Wild-type Research Articles

Exploring distinct modes of inter-spike cross-linking for enhanced neutralization by SARS-CoV-2 antibodies

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its Omicron subvariants drastically amplifies transmissibility, infectivity, and immune escape, mainly due to their resistance to most neutralizing antibodies. Thus, exploring the mechanisms underlying antibody evasion is crucial. Although the full-length native form of antibody, immunoglobulin G (IgG), offers valuable insights into the neutralization, structural investigations primarily focus on the fragment of antigen-binding (Fab). Here, we employ single-particle cryo-electron microscopy (cryo-EM) to characterize a W328-6H2 antibody, in its native IgG form complexed with severe acute respiratory syndrome (SARS), severe acute respiratory syndrome coronavirus 2 wild-type (WT) and Omicron variant BA.1 spike protein (S). Three high-resolution structures reveal that the full-length IgG forms a centered head-to-head dimer of trimer when binds fully stoichiometrically with both SARS and WT S, while adopting a distinct offset configuration with Omicron BA.1 S. Combined with functional assays, our results suggest that, beyond the binding affinity between the RBD epitope and Fab, the higher-order architectures of S trimer and full-length IgG play an additional role in neutralization, enriching our understanding of enhanced neutralization by SARS-CoV-2 antibodies.

SMAD5 as a novel gene for familial pulmonary arterial hypertension.

Genetic diagnostic testing of 325 pulmonary arterial hypertension (PAH) patients using a PAH specific gene panel including 18 known PAH genes revealed mutations in 23 %. Further PAH candidate genes were sequenced in the remaining patients exposing two SMAD5 variants, which were clinically and functionally characterized. We first recorded familial cosegregation and clinical parameters. Functional tests were performed following transient over-expression of the two SMAD5 variants in pulmonary artery smooth muscle cells (PAMSCs). Expression of these variants was confirmed by qPCR, Sanger sequencing and Western blotting. Cell viability was evaluated using cell counting kit 8, cell proliferation by BrdU and apoptosis by annexin V assay. Both SMAD5 missense variants were absent in healthy controls and predicted to be pathogenic. The variant c.1175T>C p.(Leu392Pro) was identified in a heritable PAH patient and her healthy son. The mother had died of suspected PAH aged 42. Expression of this variant in PAMSCs led to significantly higher cell viability due to higher proliferation in comparison to SMAD5 wild type cells. The second variant c.277T>A p.(Trp93Arg) was identified in a patient with congenital heart disease associated PAH with a surgically repaired ventricle septal defect. Its expression led to significant lower cell viability due to increased apoptosis in comparison to wild type SMAD5 cells. Taking into account familial aggregation, clinical findings and functional evidence both variants could be classified as likely pathogenic. This is the first description of SMAD5 as a potential novel PAH gene for genetic diagnostic testing.

MsDUF3700 overexpression enhances aluminum tolerance in alfalfa shoots.

This study identified a gene associated with aluminum stress through GWAS, which regulates aluminum tolerance in alfalfa by contributing to the antioxidant system. Aluminum (Al) ions precipitate in acidic soils with a pH < 5.5, where they are absorbed alongside other nutrients by plants, negatively impacting plant growth. Alfalfa, the most widely grown perennial legume forage in the world, is especially vulnerable to acidic soil conditions. Our research pinpointed MsDUF3700 as a potential gene linked to Al-response traits via genome-wide association analysis in Medicago sativa. MsDUF3700 encodes the domain of unknown function (DUF). We observed higher expression of MsDUF3700 in Al-tolerant alfalfa compared to Al-sensitive ecotypes. MsDUF3700-overexpressing transgenic alfalfa (MsDUF3700-OE) showed shorter root elongation and higher Al accumulation in roots than wild type (WT) under Al conditions. However, the shoots of MsDUF3700-OE lines showed enhanced growth rates under both normal and Al stress conditions. Under Al stress, MsDUF3700-OE lines showed increased H2O2 and malondialdehyde (MDA) levels in the roots, alongside reduced catalase activity, In contrast, the shoots showed an inverse trend. In addition, we found that MsDUF3700-OE alfalfa plants had high Al accumulation in the roots and low Al accumulation in the shoots. Transcripts of MsALS3 and MsPALT1, homologs of Al translocation in alfalfa, were downregulated, while MsNrat1, a homolog of transporters absorb Al, was upregulated in the roots of MsDUF3700-OE in alfalfa. Our research indicates that MsDUF3700 plays a role in aluminum stress by participating in antioxidative defense and facilitating aluminum transport from roots to shoots.

Sn-Position-Resolved Quantification of Aminophospholipids by Isotopic N,N-Dimethyl Leucine Labeling and High-Resolution Ion Mobility Mass Spectrometry.

Aminophospholipids (APLs), composed of phosphatidylethanolamines (PEs) and phosphatidylserines (PSs), are vital components of mammalian cell membranes and lipoproteins, participating in both homeostasis and cellular signaling. Their structural changes, including the permutation of fatty acid connectivity (sn-positions), due to dysfunctional metabolic processes have been linked to many diseases. However, the accurate quantification of APLs with unambiguous fatty acyl assignment through routine label-free LC-MS/MS lipidomic analysis remains a major challenge. In this study, we explore the functionalization of the free primary amine groups of APLs using amine-reactive isotopic N,N-dimethyl leucine (iDiLeu) and employ high-resolution ion mobility MS (IM-MS) to develop a novel method for sensitive discernment and accurate quantification of APL sn-isomers. With high-resolution demultiplexing (HRdm) providing IM resolving power >200, labeled sn-isomeric pairs of APLs (ΔCCS ≈ 1%) demonstrate excellent, near baseline separation. In addition to greatly enhanced sensitivity, 5-plex iDiLeu labeling enables the construction of an internal 4-point calibration curve and therefore absolute quantification of APL sn-isomers in a single run. This strategy enabled precise annotation and quantification of 239 APLs including 60 pairs of sn-isomers in the mouse cortex. Additionally, we were able to find ratio changes in multiple APL sn-isomer pairs between wild type and APP/PS1 Alzheimer's disease (AD) model mice at different ages, indicating their strong correlation to AD progression. This strategy could provide universal utility in unraveling the alteration of APL sn-isomers, which have long been considered as the "dark matter" of traditional lipidomic analyses, leading to more precise elucidation of molecular mechanisms of various diseases.

Molecular Characterization of SHV, BLA-OXA and B-Lactamase Resistant Genes Leading to Computational Based Inhibitor Screening Against Bacillus Species

Antibiotic resistance in bacterial pathogen is a great challenge that is connected with excessive morbidity and mortality of living beings. A common motive of antibiotic resistance in bacteria is an increased abundance of Β- lactamases. There chromosomal Β- lactamases do not generally provide effective antibiotic resistance in wild type Bacilli despite evidence that the genes are not completely silenced. Under antibiotic selection pressure however, a number of strains show increased resistance suggesting mutation induced up regulation of Β- lactamases. Computer-assisted drug design has made significant progress in predicting biologically active molecules and their receptor binding conformation, with results that are sometimes more exact than those acquired through traditional methods like as high-throughput screening. The results of computational studies are sometimes more precise than the experimental possibilities, and they contribute to the improvement of the experimental array. In this study, identified pathogenic species of bacillus, and analyzed the antibiotic resistant genes. The Previous study of its characterization showed the presence of BLA-OXA and SHV genes in Bacillus cereus and Bacillus paramycoides. The sequenced antibiotics resistant genes were subjected to computational analysis for the interpretation of the potential inhibitors against BLA-OXA and SHV. The analyzed inhibitor will enable to study the diversity of antibiotic resistant mechanism and to minimize the resistance of bacterial species against antibiotics.

Functional ultrasound and brain connectivity reveal central nervous system compromise in Trembler-J mice model of Charcot-Marie-Tooth disease

The Charcot-Marie-Tooth-1E (CMT1E) disease is typically described as a peripheral neuropathy in humans, causing decreased nerve conduction, spastic paralysis, and tremor. The Trembler-J (TrJ) mice serve as a high fidelity model of this disease. Here, we use functional ultrasound (fUS) and functional connectivity (FC) to analyze TrJ mice’s brain activity during sensory stimulation and resting state experiments against wild type (WT) mice - the healthy counterpart. fUS is an imaging technique that measures cerebral blood volume (CBV) temporal changes. We study these changes in the primary somatosensory cortex barrel field (S1BF) of both mice populations during periodic vibrissae stimulation, measuring the number of pixels that correlate to the stimulation (i.e., the size of the activation area), the average correlation of these pixels (i.e., the response strength), and the CBV’s rate of change for each stimulation (i.e., the hemodynamic response). Then, we construct a FC matrix for each genotype and experiment by correlating the CBV signals from the eight cortical regions defined by the Paxinos and Franklin atlas. Our results show that TrJ mice have significantly diminished neurovascular responses and altered brain connectivity with respect to WT mice, pointing to central nervous system effects that could shift our understanding of the CMT1E disease.

GH3 Gene Family Identification in Chinese White Pear (Pyrus bretschneideri) and the Functional Analysis of PbrGH3.5 in Fe Deficiency Responses in Tomato

Iron (Fe) deficiency poses a major threat to pear (Pyrus spp.) fruit yield and quality. The Gretchen Hagen 3 (GH3) plays a vital part in plant stress responses. However, the GH3 gene family is yet to be characterized, and little focus has been given to the function of the GH3 gene in Fe deficiency responses. Here, we identified 15 GH3 proteins from the proteome of Chinese white pear (Pyrus bretschneideri) and analyzed their features using bioinformatics approaches. Structure domain and motif analyses showed that these PbrGH3s were relatively conserved, and phylogenetic investigation displayed that they were clustered into two groups (GH3 I and GH3 II). Meanwhile, cis-acting regulatory element searches of the corresponding promoters revealed that these PbrGH3s might be involved in ABA- and drought-mediated responses. Moreover, the analysis of gene expression patterns exhibited that most of the PbrGH3s were highly expressed in the calyxes, ovaries, and stems of pear plants, and some genes were significantly differentially expressed in normal and Fe-deficient pear leaves, especially for PbrGH3.5. Subsequently, the sequence of PbrGH3.5 was isolated from the pear, and the transgenic tomato plants with PbrGH3.5 overexpression (OE) were generated to investigate its role in Fe deficiency responses. It was found that the OE plants were more sensitive to Fe deficiency stress. Compared with wild-type (WT) plants, the rhizosphere acidification and ferric reductase activities were markedly weakened, and the capacity to scavenge reactive oxygen species was prominently impaired in OE plants under Fe starvation conditions. Moreover, the expressions of Fe-acquisition-associated genes, such as SlAHA4, SlFRO1, SlIRT1, and SlFER, were all greatly repressed in OE leaves under Fe depravation stress, and the free IAA level was dramatically reduced, while the conjugated IAA contents were notably escalated. Combined, our findings suggest that pear PbrGH3.5 negatively regulates Fe deficiency responses in tomato plants, and might help enrich the molecular basis of Fe deficiency responses in woody plants.

Insights into the functional mechanisms of three terpene synthases from Lavandula angustifolia (Lavender)

Lavender species are of significant economic value being cultivated extensively worldwide for their essential oils (EOs), which include terpenes that play crucial roles in the cosmetic, personal care, and pharmaceutical industries. The terpene synthases in lavender, such as Lavandula angustifolia linalool synthase (LaLINS), limonene synthase (LaLIMS), and bergamotene synthase (LaBERS), are key enzymes in terpene biosynthesis. However, the functional mechanisms underlying these enzymes remain poorly understood. Here, we used AlphaFold2 to predict the three-dimensional structures of LaLINS, LaLIMS, and LaBERS. The hydrodynamic radii of LaLINS, LaLIMS, and LaBERS were 5.7 ± 0.2, 6.2 ± 0.3, and 5.4 ± 0.2 nm, respectively. Mutations D320A or D324A led to a complete loss of activity in LaLINS compared to the wild-type (WT) enzyme; similarly, mutations D356A or D360A abolished activity in LaLIMS, and D291A or D295A eliminated activity in LaBERS. Furthermore, the genes LaLINS, LaLIMS, and LaBERS exhibited significantly higher expression levels in leaves compared to stems and flowers, with peak expression occurring at 8:00 a.m. Our findings contribute to a deeper understanding of terpene biosynthesis in lavender and offer insights for improving essential oil production through genetic engineering.

3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase

Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

Caspase-8-dependent autophagy regulates neutrophil infiltration in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a subtype of head and neck cancer that arises in the multilayered epithelia of the mouth and lips. Although inactivating mutations in CASP8 are frequently found in human OSCC their role in the disease is unknown. To investigate this, we deleted Casp8 in the oral epithelium of adult mice. Loss of Caspase-8 resulted in defects in the tongue epithelial barrier and triggered a neutrophil-rich immune infiltrate distinct from that observed on epidermal Casp8 deletion. Oral Casp8 deletion led to activation of autophagy. Inhibition of autophagy partially rescued epithelial integrity in Casp8-/- mice, while induction of autophagy in wild type mice resulted in oral barrier defects and excessive neutrophil infiltration. On treatment with the carcinogen 4-nitroquinoline-1-oxide Casp8-/- mice showed increased susceptibility to developing oral tumors. Depletion of neutrophils reduced tumor incidence, which correlated with a reduction in reactive oxygen species and decreased epithelial DNA damage. Our findings establish a functional link between epithelial integrity, autophagy, and the tumor immune microenvironment, placing Caspase-8 at the center of these processes.

Altered hypoxia- and redox-related transcriptional signatures in mitochondrial-DNA-depleted PC-3 cells

Rho 0 (ρ0) cells are widely used as a tool to investigate how the absence of respiring mitochondria affects a variety of physiological and pathological processes. Prominently, ρ0 cells have been used to study the role of mitochondrial reactive oxygen species (ROS) production and/or mitochondrial respiration in the stabilization of the hypoxia-inducible factor (HIF) in hypoxia. In this study, we cultured ρ0 and WT PC-3 cells in 5% O2 (physioxia) and Plasmax medium for 2 weeks prior to transcriptomic and functional analyses. RNA-seq showed that ρ0 PC-3 cells exhibit impaired induction of HIF-regulated genes when exposed to hypoxia, compared to wild-type (WT) cells. Surprisingly, when comparing the transcriptomes of ρ0 and WT cells in physioxia (5% O2), we found a strong presence of HIF-related gene signatures in ρ0 cells compared to WT. Among the HIF targets found to be upregulated in ρ0 cells are CA9, EGLN3, EPAS1, HK2, ENO2, and SLC2A1. Moreover, several Nrf2 targets were upregulated in ρ0 cells, including NQO1, HMOX1, GPX2, and SLC7A11, which is in line with ρ0 cells showing a significantly higher H2O2 efflux rate than WT cells. Given the alterations in HIF-dependent and Nrf2-dependent gene expression and basal ROS production observed in ρ0 PC-3 cells, we conclude that caution should be taken when interpreting the results from experiments that focus on ROS production and HIF signaling using ρ0 cells as a model.

Very broad distribution of β sheet registries of the HIV gp41 fusion peptide supports mutational robustness for fusion and infection

HIV, like other membrane-enveloped viruses, has protein spikes that include a fusion peptide (Fp) segment that binds the host cell membrane and plays a critical role in fusion (joining) viral and cell membranes. The HIV Fp is the ~23 N-terminal residues of the gp41 spike protein. Fp adopts intermolecular antiparallel β sheet structure when lipid fraction cholesterol ≈0.3, which is comparable to host cells. Rotational-echo double-resonance NMR was applied to probe the registries (alignments) of adjacent Fp molecules in membrane-bound sheets. The data were fitted to determine quantitative populations, f(t)'s, of individual antiparallel registries indexed by t, the number of residues in the registry. Both wild-type (WT) and fusion-defective V2E Fp sheets have broad but very different registry distributions, each with at least eight populated registries with f(t) > 0.02, and [Formula: see text] and ⟨t⟩V2E = 18.5. The broad WT distribution likely improves mutational robustness for HIV, as Fp is a neutralization epitope of the immune system, and Fp mutations are required for immune evasion during chronic HIV infection. V2E fusion is reduced because longer Fp sheets increase separation between initially apposed membranes. The f(t)WT were well-fitted to free energies that were sums of contributions from sheet length, aligned leucines, and sidechain membrane insertion. The f(t)V2E's were similarly well-fitted except there wasn't the insertion contribution. Relative to V2E, WT fusion is enhanced by deeper membrane insertion of Fp with accompanying greater dislocation of neighboring lipids. This study provides a rare quantitative determination of broad molecular structural distributions by experiment.

Platelet-Derived Growth Factor Subunit A Strengthens the Neurovascular Unit and Inhibits Retinal Vascular Regression Under Hyperoxic Conditions

Retinopathy of prematurity (ROP) is primarily caused by the exposure of preterm infants with underdeveloped blood vessels to high oxygen concentrations. This damages the astrocytes that promote normal vascular development, leading to avascularity, pathological neovascularization, and retinal detachment, and even blindness as the disease progresses. In this study, the aim was to investigate the differences in the characteristics of astrocytes and blood vessels between wild-type (WT) and genetically modified mice overexpressing platelet-derived growth factor subunit A (PDGF-A) in the retina immediately after high oxygen exposure, a protocol in the oxygen-induced retinopathy (OIR) model of ROP. Our results showed that PDGF-A mice exhibited an increased population of astrocytes and higher vascular density than WT mice and that PDGF-A strengthened the resistance to hyperoxic conditions. In the OIR model, PDGF-A mice had reduced avascular zone areas following hyperoxia exposure. Furthermore, immunostaining for NG2 and CD31 showed that pericytes tended to regress earlier than endothelial cells, particularly at the vessel edges in both WT and transgenic mice, indicating relatively higher susceptibility to hyperoxia-induced damage. These findings suggest that PDGF-A plays a crucial role in stabilizing retinal vessels and may serve as a novel therapeutic target for ROP, highlighting the potential significance of PDGF-A in the pathological mechanisms of retinal diseases.

Exploring the Allosteric Response of Fascin to Its Inhibitor.

Fascin is a major actin-binding protein (ABP) for stabilizing filopodia to support efficient adhesion and migration of cancer cells. Fascin is also highly expressed in metastatic tumors. Disrupting the actin-binding site (ABS) on fascin constitutes a critical approach to hindering tumor metastasis. The G2 series of small molecules was formulated with the specific purpose of obstructing the binding pocket of fascin. The determination of inhibitor-induced structural dynamics in fascin is crucial for a comprehensive of its biological functions and the strategic development of pharmacological interventions. In this study, we utilized both equilibrium and dynamical-nonequilibrium molecular dynamics (D-NEMD) to elucidate the molecular mechanisms responsible for transmitting structural changes when removing the G2 inhibitor, in both the wild type (WT) and its variants. Our findings indicate that when G2 is removed, structural dynamics in fascin originate from the G2 binding pocket of fascin and propagate signals through the conformational transformation that spans all four β-trefoil domains. Although different mutant variants demonstrated comparable conformational networks, they showed varying response times. However, the signaling pathways in mutants remained consistent in comparison to the WT fascin. This study provides valuable insights into the structural features and communication pathways of fascin and provides avenues for the development of targeted inhibitors with promising prospects in cancer therapy.

Synergistic role of Rubisco inhibitor release and degradation inphotosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) exhibits catalytic promiscuity, resulting in error-prone reactions and the formation of inhibitory sugar phosphates. Specifically, Xylulose-1,5-bisphosphate (XuBP) acts as an inhibitor by binding to the active site of Rubisco, thereby impairing its catalytic function. Thermolabile Rubisco activase (Rca) facilitates the release of such inhibitors, including XuBP, by remodelling Rubisco. In Arabidopsis thaliana, the phosphatase pair CbbYA and CbbYB subsequently hydrolyses XuBP to prevent its rebinding to Rubisco. To explore the functional interplay between these components in maintaining photosynthesis, cbbya, cbbyb and cbbyab mutants were crossed with RCA knockdown (rca-2) lines. Additionally, both RCA and CBBYA were overexpressed in wild-type (WT) Arabidopsis thaliana. Phenotypic analyses revealed an exacerbation in decreased growth and photosynthetic efficiency in the cbbyab rca-2 double mutants compared with the control mutants (cbbyab and rca-2), indicating a negative genetic interaction. Furthermore, the co-overexpression of RCA and CBBYA did not improve photosynthesis under short-term heat stress, and light reactions were adversely affected relative to the WT. These findings illustrate the synergistic roles of Rca, CbbYA and CbbYB in maintaining carbon fixation and promoting plant growth in Arabidopsis thaliana. Thus, the coordinated regulation of Rca and CbbY enzymes is crucial for optimizing photosynthetic efficiency.

Dysregulation of protein degradation and alteration of secretome in α-synuclein-exposed astrocytes: implications for dopaminergic neuronal dysfunction

BackgroundA key factor in the propagation of α-synuclein pathology is the compromised protein quality control system. Variations in membrane association and astrocytic uptake between different α-synuclein forms suggest differences in exocytosis or membrane cleavage, potentially impacting the secretome's influence on dopaminergic neurons. We aimed to understand differences in protein degradation mechanisms of astrocytes for both wild-type (WT) and mutant forms of α-synuclein, specifically during periods of reduced degradation efficiency. We also investigated α-synuclein release into the secretome and its effects on healthy dopaminergic neurons.MethodsCellular models used were rat primary astrocytes alongside hiPSC-derived astrocytes, whose impact on rat primary dopaminergic neurons and the human SH-SY5Y cell line was investigated. We examined the release and accumulation of α-synuclein resulting from impaired degradatory pathways, including matrix metalloprotease-MMP9, the ubiquitin proteasomal pathway-UPS, and the autophagy-lysosomal pathway-ALP, using immunocytochemical analysis and flow cytometry. Additionally, we explored the effect of astrocytic secretome on dopaminergic-neuronal survival, neurite collapse and function.ResultsAt early stages, astrocytes were able to deal efficiently with monomeric α-synuclein (via UPS), and larger aggregates (through MMP9 and autophagy), clearing extracellular α-synuclein and maintaining neuronal health. However, extended exposure to extracellular monomeric and aggregated α-synuclein compromised their proteasomal activity, inhibiting MMP9 and destabilizing autophagy, transforming astrocytes from protectors to promoters of neurodegeneration. This study is the first to elucidate the astrocytes' preferred degradation pathways for both monomeric and aggregated forms of α-synuclein, along with the subsequent effects of these payloads on the cellular degradation machinery. The astrocytic transformation is characterized by α-synuclein expulsion, increased release of inflammatory cytokines, and diminished secretion of growth factors leading to dopaminergic neuronal apoptosis and dysfunction, particularly neurite collapse, intracellular Ca2+ response and vesicular dopamine release. The presence of phosphorylated and nitrated α-synuclein species in astrocytes also suggests their potential involvement in modifying both forms of the protein.ConclusionThe initial protective action of astrocytes in clearing and degrading extracellular α-synuclein is severely compromised at latter stages, leading to astrocytic dysfunction and impairing neuron-glia cross-talk. This study underscores the criticality of integrating astrocytes into treatment paradigms in synucleinopathies.

Regulatory T cells crosstalk with tumor cells and endothelium through lymphotoxin signaling

Regulatory T cells (Tregs) with multifaceted functions suppress anti-tumor immunity by signaling surrounding cells. Here we report Tregs use the surface lymphotoxin (LT)α1β2 to preferentially stimulate LT beta receptor (LTβR) nonclassical NFκB signaling on both tumor cells and lymphatic endothelial cells (LECs) to accelerate tumor growth and metastasis. Selectively targeting LTβR nonclassical NFκB pathway inhibits tumor growth and migration in vitro. Leveraging in vivo Treg LTα1β2 interactions with LTβR on tumor cells and LECs, transfer of wild type but not LTα-/- Tregs promotes B16F10 melanoma growth and tumor cell-derived chemokines in LTβR-/- mice; and increases SOX18 and FLRT2 in lymphatic vessels of LTβR-/- melanoma. Blocking the nonclassical pathway suppresses tumor growth and lymphatic metastasis by reducing chemokine production, restricting Treg recruitment to tumors, and retaining intratumoral IFNγ+ CD8 T cells. Our data reveals that Treg LTα1β2 promotes LTβR nonclassical NFκB signaling in tumor cells and LECs providing a rational strategy to prevent Treg promoted tumor growth and metastasis.

The co-chaperone DNAJA2 buffers proteasomal degradation of cytosolic proteins with missense mutations

Mutations can disrupt a protein's native function by causing misfolding, which is generally handled by an intricate protein quality control network. To better understand the triaging mechanisms of misfolded cytosolic proteins, we screened a human mutation library to identify a panel of unstable mutations. The degradation of these mutated cytosolic proteins is largely dependent on the ubiquitin proteasome system. Using BioID proximity labelling, we found that the co-chaperones DNAJA1 and DNAJA2 are key interactors with one of the mutated proteins. Notably, the absence of DNAJA2 increases the turnover of the mutant but not the wild-type protein. Our work indicates that specific missense mutations in cytosolic proteins can promote enhanced interactions with molecular chaperones. Assessment of the broader panel of cytosolic mutant proteins shows that the co-chaperone DNAJA2 exhibits two distinct behaviours: acting to stabilize a wide array of cytosolic proteins including wild type variants, and specifically “buffer” some mutant proteins to reduce their turnover. Our work illustrates how distinct elements of the protein homeostasis network are utilized in the presence of a cytosolic misfolded protein.

High-resolution imaging atlas reveals the context-dependent role of pancreatic sympathetic innervation in diabetic mice.

A better understanding of how sympathetic nerves impact pancreatic function is helpful for understanding diabetes. However, there is still uncertainty and controversy surrounding the roles of sympathetic nerves within the pancreas. To address this, we utilize high-resolution imaging and advanced three-dimensional (3D) reconstruction techniques to study the patterns of sympathetic innervation and morphology in the islets of adult wild-type (WT) and diabetic mice. Our data show that more than ~30% of α/β-cells are innervated by sympathetic nerves in both WT and diabetic mice. Additionally, sympathetic innervated α/β-cells are reduced in diet-induced obese (DIO) mice, whereas sympathetic innervated β-cells are increased in db/ db mice. In addition, in situ chemical pancreatic sympathetic denervation (cPSD) improves glucose tolerance in WT and db/ db mice but decreases glucose tolerance in DIO mice. In situ cPSD also enhances insulin sensitivity in diabetic mice without affecting WT mice. Overall, our findings advance our understanding of diabetes by highlighting the distinctive impact of pancreatic sympathetic innervation on glucose regulation.

Bacillus quorum quenching shapes the citrus mycobiome through interkingdom signaling

Microbiomes are sustained through infinite yet mutually interacting microbial communities, with bacteria and fungi serving as the major constituents. In recent times, microbial interventions have become popular for microbiome manipulation to achieve sustainable goals. Whether and how the introduced biocontrol agent drives fungal microbial assemblages (mycobiome) and the role of interkingdom signaling in shaping the microbiome structure and function remain poorly understood. Here, we implemented wild-type (WT) Bacillus subtilis L1–21 and its quorum quenching (QQ) mutants (L1–21Δytnp, L1–21Δyxel) individually and as consortia to explore the enrichment patterns of key mycobiome members in Huanglongbing (HLB) infected citrus compartments including leaf endosphere, root endosphere, and rhizosphere soil. The application of WT and its QQ mutants produced differential mycobiome enrichment across citrus compartments. Our findings reveal that application of WT B. subtilis enriched beneficial fungi such as Trichoderma (15.82 %) in leaf endosphere. In contrast, pathogenic fungi Fusarium (47.5 %) and Gibberella (0.47 %) involved in citrus root decline were adundant in the L1–21Δytnp treated root endosphere while Nigrospora (11 %) was predominant in L1–21Δyxel treated leaf endosphere, affirming the role of bacterial quorum sensing (QS) molecules in shaping the fungal community composition. In general, based on the fungal functional prediction, fungal pathogens were highly abundant in mutant-treated plants, particularly in leaf endosphere (L1–21Δytnp: 25 %; L1–21Δyxel: 36.35 %) compared to WT (20.93 %). Additionally, some fungal members exhibited strong compartment specificity and both mutants induced distinct mycobiome shifts in rhizosphere soil, leaf, and root endopshere. In conclusion, B. subtilis QQ modifies bacterial QS networks facilitating beneficial fungi to establish, while loss of QQ leads to enrichment of pathogenic fungal groups. Our study provides a direct link of perception and regulation of mycobiome through bacterial-based QS and QQ system, and its association with disease outcomes.