Inhibitory Protein Research Articles

Network and systems biology approaches help investigate gene regulatory interactions between Salmonella disease and host in chickens: Model-based in silico evidence combined with gene expression assays.

Salmonella enteritidis (SE), a previously widespread infectious disease, is still cited as a major factor in economic losses in commercial chicken production. The host's genetic immune system determines the pathogenicity of a particular bacterium. To shed light on this topic, it was necessary to understand the key candidate genes essential for regulating susceptibility and resistance to the target disease. The field of poultry farming in particular has benefited greatly from the connection between quantitative and molecular genetics. This study aims to identify the most important immune-related genes and their signalling pathways (gene ontology, co-expression and interactions) and to analyse their accumulation in host-resistant SE diseases by combining gene expression assays with model-based in silico evidence. A two-step experimental design is followed. To start, we used free computational tools and online bioinformatics resources, including predicting gene function using a multiple association network integration algorithm (geneMania), the Kyoto Encyclopedia of Genes and Genomes, the Annotation, Visualization and Integrated Discovery (DAVID) database and the stimulator of interferon genes. Natural resistance-associated macrophage protein 1 (NRAMP1), Toll-like receptor 4 (TLR4), interferon-γ (IFNγ), immunoglobulin Y (IgY) and interleukin 8 (IL8) were among the five genes whose expression levels in liver, spleen, and cecum were evaluated at 1107 SE after 48h of inoculation. This molecular study was developed in the second phase of research to validate the in silico observations. Next, we use five promising biomarkers for relative real-time polymerase chain reaction (PCR) quantification: TLR4, IL8, NRAMP1, IFNγ and IgY genes in two case and control assays. The 2-∆∆Ct Livak and Schmittgen method was used to compare the expression of genes in treated and untreated samples. This method normalizes the expression of the target gene to that of actin, an internal control and estimates the change in expression relative to the untreated control. Internal control was provided by the Beta actin gene. Next, statistically, the postdoc test was used for the evaluation of treatments using SAS version 9.4, and p values of 0.05 and 0.01 were chosen for significant level. Interestingly, the results of our study suggest the involvement of various factors in the host immune response to Salmonella. These include inducible nitric oxide synthase, NRAMP1, immunoglobulin light chain (IgL), transforming growth factor B family (TGFb2, TGFb3 and TGFb4), interleukin 2 (IL2), apoptosis inhibitor protein 1 (IAP1), TLR4, myeloid differentiation protein 2 (MD2), IFNγ, caspase 1 (CASP1), lipopolysaccharide-induced tumour necrosis factor (LITAF), cluster of differentiation 28 (CD28) and prosaposin (PSAP). The summary of gene ontology and related genes found for SE resistance was surprisingly comprehensive and covered the following topics: positive regulation of endopeptidase activity, interleukin-8 production, chemokine production, interferon-gamma production, interleukin-6 production, positive regulation of mononuclear cell proliferation and response to interferon-gamma. The role of these promising biomarkers in our networks against SE susceptibility is essentially confirmed by these results. After 48h, the spleen showed significant expression of the tissue-specific gene expression patterns for NRAMP1 and IL8 in the cecum, spleen and liver. Based on this information, this report searches for resistance and susceptibility lineages in most genomic regions for SE. In conclusion, the development of an appropriate selection program to improve resistance to salmonellosis can be facilitated by a comprehensive understanding of the immune responses of the chicken immune system after SE exposure.

Optimizing aldosterone receptor antagonist therapy by sodium zirconium cyclosilicate in heart failure - OPRA-HF trial

Abstract Background Heart failure remains a significant health burden, characterized by disabling symptoms and mortality rates comparable to cancer. Mineralocorticoid antagonists (MRAs), pivotal in treating heart failure with reduced ejection fraction (HFrEF), are underutilized due to concerns of hyperkalemia. Sodium Zirconium Cyclosilicate (SZC) enables optimized MRA therapy by reducing hyperkalemia. However, prospective studies on the efficacy and safety of SZC in patients with HFrEF and high hyperkalemia risk are lacking, particularly in the context of contemporary heart failure quadruple-therapy including angiotensin receptor neprilysin inhibitor (ARNI) and Sodium-Glucose Transport Protein 2 (SGLT2) Inhibitors. Purpose The OPRA-HF trial aims to assess the efficacy and safety of SZC in optimizing MRA treatment in symptomatic patients with HFrEF with risk for hyperkalemia on top of optimal guideline-directed medical therapy including ARNI and SGLT2 Inhibitors. Methods This investigator-initiated multicentred, randomized, placebo-controlled, and double-blinded trial include patients with HFrEF on suboptimal MRA treatment due to (1) history of hyperkalemia due to MRA, or (2) risk of hyperkalemia due to reduced renal function (eGFR 30-45 ml/min/m2) and current potassium 4.5-5-0 mmol/L. Patients undergo a Run-In phase with SZC, initiating or up-titrating MRA to target doses for 4-7 weeks and SZC was initiated in those patients that became hyperkalemic (K+>5 mEq/L. Those normokalemic and tolerating MRA ≥ 25 mg daily or at least 25 mg dose increase vs before run-in were randomized to SZC (5g or 10g) or placebo for 6 months. Study is powered to randomize 110 patients. Primary outcomes include maintaining daily MRA dose ≥ 25 mg or dose increase by 25 mg with normal potassium levels without rescue therapy. Secondary outcomes include evaluating the safety and tolerability of SZC as well as impact of SZC on quality of life. Results In this ongoing trial, so far 40 patients have been randomized. Of these, mean age was 74.3 years, 88.9% men. Most patients had ischemic etiology (54.2%), 36.1% had diabetes mellitus, 52.8% had hypertension.n. At screening 94.4% had beta-blockers, 35% ACEI/ARB, 60% ARNI and 72.2% had SGLT2 inhibitors, 33.3% had no MRAs and 35,6% of patients had less than 25 mg MRA daily,62,2% had 25 mg MRA daily, mean p-potassium was 4.7+0.4 mmol/L, and eGFR was 57.3 ml/min/1.73m². After run-in, 85.2% were on 50 mg MRA daily. A substantial number (51.2%) of eligible patients with previous hyperkalemia had Run-In failure as they now successfully tolerated target dose of MRA at 50 mg daily without recurrent hyperkalemia. SZC dosing was 5g once daily in 78.9% of patients. Conclusion This ongoing trial will provide evidence for the use of SZC to optimize MRA treatment in contemporary HFrEF patients. Preliminary findings suggest SZC effectively maintains normokalemia, enabling MRA optimization, potentially improving patients’ otcome.

Recent advances in mRNA-based cancer vaccines encoding immunostimulants and their delivery strategies

The high prevalence of drug resistance, relapse, and unfavorable response rate of conventional cancer therapies necessitate the development of more efficient treatment modalities. Immunotherapy represents a novel therapeutic approach to cancer treatment in which the immune system's potential is harnessed to recognize and eliminate tumor cells. mRNA cancer vaccines, as a burgeoning field of immunotherapy, have recently drawn particular attention, and among mRNAs encoding tumor-associated antigens, tumor-specific antigens, and immune stimulatory factors, the latter has been relatively less explored. These immunostimulatory mRNAs encode a range of proteins, including stimulatory ligands, receptors, enzymes, pro-inflammatory cytokines, and inhibitory binding proteins, which collectively augment the host immune system's ability against cancerous cells. In this review, we aimed to provide a comprehensive account of mRNA-based cancer vaccines encoding immune stimulants, encompassing their current status, mechanisms of action, delivery strategies employed, as well as recent advances in preclinical and clinical studies. The potential challenges, strategies and future perspectives have also been discussed.

Dissection of signaling pathways regulating TrkB-dependent gephyrin clustering

IntroductionThe TrkB receptor is known for its role in regulating excitatory neuronal plasticity. However, accumulating evidence over the past decade has highlighted the involvement of TrkB in regulating inhibitory synapse stability and plasticity, particularly through regulation of the inhibitory scaffold protein gephyrin, although with contradicting results.MethodsIn this study, we extended on these findings by overexpressing rat TrkB mutants deficient in either Shc-or PLCγ-dependent signaling, as well as a kinase-dead mutant, to dissect the contributions of specific TrkB-dependent signaling pathways to gephyrin clustering.ResultsOur results demonstrate that TrkB signaling is required for gephyrin clustering on the perisomatic area of granule cells in the dentate gyrus in vivo. To further investigate, we expressed TrkB wild-type and mutants in hippocampal neurons in vitro.DiscussionUnder basal conditions, TrkB-Shc signaling was important for the reduction of gephyrin cluster size, while TrkB-PLCγ signaling accounts for gephyrin clustering specifically at synaptic sites. Concomitant, impaired PLCγ signaling was associated with disinhibition of transduced neurons. Moreover, chemically induced inhibitory long-term potentiation (chem iLTP) depended on TrkB signaling and the activation of both Shc and PLCγ pathways.ConclusionOur findings suggest a complex, pathway-specific regulation of TrkB-dependent gephyrin clustering, both under basal conditions and during chem iLTP.

DNA methylation shapes the Polycomb landscape during the exit from naive pluripotency.

In mammals, 5-methylcytosine (5mC) and Polycomb repressive complex 2 (PRC2)-deposited histone 3 lysine 27 trimethylation (H3K27me3) are generally mutually exclusive at CpG-rich regions. As mouse embryonic stem cells exit the naive pluripotent state, there is massive gain of 5mC concomitantly with restriction of broad H3K27me3 to 5mC-free, CpG-rich regions. To formally assess how 5mC shapes the H3K27me3 landscape, we profiled the epigenome of naive and differentiated cells in the presence and absence of the DNA methylation machinery. Surprisingly, we found that 5mC accumulation is not required to restrict most H3K27me3 domains. Instead, this 5mC-independent H3K27me3 restriction is mediated by aberrant expression of the PRC2 antagonist Ezhip (encoding EZH inhibitory protein). At the subset of regions where 5mC appears to genuinely supplant H3K27me3, we identified 163 candidate genes that appeared to require 5mC deposition and/or H3K27me3 depletion for their activation in differentiated cells. Using site-directed epigenome editing to directly modulate 5mC levels, we demonstrated that 5mC deposition is sufficient to antagonize H3K27me3 deposition and confer gene activation at individual candidates. Altogether, we systematically measured the antagonistic interplay between 5mC and H3K27me3 in a system that recapitulates early embryonic dynamics. Our results suggest that H3K27me3 restraint depends on 5mC, both directly and indirectly. Our study also implies a noncanonical role of 5mC in gene activation, which may be important not only for normal development but also for cancer progression, as oncogenic cells frequently exhibit dynamic replacement of 5mC for H3K27me3 and vice versa.

Uncoupling of mTORC1 from E2F activity maintains DNA damage and senescence

DNA damage is a primary trigger for cellular senescence, which in turn causes organismal aging and is a promising target of anti-aging therapies. Most DNA damage occurs when DNA is fragile during DNA replication in S phase, but senescent cells maintain DNA damage long-after DNA replication has stopped. How senescent cells induce DNA damage and why senescent cells fail to repair damaged DNA remain open questions. Here, we combine reversible expression of the senescence-inducing CDK4/6 inhibitory protein p16INK4 (p16) with live single-cell analysis and show that sustained mTORC1 signaling triggers senescence in non-proliferating cells by increasing transcriptional DNA damage and inflammation signaling that persists after p16 is degraded. Strikingly, we show that activation of E2F transcriptional program, which is regulated by CDK4/6 activity and promotes expression of DNA repair proteins, repairs transcriptionally damaged DNA without requiring DNA replication. Together, our study suggests that senescence can be maintained by ongoing mTORC1-induced transcriptional DNA damage that cannot be sufficiently repaired without induction of protective E2F target genes.

Magnolol Inhibits High Fructose-Induced Podocyte Inflammation via Downregulation of TKFC/Sp1/HDAC4/Notch1 Activation

Background/Objectives: High fructose has been implicated as an important trigger of kidney inflammation in patients and experimental models. Magnolol, isolated from Magnolia officinalis, has an anti-inflammatory effect, but its protective role in podocytes remains underexplored. This study explored the protective effects and underlying mechanism of magnolol against high fructose-induced podocyte inflammation. Methods: The effects of magnolol on high fructose-induced podocyte inflammation were assessed in male Sprague Dawley rats administered 10% (w/v) fructose water for 12 weeks and heat-sensitive human podocyte cell lines (HPCs) exposed to 5 mM fructose. Podocyte foot processes were examined using transmission electron microscopy. The expression levels of nephrin, podocin, tumor necrosis factor-α (TNF-α), Notch1 intracellular domain (NICD1), triokinase/FMN cyclase (TKFC), specificity protein 1 (Sp1) and histone deacetylase 4 (HDAC4) were determined by Western blot, immunofluorescence and real-time quantitative polymerase chain reaction (qRT-PCR). The chromatin immunoprecipitation (ChIP) assay was performed to evaluate the interaction between Sp1 and the promoter region of HDAC4. Results: Magnolol mitigated the impairment of glomerular filtration function in high fructose-fed rats. Besides, it significantly alleviated the inflammatory responses in glomeruli and HPCs, evidenced by decreased protein levels of TNF-α and NICD1. Increased protein levels of TKFC, Sp1 and HDAC4 were observed in high fructose-stimulated HPCs and rat glomeruli. TMP195, an HDAC4 inhibitor, reduced TNF-α and NICD1 protein levels in high fructose-exposed HPCs. The increased Sp1 was shown to associate with the promoter region of HDAC4, promoting HDAC4 protein expression in high fructose-exposed HPCs. The knockdown of TKFC in HPCs by TKFC siRNA decreased Sp1, HDAC4 and NICD1 protein levels, alleviating podocyte inflammatory response. Furthermore, magnolol inhibited TKFC/Sp1/HDAC4/Notch1 activation in vivo and in vitro. Conclusions: Magnolol attenuated high fructose-induced podocyte inflammation possibly through the suppression of TKFC/Sp1/HDAC4/Notch1 activation, providing new evidence for its potential role in podocyte protection.

Deciphering heart failure: an integrated proteomic and transcriptomic approach with experimental validation.

This study analyzed transcriptomic and proteomic data to identify molecular changes during heart failure (HF). Additionally,we embarked on an exploration of the prospect of therapeutic intervention through the manipulation of proteins implicated in ferroptosis. Three publicly available microarray datasets (GSE135055, GSE147236, GSE161472) profiling left ventricular samples from HF patients and healthy controls were obtained. Differentially expressed genes were identified in each dataset and cross-analyzed to determine shared gene signatures. Enrichment analysis of Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and gene set enrichment analysis were performed. Differentially expressed proteins were obtained from published proteomic studies and integrated with the transcriptomic results. To validate findings, a HF mouse model was generated and ferroptosis-related proteins were evaluated. Additionally, the effect of suppression of ferroptosis on hypoxia-induced ischemia model in HL-1 cardiomyocytes was assessed by knocking down Acyl-CoA synthetase long-chain family member 4 (ACSL4) using small interfering RNA (siRNA).Cross-analysis of differentially expressed genes (DEGs) in the GSE135055, GSE147236 and GSE161472 datasets revealed 224 up-regulated and 187 down-regulated potential genes which showed high enrichment in immune, inflammatory and metabolic pathways. Notably, four proteins, among them ACSL4, displayed consistent alterations at both the transcriptional and protein levels. In the HF mouse model, ACSL4 exhibited an elevation, whereas negative regulators of ferroptosis witnessed a decrement. Subsequently, knockdown of ACSL4 in a hypoxia-induced ischemic HL-1 cardiomyocyte cell model upregulated the expression of ferroptosis inhibitory protein and decreased the levels of reactive oxygen species (ROS), malondialdehyde (MDA)., and free iron and increased cell viability. Comprehensive multi-omics analysis revealed that the expression of the molecular target ACSL4 was increased in HF. Targeting ACSL4 to inhibit ferroptosis may represent a novel therapeutic strategy for HF treatment.

Modular Nanotransporters Deliver Anti-Keap1 Monobody into Mouse Hepatocytes, Thereby Inhibiting Production of Reactive Oxygen Species.

Background/Objectives: The study of oxidative stress in cells and ways to prevent it attract increasing attention. Antioxidant defense of cells can be activated by releasing the transcription factor Nrf2 from a complex with Keap1, its inhibitor protein. The aim of the work was to study the effect of the modular nanotransporter (MNT) carrying an R1 anti-Keap1 monobody (MNTR1) on cell homeostasis. Methods: The murine hepatocyte AML12 cells were used for the study. The interaction of fluorescently labeled MNTR1 with Keap1 fused to hrGFP was studied using the Fluorescence-Lifetime Imaging Microscopy-Förster Resonance Energy Transfer (FLIM-FRET) technique on living AML12 cells transfected with the Keap1-hrGFP gene. The release of Nrf2 from the complex with Keap1 and its levels in the cytoplasm and nuclei of the AML12 cells were examined using a cellular thermal shift assay (CETSA) and confocal laser scanning microscopy, respectively. The effect of MNT on the formation of reactive oxygen species was studied by flow cytometry using 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. Results: MNTR1 is able to interact with Keap1 in the cytoplasm, leading to the release of Nrf2 from the complex with Keap1 and a rapid rise in Nrf2 levels both in the cytoplasm and nuclei, ultimately causing protection of cells from the action of hydrogen peroxide. The possibility of cleavage of the monobody in endosomes leads to an increase in the observed effects. Conclusions: These findings open up a new approach to specifically modulating the interaction of intracellular proteins, as demonstrated by the example of the Keap1-Nrf2 system.

Targeting VPS18 hampers retromer trafficking of PD-L1 and augments immunotherapy.

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) or programmed cell death-ligand 1 (PD-L1) have achieved impressive antitumor clinical outcomes. However, the limited response rates suggest the incomplete understanding of PD-L1 regulation. Here, we demonstrate that vacuole protein sorting 11 and 18 (VPS11/18), two key players in vesicular trafficking, positively regulate PD-L1 and confer resistance to immune checkpoint blockade therapy. VPS11/18 interact with PD-L1 in endosome recycling accompanied by promoting PD-L1 glycosylation and protein stability. VPS18 deficiency enhances antitumor immune response. Pharmacological inhibition by VPS18 inhibitor RDN impaired PD-L1 member trafficking and protein stability. Combination treatment of RDN and anti-cytotoxic T lymphocyte-associated antigen 4 synergistically enhances antitumor efficacy in aggressive and drug-resistant tumors. RDN exerted lung-preferred distribution and good bioavailability, suggesting a favorable drug efficacy. Together, our study links VPS18/11-mediated trans-Golgi network recycling of PD-L1 and points to a promising treatment strategy for the enhancement of antitumor immunity.

Description and functional validation of human enteroendocrine cell sensors.

Enteroendocrine cells (EECs) are gut epithelial cells that respond to intestinal contents by secreting hormones, including the incretins glucagon-like peptide 1 (GLP-1) and gastric inhibitory protein (GIP), which regulate multiple physiological processes. Hormone release is controlled through metabolite-sensing proteins. Low expression, interspecies differences, and the existence of multiple EEC subtypes have posed challenges to the study of these sensors. We describe differentiation of stomach EECs to complement existing intestinal organoid protocols. CD200 emerged as a pan-EEC surface marker, allowing deep transcriptomic profiling from primary human tissue along the stomach-intestinal tract. We generated loss-of-function mutations in 22 receptors and subjected organoids to ligand-induced secretion experiments. We delineate the role of individual human EEC sensors in the secretion of hormones, including GLP-1. These represent potential pharmacological targets to influence appetite, bowel movement, insulin sensitivity, and mucosal immunity.

P57Kip2 Phosphorylation Modulates Its Localization, Stability, and Interactions.

p57Kip2 is a member of the cyclin-dependent kinase (CDK) Interacting Protein/Kinase Inhibitory Protein (CIP/Kip) family that also includes p21Cip1/WAF1 and p27Kip1. Different from its siblings, few data are available about the p57Kip2 protein, especially in humans. Structurally, p57Kip2 is an intrinsically unstructured protein, a characteristic that confers functional flexibility with multiple transient interactions influencing the metabolism and roles of the protein. Being an IUP, its localization, stability, and binding to functional partners might be strongly modulated by post-translational modifications, especially phosphorylation. In this work, we investigated by two-dimensional analysis the phosphorylation pattern of p57Kip2 in different cellular models, revealing how the human protein appears to be extensively phosphorylated, compared to p21Cip1/WAF1 and p27Kip1. We further observed clear differences in the phosphoisoforms distributed in the cytosolic and nuclear compartments in asynchronous and synchronized cells. Particularly, the unmodified form is detectable only in the nucleus, while the more acidic forms are present in the cytoplasm. Most importantly, we found that the phosphorylation state of p57Kip2 influences the binding with some p57Kip2 partners, such as CDKs, LIMK1 and CRM1. Thus, it is necessary to completely identify the phosphorylated residues of the protein to fully unravel the roles of this CIP/Kip protein, which are still partially identified.

Inflammasome protein scaffolds the DNA damage complex during tumor development.

Inflammasome sensors activate cellular signaling machineries to drive inflammation and cell death processes. Inflammasomes also control the development of certain diseases independently of canonical functions. Here, we show that the inflammasome protein NLR family CARD domain-containing protein 4 (NLRC4) attenuated the development of tumors in the Apcmin/+ mouse model. This response was independent of inflammasome signaling by NLRP3, NLRP6, NLR family apoptosis inhibitory proteins, absent in melanoma 2, apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1 and caspase-11. NLRC4 interacted with the DNA-damage-sensing ataxia telangiectasia and Rad3-related (ATR)-ATR-interacting protein (ATRIP)-Ewing tumor-associated antigen 1 (ETAA1) complex to promote the recruitment of the checkpoint adapter protein claspin, licensing the activation of the kinase checkpoint kinase-1 (CHK1). Genotoxicity-induced activation of the NLRC4-ATR-ATRIP-ETAA1 complex drove the tumor-suppressing DNA damage response and CHK1 activation, and further attenuated the accumulation of DNA damage. These findings demonstrate a noninflammatory function of an inflammasome protein in promoting the DNA damage response and mediating protection against cancer.

Selective activation of PPARα by pemafibrate mitigates peritoneal inflammation and fibrosis through suppression of NLRP3 inflammasome and modulation of inflammation

Peritoneal inflammation and fibrosis remain major challenges to the long-term maintenance of peritoneal dialysis. Pemafibrate, a selective peroxisome proliferator-activated receptor α (PPARα) modulator, has been implicated in the management of fibrosis-related disorders. We investigated whether pemafibrate ameliorates peritoneal inflammation and fibrosis and explored the underlying mechanisms in mice with methylglyoxal (MGO)-induced peritoneal fibrosis (MGO mice). MGO mice exhibited peritoneal fibrosis with increased expression of mesenchymal markers, transforming growth factor-β1 (TGF-β1), and substantial deposition of extracellular matrix (ECM) proteins. Additionally, MGO mice exhibited peritoneal inflammation as indicated by elevated tumor necrosis factor-α expression and macrophage infiltration in peritoneal tissue. These effects were mitigated by pemafibrate treatment, which also restored peritoneal membrane function. Furthermore, pemafibrate promoted anti-inflammatory macrophage polarization in both mice and THP-1 cells. In human peritoneal mesothelial cells (HPMCs), pemafibrate effectively inhibited interferon-γ-induced production of TGF-β1 and ECM while suppressing the proinflammatory cytokines nuclear factor-κB (NF-κB) and activator protein 1. The NF-κB inhibitory effect of pemafibrate involved stabilization of the NF-κB inhibitory protein IkBα. Notably, pemafibrate hindered activation of the NLR family pyrin domain containing 3/caspase-1 axis in interferon-γ-stimulated THP-1 cells. These findings suggest that pemafibrate ameliorates peritoneal inflammation and fibrosis, making it a promising candidate for peritoneal fibrosis therapy.

12231 PAPP-A As A Potential Target In Thyroid Eye Disease

Abstract Disclosure: C.A. Conover: None. L.K. Bale: None. D. Hamadi: None. M. Stan: ; Tourmaline, Genentech, Third Rock Ventures, ArgenX, Septerna, OrthoDi. ; Horizon, Immunovant, Lassen, Sling. Background: Proptosis in Thyroid Eye Disease (TED) can result in facial disfigurement and visual dysfunction. Studies revealed an interaction between thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor-I receptor (IGF-IR) that is essential in promoting TED pathogenesis. Treatment with Insulin-like growth factor I receptor (IGF-IR) inhibitors has been shown to be effective in reducing proptosis but with side effects. PAPP-A is metalloprotease that can increase pericellular IGF bioavailability through specific cleavage of inhibitory IGF binding proteins, in particular IGFBP-4. We propose that inhibition of IGF-IR indirectly and more selectively with PAPP-A inhibitory antibodies attenuates IGF-IR signaling in TED with fewer side effects. Methods: Informed consent was obtained from TED patients undergoing surgery in Mayo Clinic operating suites in Rochester, MN. Retro-orbital tissue was collected from 19 TED patients and 2 control subjects for fibroblast isolation and culture and was performed in a sterile tissue culture facility. TED and control fibroblasts were treated with pro-inflammatory cytokines, and flow separation of CD34- and CD34+ orbital fibroblasts was done, the latter representing infiltrating fibrocytes into the orbit in TED. Main outcome measured were PAPP-A expression and proteolytic activity, IGF-I stimulation of phosphatidylinositol 3 kinase/Akt pathway and inhibition by immuno-neutralizing antibodies against PAPP-A, CD34+ status and associated PAPP-A and IGF-IR expression. Results: Primary cultured cells showed that PAPP-A is expressed in orbital fibroblasts from TED patients but not in control subject fibroblasts and were markedly increased by pro-inflammatory cytokines stimulation. IGF-IR expression was not affected by cytokine treatment. Inhibition of PAPP-A’s proteolytic activity suppressed IGF-IR activation in orbital fibroblasts from TED patients. CD34+ orbital fibroblasts represent 80% of cells in culture from TED patients, and approximately 70% of these cells were accountable for PAPP-A and IGF-IR expression. Conclusion: These results support a role for PAPP-A in TED pathogenesis and indicate the potential for novel therapeutic targeting of the IGF axis. Presentation: 6/2/2024

Multifocal osteonecrosis due to the synergistic impact of inherited thrombophilia, autoimmunity and pregnancy: a case report.

Multifocal osteonecrosis is a rare entity which can lead to bone fragility, fractures and considerable morbidity. The objective of this report is to describe the presentation, evaluation and management of a case of multifocal osteonecrosis. A 34-year-old lady presented with multiple fractures and bone pain which was exacerbated during pregnancy. She was found to have osteonecrosis involving multiple sites. Investigation revealed that she had an undefined autoimmune condition with antinuclear antibody and anti-Ro (SS-A) positivity, not fulfilling criteria for diagnosis of Sjogren syndrome. She had low Protein S levels for age and gender (confirmed on 2 occasions 12 weeks apart outside pregnancy). She had a novel variant in the PROS1 gene (NM_000313.4:c.1513G>A; p.Gly505Ser) which is associated with heterozygous Protein S (PS) deficiency. She had a good response to treatment with anticoagulation. The combination of inherited thrombophilia, autoimmunity and the thrombophilic state induced by pregnancy probably contributed to the severe phenotype in this patient. The variant was identified in the Lamin G1 domain, which is instrumental in PS binding to tissue factor pathway inhibitor and C4 binding protein, thus playing a role in both the coagulation and complement pathway, and could explain the coexistence of autoimmunity with thrombophilia. This variant is present in 0.034% of South Asians in the gnomAD population database, and may represent an important cause of inherited thrombophilia in this group.

A new Bowman-Birk type protease inhibitor regulated by MeJA pathway in maize exhibits anti-feedant activity against the Ostrinia furnacalis.

Jasmonic acid (JA), an important plant hormone, plays a crucial role in defending against herbivorous insects. In this study, we have identified a new Bowman-Birk type protease inhibitor (BBTI) protein in maize that is regulated by the JA pathway and exhibits significant antifeedant activity, which is notably induced by exogenous Methyl Jasmonate and Ostrinia furnacalis feeding treatments. Bioinformatics analysis revealed significant differences in the BBTI protein among different maize inbred lines, except for the conserved domain. Prokaryotic and eukaryotic expression systems were constructed and expressed, and combined with bioassays, it was demonstrated that the antifeedant activity of BBTI is determined by protein modifications and conserved domains. Through RT-qPCR detection of BBTI and JA regulatory pathway-related genes' temporal expression in different maize inbred lines, we identified the regulatory mechanism of BBTI synthesis under the JA pathway. This study successfully cloned and identified the MeJA-induced anti-feedant activity gene BBTI and conducted functional validation in different maize inbred lines, providing valuable insights into the response mechanism of insect resistance induced by the plant JA pathway. The increased expression of the anti-feedant activity gene BBTI through exogenous MeJA induction may offer a potential new strategy for mediating plant defense against Lepidoptan insects.

Effects of a Protease Inhibitor Protein on Buchnera aphidicola and Gene Expression in Pea Aphids (Acyrthosiphon pisum)

ABSTRACTAphids are serious insect pests for agricultural and horticultural crops and may cause the major economic losses. Insecticides used to control aphids have caused environmental pollution and the insecticide residues in agricultural products. A new protease inhibitor gene from bacterium Xenorhabdus bovienii (Xbpi‐1) and the protease inhibitor protein expressed by the gene against the pea aphid (Acyrthosiphon pisum Harris; Hemiptera: Aphididae) have been reported, however, effects of the PIP on the symbiotic bacteria, Buchnera aphidicola, and gene expression in pea aphids are unknown. By assessing the quantity of B. aphidicola, the primary symbiotic bacterium, in the aphid fed on an artificial diet containing 100 and 500 μg/mL Xbpi‐1, we observed a substantial reduction in its population by 27% and 46%, respectively, as analysed by real‐time quantitative reverse transcription PCR (qRT‐PCR). Furthermore, the growth of other aphid‐associated bacteria was also significantly inhibited by Xbpi‐1. To elucidate the mechanisms at the gene level, we conducted transcriptome analysis and identified differentially expressed genes (DEGs). Subsequent Gene Ontology (GO) analysis of the 213 DEGs shed light on the impact of Xbpi‐1 on aphid metabolism processes and gene expression. Notably, the results highlighted several aphid nutrient metabolism pathways affected by Xbpi‐1, which are relevant to vector‐borne diseases. These pathways encompass crucial factors such as heat shock proteins, cuticle proteins and proteases. The results from this study revealed that the PIP had a novel mechanism against pea aphids by having adverse effects on the primary symbiotic bacteria in pea aphids and affecting aphid gene expression, showing that the PIP may be a promise bioinsecticide for aphid control in the future.

Senolytic therapy preserves blood-brain barrier integrity and promotes microglia homeostasis in a tauopathy model

Cellular senescence, characterized by expressing the cell cycle inhibitory proteins, is evident in driving age-related diseases. Senescent cells play a crucial role in the initiation and progression of tau-mediated pathology, suggesting that targeting cell senescence offers a therapeutic potential for treating tauopathy associated diseases. This study focuses on identifying non-invasive biomarkers and validating their responses to a well-characterized senolytic therapy combining dasatinib and quercetin (D + Q), in a widely used tauopathy mouse model, PS19. We employed human-translatable MRI measures, including water extraction with phase-contrast arterial spin tagging (WEPCAST) MRI, T2 relaxation under spin tagging (TRUST), longitudinally assessed brain physiology and high-resolution structural MRI evaluated the brain regional volumes in PS19 mice. Our data reveal increased BBB permeability, decreased oxygen extraction fraction, and brain atrophy in 9-month-old PS19 mice compared to their littermate controls. (D + Q) treatment effectively preserves BBB integrity, rescues cerebral oxygen hypometabolism, attenuates brain atrophy, and alleviates tau hyperphosphorylation in PS19 mice. Mechanistically, D + Q treatment induces a shift of microglia from a disease-associated to a homeostatic state, reducing a senescence-like microglial phenotype marked by increased p16/Ink4a. D + Q-treated PS19 mice exhibit enhanced cue-associated cognitive performance in the tracing fear conditioning test compared to the vehicle-treated littermates, implying improved cognitive function by D + Q treatment. Our results pave the way for application of senolytic treatment as well as these noninvasive MRI biomarkers in clinical trials in tauopathy associated neurological disorders.

Construction of the technological process and design development of a multifunctional unit for processing vital soybeans with ultrasound. Feasibility of water-crude suspension

Cereals and legumes have a porous structure, being an inert carrier during mass exchange processes; they contain voids in the form of pores and stomata, on the surface of which there are foreign extractable substances in a solid state, in particular, trypsin and urease inhibitor proteins. These substances, due to their harmful effect on the final product, are subject to removal by extraction. One of the methods for inactivating anti-nutritional substances is ultrasonic treatment of soybeans.The article discusses the technology of inactivating inhibitors that worsen food and protein value of vital soybeans. The developed technology was aimed at dissolving a solid substance - soybean inhibitor protein in a liquid, carried out due to molecular diffusion, intensifying the processes of mass transfer and mass return. When creating a suspension by mixing the solid (crushed soybean grain) and liquid (water) phases, water molecules, which are a solvent, penetrate under the action of capillary forces into the pores and stomata of the treated body, begin to penetrate. On this basis, technological directions of research have been established, a sequence has been built and the physical essence of operations has been substantiated, consisting of four main ones - grinding soybean grain, creating a “water-soybean” suspension, ultrasonic (US) treatment of soybeans, drying. As a result of the conducted research, optimal technological parameters have been established for intensifying the process of inactivation of anti-nutritional substances contained in native soybeans: the degree of grinding of soybean grains is 0.5-1.0 mm; the ratio of water-soy suspension (1:1). The efficiency of the developed technological process will be factors affecting the quality of the resulting products, the duration and energy intensity of the process, resource conservation, simplicity of design and low cost of manufacture, the possibility of automation.