but its role in AD remains unclear. MethodsHMGB2−/− mice were generated and treated with β-aminopropionitrile and angiotensin II (Ang II) to establish an AD model. An F12 gel containing AAV9-HMGB2 was applied to overexpress HMGB2 in mice. Pathological changes in the aorta were assessed by visualizing vascular collagen deposition and elastic fiber fracture via H&E, Masson and EVG staining. HMGB2 expression was measured by Western blotting and immunohistochemistry. MTS, CCK-8 and EdU assays were used to test cell proliferation. ResultsHMGB2 expression was increased in samples from AD patients, samples from AD mouse modeland human aortic smooth muscle cells (HASMCs). HMGB2 promoted HASMC proliferation. Immunofluorescence staining and plasma membrane protein isolation revealed that HMGB2 decreased GLUT1 expression and promoted GLUT4 translocation. HMGB2 was also found to inhibit the expression of SIRT1/PGC-1α, but blocking the PPAR-γ pathway attenuated this effect. HMGB2−/− significantly reduced the incidence and mortality rates of AD, whereas treatment with AAV9-HMGB2 exacerbated AD. ConclusionsThis study suggests that HMGB2 promotes HASMC proliferation and vascular remodeling by regulating glucose metabolism through the PPAR-γ/SIRT1/PGC-1α pathway. HMGB2 knockdown reduces, while HMGB2 overexpression promotes, the occurrence of AD in mice. This study may help elucidate the underlying mechanisms and provide a new preventive target for AD.

ALYREF recruits ELAVL1 to promote colorectal tumorigenesis via facilitating RNA m5C recognition and nuclear export

ALYREF can recognize 5-methylcytosine (m5C) decoration throughout RNAs to regulate RNA metabolism. However, its implications in cancer and precise regulatory mechanisms remain largely elusive. Here, we demonstrated that ALYREF supported colorectal cancer (CRC) growth and migration. Integrated analysis of ALYREF-RIP-Bis-seq and transcriptome profiles identified ribosomal protein S6 kinase B2 (RPS6KB2) and regulatory-associated protein of mTOR (RPTOR) as ALYREF’s possible downstream effectors. Mechanistically, ALYREF formed a complex with ELAV like RNA binding protein 1 (ELAVL1) to cooperatively promote m5C recognition and nuclear export of the two mRNAs. Moreover, ALYREF protein was highly expressed in tumor tissues of CRC patients, which predicted their poor prognosis. E2F transcription factor 6 (E2F6)-mediated transactivation gave a molecular insight into ALYREF overexpression. Collectively, ALYREF recruits ELAVL1 to collaboratively facilitate m5C recognition and nuclear export of RPS6KB2 and RPTOR transcripts for colorectal tumorigenesis, providing RNA m5C methylation as promising therapeutic targets and prognostic biomarkers for CRC.

BMP3b regulates bone mass by inhibiting BMP signaling

Bone morphogenetic protein 3b (BMP3b), also known as growth differentiation factor 10 (GDF10), is a non-osteogenic BMP highly expressed in the skeleton. Although in vitro studies have shown that BMP3b suppresses osteoblast differentiation, the physiological role of BMP3b in regulating bone mass in vivo remains unknown. Here, we show that BMP3b deletion in mice leads to a high bone mass phenotype via an unexpected novel mechanism involving de-repression of canonical BMP/Smad signaling. BMP3b null mice were viable, and exhibited no significant difference in body size compared to wildtype control. Trabecular bone parameters assessed by histomorphometry and μCT, revealed a significant increase in bone volume and bone mineral density. Expression of osteoblast-differentiation genes were elevated in bone tissue of BMP3b null mice, whereas expression of osteoclast-related genes remained unchanged. Consistent with this, Bmp3b was highly expressed in osteoblasts relative to osteoclast cells. Ex-vivo culture of primary bone marrow mesenchymal stem cells (BMSCs) and primary bone marrow-derived osteoclasts revealed that inactivation of BMP3b enhances osteogenesis without affecting osteoclastogenesis. Mechanistically, we found that BMP3b suppressed BMP4-induced Smad1/5 phosphorylation and inhibited the activity of a BMP4-driven Id-1 luciferase reporter. Protein-protein interaction assays revealed that BMP3b competitively interfered with the association of BMP4 and BMP type I receptors. These findings suggest that BMP3b regulates bone mass by acting as a BMP receptor antagonist. Thus, maintenance of bone mass involves antagonism of canonical BMP/Smad signaling by a member of the BMP family.

Inorganic nitrogen inhibits symbiotic nitrogen fixation through blocking NRAMP2-mediated iron delivery in soybean nodules

Symbiotic nitrogen fixation (SNF) in legume-rhizobia serves as a sustainable source of nitrogen (N) in agriculture. However, the addition of inorganic N fertilizers significantly inhibits SNF, and the underlying mechanisms remain not-well understood. Here, we report that inorganic N disrupts iron (Fe) homeostasis in soybean nodules, leading to a decrease in SNF efficiency. This disruption is attributed to the inhibition of the Fe transporter genes Natural Resistance-Associated Macrophage Protein 2a and 2b (GmNRAMP2a&2b) by inorganic N. GmNRAMP2a&2b are predominantly localized at the tonoplast of uninfected nodule tissues, affecting Fe transfer to infected cells and consequently, modulating SNF efficiency. In addition, we identified a pair of N-signal regulators, nitrogen-regulated GARP-type transcription factors 1a and 1b (GmNIGT1a&1b), that negatively regulate the expression of GmNRAMP2a&2b, which establishes a link between N signaling and Fe homeostasis in nodules. Our findings reveal a plausible mechanism by which soybean adjusts SNF efficiency through Fe allocation in response to fluctuating inorganic N conditions, offering valuable insights for optimizing N and Fe management in legume-based agricultural systems.

Genetically engineered human induced pluripotent stem cells for the production of brain-targeting extracellular vesicles

BackgroundExtracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising, natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture, human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However, hiPSC-EVs only have a moderate ability for brain delivery. Herein, we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor.MethodsAn RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging.ResultsHere we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype, express pluripotency markers, and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways, compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency.ConclusionTaken together, we have established stable, genetically engineered hiPSCs for producing EVs with RVG expression, offering the improved ability for brain-targeted drug delivery.

FengycinA-M3 Inhibits Listeria monocytogenes by Binding to Penicillin-Binding Protein 2B Targets to Disrupt Cell Structure.

The contamination of food with Listeria monocytogenes threatens food safety and human health, and developing a novel, green, and safe antimicrobial substance will offer a new food preservation strategy. FengycinA-M3 is a novel lipid peptide with low cytotoxicity and resistance and has effective antibacterial activity against L. monocytogenes with a minimum inhibitory concentration (MIC) of 4 µg/mL. Further combined transcriptomics and proteomics analysis yielded 20 differentially expressed genes (DEGs). The MICs of the combined use of FengycinA-M3 and Cefalexin on L. monocytogenes were further determined as FengycinA-M3 (2 µg/mL) and Cefalexin (8 µg/mL) using the checkerboard method. In addition, FengycinA-M3 was found to play a role in delaying pork deterioration. This study explored the inhibitory effect of FengycinA-M3 on L. monocytogenes and its mechanism of action. FengycinA-M3 interacted with penicillin-binding protein 2B on the cell membrane of L. monocytogenes, destroying the permeability of the membrane, causing cell membrane rupture, thereby inhibiting the growth of L. monocytogenes. Overall, FengycinA-M3 is a promising candidate for preventing the emergence and spread of L. monocytogenes with potential applications in food processing.

Polymorphic Supramolecular Therapeutic Platforms with Precise Dye/Drug Ratio to Perform Synergistic Chemo-Photo Anti-Tumor Therapy and Long-Term Immune Protection.

Malignant tumor has become one of the hellish killers threatening the health of people around the world, its diagnosis and treatment has become the concerns of public. However, the optimal therapeutic dose, undesired side-effect, and long-term immune activation werekey and bottleneck problems in tumor treatment. Herein, different batches of supramolecular therapeutic platforms, including vesicles, spherical nanoparticles, and cylindrical nanorods, with precise ratios of dye to drug (1:2) and multiple stimulus responsiveness wereconstructed by host-guest complexation between cyanine-camptothecin conjugates (IR780-CPT2) and β-cyclodextrin (β-CD) pendent hydrophilic copolymers. The reduction responsiveness, near-infrared photothermal conversion and singlet oxygen (1O2) generation performances endowed these platforms excellent cancer cells killing effect in both of in vitro cellular experiments and in vivo mice models. More importantly, without affecting the weight of mice, the maturation of dendritic cells, proliferation of T cells, up-regulation of high mobility group protein B1, and reduction of immunosuppressive regulatory T cells weredetected after employing a synergistic chemo-photo therapy, demonstrating the body's immune effect was successfully activated. Thus, during the treatment of primary tumor, the distal tumor was also inhibited. Webelieve this work could provide a distinctive way to fabricate supramolecular theranostic platforms with different morphologies and improve antitumor and antimetastasis capabilities.