Immune protection of three serine protease inhibitors vaccine in mice against Rhipicephalus sanguineus.

Bioactive molecules in tick saliva are considered to be key to successful feeding and further the transmission of tick-borne pathogens. Problems such as pathogen transmission and animal weight loss result in tick infestation can cause tremendous economic losses to the livestock industry. Therefore, the development of a universal tick vaccine is urgently needed. In this paper, three serine protease inhibitor (serpin) proteins RMS-3, L7LRK7 and L7LTU1 were analyzed with bioinformatics methods. Subsequently the proteins were expressed and purified, and inoculated into Kunming mice for immune protection analysis. The amino acid sequence similarities between RMS-3, L7LRK7 and L7LTU1 were up to 90% in Rhipicephalus sanguineus. The recombinant RMS-3 + L7LRK7 + L7LTU1 showed anticoagulant reaction function and could inhibit the activity of CD4+ lymphocytes, when inoculated into Kunming mice. Additionally, After the immunized mice were challenged with Rhipicephalus sanguineus, the percentage of larvae and nymphs that were fully engorged dropped to 40.87% (P < 0.05) and 87.68% (P > 0.05) in the RmS-3 + L7LRK7 immune group, 49.57% (P < 0.01) and 52.06% (P < 0.05) in the RmS-3 + L7LTU1 group, and 45.22% (P < 0.05) and 60.28% (P < 0.05) in the RmS-3 + L7LRK7 + L7LTU1 immune group, in comparison with the control group. These data indicate that RmS-3 + L7LRK7 + L7LTU1 has good immune protection and has the potential to be developed into a vaccine against the larvae and nymphs of R. sanguineus.

DPM1 modulates desmosomal adhesion and epidermal differentiation through SERPINB5.

Glycosylation is essential to facilitate cell-cell adhesion and differentiation. We determined the role of the dolichol phosphate mannosyltransferase (DPM) complex, a central regulator for glycosylation, for desmosomal adhesive function and epidermal differentiation. Deletion of the key molecule of the DPM complex, DPM1, in human keratinocytes resulted in weakened cell-cell adhesion, impaired localization of the desmosomal components desmoplakin and desmoglein-2, and led to cytoskeletal organization defects in human keratinocytes. In a 3D organotypic human epidermis model, loss of DPM1 caused impaired differentiation with abnormally increased cornification, reduced thickness of non-corneal layers, and formation of intercellular gaps in the epidermis. Using proteomic approaches, SERPINB5 was identified as a DPM1-dependent interaction partner of desmoplakin. Mechanistically, SERPINB5 reduced desmoplakin phosphorylation at serine 176, which was required for strong intercellular adhesion. These results uncover a novel role of the DPM complex in connecting desmosomal adhesion with epidermal differentiation.

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

A Novel Kunitz-Type Serine Protease Inhibitor (HcKuSPI) is Involved in Antibacterial Defense in Innate Immunity and Participates in Shell Formation of Hyriopsis cumingii.

Serine protease inhibitors (SPIs) are abundantly reported for its inhibition against specific proteases involved in the immune responses, but SPI data related to calcareous shells are scarce. Previously, our research group has reported the proteome analysis of non-nucleated pearl powder, and a candidate matrix protein containing two Kunitz domains in the acid soluble fraction caught our attention. In the present study, the full-length cDNA sequence of HcKuSPI was obtained from Hyriopsis cumingii. HcKuSPI was specifically expressed in the mantle, with hybridization signals mainly concentrated to dorsal epithelial cells at the mantle edge and weak signals at the mantle pallium, suggesting HcKuSPI was involved in shell formation. HcKuSPI expression in the mantle was upregulated after Aeromonas hydrophila and Staphylococcus aureus challenge to extrapallial fluids (EPFs). A glutathione S transferase (GST)-HcKuSPI recombinant protein showed strong inhibitory activity against the proteases, trypsin and chymotrypsin. Moreover, HcKuSPI expression in an experimental group was significantly higher when compared with a control group during pellicle growth and crystal deposition in shell regeneration processes, while the organic shell framework of newborn prisms and nacre tablets was completely destroyed after HcKuSPI RNA interference (RNAi). Therefore, HcKuSPI secreted by the mantle may effectively neutralize excess proteases and bacterial proteases in the EPF during bacterial infection and could prevent matrix protein extracellular degradation by suppressing protease proteolytic activity, thereby ensuring a smooth shell biomineralization. In addition, GST-HcKuSPI was also crucial for crystal morphology regulation. These results have important implications for our understanding of the potential roles of SPIs during shell biomineralization.

Effects of Trichinella spiralis and its serine protease inhibitors on autophagy of host small intestinal cells.

In eukaryotes, autophagy is induced as an innate defense mechanism against pathogenic microorganisms by self-degradation. Although trichinellosis is a foodborne zoonotic disease, there are few reports on the interplay between Trichinella spiralissurvival strategies and autophagy-mediated host defense. Therefore, this study focused on the association between T. spiralis and autophagy of host small intestinal cells. In this study, the autophagy-related indexes of host small intestinal cells after T. spiralis infection were detected using transmission electron microscopy, hematoxylin and eosin staining, immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blotting. The results showed that autophagosomes and autolysosomes were formed in small intestinal cells, intestinal villi appeared edema, epithelial compactness was decreased, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was expressed in lamina propria stromal cells of small intestine, and the expression of autophagy-related genes and proteins was changed significantly, indicating that T. spiralis induced autophagy of host small intestinal cells. Then, the effect of T. spiralis on autophagy-related pathways was explored by Western blotting. The results showed that the expression of autophagy-related pathway proteins was changed, indicating that T. spiralis regulated autophagy by affecting autophagy-related pathways. Finally, the roles of T. spiralis serine protease inhibitors (TsSPIs), such as T. spiralis Kazal-type SPI (TsKaSPI) and T. spiralis Serpin-type SPI (TsAdSPI), were further discussed in vitro and in vivo experiments. The results revealed that TsSPIs induced autophagy by influencing autophagy-related pathways, and TsAdSPI has more advantages. Overall, our results indicated that T. spiralis induced autophagy of host small intestinal cells, and its TsSPIs play an important role in enhancing autophagy flux by affecting autophagy-related pathways. These findings lay a foundation for further exploring the pathogenesis of intestinal dysfunction of host after T. spiralis infection, and also provide some experimental and theoretical basis for the prevention and treatment of trichinellosis.

SPINK2 Protein Expression Is an Independent Adverse Prognostic Marker in AML and Is Potentially Implicated in the Regulation of Ferroptosis and Immune Response.

There is an urgent need for the identification as well as clinicopathological and functional characterization of potent prognostic biomarkers and therapeutic targets in acute myeloid leukemia (AML). Using immunohistochemistry and next-generation sequencing, we investigated the protein expression as well as clinicopathological and prognostic associations of serine protease inhibitor Kazal type 2 (SPINK2) in AML and examined its potential biological functions. High SPINK2 protein expression was an independent adverse biomarker for survival and an indicator of elevated therapy resistance and relapse risk. SPINK2 expression was associated with AML with an NPM1 mutation and an intermediate risk by cytogenetics and European LeukemiaNet (ELN) 2022 criteria. Furthermore, SPINK2 expression could refine the ELN2022prognostic stratification. Functionally, an RNA sequencing analysis uncovered a potential link of SPINK2 with ferroptosis and immune response. SPINK2 regulated the expression of certain P53 targets and ferroptosis-related genes, including SLC7A11 and STEAP3, and affected cystine uptake, intracellular iron levels and sensitivity to erastin, a specific ferroptosis inducer. Furthermore, SPINK2 inhibition consistently increased the expression of ALCAM, an immune response enhancer and promoter of T-cell activity. Additionally, we identified a potential small-molecule inhibitor of SPINK2, which requires further characterization. In summary, high SPINK2 protein expression was a potent adverse prognostic marker in AML and might represent a druggable target.

Complete non-proline backbone resonance assignments of the S. aureus neutrophil serine protease inhibitor, EapH1.

The S. aureus extracellular adherence protein (Eap) and its homologs, EapH1 and EapH2, serve roles in evasion of the human innate immune system. EapH1 binds with high-affinity and inhibits the neutrophil azurophilic granule proteases neutrophil elastase, cathepsin-G and proteinase-3. Previous structural studies using X-ray crystallography have shown that EapH1 binds to neutrophil elastase and cathepsin-G using a globally similar binding mode. However, whether the same holds true in solution is unknown and whether the inhibitor experiences dynamic changes following binding remains uncertain. To facilitate solution-phase structural and biochemical studies of EapH1 and its complexes with neutrophil granule proteases, we have characterized EapH1 by multidimensional NMR spectroscopy. Here we report a total of 100% of the non-proline backbone resonance assignments of EapH1 with BMRB accession number 50,304.

The under-appreciated world of the serpin family of serine proteinase inhibitors.

In the practice of medicine, many fundamental biological pathways that require tight on/off control, such as inflammation and circulatory homeostasis, are regulated by serine proteinases, but we rarely consider the unique protease inhibitors that, in turn, regulate these proteases. The serpins are a family of proteins with a shared tertiary structure, whose members largely act as serine protease inhibitors, found in all forms of life, ranging from viruses, bacteria, and archaea to plants and animals. These proteins represent up to 2-10% of proteins in the human blood and are the third most common protein family.

Amino Acid Substitutions at P1 Position Change the Inhibitory Activity and Specificity of Protease Inhibitors BmSPI38 and BmSPI39 from Bombyx mori.

It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and activity. BmSPI38 and BmSPI39 with unique structure and activity may be good models for studying the relationship between the structure and function of small-molecule TIL-type protease inhibitors. In this study, site-directed saturation mutagenesis at the P1 position was conducted to investigate the effect of P1 sites on the inhibitory activity and specificity of BmSPI38 and BmSPI39. In-gel activity staining and protease inhibition experiments confirmed that BmSPI38 and BmSPI39 could strongly inhibit elastase activity. Almost all mutant proteins of BmSPI38 and BmSPI39 retained the inhibitory activities against subtilisin and elastase, but the replacement of P1 residues greatly affected their intrinsic inhibitory activities. Overall, the substitution of Gly54 in BmSPI38 and Ala56 in BmSPI39 with Gln, Ser, or Thr was able to significantly enhance their inhibitory activities against subtilisin and elastase. However, replacing P1 residues in BmSPI38 and BmSPI39 with Ile, Trp, Pro, or Val could seriously weaken their inhibitory activity against subtilisin and elastase. The replacement of P1 residues with Arg or Lys not only reduced the intrinsic activities of BmSPI38 and BmSPI39, but also resulted in the acquisition of stronger trypsin inhibitory activities and weaker chymotrypsin inhibitory activities. The activity staining results showed that BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) had extremely high acid-base and thermal stability. In conclusion, this study not only confirmed that BmSPI38 and BmSPI39 had strong elastase inhibitory activity, but also confirmed that P1 residue replacement could change their activity and inhibitory specificity. This not only provides a new perspective and idea for the exploitation and utilization of BmSPI38 and BmSPI39 in biomedicine and pest control, but also provides a basis or reference for the activity and specificity modification of TIL-type protease inhibitors.

Proteomic characterization of Shiitake (Lentinula edodes) post-harvest fruit bodies grown on hardwood logs and isolation of an antibacterial serine protease inhibitor.

Lentinula edodes (Shiitake) is one of the most heavily cultivated mushrooms in the world with proven antioxidant and antibacterial properties, among others. Evidence indicates that the choice of mushroom cultivation technique strongly influences the production of bioactive compounds, but to date the nature of many of these compounds has not been fully established. This work focuses on the proteomic characterization of L. edodes to highlight the main active processes two days after harvest and elucidates the proteins involved in the known antioxidant and antibacterial proprieties of Shiitake fruit bodies cultivated on oak logs. A label-free approach allowed us to identify a total of 2702 proteins which were mainly involved in carbohydrate and protein metabolism, cell growth and replication, indicating that several developmental processes remain active in fruit bodies post-harvest. Proteins with antioxidant activities were identified, indicating the contribution of proteins to the antioxidant properties of L. edodes extracts. Antibacterial assays also reveal the activity of a serine protease inhibitor that strongly accumulates in the post-harvest fruit body grown on oak logs. Overall, this study contributes to the understanding of the impact of the log cultivation method on the production of Shiitake mushrooms richest in high-value bioactive compounds.

Tick Salivary Kunitz-Type Inhibitors: Targeting Host Hemostasis and Immunity to Mediate Successful Blood Feeding.

Kunitz domain-containing proteins are ubiquitous serine protease inhibitors with promising therapeutic potential. They target key proteases involved in major cellular processes such as inflammation or hemostasis through competitive inhibition in a substrate-like manner. Protease inhibitors from the Kunitz superfamily have a low molecular weight (18-24 kDa) and are characterized by the presence of one or more Kunitz motifs consisting of α-helices and antiparallel ß-sheets stabilized by three disulfide bonds. Kunitz-type inhibitors are an important fraction of the protease inhibitors found in tick saliva. Their roles in inhibiting and/or suppressing host homeostatic responses continue to be shown to be additive or synergistic with other protease inhibitors such as cystatins or serpins, ultimately mediating successful blood feeding for the tick. In this review, we discuss the biochemical features of tick salivary Kunitz-type protease inhibitors. We focus on their various effects on host hemostasis and immunity at the molecular and cellular level and their potential therapeutic applications. In doing so, we highlight that their pharmacological properties can be exploited for the development of novel therapies and vaccines.

Expression of Trichinella spiralis serpin Tsp_01570 in Pichia pastoris: a first insight of its biomodulatory activity.

Serpins represent one of the most diverse families of serine protease inhibitors. Despite their complexity, they are virtually found in all organisms and play an important role in homeostasis processes such as blood coagulation, inflammation, fibrinolysis, immune responses, chromatin condensation, tumor suppression, and apoptosis. There has recently been particular interest in studying serpin functions in infection and inflammation, especially since more serpins from parasites have been identified and characterized. Among helminths, Trichinella spiralis is one of the few parasites with an extremely strong ability to induce host immune suppression. Previous studies show that serpins are present in Trichinella and are expressed differentially at different parasite stages. More interesting, there is evidence of a recombinant serpin from Trichinella pseudospiralis that alters macrophage polarization in vitro. This finding could be relevant to comprehend the modulation process of the immune response. We expressed Tsp_01570, a putative serpin gene from Trichinella spiralis, in the eukaryotic system Pichia pastoris SMD1168H and evaluated its presence at different parasite stages, finding the serine protease inhibitor in the crude extract of adult worms. The effect of recombinant serpin on THP-1 cells was tested by quantification of IL-12p40, TNF-α, IL-4, and IL-10 cytokines released by ELISA. We also evaluated the expression of the M1 markers, CCR7 and CD86, and the M2 markers, CD163 and CD206, by immunofluorescence staining. This study represents the first insight in elucidating the importance of serpin Tsp_01570 as a potential molecular modulator.

Reduced cell invasion may be a characteristic of placental defects in pregnant women of advanced maternal age at single-cell level.

The mechanisms underlying pregnancy complications caused by advanced maternal age (AMA) remain unclear. We analyzed the cellular signature and transcriptomes of human placentas in AMA women to elucidate these mechanisms. Placental tissues from two AMA women and two controls were used for single-cell RNA-sequencing (scRNA-seq). Controls consisted of AMA women who did not experience any pregnancy complications and pregnant women below the age of 35 years without pregnancy complications. Trophoblast cells were obtained from the placentas of another six pregnant women (three AMA women and three controls), and in-vitro transwell assays were conducted to observe the cell invasion ability. Thirty additional samples (from 15 AMA women and 15 controls) were analyzed to verify the specific expression of serine protease inhibitor clade E member 1 (SERPINE1). Preliminary study of the role of SERPINE1 in cell invasion was carried out with HTR8-S/Vneo cells. High-quality transcriptomes of 27 |607 cells were detected. Three types of trophoblast cells were detected, which were further classified into eight subtypes according to differences in gene expression and Gene Ontology (GO) function. We identified 110 differentially expressed genes (DEGs) in trophoblast cells between the AMA and control groups, and the DEGs were enriched in multiple pathways related to cell invasion. In-vitro transwell assays suggested that the invading trophoblast cells in AMA women were reduced. SERPINE1 was specifically expressed in the trophoblast, and its expression was higher in AMA women (P<0.05). Transfection of human SERPINE1 (hSERPINE1) into HTR8-S/Vneo trophoblast cells showed fewer invading cells in the hSERPINE1 group. Impaired cell invasion may underlie the increased risk of adverse pregnancy outcomes in AMA women. Abnormal expression of SERPINE1 in extravillous trophoblast (EVT) cells appears to play an important role.

Structural study of the uPA-nafamostat complex reveals a covalent inhibitory mechanism of nafamostat.

Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases produced during the coagulation cascade and inflammation. Previous studies showed that NM was a highly safe drug for the treatment of different cancers, but the precise functions and mechanisms of NM are not clear. In this study, we determined a series of crystal structures of NM and its hydrolysates in complex with a serine protease (urokinase-type plasminogen activator [uPA]). These structures reveal that NM was cleaved by uPA and that a hydrolyzed product (4-guanidinobenzoic acid [GBA]) remained covalently linked to Ser195 of uPA, and the other hydrolyzed product (6-amidino-2-naphthol [6A2N]) released from uPA. Strikingly, in the inactive uPA (uPA-S195A):NM structure, the 6A2N side of intact NM binds to the specific pocket of uPA. Molecular dynamics simulations and end-point binding free-energy calculations show that the conf1 of NM (6A2N as P1 group) in the uPA-S195A:NM complex may be more stable than conf2 of NM (GBA as P1 group). Moreover, in the structure of uPA:NM complex, the imidazole group of His57 flips further away from Ser195 and disrupts the stable canonical catalytic triad conformation. These results not only reveal the inhibitory mechanism of NM as an efficient serine protease inhibitor but also might provide the structural basis for the further development of serine protease inhibitors.

Ulinastatin Alleviates Rhabdomyolysis-Induced Acute Kidney Injury by Suppressing Inflammation and Apoptosis via Inhibiting TLR4/NF-κB Signaling Pathway.

Acute kidney injury (AKI) is an important complication of rhabdomyolysis (RM), but there is lack of effective treatments. Ulinastatin (UTI) is a broad-spectrum serine protease inhibitor isolated and purified from human urine with strong anti-inflammatory and cytoprotective actions. The aim of this research was to investigate the effect and potential mechanism of UTI on RM-induced AKI (RM-AKI). We established RM-induced AKI model and myoglobin (Mb)-stimulated NRK-52E cell model. In vivo, twenty-four rats were randomly divided into three groups (n = 8): control, RM-AKI, and RM-AKI + UTI. In vitro, the NRK-52E cells were divided into six groups according to the different treatment method. Mb-stimulated NRK-52E cells were treated with UTI or si-TLR4 transfection to characterize the mechanisms of UTI in RM-AKI. Indicators of the kidney injury, cell viability, cell cycle, oxidative stress, inflammation, apoptosis, and TLR4/NF-κB signaling pathway were assessed. In vivo and in vitro, UTI significantly decreased the expression of TLR4 and p65. In vivo, UTI significantly improved renal function and reduced inflammatory reaction and kidney injury. In vitro, UTI protected NRK-52E cells from Mb stimulation by suppressing cell cytotoxicity, cell cycle inhibition, overproduction of ROS, inflammation, and apoptosis. Additionally, UTI played a protective role by downregulating the TLR4 expression. The results indicate that UTI alleviates RM-AKI by suppressing the inflammatory response and apoptosis via inhibiting TLR4/NF-κB signaling pathway. Our study provides a new mechanism for the protective effect of UTI on RM-AKI.

Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor.

Serine protease inhibitors (SPIs) act in diverse biological processes in insects such as immunity, development, and digestion by preventing the unwanted proteolysis. So far, the repertoire of genes encoding SPIs has been identified from few insect species. In this study, 62 SPI genes were identified from the genome of the yellow mealworm, Tenebrio molitor. According to their modes of action, they were classified into three families, serpin (26), canonical SPI (31), and α-macroglobulins (A2M) (5). These SPIs feature eight domains including serpin, Kazal, TIL, Kunitz, WAP, Antistasin, pacifastin, and A2M. In total, 39 SPIs contain a single SPI domain, while the others encode at least two inhibitor units. Based on the amino acids in the cleaved reactive sites, the abilities of these SPIs to inhibit trypsin, chymotrypsin, or elastase-like enzymes are predicted. The expression profiling based on the RNA-seq data showed that these genes displayed stage-specific expression patterns during development, suggesting to us their significance in development. Some of the SPI genes were exclusively expressed in particular tissues such as hemocyte, fat body, gut, ovary, and testis, which may be involved in biological processes specific to the indicated tissues. These findings provide necessary information for further investigation of insect SPIs.

Structure-Based Design of High-Affinity and Selective Peptidomimetic Hepsin Inhibitors.

In many solid tumors, increased upregulation of transmembrane serine proteases (TTSPs) leads to an overactivation of growth factors, which promotes tumor progression. Here, we have used a combinatorial methodology to develop high-affinity tetrapeptidic inhibitors. A previous virtual screening of 8000 peptide combinations against the crystal structure of the TTSP hepsin identified a series of recognition sequences, customized for the non-prime substrate binding (P) sites of this serine protease. A combination of the top recognition sequences with an electrophilic warhead resulted in highly potent inhibitors with good selectivity against coagulation proteases factor Xa and thrombin. Structure-activity relationships of two selected compounds were further elucidated by investigation of their stability in biological fluids as well as the influence of the warhead and truncated inhibitors on the inhibitory potency. Overall, this methodology yielded compounds as selective inhibitors for potential cancer drug development, where hepsin is overexpressed.

Physicochemical Properties and Elimination of the Activity of Anti-Nutritional Serine Protease Inhibitors from Mulberry Leaves.

Mulberry leaf is an excellent protein resource that can be used as feed additive for livestock and poultry. Nevertheless, the use of mulberry leaves in animal diets is limited by its protease inhibitors, tannic acid and other anti-nutritional factors. This study systematically analyzed the type and activity of serine protease inhibitors (SPIs) from the leaves of 34 mulberry varieties, aiming to reveal the physicochemical properties and inactivation mechanism of SPIs. The types and activities of trypsin inhibitors (TIs) and chymotrypsin inhibitors (CIs) exhibited polymorphisms among different mulberry varieties. The highest number of types of inhibitors was detected in Jinshi, with six TIs (TI-1~TI-6) and six CIs (CI-1~CI-6). TIs and CIs exhibited strong thermal and acid-base stability. High-temperature and high-pressure treatment could reduce the activities of TIs and CIs to a certain extent. ß-mercaptoethanol treatment could completely abolish TIs and CIs, suggesting that the disulfide bridges were critical for their inhibitory activities. The Maillard reaction could effectively eliminate the inhibitory activities of TI-1~TI-4 and CI-1~CI-4. This study reveals the physicochemical properties and inactivation mechanisms of the anti-nutritional SPIs from mulberry leaves, which is helpful to exploit mulberry-leaf food with low-activity SPIs, promote the development and utilization of mulberry-leaf resources in animal feed and provide reference for mulberry breeding with different functions.

Regulatory effects of Trichinella spiralis and a serine protease inhibitor on the endoplasmic reticulum stress response of intestinal epithelial cells.

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause an endoplasmic reticulum stress (ERS) response. If ERS continues or cannot be alleviated, it will cause the production of proapoptotic factors and eventually lead to apoptosis. Therefore, this study mainly explored whether Trichinella spiralis Kazal-type serine protease inhibitor (TsKaSPI) contributed to the invasion of intestinal epithelial cells during the infectious stage of T. spiralis by regulating ERS. First, in the T. spiralis infection model, H&E staining was used to analyse the damage to jejunum tissue, a TUNEL assay was used to examine cell apoptosis, and the expression of ERS-related and apoptosis-related molecules was also measured. The results showed that ERS occurred during the intestinal phase of T. spiralis infection, while remission began during the cyclic phase. Then, we selected TsKaSPI, one of the important components of T. spiralis ES antigens, for in vitro experiments. The results showed that TsKaSPI could induce apoptosis in a porcine small intestinal epithelial cell line (IPEC cells) by activating ERS and promote activation of the NF-κB signalling pathway. Inhibition experiments confirmed that the occurrence of ERS was accompanied by the activation of NF-κB, and the two processes regulated each other. Finally, we conducted in vivo experiments and administered TsKaSPI to mice. The results confirmed that TsKaSPI could activate ERS and lead to apoptosis in intestinal epithelial cells. In conclusion, T. spiralis infection and TsKaSPI can promote cell apoptosis by activating the ERS response in intestinal epithelial cells and activate the NF-κB signalling pathway to promote the occurrence and development of inflammation.

Low-intensity blast induces acute glutamatergic hyperexcitability in mouse hippocampus leading to long-term learning deficits and altered expression of proteins involved in synaptic plasticity and serine protease inhibitors.

Neurocognitive consequences of blast-induced traumatic brain injury (bTBI) pose significant concerns for military service members and veterans with the majority of "invisible injury." However, the underlying mechanism of such mild bTBI by low-intensity blast (LIB) exposure for long-term cognitive and mental deficits remains elusive. Our previous studies have shown that mice exposed to LIB result in nanoscale ultrastructural abnormalities in the absence of gross or apparent cellular damage in the brain. Here we tested the hypothesis that glutamatergic hyperexcitability may contribute to long-term learning deficits. Using brain slice electrophysiological recordings, we found an increase in averaged frequencies with a burst pattern of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal CA3 neurons in LIB-exposed mice at 1- and 7-days post injury, which was blocked by a specific NMDA receptor antagonist AP5. In addition, cognitive function assessed at 3-months post LIB exposure by automated home-cage monitoring showed deficits in dynamic patterns of discrimination learning and cognitive flexibility in LIB-exposed mice. Collected hippocampal tissue was further processed for quantitative global-proteomic analysis. Advanced data-independent acquisition for quantitative tandem mass spectrometry analysis identified altered expression of proteins involved in synaptic plasticity and serine protease inhibitors in LIB-exposed mice. Some were correlated with the ability of discrimination learning and cognitive flexibility. These findings show that acute glutamatergic hyperexcitability in the hippocampus induced by LIB may contribute to long-term cognitive dysfunction and protein alterations. Studies using this military-relevant mouse model of mild bTBI provide valuable insights into developing a potential therapeutic strategy to ameliorate hyperexcitability-modulated LIB injuries.