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Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta ( Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated Hexb tdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS. Microglia have key roles in central nervous system (CNS) disease and homeostasis but their study can be challenging. Prinz and colleagues identify hexosaminidase subunit beta ( Hexb ) to be specifically expressed by microglia and stable even under inflammatory conditions.

Metal–organic frameworks (MOFs) have recently garnered consideration as an attractive solid substrate because the highly tunable MOF framework can not only serve as an inert host but also enhance the selectivity, stability, and/or activity of the enzymes. Herein, we demonstrate the advantages of using a mechanochemical strategy to encapsulate enzymes into robust MOFs. A range of enzymes, namely β-glucosidase, invertase, β-galactosidase, and catalase, are encapsulated in ZIF-8, UiO-66-NH 2 , or Zn-MOF-74 via a ball milling process. The solid-state mechanochemical strategy is rapid and minimizes the use of organic solvents and strong acids during synthesis, allowing the encapsulation of enzymes into three prototypical robust MOFs while maintaining enzymatic biological activity. The activity of encapsulated enzyme is demonstrated and shows increased resistance to proteases, even under acidic conditions. This work represents a step toward the creation of a suite of biomolecule-in-MOF composites for application in a variety of industrial processes. Metal–organic frameworks (MOFs) are attractive for encapsulating enzymes for industrial purposes because they can increase selectivity, stability, and/or activity of the enzymes. Here, the authors developed an economical solid-state mechanochemical method to encapsulate enzymes during MOF synthesis.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of “zero functional G” at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins (“zero arrestin”), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at “zero functional G”: arrestin recruitment and internalization, but—unexpectedly—complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2. Arrestins terminate signaling from GPCRs, but several lines of evidence suggest that they are also able to transduce signals independently of G proteins. Here, the authors systematically ablate G proteins in cell lines, and show that arrestins are unable to act as genuine signal initiators.

Betaglycan (BG) is a transmembrane co-receptor of the transforming growth factor-β (TGF-β) family of signaling ligands. It is essential for embryonic development, tissue homeostasis and fertility in adults. It functions by enabling binding of the three TGF-β isoforms to their signaling receptors and is additionally required for inhibin A (InhA) activity. Despite its requirement for the functions of TGF-βs and InhA in vivo, structural information explaining BG ligand selectivity and its mechanism of action is lacking. Here, we determine the structure of TGF-β bound both to BG and the signaling receptors, TGFBR1 and TGFBR2. We identify key regions responsible for ligand engagement, which has revealed binding interfaces that differ from those described for the closely related co-receptor of the TGF-β family, endoglin, thus demonstrating remarkable evolutionary adaptation to enable ligand selectivity. Finally, we provide a structural explanation for the hand-off mechanism underlying TGF-β signal potentiation. Betaglycan is a co-receptor for selective TGF-β family ligands. Here, the authors solve its structure in complex with TGF-β and the signaling receptors, which explains its ligand selectivity and reveals its mechanism in potentiating TGF-β signaling.

Background Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher’s exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.

Aims/hypothesisMacrophage levels are elevated in pancreatic islets, and the resulting inflammatory response is a major contributor to beta cell failure during obesity and type 2 diabetes mellitus. Previous studies by us and others have reported that exosomes released by macrophages play important roles in mediating cell-to-cell communication, and represent a class of inflammatory factors involved in the inflammatory process associated with type 2 diabetes mellitus. However, to date, no reports have demonstrated the effect of macrophage-derived exosomes on beta cells, and little is known regarding their underlying mechanisms in beta cell injury. Thus, we aimed to study the impact of macrophage-derived exosomes on islet beta cell injury in vitro and in vivo.MethodsThe phenotypic profiles of islet-resident macrophages were analysed in C57BL/6J mice fed a high-fat diet (HFD). Exosomes were collected from the medium of cultured bone marrow-derived macrophages (BMDMs) and from isolated islet-resident macrophages of HFD-fed mice (HFD-Exos). The role of exosomes secreted by inflammatory M1 phenotype BMDMs (M1-Exos) and HFD-Exos on beta cell function was assessed. An miRNA microarray and quantitative real-time PCR (qPCR) were conducted to test the level of M1-Exos-derived miR-212-5p in beta cells. Then, miR-212-5p was overexpressed or inhibited in M1-Exos or beta cells to determine its molecular and functional impact.ResultsM1-polarised macrophages were enriched in the islets of obese mice. M1 macrophages and islet-resident macrophages of HFD-fed mice impaired beta cell insulin secretion in an exosome-dependent manner. miR-212-5p was notably upregulated in M1-Exos and HFD-Exos. Enhancing the expression of miR-212-5p impaired beta cell insulin secretion. Blocking miR-212-5p elicited a significant improvement in M1-Exos-mediated beta cell insulin secretion during injury. Mechanistically, M1-Exos mediated an intercellular transfer of the miR-212-5p, targeting the sirtuin 2 gene and regulating the Akt/GSK-3β/β-catenin pathway in recipient beta cells to restrict insulin secretion.Conclusions/interpretationA novel exosome-modulated mechanism was delineated for macrophage-beta cell crosstalk that drove beta cell dysfunction and should be explored for its therapeutic utility.

Antimicrobial resistance is a growing global concern that compromises the efficacy of antibiotics, particularly due to the misuse and overuse of these agents. Beta-lactam antibiotics, widely used for their broad-spectrum activity, are increasingly threatened by bacterial resistance mechanisms. Recent studies showed that resistant Gram-negative bacteria cause many hospital infections, which makes the search for new treatments very urgent. This review explores recent advancements in the development of novel beta-lactam antibiotics, including new drug combinations and structural modifications designed to overcome beta-lactamase degradation and effectively target mutated penicillin-binding proteins (PBPs). One of the main resistance mechanisms in Gram-negative bacteria is the production of beta-lactamases, which break the beta-lactam ring and stop the drug from working. New ideas include adding more than one beta-lactam ring in the drug, combining with strong beta-lactamase inhibitors, and making hybrid structures. These strategies have shown promising results in preclinical evaluations. Novel beta-lactam antibiotics demonstrate significant potential in combating resistant bacterial strains. Future directions should emphasize large-scale in vivo validation, the incorporation of novel β-lactamase inhibitors, and the development of advanced drug delivery systems. Integration of these strategies may enhance the clinical applicability of β-lactams and provide sustainable solutions to address the global burden of antimicrobial resistance.

Cervical cancer is the major cause of cancer related deaths in women, especially in developing countries and Human Papilloma Virus infection in conjunction with multiple deregulated signaling pathways leads to cervical carcinogenesis. TGF-β signaling in later stages of cancer is known to induce epithelial to mesenchymal transition promoting tumor growth. Phytochemicals, curcumin and emodin, are effective as chemopreventive and chemotherapeutic compounds against several cancers including cervical cancer. The main objective of this work was to study the effect of curcumin and emodin on TGF-β signaling pathway and its functional relevance to growth, migration and invasion in two cervical cancer cell lines, SiHa and HeLa. Since TGF-β and Wnt/β-catenin signaling pathways are known to cross talk having common downstream targets, we analyzed the effect of TGF-β on β-catenin (an important player in Wnt/β-catenin signaling) and also studied whether curcumin and emodin modulate them. We observed that curcumin and emodin effectively down regulate TGF-β signaling pathway by decreasing the expression of TGF-β Receptor II, P-Smad3 and Smad4, and also counterbalance the tumorigenic effects of TGF-β by inhibiting the TGF-β-induced migration and invasion. Expression of downstream effectors of TGF-β signaling pathway, cyclinD1, p21 and Pin1, was inhibited along with the down regulation of key mesenchymal markers (Snail and Slug) upon curcumin and emodin treatment. Curcumin and emodin were also found to synergistically inhibit cell population and migration in SiHa and HeLa cells. Moreover, we found that TGF-β activates Wnt/β-catenin signaling pathway in HeLa cells, and curcumin and emodin down regulate the pathway by inhibiting β-catenin. Taken together our data provide a mechanistic basis for the use of curcumin and emodin in the treatment of cervical cancer.

β-lactam antibiotics have been prescribed for most bacterial infections since their discovery. However, resistance to β-lactams, mediated by β-lactamase (Bla) enzymes such as extended spectrum β-lactamases (ESBLs), has become widespread. Bla inhibitors can restore the efficacy of β-lactams against resistant bacteria, an approach which preserves existing antibiotics despite declining industry investment. However, the effects of combination treatment on selection for β-lactam resistance are not well understood. Bla production confers both private benefits for resistant cells and public benefits which faster-growing sensitive cells can also exploit. These benefits may be differentially impacted by Bla inhibitors, leading to non-intuitive selection dynamics. In this study, we demonstrate strain-to-strain variation in effective combination doses, with complex growth dynamics in mixed populations. Using modeling, we derive a criterion for the selection outcome of combination treatment, dependent on the burden and effective private benefit of Bla production. We then use engineered strains and natural isolates to show that strong private benefits of Bla are associated with increased selection for resistance. Finally, we demonstrate that this parameter can be coarsely estimated using high-throughput phenotyping of clonal populations. Our analysis shows that quantifying the phenotypic responses of bacteria to combination treatment can facilitate resistance-minimizing optimization of treatment. The authors derive a criterion for when β-lactam/β-lactamase inhibitor combinations can select against a β-lactam-resistant bacterial strain. In particular, the private benefit of resistance is shown to be estimable from clonal growth curves and predictive of selection outcomes.

Measurement of human plasma amyloid-β biomarkers using immunoprecipitation coupled with mass spectrometry reliably predicts individual brain amyloid-β status and has potential clinical utility. Plasma marker predicts amyloid-β pathology in the brain Alzheimer's disease is characterized by the deposition of amyloid-β (Aβ) peptide in the brain. The only available methods to reliably determine the levels of Aβ deposition are Aβ-PET imaging or measurement of Aβ levels in the cerebrospinal fluid. Therefore, identifying a blood-based biomarker that can be assessed in a minimally invasive and cost-effective manner is highly desirable. Katsuhiko Yanagisawa and colleagues use immunoprecipitation and mass spectrometry to measure the levels of several Aβ-related peptide fragments in blood. The APP 669–711 /Aβ 1–42 and Aβ 1–40 /Aβ 1–42 ratios and a composite score reliably predict individual levels of Aβ deposition in the brain. These results highlight the potential clinical utility of plasma biomarkers in predicting brain Aβ burden at an individual level. To facilitate clinical trials of disease-modifying therapies for Alzheimer’s disease, which are expected to be most efficacious at the earliest and mildest stages of the disease 1 , 2 , supportive biomarker information is necessary. The only validated methods for identifying amyloid-β deposition in the brain—the earliest pathological signature of Alzheimer’s disease—are amyloid-β positron-emission tomography (PET) imaging or measurement of amyloid-β in cerebrospinal fluid. Therefore, a minimally invasive, cost-effective blood-based biomarker is desirable 3 , 4 . Despite much effort 3 , 4 , 5 , 6 , 7 , to our knowledge, no study has validated the clinical utility of blood-based amyloid-β markers. Here we demonstrate the measurement of high-performance plasma amyloid-β biomarkers by immunoprecipitation coupled with mass spectrometry. The ability of amyloid-β precursor protein (APP) 669–711 /amyloid-β (Aβ) 1–42 and Aβ 1–40 /Aβ 1–42 ratios, and their composites, to predict individual brain amyloid-β-positive or -negative status was determined by amyloid-β-PET imaging and tested using two independent data sets: a discovery data set (Japan, n  = 121) and a validation data set (Australia, n  = 252 including 111 individuals diagnosed using 11 C-labelled Pittsburgh compound-B (PIB)-PET and 141 using other ligands). Both data sets included cognitively normal individuals, individuals with mild cognitive impairment and individuals with Alzheimer’s disease. All test biomarkers showed high performance when predicting brain amyloid-β burden. In particular, the composite biomarker showed very high areas under the receiver operating characteristic curves (AUCs) in both data sets (discovery, 96.7%, n  = 121 and validation, 94.1%, n  = 111) with an accuracy approximately equal to 90% when using PIB-PET as a standard of truth. Furthermore, test biomarkers were correlated with amyloid-β-PET burden and levels of Aβ 1–42 in cerebrospinal fluid. These results demonstrate the potential clinical utility of plasma biomarkers in predicting brain amyloid-β burden at an individual level. These plasma biomarkers also have cost–benefit and scalability advantages over current techniques, potentially enabling broader clinical access and efficient population screening.