Tandem mass tag-based proteomic analysis of granulosa and theca interna cells of the porcine ovarian follicle following in vitro treatment with vitamin D3 and insulin alone or in combination.

This study was performed to investigate the proteomic basis underlying the interaction between vitamin D3 (VD) and insulin (I) within ovarian follicle using the pig as a model. Porcine antral follicles were incubated in vitro for 12 h with VD alone and I alone or in combination (VD + I) or with no treatment as the control (C). In total, 7690 and 7467 proteins were identified in the granulosa and theca interna compartments, respectively. Comparative proteomic analysis revealed 97 differentially abundant proteins (DAPs) within the granulosa layer and 11 DAPs within the theca interna layer. In the granulosa compartment, VD affected proteome leading to the promotion of cell proliferation, whereas I influenced mainly proteins related to cellular adhesion. The VD + I treatment induced granulosa cell proliferation probably via the DAPs involved in DNA synthesis and the cell cycle regulation. In the theca interna layer, VD alone or in co-treatment with I affected DAPs associated with cholesterol transport and lipid and steroid metabolic processes that was further confirmed by diminished lipid droplet accumulation. SIGNIFICANCE: The application of quantitative proteomics demonstrated for the first time the complexity of VD and I interactions in porcine ovarian follicle, providing a framework for understanding the molecular mechanisms underlying their cross-talk. Although identified DAPs were related to crucial ovarian processes, including the granulosa cell proliferation and cholesterol transport in the theca interna layer, novel molecular pathways underlying these processes have been proposed. The identified unique proteins may serve as indicators of VD and I interactions in both follicle layers, and could be useful biomarkers of ovarian pathologies characterized by impaired VD and I levels, such as polycystic ovary syndrome.

Insights into the structure and activation mechanism of some class B1 GPCR family members.

In humans, 15 genes encode the class B1 family of GPCRs, which are polypeptide hormone receptors characterized by having a large N-terminal extracellular domain (ECD) and receive signals from outside the cell to activate cellular response. For example, the insulinotropic polypeptide (GIP) stimulates the glucose-dependent insulinotropic polypeptide receptor (GIPR), while the glucagon receptor (GCGR) responds to glucagon by increasing blood glucose levels and promoting the breakdown of liver glycogen to induce the production of insulin. The glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) elicit a response from glucagon-like peptide receptor types 1 and 2 (GLP1R and GLP2R), respectively. Since these receptors are implicated in the pathogenesis of diabetes, studying their activation is crucial for the development of effective therapies for the condition. With more structural information being revealed by experimental methods such as X-ray crystallography, cryo-EM, and NMR, the activation mechanism of class B1 GPCRs becomes unraveled. The available crystal and cryo-EM structures reveal that class B1 GPCRs follow a two-step model for peptide binding and receptor activation. The regions close to the C-termini of hormones interact with the N-terminal ECD of the receptor while the regions close to the N-terminus of the peptide interact with the TM domain and transmit signals. This review highlights the structural details of class B1 GPCRs and their conformational changes following activation. The roles of MD simulation in characterizing those conformational changes are briefly discussed, providing insights into the potential structural exploration for future ligand designs.

High-Density Lipoprotein-Associated Paraoxonase-1 (PON-1) and Scavenger Receptor Class B Type 1 (SRB-1) in Coronary Artery Disease: Correlation with Disease Severity.

Background: Coronary artery disease (CAD) is the leading cause of death worldwide. High-Density lipoprotein (HDL) is a well-established marker associated with CAD. The current research goes beyond the conventional HDL-C measurement in previous studies and dives into the functional intricacies of HDL. By understanding how HDL works, rather than just how much of it exists, we can better tailor diagnostic and therapeutic strategies for CAD and related conditions. Hence, the current study quantifies the serum levels of two novel HDL-associated markers, Paraoxonase-1 (PON-1) and Scavenger Receptor Class B Type 1 (SRB-1), in CAD cases vs. controls. Methods: A total of 92 subjects, including 69 CAD and 23 healthy controls, were included, based on the prevalence of the disease. Further, based on the severity of the disease, CAD cases were subcategorized as CAD-I, -II, and -III. Serum PON-1 and SRB-1 levels were measured and compared between patient and control groups. Results: The levels of PON-1 and SRB-1 (32.6 ng/mL and 12.49 ng/mL) were significantly lower in CAD patients vs. the healthy control, at 60.36 ng/mL and 15.85 ng/mL, respectively (p < 0.000). A further intergroup comparison showed a statistically significant difference between the CAT-I and -III for PON-1 (p < 0.025), the CAT-I and -III, and CAT-II and -III for SRB-1 (p < 0.000). The receiver operating characteristics (ROC) curve showed cutoff values of 48.20 ng/mL and 14.90 ng/mL for PON-1 and SRB-1. Conclusions: The current study found that serum levels of HDL-associated PON-1 and SRB-1 are significantly lower in CAD cases, and were also inversely related to the increasing severity of coronary artery disease. This inference implies that serum PON-1 and SRB-1 could be used as non-invasive tools for the identification of coronary atherosclerosis and risk assessment in CAD cases.

Isoquinoline small molecule ligands are agonists and probe-dependent allosteric modulators of the glucagon subfamily of GPCRs.

Class B1 G protein-coupled receptors (GPCRs) are peptide hormone receptors and well validated therapeutic targets, however development of non-peptide drugs targeting this class of receptors is challenging. Recently, a series of isoquinoline-based derivates were reported in the patent literature as allosteric ligands for the glucagon receptor subfamily, and two compounds, LSN3451217 and LSN3556672, were used to facilitate structural studies with the glucagon-like peptide-1 receptor (GLP-1R) and glucose dependent insulinotropic peptide receptor (GIPR) bound to orthosteric agonists. Here we pharmacologically characterized stereoisomers of LSN3451217 and LSN3556672, across the class B1 GPCR family. This revealed LSN3556672 isomers are agonists for the glucagon receptor (GCGR), GLP-1R, GIPR and the calcitonin receptor (CTR), albeit the degree of agonism varied at each receptor. In contrast, LSN3451217 isomers were more selective agonists at the GLP-1R, with lower potency at the GCGR and CTR and no activity at the GIPR. All compounds also modulated peptide-mediated cyclic adenosine monophosphate (cAMP) signaling at the GIPR, and to a lesser extent the GLP-1R, in a probe-dependent manner, with modest positive allosteric modulation observed for some peptides, and negligible effects observed with other peptides. In contrast neutral or weak negative/positive allosteric modulation was observed with peptides assessed at the GCGR and CTR. This study expands our knowledge on class B1 GPCR allosteric modulation and may have implications for future structural and drug discovery efforts targeting the class B1 GPCR subfamily.

Altered desensitization and internalization patterns of rodent versus human glucose-dependent insulinotropic polypeptide (GIP) receptors. An important drug discovery challenge.

BACKGROUND AND PURPOSE: The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus. EXPERIMENTAL APPROACH: The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel. KEY RESULTS: The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for Gαs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. 'Tail-swapped' human, rat and mouse GIPRs were all fully functional in their Gαs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail. CONCLUSIONS AND IMPLICATIONS: Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds.

Class B1 GPCRs: Insights into Multi-Receptor Pharmacology for the Treatment of Metabolic Disease.

The secretin-like, class B1 sub-family of seven transmembrane-spanning G protein coupled receptors (GPCRs) consists of 15 members that coordinate important physiological processes. These receptors bind peptide ligands and utilize a distinct mechanism of activation that is driven by evolutionarily conserved structural features. For the class B1 receptors, the C-terminus of the cognate ligand is initially recognized by the receptor via a large N-terminal extracellular domain that forms a hydrophobic ligand binding groove. This binding enables the N-terminus of the ligand to engage deep into a large volume, open transmembrane pocket of the receptor. Importantly, the phylogenetic basis of this ligand-receptor activation mechanism has provided opportunities to engineer analogues of several class B1 ligands for therapeutic use. Among the most successful of these are drugs targeting the glucagon-like peptide-1 (GLP-1) receptor for the treatment of type 2 diabetes and obesity. Recently, multi-functional agonists possessing activity at the GLP-1 receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor, such as tirzepatide, and others that also contain glucagon receptor activity, have been developed. In this article, we review members of the class B1 GPCR family with focus on receptors for GLP-1, GIP, and glucagon, including their signal transduction and receptor trafficking characteristics. The metabolic importance of these receptors is also highlighted, along with the benefit of poly-pharmacologic ligands. Further, key structural features and comparative analyses of high-resolution cryogenic electron microscopy structures for these receptors in active-state complex with either native ligands or multi-functional agonists are provided, supporting the pharmacological basis of such therapeutic agents.

Coupled single-cell and bulk RNA-seq analysis reveals the engulfment role of endothelial cells in atherosclerosis.

The clearance of apoptotic cell debris, containing professional phagocytosis and non-professional phagocytosis, is essential for maintaining the homeostasis of healthy tissues. Here, we discovered that endothelial cells could engulf apoptotic cell debris in atherosclerotic plaque. Single-cell RNA sequencing (RNA-seq) has revealed a unique endothelial cell subpopulation in atherosclerosis, which was strongly associated with vascular injury-related pathways. Moreover, integrated analysis of three vascular injury-related RNA-seq datasets showed that the expression of scavenger receptor class B type 1 (SR-B1) was up-regulated and specifically enriched in the phagocytosis pathway under vascular injury circumstances. Single-cell RNA-seq and bulk RNA-seq indicate that SR-B1 was highly expressed in a unique endothelial cell subpopulation of mouse aorta and strongly associated with the reorganization of cellular adherent junctions and cytoskeleton which were necessary for phagocytosis. Furthermore, SR-B1 was strongly required for endothelial cells to engulf apoptotic cell debris in atherosclerotic plaque of both mouse and human aorta. Overall, this study demonstrated that apoptotic cell debris could be engulfed by endothelial cells through SR-B1 and associated with the reorganization of cellular adherent junctions and cytoskeleton.

Pseudomonas aeruginosa producing FIM-1-metallo-ß-lactamase: emergence and cross-transmission in a long-term acute care rehabilitation hospital.

FIM-1 metallo-ß-lactamase was previously detected in sporadic Pseudomonas aeruginosa clinical isolates. Here, we report on FIM-1-positive P. aeruginosa from two patients who had shared the same ward in a long-term acute care rehabilitation hospital. Whole-genome sequencing analysis revealed close relatedness of these isolates, which belonged to an ST235 sublineage (clade 8/14) different from those previously reported. Results highlighted the occurrence of clonal diversity among FIM-positive strains and the possibility of their cross-transmission in some healthcare settings.

Ligand-dependent interactions between SR-B1 and S1PR1 in macrophages and atherosclerotic plaques.

HDLs carry sphingosine-1-phosphate (S1P) and stimulate signaling pathways in different cells including macrophages and endothelial cells, involved in atherosclerotic plaque development. HDL signaling via S1P relies on the HDL receptor scavenger receptor class B, type I (SR-B1) and the sphingosine-1-phosphate receptor 1 (S1PR1), which interact when both are heterologously overexpressed in the HEK293 cell line. In this study, we set out to test if SR-B1 and S1PR1 interacted in primary murine macrophages in culture and atherosclerotic plaques. We used knock-in mice that endogenously expressed S1PR1 tagged with eGFP-(S1pr1eGFP/eGFP mice), combined with proximity ligation analysis to demonstrate that HDL stimulates the physical interaction between SR-B1 and S1PR1 in primary macrophages, that this is dependent on HDL-associated S1P and can be blocked by an inhibitor of SR-B1's lipid transfer activity or an antagonist of S1PR1. We also demonstrate that a synthetic S1PR1-selective agonist, SEW2871, stimulates the interaction between SR-B1 and S1PR1 and that this was also blocked by an inhibitor of SR-B1's lipid transport activity. Furthermore, we detected abundant SR-B1/S1PR1 complexes in atherosclerotic plaques of S1pr1eGFP/eGFP mice that also lacked apolipoprotein E. Treatment of mice with the S1PR1 antagonist, Ex26, for 12 h disrupted the SR-B1-S1PR1 interaction in atherosclerotic plaques. These findings demonstrate that SR-B1 and S1PR1 form ligand-dependent complexes both in cultured primary macrophages and within atherosclerotic plaques in mice and provide mechanistic insight into how SR-B1 and S1PR1 participate in mediating HDL signaling to activate atheroprotective responses in macrophages.

Systemic Chemotherapy for Advanced Hepatocellular Carcinoma in Patients With Child-Pugh class B.

Background/Aim: Numerous agents, including immune checkpoint inhibitors, are now available for hepatocellular carcinoma (HCC) treatment. Most trials involving systemic chemotherapy have included patients with Child-Pugh class A, while excluding or minimally enrolling those with Child-Pugh class B, due to liver dysfunction-related mortality. This study aimed to identify prognostic factors for survival in Child-Pugh class B patients receiving sorafenib (SOR), lenvatinib (LEN), atezolizumab plus bevacizumab (ATZ+BEV), or hepatic arterial infusion chemotherapy (HAIC). Patients and Methods: From December 2003 to June 2023, 137 patients with advanced HCC receiving systemic chemotherapies (SOR: n=43, LEN: n=16, ATZ+BEV: n=18, HAIC: n=60) were enrolled. Results: Overall survival (OS) and response rates did not differ significantly across treatments (SOR: 8.3 months, LEN: 10.2 months, ATZ+BEV: 8.5 months, HAIC: 7.3 months). Patients on HAIC and LEN had a lower rate of discontinuing treatment within three months compared to those on ATZ+BEV and SOR. HAIC was associated with fewer changes in ALBI score and better preservation of liver function. Multivariate logistic regression identified serum α-fetoprotein >400 ng/ml [hazard ratio (HR)=1.94; p=0.001], tumor count >5 (HR=1.55; p=0.043), and Child-Pugh score (HR=2.53; p=0.002) as independent predictors of OS. Conclusion: OS and response rates were similar across systemic chemotherapies. Prognosis for HCC in Child-Pugh class B patients was associated with liver function, necessitating further research for optimal treatment.

Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus.

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

Scavenger Receptor Class B Type I Deficiency Induces Iron Overload and Ferroptosis in Renal Tubular Epithelial Cells via Hypoxia-Inducible Factor-1α/Transferrin Receptor 1 Signaling Pathway.

Aims: Scavenger receptor class B type I (SRBI) promotes cell cholesterol efflux and the clearance of plasma cholesterol. Thus, SRBI deficiency causes abnormal cholesterol metabolism and hyperlipidemia. Studies have suggested that ferroptosis is involved in lipotoxicity; however, whether SRBI deficiency could induce ferroptosis remains to be investigated. Results: We knocked down or knocked out SRBI in renal HK-2 cells and C57BL/6 mice to determine the expression levels of ferroptosis-related regulators. Our results demonstrated that SRBI deficiency upregulates transferrin receptor 1 (TFR1) expression and downregulates ferroportin expression, which induces iron overload and subsequent ferroptosis in renal tubular epithelial cells. TFR1 is known to be regulated by hypoxia-inducible factor-1α (HIF-1α). Next, we investigated whether SRBI deletion affected HIF-1α. SRBI deletion upregulated the mRNA and protein expression of HIF-1α, and promoted its translocation to the nucleus. To determine whether HIF-1α plays a key role in SRBI-deficiency-induced ferroptosis, we used HIF-1α inhibitor and siHIF-1α in HK-2 cells, and found that downregulation of HIF-1α prevented SRBI-silencing-induced TFR1 upregulation and iron overload, and eventually reduced ferroptosis. The underlying mechanism of HIF-1α activation was explored next, and the results showed that SRBI knockout or knockdown may upregulate the expression of HIF-1α, and promote HIF-1α translocation from the cytoplasm into the nucleus via the PKC-ß/NF-κB signaling pathway. Innovation and Conclusion: Our study showed, for the first time, that SRBI deficiency induces iron overload and subsequent ferroptosis via the HIF-1α/TFR1 pathway.

Strategies to Name Metallo-ß-Lactamases and Number Their Amino Acid Residues.

Metallo-ß-lactamases (MBLs), also known as class B ß-lactamases (BBLs), are Zn(II)-containing enzymes able to inactivate a broad range of ß-lactams, the most commonly used antibiotics, including life-saving carbapenems. They have been known for about six decades, yet they have only gained much attention as a clinical problem for about three decades. The naming conventions of these enzymes have changed over time and followed various strategies, sometimes leading to confusion. We are summarizing the naming strategies of the currently known MBLs. These enzymes are quite diverse on the amino acid sequence level but structurally similar. Problems trying to describe conserved residues, such as Zn(II) ligands and other catalytically important residues, which have different numbers in different sequences, have led to the establishment of a standard numbering scheme for BBLs. While well intended, the standard numbering scheme is not trivial and has not been applied consistently. We revisit this standard numbering scheme and suggest some strategies for how its implementation could be made more accessible to researchers. Standard numbering facilitates the comparison of different enzymes as well as their interaction with novel antibiotics and BBL inhibitors.

Large-Scale Identification of Lysine Crotonylation Reveals Its Potential Role in Oral Squamous Cell Carcinoma.

Purpose: Lysine crotonylation, an emerging posttranslational modification, has been implicated in the regulation of diverse biological processes. However, its involvement in oral squamous cell carcinoma (OSCC) remains elusive. This study aims to reveal the global crotonylome in OSCC under hypoxic conditions and explore the potential regulatory mechanism of crotonylation in OSCC. Methods: Liquid-chromatography fractionation, affinity enrichment of crotonylated peptides, and high-resolution mass spectrometry were employed to detect differential crotonylation in CAL27 cells cultured under hypoxia. The obtained data were further subjected to bioinformatics analysis to uncover the involved biological processes and pathways of the dysregulated crotonylated proteins. A site-mutated plasmid was utilized to investigate the effect of crotonylation on Heat Shock Protein 90 Alpha Family Class B Member 1 (HAP90AB1) function. Results: A large-scale crotonylome analysis revealed 1563 crotonylated modification sites on 605 proteins in CAL27 cells under hypoxia. Bioinformatics analysis revealed a significant decrease in histone crotonylation levels, while up-regulated crotonylated proteins were mainly concentrated in non-histone proteins. Notably, glycolysis-related proteins exhibited prominent up-regulation among the identified crotonylated proteins, with HSP90AB1 displaying the most significant changes. Subsequent experimental findings confirmed that mutating lysine 265 of HSP90AB1 into a silent arginine impaired its function in promoting glycolysis. Conclusion: Our study provides insights into the crotonylation modification of proteins in OSCC under hypoxic conditions and elucidates the associated biological processes and pathways. Crotonylation of HSP90AB1 in hypoxic conditions may enhance the glycolysis regulation ability in OSCC, offering novel perspectives on the regulatory mechanism of crotonylation in hypoxic OSCC and potential therapeutic targets for OSCC treatment.

Serum opacity factor normalizes erythrocyte morphology in Scarb1-/- mice in an HDL-free cholesterol-dependent way.

Compared with WT mice, HDL receptor-deficient (Scarb1-/-) mice have higher plasma levels of free cholesterol (FC)-rich HDL and exhibit multiple pathologies associated with a high mol% FC in ovaries, platelets, and erythrocytes, which are reversed by lowering HDL. Bacterial serum opacity factor (SOF) catalyzes the opacification of plasma by targeting and quantitatively converting HDL to neo HDL (HDL remnant), a cholesterol ester-rich microemulsion, and lipid-free APOA1. SOF delivery with an adeno-associated virus (AAVSOF) constitutively lowers plasma HDL-FC and reverses female infertility in Scarb1-/- mice in an HDL-dependent way. We tested whether AAVSOF delivery to Scarb1-/- mice will normalize erythrocyte morphology in an HDL-FC-dependent way. We determined erythrocyte morphology and FC content (mol%) in three groups-WT, untreated Scarb1-/- (control), and Scarb1-/- mice receiving AAVSOF-and correlated these with their respective HDL-mol% FC. Plasma-, HDL-, and tissue-lipid compositions were also determined. Plasma- and HDL-mol% FC positively correlated across all groups. Among Scarb1-/- mice, AAVSOF treatment normalized reticulocyte number, erythrocyte morphology, and erythrocyte-mol% FC. Erythrocyte-mol% FC positively correlated with HDL-mol% FC and with both the number of reticulocytes and abnormal erythrocytes. AAVSOF treatment also reduced FC of extravascular tissues to a lesser extent. HDL-FC spontaneously transfers from plasma HDL to cell membranes. AAVSOF treatment lowers erythrocyte-FC and normalizes erythrocyte morphology and lipid composition by reducing HDL-mol% FC.

An updated patent review of metallo-ß-lactamase inhibitors (2020-2023).

INTRODUCTION: Metallo-ß-lactamases (MBLs) are enzymes produced by bacteria that confer resistance to most ß-lactam antibiotics, including carbapenems, which have the broadest spectrum of activity. This resistance mechanism poses a significant threat to public health as it drastically reduces treatment options for severe bacterial infections. Developing effective inhibitors against MBLs is crucial to restore susceptibility to ß-lactam antibiotics. AREAS COVERED: This review aims to provide an updated analysis of patents describing novel MBL inhibitors and their potential therapeutic applications that were filed between January 2020 and May 2023. EXPERT OPINION: Significant advancements were made in the development of selective MBL inhibitors with zinc-binding and zinc-chelating mechanisms of action. Dual inhibitors, targeting simultaneously both serine-ß-lactamases (SBLs) and MBLs, represent an interesting alternative approach that is increasingly pertinent for the treatment of infections involving multiple ß-lactamases from different Ambler classes. Most examples of MBL-specific inhibitors were focused on the treatment of MBL-mediated infections in Enterobacterales, where IMP-1 was a more difficult target compared with VIM-1 or NDM-1, and much less on Pseudomonas aeruginosa or Acinetobacter baumannii, which are more challenging to address.

Exploring LoRaWAN Traffic: In-Depth Analysis of IoT Network Communications.

In the past decade, Long-Range Wire-Area Network (LoRaWAN) has emerged as one of the most widely adopted Low Power Wide Area Network (LPWAN) standards. Significant efforts have been devoted to optimizing the operation of this network. However, research in this domain heavily relies on simulations and demands high-quality real-world traffic data. To address this need, we monitored and analyzed LoRaWAN traffic in four European cities, making the obtained data and post-processing scripts publicly available. For monitoring purposes, we developed an open-source sniffer capable of capturing all LoRaWAN communication within the EU868 band. Our analysis discovered significant issues in current LoRaWAN deployments, including violations of fundamental security principles, such as the use of default and exposed encryption keys, potential breaches of spectrum regulations including duty cycle violations, SyncWord issues, and misaligned Class-B beacons. This misalignment can render Class-B unusable, as the beacons cannot be validated. Furthermore, we enhanced Wireshark's LoRaWAN protocol dissector to accurately decode recorded traffic. Additionally, we proposed the passive reception of Class-B beacons as an alternative timebase source for devices operating within LoRaWAN coverage under the assumption that the issue of misaligned beacons can be addressed or mitigated in the future. The identified issues and the published dataset can serve as valuable resources for researchers simulating real-world traffic and for the LoRaWAN Alliance to enhance the standard to facilitate more reliable Class-B communication.

Deficiency of SR-B1 reduced the tumor load of colitis-induced or APCmin /+ -induced colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common tumors in the world. Cholesterol plays an important role in the pathogenesis of tumors. One of the cholesterol transporters, scavenger receptor class B type 1 (SR-B1), a multi-ligand membrane receptor protein, is expressed in the intestines which also highly expressed in various tumors. But the potential mechanism of SR-B1 in CRC development has not been reported. AIMS: This study aimed to clarify the importance of SR-B1 in the development and prognosis of CRC as much as possible to provide a possible strategy in CRC treatment. MATERIALS & METHODS: In this study, we used SR-B1 gene knockdown mice to study the effect of SR-B1 on colitis-induced or APCmin/+ -induced CRC. The expression of related molecules were detected through the immunohistochemistry and hematoxylin-eosin staining, western blot analysis, and Flow cytometry. The gene expression and microbiota in microenvironment of CRC mice were analyzed through eukaryotic mRNA sequencing and 16S rRNA high-throughput sequencing. RESULTS: The results showed that SR-B1 knockdown reduced the tumor load of colitis-induced or APCmin/+ -induced CRC. SR-B1 knockdown improved the immune microenvironment by affecting the level of tumor-associated macrophage (TAM), mononuclear myeloid-derived suppressor cells (M-MDSCs), granulocytic myeloid-derived suppressor cells (G-MDSCs), programmed cell death-ligand 1 (PD-L1), and human leukocyte antigen class I-B (HLA-B), and also reduced the level of low-density lipoprotein receptor (LDL-R), and increased the level of ATP binding cassette transporter A1 (ABCA1) to regulate the cholesterol metabolism, and regulated the expression of related genes and intestinal microbiota. SR-B1 knockdown can also trigger the anti-CRC effect of anti-PD 1 in colitis-induced CRC. DISCUSSION: SR-B1 deficiency significantly improved the immunity in tumor microenvironment of colitis-induced or APCmin/+ -induced CRC. In addition, the microbiota changes caused by SR-B1 deficiency favor improving the immune response to chemotherapeutic drugs and anti-PD1 therapy. The mechanism of action of SR-B1 deficiency on the development of CRC still needs further in-depth research. CONCLUSION: This study provides a new treatment strategy for treating CRC by affecting the expression of SR-B1 in intestine.

[Association Analysis Between Methylation of SCARB1 Gene Promoter and Coronary Heart Disease].

Objective To explore the relationship between scavenger receptor class B member 1 (SCARB1) gene promoter methylation and the pathogenesis of coronary artery disease. Methods A total of 120 patients with coronary heart disease treated in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine from December 2018 to May 2020 were selected as the case group,while 140 gender and age matched healthy participants were randomly selected as the control group for a case-control study.The methylation status was detected by high-throughput target sequencing after bisulfite converting,and the methylation of CpG sites in the promoter region of SCARB1 gene was compared between the two groups. Results The case group showed higher methylation level of SCARB1+67 and lower methylation level of SCARB1+134 than the control group (both P<0.001),and the differences remained statistically significant in men (both P<0.001) and women (both P<0.001).The overall methylation level in the case group was lower than that in the control group [(80.27±2.14)% vs.(81.11±1.27)%;P=0.006],while this trend was statistically significant only in men (P=0.002). Conclusion The methylation of SCARB1 gene promotor is associated with the pathogenesis and may participate in the occurrence and development of coronary heart disease.

Lycopene Accumulation in Transgenic Mice Lacking One or Both Carotenoid Cleaving Enzymes.

BACKGROUND: ß-carotene oxygenase 1 (BCO1) and ß-carotene oxygenase 2 (BCO2) are responsible for the cleavage of carotenoids in mammals. OBJECTIVE: The goals of this study were to (1) establish the relative contribution of each enzyme on lycopene accumulation in mice and (2) examine the role of lycopene on gene expression in the gut of wild type (WT) mice. METHODS: We utilized male and female WT, Bco1-/-, Bco2-/-, and Bco1-/-Bco2-/- double knockout (DKO) mice. We gavaged the mice with either 1 mg of lycopene resuspended in cottonseed oil or vehicle as a control group daily for 2 wk. In a second study, we evaluated the effect of dietary vitamin A on lycopene absorption and intestinal gene expression by RT-PCR. We also quantified lycopene concentration isomer distribution by high performance liquid chromatography. RESULTS: Of the 11 tissues measured, the liver accounted for 94 to 98% of the lycopene content across genotypes. We did not observe sex differences between genotypes, although hepatic lycopene levels in Bco1-/- mice were approximately half in comparison to the other genotypes; Bco1-/- verses Bco2-/- (P < 0.0001), DKO mice (P < 0.001), WT (ns). Analyses of mitochondrial lycopene content revealed a 3- to 5-fold enrichment compared with total hepatic content (P < 0.05) in all genotypes and sexes. In our second study, WT mice fed a vitamin A-deficient diet (VAD) accumulated greater amounts of lycopene in the liver than those fed a vitamin A-sufficient diet (VAS) (P < 0.01). These changes were accompanied by an upregulation of the vitamin A-responsive transcription factor intestine specific homeobox (ISX) in mice fed VAD + lycopene and VAS + lycopene diets compared with VAD control-fed mice (P < 0.05). CONCLUSIONS: Our data suggest that BCO2 is the primary lycopene cleavage enzyme in mice. Lycopene concentration was enriched in the mitochondria of hepatocytes independently of genotype, and lycopene stimulated vitamin A signaling in WT mice.