Amino-terminal Fragment Research Articles

N-terminal pro-B-type natriuretic peptide levels vary by ethnicity and are associated with insulin sensitivity after gestational diabetes mellitus

BackgroundIndividuals of South Asian origin have a greater risk of cardiovascular disease after gestational diabetes mellitus (GDM) than European individuals. B-type natriuretic peptide (BNP) and the amino-terminal fragment of its prohormone (NT-proBNP) are commonly used for heart failure screening and diagnosis, but biologically BNP exerts several beneficial cardiovascular effects primarily by counteracting the renin-angiotensin-aldosterone-system. We asked whether ethnic differences in circulating NT-proBNP levels could be explained by the differences in cardiometabolic and inflammatory risk markers?MethodsWe examined 162 South Asian and 107 Nordic women in Norway 1–3 years after GDM with a clinical examination, fasting blood samples and an oral glucose tolerance test. We measured the levels of NT-proBNP, high-sensitivity cardiac troponin T, high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), leptin, adiponectin and markers of insulin sensitivity, such as the Matsuda insulin sensitivity index (ISI). Finally, we tried to identify which independent covariate best mediated the ethnic differences in NT-proBNP.ResultsThe mean (SD) age was 35.3 (4.5) years, BMI 29.1 (6.0) kg/m2, waist-height ratio 0.60 (0.08) and 164 women (61%) had prediabetes/diabetes. Notably, South Asian women had lower levels of NT-proBNP than Nordic women in both the normoglycemic and prediabetes/diabetes groups (median (IQR) 26 (15–38) vs. 42 (22–66) ng/L, p < 0.001). Higher NT-proBNP levels were associated with greater insulin sensitivity in both South Asian and Nordic women (p = 0.005 and p < 0.001). South Asian women had higher levels of hsCRP (median (IQR) 2.2 (1.1–4.4) vs. 1.2 (0.3–4.2) mg/L), IL-6 (2.3 (1.5–3.2) vs. 1.5 (1.5–2.5) pg/mL), leptin (1647 (1176–2480) vs. 1223 (876–2313) pmol/L), and lower adiponectin levels (7.2 (5.3–9.3) vs. 10.0 (7.2–13.5) mg/L) and Matsuda ISI (2.4 (1.7–3.7) vs. 4.2 (2.9–6.1), pall<0.01) than Nordic women. Even after adjusting for these differences, higher NT-proBNP levels remained associated with insulin sensitivity (22% higher NT-proBNP per SD Matsuda ISI, p = 0.015). Insulin sensitivity and adiponectin mediated 53% and 41% of the ethnic difference in NT-proBNP.ConclusionsNT-proBNP levels are lower in South Asian than in Nordic women after GDM. Lower NT-proBNP levels correlate with impaired insulin sensitivity. Lower NT-proBNP levels in South Asian women could, therefore, be attributed to impaired insulin sensitivity rather than total body fat.

The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator.

The active form of the murine urokinase-type plasminogen activator (muPA) is formed by a 27-residue disordered light chain connecting the amino-terminal fragment (ATF) with the serine protease domain. The two chains are tethered by a disulfide bond between C1CT in the disordered light chain and C122CT in the protease domain. Previous work showed that the presence of the disordered light chain affected the inhibition of the protease domain by antibodies. Here we show that the disordered light chain induced a 3.7-fold increase in kcat of the protease domain of muPA. In addition, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and accelerated molecular dynamics (AMD) were performed to identify the interactions between the disordered light chain and the protease domain. HDX-MS revealed that the light chain is contacting the 110s, the turn between the β10- and β11-strand, and the β7-strand. A reduction in deuterium uptake was also observed in the activation loop, the 140s loop and the 220s loop, which forms the S1-specificty pocket where the substrate binds. These loops are further away from where the light chain seems to be interacting with the protease domain. Our results suggest that the light chain most likely increases the activity of muPA by allosterically favoring conformations in which the specificity pocket is formed. We propose a model by which the allostery would be transmitted through the β-strands of the β-barrels to the loops on the other side of the protease domain.

A palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis.

Gasdermin D (GSDMD) is the executor of pyroptosis, which is important for host defence against pathogen infection. Following activation, caspase-mediated cleavage of GSDMD releases an amino-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and release of proinflammatory cytokines. The spatial and temporal regulation of this process in cells remains unclear. Here we identify GSDMD as a substrate for reversible S-palmitoylation on C192 during pyroptosis. The palmitoyl acyltransferase DHHC7 palmitoylates GSDMD to direct its cleavage by caspases. Subsequently, palmitoylation of GSDMD-NT promotes its translocation to the plasma membrane, where APT2 depalmitoylates GSDMD-NT to unmask the C192 residue and promote GSDMD-NT oligomerization. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, leading to increased survival of mice with lipopolysaccharide-induced lethal septic shock and increased sensitivity to bacterial infection. Our findings reveal a model through which a palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation during pyroptosis.

In vitro and in vivo anti-tumor effect of Trichobakin fused with urokinase-type plasminogen activator ATF-TBK.

Trichobakin (TBK), a member of type I ribosome-inactivating proteins (RIPs), was first successfully cloned from Trichosanthes sp Bac Kan 8-98 in Vietnam. Previous study has shown that TBK acts as a potential protein synthesis inhibitor; however, the inhibition efficiency and specificity of TBK on cancer cells remain to be fully elucidated. In this work, we employed TBK and TBK conjugated with a part of the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), which contains the Ω-loop that primarily interacts with urokinase-type plasminogen activator receptor, and can be a powerful carrier in the drug delivery to cancer cells. Four different human tumor cell lines and BALB/c mice bearing Lewis lung carcinoma cells (LLC) were used to evaluate the role of TBK and ATF-TBK in the inhibition of tumor growth. Here we showed that the obtained ligand fused RIP (ATF-TBK) reduced the growth of four human cancer cell lines in vitro in the uPA receptor level-dependent manner, including the breast adenocarcinoma MDA-MB 231 cells and MCF7 cells, the prostate carcinoma LNCaP cells and the hepatocellular carcinoma HepG2 cells. Furthermore, the conjugate showed anti-tumor activity and prolonged the survival time of tumor-bearing mice. The ATF-TBK also did not cause the death of mice with doses up to 48mg/kg, and they were not significantly distinct on parameters of hematology and serum biochemistry between the control and experiment groups. In conclusion, ATF-TBK reduced the growth of four different human tumor cell lines and inhibited lung tumor growth in a mouse model with little side effects. Hence, the ATF-TBK may be a target to consider as an anti-cancer agent for clinical trials.

Diagnosis of acute myocardial infarction before surgery: high-sensitivity troponin T or amino-terminal fragment of pro-brain natriuretic peptide type B?

Currently, there are data in the literature about the importance of the aminoterminal fragment of pro-brain natriuretic peptide type B in the diagnosis of patients with acute myocardial infarction.The aim. To study preoperative levels of aminoterminal fragment of pro-brain natriuretic peptide type B and high-sensitivity troponin T in patients with acute myocardial infarction and to assess the significance of aminoterminal fragment of pro-brain natriuretic peptide type B in patients with this pathology.Materials and methods. We retrospectively analyzed 50 medical records of patients diagnosed with acute myocardial infarction.The results. Study of preoperative levels of aminoterminal fragment of pro-brain natriuretic peptide type B in patients with acute myocardial infarction showed that the values of aminoterminal fragment of pro-brain natriuretic peptide type B at hospital admission ranged from 127 to 600 pg/ml; correlated with left ventricular ejection fraction (r =-0,42, p &lt; 0,05), severity of acute heart failure (r =+0,57, p &lt; 0,05), and outcome of acute myocardial infarction (r =+0,45, p &lt; 0,05). Assessment of the value of aminoterminal fragment of pro-brain natriuretic peptide type B in patients with acute myocardial infarction on the basis of comparison with high-sensitivity troponin T revealed no advantages of its use in this pathology as a marker of acute cardiomyocyte damage: aminoterminal fragment of pro-brain natriuretic peptide type B has less diagnostic and prognostic value than high-sensitivity troponin T. Diagnosis of acute myocardial infarction before surgery: highly sensitive troponin T or aminoterminal fragment of pro-brain natriuretic peptide type B?

Highly Potent Peptide Therapeutics To Prevent Protein Aggregation in Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder resulting from a significant amplification of CAG repeats in exon 1 of the Huntingtin (Htt) gene. More than 36 CAG repeats result in the formation of a mutant Htt (mHtt) protein. These amino-terminal mHtt fragments lead to the formation of misfolded proteins, which then form aggregates in the relevant brain regions. Therapies that can delay the progression of the disease are imperative to halting the course of the disease. Peptide-based drug therapies provide such a platform. Inhibitory peptides were screened against monomeric units of both wild type (Htt(Q25)) and mHtt fragments, Htt(Q46) and Htt(Q103). Fibril kinetics was studied by utilizing the Thioflavin T (ThT) assay. Atomic force microscopy was also used to study the influence of the peptides on fibril formation. These experiments demonstrate that the chosen peptides suppress the formation of fibrils in mHtt proteins and can provide a therapeutic lead for further optimization and development.

Amino-terminal proteolytic fragment of the axon growth inhibitor Nogo-A (Rtn4A) is upregulated by injury and promotes axon regeneration

After adult mammalian central nervous system injury, axon regeneration is extremely limited or absent, resulting in persistent neurological deficits. Axon regeneration failure is due in part to the presence of inhibitory proteins, including NogoA (Rtn4A), from which two inhibitory domains have been defined. When these inhibitory domains are deleted, but an amino-terminal domain is still expressed in a gene trap line, mice show axon regeneration and enhanced recovery from injury. In contrast, when there is no amino-terminal Nogo-A fragment in the setting of inhibitory domain deletion, then axon regeneration and recovery are indistinguishable from WT. These data indicated that an amino-terminal Nogo-A fragment derived from the gene trap might promote axon regeneration, but this had not been tested directly and production of this fragment without gene targeting was unclear. Here, we describe posttranslation production of an amino-terminal fragment of Nogo-A from the intact gene product. This fragment is created by proteolysis near amino acid G214-N215 and levels are enhanced by axotomy. Furthermore, this fragment promotes axon regeneration in vitro and acts cell autonomously in neurons, in contrast to the inhibitory extracellular action of other Nogo-A domains.Proteins interacting with the amino-terminal Nogo-A fragment by immunoprecipitation include HSPA8 (HSC70, HSP7C). Suppression of HSPA8 expression by shRNA decreases axon regeneration from cerebral cortical neurons and overexpression increases axon regeneration. Moreover, the amino-terminal Nogo-A fragment increases HSPA8 chaperone activity. These data provide an explanation for varied results in different gene-targeted Nogo-A mice, as well as revealing an axon regeneration promoting domain of Nogo-A.

Combined and sequential therapies with anabolic and antiresorptive drugs in the management of patients with postmenopausal osteoporosis

The aim of osteoporosis therapy is to reduce the increased fracture risk associated with osteoporosis-related bone fragility. Prevention of fragility fracture relies on balanced nutrition, weight-bearing and balance-improving physical exercises, and pharmacological therapies. Among the latter, the antiresorptive drugs are the most widely used. Bone formation stimulators (anabolics) are second line-therapy with reversible effects once discontinued. For patients at very high risk or imminent risk of fracture, the question arises of whether combining drugs with different modes of action, or using sequential regimens with these agents, could achieve early, higher-magnitude antifracture efficacy than is obtained with usual antiresorptives, and sustained antifracture efficacy. As regards combination therapies, until we have clear evidence that using drugs together provides greater fracture risk reduction than monotherapy, these therapies are not recommended. Anabolic treatments like teriparatide, the amino-terminal fragment of parathyroid hormone, abaloparatide, an analog of parathyroid hormone related protein, and romosozumab, the monoclonal antibody against sclerostin, decrease vertebral and non-vertebral fracture risk and are more efficacious in fracture risk reduction than antiresorptives, as shown in head-to-head trials. However, an anabolic agent should be followed by an antiresorptive drug to maintain and even further increase its antifracture efficacy, which is otherwise rapidly reversible. Because of their early, high-magnitude and sustained antifracture efficacy, such sequential regimens should become the standard of care for patients at very high or imminent risk of fracture.

Abstract 2703: Evaluation of the effect of tumor-targeted and immunomodulating nanoparticles on the activity of cytotoxic T cells using CAR T cells as an in vitro model

Abstract In solid tumors, there are multiple barriers that prevent anticancer agents and T cells from being able to be delivered into and reach tumor cells. For effective antitumor activity, it is necessary to have direct interactions between cytotoxic T cells and tumor cells. Hyaluronic acid and Iron oxide both have distinctive properties that are advantageous for the development of biocompatible and biodegradable nanoparticle drug delivery systems for cancer. We have developed several targeting and immunomodulatory ligands that can be conjugated to the nanoparticles while carrying chemotherapy or molecular targeted agents for targeted cancer therapy as shown in Table 1. Although the therapeutic efficacy of those targeted nanoparticles has been demonstrated in mouse tumor models, the effect of nanoparticles and their payload drugs on the activities of tumor specific cytotoxic T cells is still unclear. To determine the effect of those nanoparticles on T cell immune responses, CAR T cells provided a well-suited in vitro cellular model. In this study, anti-mesothelin CAR T cells generated from lentiviral CAR T vector transduced T cells were incubated with nanoparticle/drugs and free drugs. The effect of various nanoparticles on CAR T cell viability, and in vitro cytotoxicity on pancreatic cancer cells was evaluated by live/dead, Alamarblue and SRB proliferation Assays. Our results showed that the incubation of CAR T cells with targeted nanoparticles/drugs did not reduce CAR T cells' viability and did not inhibit T cells' activity in killing tumor cells. The ATFmmp14-HANP/SN38 nanoparticle showed an enhanced effect of CAR T cells. Thus, this nanoparticle/drug has the potential for the development of combination therapy with CAR T cells to improve the CAR T cell delivery while synergizing anti-tumor effect by targeted drug delivery, stroma modulation, and tumor cell killing by drug and CAR T cells. Table 1. Nanoparticle formulations overview Nanoparticle Types Target Payload Mechanism of action Observed Results (in vivo) Observed Results (in vitro) ATFmmp14-HANP/SN38:ATFmmp14 conjugated HANP uPAR CD44 SN38 (an active metabolite of irinotecan) Inhibits DNA topoiso-merase I, causing DNA single strand breaks and DNA damage. Significant tumor growth inhibition in human pancreatic and breast cancer PDX models. HANP more effective towards pancreatic cell line and less toxic against CAR T cells: Greater anti-tumor with combination HANP/GKT831: HANP CD44 Setanaxib (GKT831:an inhibitor of NOX1 and NOX4 Reduce the level of reactive oxygen species (ROS): DNA repair downregulation, increases DNA damage Efficient delivery into mouse breast PDX tumors and sensitizing tumor cells to radiotherapy and DNA damaging agents. Nontoxic towards CAR T cells: limited additional therapeutic effects PDY1-HANP/Avasimibe: PD-L1 blocking PDY1 peptide conjugated HANP PD-L1 CD44 Avasimibe (Ava), an inhibitor of acyl-Coenzyme A:cholesterolacyltransferase Decreases intracellular cholesterol.Inhibit tumor cell growth, Activate cytotoxic T cells Efficient delivery into mouse colon tumors, Inhibition of tumor growth: Increased levels of CD8+ T cells and CD68+ Macrophages in tumors Nontoxic towards pancreatic cell lines and CAR T cells: limited additional therapeutic effects ATFmmp14-IONP-DOX: ATFmmp14 conjugated IONP uPAR Doxorubicin Direct DNA damage and inhibits DNA repair by inhibiting topoisomerase Significantly inhibited tumor growth in human pancreatic and breast cancer PDX model ATFmmp14-IONP/DOX more effective towards pancreatic cancer cell lines and less toxic against CAR T cells: slightly increased killing of tumor cells with combination ATFmmp14: Amino terminal fragment (ATF) of uPA fused with MMP14 catalytic domain; PD1Y: PD1 peptide mimetic that binds to and inhibit PD-L1 HANP: Hyaluronic acid nanoparticle; IONP: Iron oxide nanoparticle Citation Format: Kory Wells, Tongrui Lui, Lei Zhu, Chrystal Paulos, Lily Yang. Evaluation of the effect of tumor-targeted and immunomodulating nanoparticles on the activity of cytotoxic T cells using CAR T cells as an in vitro model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2703.

Big data mining, rational modification, and ancestral sequence reconstruction inferred multiple xylose isomerases for biorefinery.

The isomerization of xylose to xylulose is considered the most promising approach to initiate xylose bioconversion. Here, phylogeny-guided big data mining, rational modification, and ancestral sequence reconstruction strategies were implemented to explore new active xylose isomerases (XIs) for Saccharomyces cerevisiae. Significantly, 13 new active XIs for S. cerevisiae were mined or artificially created. Moreover, the importance of the amino-terminal fragment for maintaining basic XI activity was demonstrated. With the mined XIs, four efficient xylose-utilizing S. cerevisiae were constructed and evolved, among which the strain S. cerevisiae CRD5HS contributed to ethanol titers as high as 85.95 and 94.76 g/liter from pretreated corn stover and corn cob, respectively, without detoxifying or washing pretreated biomass. Potential genetic targets obtained from adaptive laboratory evolution were further analyzed by sequencing the high-performance strains. The combined XI mining methods described here provide practical references for mining other scarce and valuable enzymes.

A hepatokine derived from the ER protein CREBH promotes triglyceride metabolism by stimulating lipoprotein lipase activity.

The endoplasmic reticulum (ER)-tethered, liver-enriched stress sensor CREBH is processed in response to increased energy demands or hepatic stress to release an amino-terminal fragment that functions as a transcription factor for hepatic genes encoding lipid and glucose metabolic factors. Here, we discovered that the carboxyl-terminal fragment of CREBH (CREBH-C) derived from membrane-bound, full-length CREBH was secreted as a hepatokine in response to fasting or hepatic stress. Phosphorylation of CREBH-C mediated by the kinase CaMKII was required for efficient secretion of CREBH-C through exocytosis. Lipoprotein lipase (LPL) mediates the lipolysis of circulating triglycerides for tissue uptake and is inhibited by a complex consisting of angiopoietin-like (ANGPTL) 3 and ANGPTL8. Secreted CREBH-C blocked the formation of ANGPTL3-ANGPTL8 complexes, leading to increased LPL activity in plasma and metabolic tissues in mice. CREBH-C administration promoted plasma triglyceride clearance and partitioning into peripheral tissues and mitigated hypertriglyceridemia and hepatic steatosis in mice fed a high-fat diet. Individuals with obesity had higher circulating amounts of CREBH-C than control individuals, and human CREBH loss-of-function variants were associated with dysregulated plasma triglycerides. These results identify a stress-induced, secreted protein fragment derived from CREBH that functions as a hepatokine to stimulate LPL activity and triglyceride homeostasis.

Duck gasdermin E is a substrate of caspase-3/-7 and an executioner of pyroptosis.

Gasdermin (GSDM)-mediated cell death is an ancient immune defensive mechanism that plays an essential role in bacteria, fungi, coral, teleost, and mammals. After being cleaved by proteases of hosts or pathogens, amino-terminal (NT) fragment of GSDMs (GSDM-NTs) form pores in the membrane structure of cells, thereby leading to pyroptotic cell death. However, the expression profile, activation mechanism and function of avian GSDMs have not been studied in depth yet. In the current study, genes encoding duck gasdermin E (duGSDME), caspase-3 (ducaspase-3) and ducaspase-7 were cloned from mRNA of a virus-challenged duck embryo. The cleavage of duGSDME by ducaspase-3/-7 was verified in the cell-free system and/or in human embryonic kidney cells (HEK293). Ducaspase-3/-7 could recognize and cleave duGSDME at 270DAVD273. Overexpression of duGSDME-NT (1-273aa) fragment led to pyroptosis-like morphological change, increased lactic dehydrogenase (LDH) release and propidium iodide uptake of HEK293 cells, which indicated that duGSDME-NTs could cause cell membrane damage. In addition, recombinantly expressed duGSDME-NT showed bactericidal activity to an enterotoxic Escherichia coli (F5+) strain. The expression level of duGSDME was low in duckling tissues. DHAV-3 challenge upregulated the expression of duGSDME and ducaspase-3 in different tissues and led to the activation of ducaspase-3 and cleavage of duGSDME. The results indicated that duGSDME is a substrate of ducapsase-3/-7, and duGSDME-NT can cause pyroptosis. In addition, duGSDME may play a role in the immune defense of ducks against infectious diseases after being cleaved by ducaspase-3. The current study provides essential information for further investigation of the mechanisms of avian innate immunity and avian diseases.

ATF6 aggravates apoptosis in early porcine embryonic development by regulating organelle homeostasis under high-temperature conditions.

Activating transcription factor 6 (ATF6), one of the three sensor proteins in the endoplasmic reticulum (ER), is an important regulator of ER stress-induced apoptosis. ATF6 resides in the ER and, upon activation, is translocated to the Golgi apparatus, where it is cleaved by site-1 protease (S1P) to generate an amino-terminal cytoplasmic fragment. Although recent studies have made progress in elucidating the regulatory mechanisms of ATF6, its function during early porcine embryonic development under high-temperature (HT) stress remains unclear. In this study, zygotes were divided into four groups: control, HT, HT+ATF6 knockdown, and HT+PF (S1P inhibitor). Results showed that HT exposure induced ER stress, which increased ATF6 protein expression and led to a decrease in the blastocyst rate. Next, ATF6 expression was knocked down in HT embryos under microinjection of ATF6 double-stranded RNA (dsRNA). Results revealed that ATF6 knockdown (ATF6-KD) attenuated the increased expression of CHOP, an ER stress marker, and Ca 2+ release induced by HT. In addition, ATF6-KD alleviated homeostasis dysregulation among organelles caused by HT-induced ER stress, and further reduced Golgi apparatus and mitochondrial dysfunction in HT embryos. AIFM2 is an important downstream effector of ATF6. Results showed that ATF6-KD reduced the occurrence of AIFM2-mediated embryonic apoptosis at HT. Taken together, our findings suggest that ATF6 is a crucial mediator of apoptosis during early porcine embryonic development, resulting from HT-induced ER stress and disruption of organelle homeostasis.

Assessing Interleukin-1β Activation During Pyroptosis.

The cytokine interleukin-1β (IL-1β) is a major mediator of inflammation in autoinflammatory disease and the host response to infection. IL-1β is stored within cells in an inert form, which requires proteolytic removal of an amino-terminal fragment in order to bind the IL-1 receptor complex and have pro-inflammatory activity. This cleavage event is canonically carried out by inflammasome-activated caspase proteases, but microbe and host proteases can also generate unique active forms. Post-translational regulation of IL-1β and the diversity in resulting products can present challenges to the evaluation of IL-1β activation. This chapter details methods and important controls for the accurate and sensitive measurement of IL-1β activation within biological samples.

An amino-terminal fragment of apolipoprotein E4 leads to behavioral deficits, increased PHF-1 immunoreactivity, and mortality in zebrafish.

Although the increased risk of developing sporadic Alzheimer's disease (AD) associated with the inheritance of the apolipoprotein E4 (APOE4) allele is well characterized, the molecular underpinnings of how ApoE4 imparts risk remains unknown. Enhanced proteolysis of the ApoE4 protein with a toxic-gain of function has been suggested and a 17 kDa amino-terminal ApoE4 fragment (nApoE41-151) has been identified in post-mortem human AD frontal cortex sections. Recently, we demonstrated in vitro, exogenous treatment of nApoE41-151 in BV2 microglial cells leads to uptake, trafficking to the nucleus and increased expression of genes associated with cell toxicity and inflammation. In the present study, we extend these findings to zebrafish (Danio rerio), an in vivo model system to assess the toxicity of nApoE41-151. Exogenous treatment of nApoE41-151 to 24-hour post-fertilization for 24 hours resulted in significant mortality. In addition, developmental abnormalities were observed following treatment with nApoE41-151 including improper folding of the hindbrain, delay in ear development, deformed yolk sac, enlarged cardiac cavity, and significantly lower heart rates. A similar nApoE31-151 fragment that differs by a single amino acid change (C>R) at position 112 had no effects on these parameters under identical treatment conditions. Decreased presence of pigmentation was noted for both nApoE31-151- and nApoE41-151-treated larvae compared with controls. Behaviorally, touch-evoked responses to stimulus were negatively impacted by treatment with nApoE41-151 but did not reach statistical significance. Additionally, triple-labeling confocal microscopy not only confirmed the nuclear localization of the nApoE41-151 fragment within neuronal populations following exogenous treatment, but also identified the presence of tau pathology, one of the hallmark features of AD. Collectively, these in vivo data demonstrating toxicity as well as sublethal effects on organ and tissue development support a novel pathophysiological function of this AD associated-risk factor.

Nafamostat Mesylate in Combination with the Mouse Amino-Terminal Fragment of Urokinase-Human Serum Albumin Improves the Treatment Outcome of Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is highly aggressive and causes a higher proportion of metastatic cases. However, therapies directed to specific molecular targets have rarely achieved clinically meaningful improvements in the outcome of TNBC therapy. A urokinase-type plasminogen activator (uPA), one of the best-validated biomarkers of breast cancer, is an extracellular proteolytic serine protease involved in many pathological and physiological processes, including tumor cell invasion and metastasis. Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases and has been used as a therapeutic agent for the treatment of TNBC. Nevertheless, NM has poor specificity for serine proteases and is easy be hydrolyzed; moreover, the inhibitory mechanism of TNBC therapy is unclear. In this study, we combine NM with a macromolecular drug delivery vehicle, mouse amino-terminal fragment of urokinase-human serum albumin (mATF-HSA), to form a complex (mATF-HSA:NM) using the dilution-incubation-purification method. mATF specifically targets uPAR overexpressed on the surface of TNBC cells; moreover, HSA prevents NM from being hydrolyzed by numerous serine proteases. mATF-HSA:NM showed stronger inhibitory effects on the proliferation and metastasis of TNBC in vitro and in vivo without significant cytotoxicity on normal cells and tissues. In addition, we demonstrated that NM mediates metastasis of TNBC cells through inhibition of uPA using a stable uPA knockdown cell line (MDA-MB231 shuPA). Overall, we have developed a macromolecular complex targeted to treat high uPAR-expressing tumor types, and mATF-HSA can potentially be used to load other types of drugs with tumor-targeting specificity for mouse tumor models and is a promising tool to study tumor biology in mouse tumor models.

Emerging Role of NLRP3 Inflammasome and Pyroptosis in Liver Transplantation.

The nucleotide-binding domain leucine-rich repeat-receptor, pyrin domain-containing-3 (NLRP3) inflammasome contributes to the inflammatory response by activating caspase-1, which in turn participates in the maturation of interleukin (IL)-1β and IL-18, which are mainly secreted via pyroptosis. Pyroptosis is a lytic type of cell death that is controlled by caspase-1 processing gasdermin D. The amino-terminal fragment of gasdermin D inserts into the plasma membrane, creating stable pores and enabling the release of several proinflammatory factors. The activation of NLRP3 inflammasome and pyroptosis has been involved in the progression of liver fibrosis and its end-stage cirrhosis, which is among the main etiologies for liver transplantation (LT). Moreover, the NLRP3 inflammasome is involved in ischemia-reperfusion injury and early inflammation and rejection after LT. In this review, we summarize the recent literature addressing the role of the NLRP3 inflammasome and pyroptosis in all stages involved in LT and argue the potential targeting of this pathway as a future therapeutic strategy to improve LT outcomes. Likewise, we also discuss the impact of graft quality influenced by donation after circulatory death and the expected role of machine perfusion technology to modify the injury response related to inflammasome activation.

2D Echocardiographic Findings, NT-ProBNP Levels, and Outcomes in Dyspnoeic Patients: Results from the Pro-BNP Investigation of Dyspnoea in the Emergency Department- a Single Centre Experience

Background: Dyspnoeic in the emergency department with multiple co-morbidities is a diagnostic challenge. Approximately 15-20% of acute dyspneic in the Emergency Department due to ADHF (acute decompensated heart failure) are misdiagnosed. B-type peptide (BNP) and its amino-terminal fragment (NT-proBNP) accurately identify HF in dyspnoeic patients. In the general population with dyspnoea, plasma pro-BNP concentrations are increased in left ventricular dilatation, hypertrophy, systolic dysfunction, or diastolic dysfunction but are unaffected by pulmonary dysfunction.&#x0D; Aims and Objectives: To study the relation between NT pro-BNP &amp; echocardiographic findings in acute dyspnoeic patients, and the relation between NT pro-BNP and In-hospital Mortality.&#x0D; Materials and Methods: Source of data- Patients admitted to the Emergency Room or cardiac intensive care unit with a history of acute dyspnea in a tertiary cardiac care center in south India, meeting inclusion &amp; exclusion criteria, were studied.&#x0D; Results: The study population is predominantly constituted of the elderly population. The most common co-morbid condition was hypertension. The present study uses an NT pro BNP level of 900pg/ml as the cut-off level. 78 patients were positive for the test. Echocardiography showed that 58% had LV systolic dysfunction, 60% had diastolic dysfunction. 42 patients had EF &gt;55%, 10 patients between 55-45%, 33 patients between 44-30% and 15 patients had EF &lt; 30%. Mortality rate was 6% in the whole study population. However, Mortality was seen in only NT pro-BNP positive group it was not statistically significant (p=0.46).&#x0D; Conclusions: NT pro-BNP correlates well with the worsening of LV systolic function; as the EF decreases, NT pro-BNP increases. Increase in NT pro-BNP levels has to be interpreted in the clinical context, and it is not a substitute for echocardiography for assessing cardiac abnormalities and dysfunction.

Allergen protease-activated stress granule assembly and gasdermin D fragmentation control interleukin-33 secretion.

Interleukin-33 (IL-33), an epithelial cell-derived cytokine that responds rapidly to environmental insult, has a critical role in initiating airway inflammatory diseases. However, the molecular mechanism underlying IL-33 secretion following allergen exposure is not clear. Here, we found that two cell events were fundamental for IL-33 secretion after exposure to allergens. First, stress granule assembly activated by allergens licensed the nuclear-cytoplasmic transport of IL-33, but not the secretion of IL-33. Second, a neo-form murine amino-terminal p40 fragment gasdermin D (Gsdmd), whose generation was independent of inflammatory caspase-1 and caspase-11, dominated cytosolic secretion of IL-33 by forming pores in the cell membrane. Either the blockade of stress granule assembly or the abolishment of p40 production through amino acid mutation of residues 309-313 (ELRQQ) could efficiently prevent the release of IL-33 in murine epithelial cells. Our findings indicated that targeting stress granule disassembly and Gsdmd fragmentation could reduce IL-33-dependent allergic airway inflammation.

Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes.

Gasdermins (GSDMs) are a family of pore-forming effectors that permeabilize the cell membrane during the cell death program pyroptosis1. GSDMs are activated by proteolytic removal of autoinhibitory carboxy-terminal domains, typically by caspase regulators1-9. However, no activator is known for one member of this family, GSDMA. Here we show that the major human pathogen group A Streptococcus (GAS) secretes a protease virulence factor, SpeB, that induces GSDMA-dependent pyroptosis. SpeB cleavage of GSDMA releases an active amino-terminal fragment that can insert into membranes to form lytic pores. GSDMA is primarily expressed in the skin10, and keratinocytes infected with SpeB-expressing GAS die of GSDMA-dependent pyroptosis. Mice have three homologues of human GSDMA, and triple-knockout mice are more susceptible to invasive infection by a pandemic hypervirulent M1T1 clone of GAS. These results indicate that GSDMA is critical in the immune defence against invasive skin infections by GAS. Furthermore, they show that GSDMs can act independently of host regulators as direct sensors of exogenous proteases. As SpeB is essential for tissue invasion and survival within skin cells, these results suggest that GSDMA can act akin to a guard protein that directly detects concerning virulence activities of microorganisms that present a severe infectious threat.