Expression Of Protease Inhibitor Research Articles

Blood Plasma Markers in Depressed Mice under Chronic Social Defeat Stress.

It has previously been shown that, in mice, chronic social defeat stress in daily agonistic interactions leads to a depression-like state similar to that in depressive patients. With this model, it has become obvious that it is possible to study peripheral markers of the depression-like state in an experiment. This paper was aimed at searching for protein markers in the blood plasma of depressed mice in the chronic social conflict model, which allows for us to obtain male mice with repeated experiences of defeat. Proteomic analysis of blood plasma samples was conducted to identify proteins differentially expressed in this state. There were changes in the expression levels of the amyloid proteins SAA1, SAA4, and SAMP and apolipoproteins APOC3, APOD, and ADIPO in the blood plasma of depressed mice compared with controls (unstressed mice). Changes in the expression of serine protease inhibitors and/or proteins associated with lipid metabolism, inflammation, or immune function [ITIH4, SPA3, A1AT5, HTP (HP), CO9, and A2MG] were also found. Here, we showed that chronic social stress is accompanied by increased levels of amyloid proteins and apolipoproteins in blood plasma. A similarity was noted between the marker protein expression changes in the depressed mice and those in patients with Alzheimer's disease. These data indicate a psychopathogenic role of chronic social stress, which can form a predisposition to neurodegenerative and/or psychoemotional disorders.

Fibrogenesis in Human Mucosa and Muscularis Precision-Cut Intestinal Slices.

In Crohn's Disease (CD), intestinal fibrosis is a prevalent yet unresolved complication arising from chronic and transmural inflammation. The histological assessment of CD intestines shows changes in tissue morphology in all the layers, including the mucosa and muscularis. This study aimed to determine the differences in fibrogenesis between mucosa and muscularis. Human precision-cut intestinal slices (hPCIS) were prepared from human intestine mucosa and muscularis and treated with TGF-β1 and/or PDGF-BB for 72 h. Gene and protein expression and matrix metalloproteinase (MMP) activity were determined. The basal gene expression of various fibrosis markers was higher in muscularis compared to mucosa hPCIS. During incubation, Pro-Collagen-1A1 secretion increased in muscularis but not in mucosa hPCIS. MMP gene expression increased during incubation in mucosa and muscularis hPCIS, except for MMP9, MMP12, and MMP13 in muscularis hPCIS. Incubation with TGF-β1 caused increased COL1A1 expression in the mucosa but not in muscularis hPCIS. In muscularis hPCIS, TGF-β1 treatment caused a decrease in MMP1 and CTSK expression, while MMP13 was increased. In the presence of TGF-β1, protease inhibitor expression was stable, except for SERPINE1, which was increased in muscularis hPCIS. We conclude that fibrogenesis is more pronounced in muscularis hPCIS compared to mucosa hPCIS, especially when stimulated with TGF-β1.

LncRNA Malat1 suppresses pyroptosis and T cell-mediated killing of incipient metastatic cells.

The contribution of antitumor immunity to metastatic dormancy is poorly understood. Here we show that the long noncoding RNA Malat1 is required for tumor initiation and metastatic reactivation in mouse models of breast cancer and other tumor types. Malat1 localizes to nuclear speckles to couple transcription, splicing and mRNA maturation. In metastatic cells, Malat1 induces WNT ligands, autocrine loops to promote self-renewal and the expression of Serpin protease inhibitors. Through inhibition of caspase-1 and cathepsin G, SERPINB6B prevents gasdermin D-mediated induction of pyroptosis. In this way, SERPINB6B suppresses immunogenic cell death and confers evasion of T cell-mediated tumor lysis of incipient metastatic cells. On-target inhibition of Malat1 using therapeutic antisense nucleotides suppresses metastasis in a SERPINB6B-dependent manner. These results suggest that Malat1-induced expression of SERPINB6B can titrate pyroptosis and immune recognition at metastatic sites. Thus, Malat1 is at the nexus of tumor initiation, reactivation and immune evasion and represents a tractable and clinically relevant drug target.

P064 High intestinal Secretory Leukocyte Protease Inhibitor (SLPI) expression correlates with abundant neutrophilic inflammation and identifies Inflammatory Bowel Disease patients with a strong IL-17 response.

Abstract Background Inflammatory bowel disease, comprising Crohn’s disease (CD) and ulcerative colitis (UC), is driven by aberrant host-microbial immune interactions. Disease heterogeneity and severity hamper effective treatment, thus new therapeutic strategies tailored to the patient’s underlying immune defect are required. In mice, upon epithelial barrier breach and microbiota driven inflammation, colonic intestinal epithelial cells increase Secretory Leukocyte Protease Inhibitor (SLPI) expression. SLPI can exert anti-microbial activity and inhibit NF-kB activation. Therefore, we hypothesized that SLPI expression is increased in the intestine of IBD patients and may differentiate a subgroup of patients with a distinctive underlying immune disease. Methods Intestinal biopsies and plasma from therapy-naive pediatric patients (age 6-18) with CD (n=41), UC (n=22) or IBD-negative controls (n=15) were collected. Intestinal SLPI protein was detected by immunohistochemistry (IHC), scored semi-quantitatively, and patients were dichotomized into ‘SLPI-high’ and ‘SLPI-low’ groups. RNA sequencing and quantitative PCR (qPCR) were performed. Plasma protein concentrations were assessed with Olink technology®. Results We observed increased SLPI mRNA and protein expression in intestinal tissue of CD and UC patients, compared to IBD-negative controls. Expression was the strongest in macroscopically inflamed tissue and more dominant in colon than in small intestinal ileum. Colonic SLPI expression was associated with endoscopic severity of inflammation, with histologic disease activity, and with clinical disease activity in CD and UC patients. Furthermore, high epithelial SLPI expression associated with abundant neutrophil infiltration and calprotectin expression. RNA-seq revealed that biopsies of SLPI-high patients had a signature of inflammatory neutrophilic inflammation including the genes OSM, IL-1B, and CXCL8, which have been associated with severe disease progression and resistance to anti-TNF treatment. Most notably, biopsies of SLPI-high patients had a signature of increased IL-17 signaling, which was supported by IHC detection of high frequencies of IL-17 producing cells in the paired biopsies and higher plasma concentrations of CCL20, MMP10 and IL-17A. Conclusion Together, these results demonstrate that intestinal SLPI expression identifies severe IBD patients with high intestinal SLPI expression and immune features of therapy resistance, who have increased IL-17A responses and subsequent neutrophilic inflammation compared to SLPI-low patients. As such, epithelial SLPI expression is a histological parameter that could support tailored treatment strategies in pediatric IBD patients at the time of diagnosis.

Inhibition of secretory leukocyte protease inhibitor (SLPI) promotes the PUMA-mediated apoptosis and chemosensitivity to cisplatin in colorectal cancer cells

BackgroundAberrant expression of Secretory Leukocyte Protease Inhibitor (SLPI) has been associated with human cancer growth and its suppression was identified as a potential target for anti-cancer drugs, particularly in colorectal cancer. However, the underlying mechanism by which SLPI affected the development of drug resistance in CRC remains unclear.ObjectiveThis study investigated the role of SLPI in the p53-up-regulated modulator of apoptosis (PUMA)-mediated CRC cells’ apoptosis and their chemosensitivity to Cisplatin.MethodsA series of qRT-PCR and western blot analyses were performed to characterize the expressions of SLPI, PUMA, and Akt in CRC lines. Tunel, transwell, and CCK-8 analyses were monitored to define the impacts of the siRNA-mediated knockdown of SLPI on CRC cell development. Furthermore, in vivo development of CRC was evaluated in nude mice infected with siSLPI or Cisplatin alone or both, and Ki67 and caspase-3 immunohistochemistry assay was monitored on multiple tissue microarray from the same cohort.ResultsOur results showed that SLPI inhibition strongly promoted the expressions of the pro-apoptotic protein PUMA, cleaved-caspase3 and Bax and reduced the cell viability of HT29 and HT116 cell lines in vitro. In addition, siSLPI knockdown effectively suppressed both Akt and FoxO3 proteins and improved the sensitivity to cisplatin chemotherapy. Xenograft tumor assay revealed a lowered growth in mice treated with Cisplatin, while combined treatment of siSLPI achieved more significant anticancer effects than Cisplatin alone.ConclusionsTaken together, these findings demonstrated that suppression of SLPI might repress the growth of human colorectal cancer cells both in vitro and in vivo. These results suggested SLPI as a novel resistance factor to Cisplatin, and a combination of Cisplatin and SLPI inhibitor be beneficial for colorectal cancer therapy.

Bacillus cereus EC9 protects tomato against Fusarium wilt through JA/ET-activated immunity.

The mechanisms of action and the limitations of effectiveness of natural biocontrol agents should be determined in order to convert them into end products that can be used in practice. Rhizosphere Bacillus spp. protect plants from various pathogens by displaying several modes of action. However, the ability of Bacillus spp. to control plant diseases depends on the interaction between the bacteria, host, and pathogen, and the environmental conditions. We found that soil drenching of tomato plants with the non-antifungal Bacillus cereus strain EC9 (EC9) enhances plant defense against Fusarium oxysporum f. sp. lycopersici (Fol). To study the involvement of plant defense-related phytohormones in the regulation of EC9-activated protection against Fol, we conducted plant bioassays in tomato genotypes impaired in salicylic acid (SA) accumulation, jasmonic acid (JA) biosynthesis, and ethylene (ET) production, and analyzed the transcript levels of pathways-related marker genes. Our results indicate that JA/ET-dependent signaling is required for EC9-mediated protection against Fol in tomato. We provide evidence that EC9 primes tomato plants for enhanced expression of proteinase inhibitor I (PI-I) and ethylene receptor4 (ETR4). Moreover, we demonstrated that EC9 induces callose deposition in tomato roots. Understanding the involvement of defense-related phytohormones in EC9-mediated defense against Fusarium wilt has increased our knowledge of interactions between non-antifungal plant defense-inducing rhizobacteria and plants.

Differential expression of inflammatory cytokines, prostaglandin synthases and secretory leukocyte protease inhibitor in the endometrium and circulation in different graded CEH-pyometra in bitch

Cystic endometrial hyperplasia (CEH)-pyometra (CEH-P) is one of the most common reproductive disorders in bitches, posing a risk to both future fertility and life. The aims of the current study were to elucidate the differential expression patterns of inflammatory mediators at transcript and protein levels in the endometrium and to assess the concentrations of key inflammatory mediators in the peripheral circulation of bitches with different graded CEH-P. A total of 25 client-owned intact mixed breed bitches of 3–10 years presented to the outpatient department of RVP-TVCC of the institute were considered for the study. Of which, 22 cases suggestive of pyometra and 3 cases of CEH obtained during routine elective ovariohysterectomy were subjected to histopathological examination. Uteri were categorized into CEH (n = 3), moderate CEH-P (mCEH-P, n = 9), severe CEH-P (sCEH-P, n = 6) and atrophic pyometra (AT-P, n = 7). A group of age matched (n = 12) bitches without pyometra served as control. Endometrial transcripts such as IL6, IL8, PTGS2, PGFS, and SLPI were expressed differentially in the CEH and CEH-P bitch. In addition, a strong immunoreactivity (IR) of IL6, IL8, PTGS2, and mPGES1 was recorded in the sCEH-P uterus, while expression of IL10 was noticed in AT-P. In circulation, serum IL6 was the most relevant marker with high sensitivity of 96.2% and specificity of 84.6% at a cut off concentration 8.5 pg/mL followed by SLPI with 95.2% sensitivity, and 84.6% specificity at cut off concentration of 1.3 ng/mL. Serum IL10, PGFM and SLPI concentration in the peripheral circulation were 1.5–2.23 fold higher in mCEH-P, 0.87–2.5 fold higher in sCEH-P and 2.9–3.5 fold higher in AT-P than that of control. It is concluded that monitoring the serum concentration of IL6, IL10 and SLPI would be useful adjunct to the established hematobiochemical parameters in the management of pyometra in the bitch with critical illness.

Inoculation of Pochonia chlamydosporia triggers a defense response in tomato roots, affecting parasitism by Meloidogyne javanica

Pochonia chlamydosporia is a soil-dwelling fungus and biological control agent of nematodes, active ingredient in commercial bionematicides. The fungus is also endophytically associated with the roots of several plant species, promoting their growth and inducing systemic resistance. In this study, different pathways and tomato defense metabolites were studied to identify mechanisms induced by P. chlamydosporia that contribute to the control of Meloidogyne javanica, at early and late developmental stages. Some defense responses activated by the fungus appeared related to the nematode life cycle. Among the evaluated biochemical analysis, root colonization of P. chlamydosporia showed an increase in the concentration of phenolic compounds, such as chlorogenic acid. In addition, the expression of some host plant genes was also modified. The interaction of the fungus with roots parasitized by M. javanica resulted in the highest expression of Phenylalanine Ammonia-Lyase (PAL), Chalcone synthase (LECHS 2), and Protease Inhibitor (PI1) genes at 24 days post-inoculation. At the second sampling time (44 days), there was an increase in the expression of the Respiratory Burst Oxidase Homolog (RBOH) gene. Fungus reduced the expression of the ACC-oxidase and Pathogenesis-Related Proteins 1 (PR-1) genes in roots. Moreover, P. chlamydosporia inoculation changed metabolites and phytohormone profiles of the gall formed by M. javanica. Plant defense response appeared to involve the jasmonic acid and phytosphingosine cascades. With this analysis, it was possible to propose new molecular mechanisms induced by the fungus that contribute to the control of M. javanica.

Adeno-associated virus 9 vector-mediated cardiac-selective expression of human secretory leukocyte protease inhibitor attenuates myocardial ischemia/reperfusion injury.

Protease enzymes contribute to the initiation of cardiac remodeling and heart failure after myocardial ischemic/reperfusion (I/R) injury. Protease inhibitors attenuate protease activity and limit left ventricular dysfunction and remodeling. Previous studies showed the cardioprotective effect of secretory leukocyte protease inhibitor (SLPI) against I/R injury. However, overexpression of SLPI gene in cardiovascular diseases has only been investigated in an in vitro experiment. Here, cardiac-selective expression of the human secretory leukocyte protease inhibitor (hSLPI) gene and its effect on I/R injury were investigated. Adeno-associated virus (AAV) serotype 9 carrying hSLPI under the control of cardiac-selective expression promoter (cardiac troponin, cTn) was intravenously administered to Sprague–Dawley rats for 4 weeks prior to coronary artery ligation. The results showed that myocardial-selective expression of hSLPI significantly reduced infarct size, cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and myoglobin levels that all served to improve cardiac function. Moreover, overexpression of hSLPI showed a reduction in inflammatory cytokines, oxidatively modified protein carbonyl (PC) content, ischemia-modified albumin (IMA), and necrosis and cardiac tissue degeneration. In conclusion, this is the first study to demonstrate cardiac-selective gene delivery of hSLPI providing cardioprotection against myocardial I/R injury in an in vivo model.

High expression of secretory leukocyte protease inhibitor (SLPI) in stage III micro-satellite stable colorectal cancer is associated with reduced disease recurrence

Secretory leukocyte protease inhibitor (SLPI) is a pleiotropic protein produced by healthy intestinal epithelial cells. SLPI regulates NF-κB activation, inhibits neutrophil proteases and has broad antimicrobial activity. Recently, increased SLPI expression was found in various types of carcinomas and was suggested to increase their metastatic potential. Indeed, we demonstrated that SLPI protein expression in colorectal cancer (CRC) liver metastases and matched primary tumors is associated with worse outcome, suggesting that SLPI promotes metastasis in human CRC. However, whether SLPI plays a role in CRC before distant metastases have formed is unclear. Therefore, we examined whether SLPI expression is associated with prognosis in CRC patients with localized disease. Using a cohort of 226 stage II and 160 stage III CRC patients we demonstrate that high SLPI protein expression is associated with reduced disease recurrence in patients with stage III micro-satellite stable tumors treated with adjuvant chemotherapy, independently of established clinical risk factors (hazard rate ratio 0.54, P-value 0.03). SLPI protein expression was not associated with disease-free survival in stage II CRC patients. Our data suggest that the role of SLPI in CRC may be different depending on the stage of disease. In stage III CRC, SLPI expression may be unfavorable for tumors, whereas SLPI expression may be beneficial for tumors once distant metastases have established.

Levels of secretory leukocyte protease inhibitor expression in acute wounds.

Even with our best practices, we are frequently unable to prevent slow and stalled wound healing-particularly in people with impaired circulation and conditions such as diabetes. As a result, greater insight into the nature of wound healing and alternative treatment approaches is needed. An avenue that may be of particular promise is increasing understanding of the role of secretory leukocyte protease inhibitor (SLPI) as there is evidence that it enhances wound healing, its expression increases in response to inflammation and infection, and it exhibits anti-protease, anti-inflammatory, antiviral antibacterial and antifungal activities. The response of SLPI levels to wounding and skin injury was assessed by taking punch skin biopsies from healthy volunteers and assessing the levels of SLPI at the site of injury at the time of wounding (baseline) as well as one, two, three, four, seven, nine and 12 weeks later. A total of 35 volunteers took part in the study. Significant elevations were found: levels of SLPI were greatly increased, 12 times that at baseline, and remained elevated at three weeks despite re-epithelialisation having occurred. These findings not only suggest that levels of SLPI rise rapidly following wounding, but that these elevations are sustained, and continue to increase even when re-epithelialisation has occurred. These results suggest that the role and potential benefits of this protease inhibitor deserve further exploration.

Quantitative proteomics analysis reveals core and variable tick salivary proteins at the tick-vertebrate host interface.

Few studies have examined tick proteomes, how they adapt to their environment, and their roles in the parasite-host interactions that drive tick infestation and pathogen transmission. Here we used a proteomics approach to screen for biologically and immunologically relevant proteins acting at the tick-host interface during tick feeding and, as proof of principle, measured host antibody responses to some of the discovered candidates. We used a label-free quantitative proteomic workflow to study salivary proteomes of (i) wild Ixodes ricinus ticks fed on different hosts, (ii) wild or laboratory ticks fed on the same host, and (iii) adult ticks cofed with nymphs. Our results reveal high and stable expression of several protease inhibitors and other tick-specific proteins under different feeding conditions. Most pathways functionally enriched in sialoproteomes were related to proteolysis, endopeptidase, and amine-binding activities. The generated catalogue of tick salivary proteins enabled the selection of six candidate secreted immunogenic peptides for rabbit immunizations, three of which induced strong and durable antigen-specific antibody responses in rabbits. Furthermore, rabbits exposed to ticks mounted immune responses against the candidate peptides/proteins, confirming their expression at the tick-vertebrate interface. Our approach provides insights into tick adaptation strategies to different feeding conditions and promising candidates for developing antitick vaccines or markers of exposure of vertebrate hosts to tick bites.

Upregulation of neuronal progranulin mediates the antinociceptive effect of trimetazidine in paclitaxel-induced peripheral neuropathy: Role of ERK1/2 signaling

Neuronal progranulin (PGRN) overexpression is an endogenous adaptive pain defense following nerve injury. It allows the survival of injured neurons to block enhanced nociceptive responses. Trimetazidine (TMZ) is widely used by cardiac patients as an anti-anginal drug, reflecting its anti-ischemic property. TMZ promotes axonal regeneration of sciatic nerves after crush injury. This study explored the interplay between PGRN and extracellular signal-regulated kinases (ERK1/2) to address mechanisms underlying neuropathic pain alleviation following paclitaxel (PTX) administration. Rats were given four injections of PTX (2 mg/kg, i.p.) every other day. Two days after the last dose, rats received TMZ (25 mg/kg) with or without the ERK inhibitor, PD98059, daily for 21 days. TMZ preserved the integrity of myelinated nerve fibers, as evidenced by an obvious reduction in axonal damage biomarkers. Accordingly, it alleviated PTX-evoked thermal, cold, and mechanical hyperalgesia/allodynia. TMZ also promoted ERK1/2 phosphorylation with a profound upsurge in PGRN content. These effects were associated with a substantial increase in Notch1 receptor gene expression and a prominent anti-inflammatory effect with a marked increase in mRNA expression of secretory leukocyte protease inhibitor. Further, TMZ decreased oxidative stress and caspase-3 activity in the sciatic nerve. Conversely, co-administration of PD98059 completely abolished these beneficial effects. Thus, the robust antinociceptive effect of TMZ is largely attributed to upregulating PGRN and Notch1 receptors via ERK1/2 activation.

Heterologous expression of a papain-like protease inhibitor (SnuCalCpI17) in the E. coli and its mode of inhibition.

The effect of the Escherichia coli (E. coli) Rosetta (DE3) system on the expression of recombinant papain-like cysteine protease inhibitors (SnuCalCpIs) was evaluated, and the inhibition mode of the expressed inhibitor was determined. SnuCalCpI08 and SnuCalCpI17, which previously had not been expressed in the E. coli BL21 (DE3) system due to rare codons of more than 10%, were successfully expressed in E. coli Rosetta (DE3) since the strain provides tRNAs for six rare codons. Initially, both inhibitors were expressed as inclusion bodies; however, the water solubility of SnuCalCpI17 could be improved by lowering the incubation temperature, reducing the IPTG concentration, and increasing the induction time. In contrast, the other inhibitor could not be solubilized in water. To validate whether the inhibitor was expressed with correct protein folding, a papain inhibition assay was performed with SnuCalCpI17. SnuCalCpI17 showed a half-maximal inhibitory concentration (IC50) of 105.671 ± 9.857µg/mL and a slow-binding inhibition mode against papain at pH 7.0 with a Kiapp of 75.80μg/mL. The slow-binding inhibitor has a slow dissociation from the inhibitor-target complex, resulting in a long residence time in vivo, and thus can effectively inhibit the target at doses far below the IC50 of the inhibitor. KEY POINTS: • Propeptide inhibitor (SnuCalCpI17) containing rare codons was expressed in E. coli Rosetta (DE3). • The slow-binding inhibition was shown by plotting the apparent first-order rate constant (kobs). • Protein-protein interaction between SnuCalCpIs and papain was verified by docking simulation.

Heterologous expression and physicochemical characteristics identification of Kunitz protease inhibitor in Brassica napus.

A Kunitz protease inhibitor gene (RTI; rti) was cloned from rapeseed and expressed in a Pichia pastoris expression system for the first time. After isolation and purification, the physical and chemical characteristics of the inhibitor were analyzed. The results showed that the induced expression level of the recombinant RTI reached 628mg/L, and the specific activity of the inhibitor reached 69.6TIU/mg protein at the shake flask fermentation level; the recombinant RTI retained more than 70% inhibitory activity between 30 and 90°C and more than 80% inhibitory activity between pH 2.0-11.0. The metal ions Cu2+ and CO2+ and the organic reagents methanol, ethanol, acetone, and chloroform inhibit its activity. The recombinant RTI interacts with trypsin in a noncompetitive manner and has a strong and specific inhibitory effect on trypsin, a typical Kunitz trypsin inhibitor from plants. Combined with its good physical and chemical properties, recombinant RTI has the potential to be developed into an insect resistance protein.

Modulation of HIV Replication in Monocyte-Derived Macrophages (MDM) by Host Antiviral Factors Secretory Leukocyte Protease Inhibitor and Serpin Family C Member 1 Induced by Steroid Hormones.

The impact of steroid hormones estrogen and progesterone on human immunodeficiency virus type 1 (HIV-1) replication is well documented. However, the exact mechanism involved in the regulation of HIV-1 replication by estrogen and progesterone is still unclear. In the present study, we wanted to elucidate the molecular mechanisms underlying the modulation of HIV-1 replication by estrogen and progesterone. To achieve this goal, we used real-time quantitative PCR arrays (PCR arrays) to identify differentially expressed host genes in response to hormone treatments that are involved in antiviral responses. Our in vitro results suggest that treatment with high doses of estrogen and progesterone promotes the expression of host antiviral factors Secretory leukocyte protease inhibitor (SLPI) and Serpin family C member 1 (SERPIN C1) among others produced in response to HIV-1 infection. SLPI is an enzyme that inhibits human leukocyte elastase, human cathepsin G, human trypsin, neutrophil elastase, and mast cell chymase. SERPIN C1 is a plasma protease inhibitor that regulates the blood coagulation cascade by the inhibition of thrombin and other activated serine proteases of the coagulation system. A dose dependent downmodulation of HIV-1 replication was observed in monocyte-derived macrophages (MDMs) pre-treated with the two proteins SLPI and SERPIN C1. Further investigations suggests that the host antiviral factors, SLPI and SERPIN C1 act at the pre-integration stage, inhibiting HIV-1 viral entry and leading to the observed downmodulation of HIV-1 replication. Our studies would help identify molecular mechanisms and pathways involved in HIV-1 pathogenesis.

Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-Inflammatory Cytokines.

BackgroundMicroglia safeguard the CNS against injuries and pathogens, and in the presence of certain harmful stimuli are capable of inducing a disease-dependent inflammatory response. When exposed to anti-inflammatory cytokines, however, these cells possess the ability to switch from an inflammatory to an immunosuppressive phenotype. Cancer cells exploit this property to evade the immune system, and elicit an anti-inflammatory microenvironment that facilitates tumor attachment and growth.ObjectiveThe tumor-supportive biological processes that are activated in microglia cells in response to anti-inflammatory cytokines released from cancer cells were explored with mass spectrometry and proteomic technologies.MethodsSerum-depleted and non-depleted human microglia cells (HMC3) were treated with a cocktail of IL-4, IL-13, IL-10, TGFβ, and CCL2. The cellular protein extracts were analyzed by LC-MS/MS. Using functional annotation clustering tools, statistically significant proteins that displayed a change in abundance between cytokine-treated and non-treated cells were mapped to their biological networks and pathways.ResultsThe proteomic analysis of HMC3 cells enabled the identification of ~10,000 proteins. Stimulation with anti-inflammatory cytokines resulted in the activation of distinct, yet integrated clusters of proteins that trigger downstream a number of tumor-promoting biological processes. The observed changes could be classified into four major categories, i.e., mitochondrial gene expression, ECM remodeling, immune response, and impaired cell cycle progression. Intracellular immune activation was mediated mainly by the transducers of MAPK, STAT, TGFβ, NFKB, and integrin signaling pathways. Abundant collagen formation along with the expression of additional receptors, matrix components, growth factors, proteases and protease inhibitors, was indicative of ECM remodeling processes supportive of cell-cell and cell-matrix adhesion. Overexpression of integrins and their modulators was reflective of signaling processes that link ECM reorganization with cytoskeletal re-arrangements supportive of cell migration. Antigen processing/presentation was represented by HLA class I histocompatibility antigens, and correlated with upregulated proteasomal subunits, vesicular/viral transport, and secretory processes. Immunosuppressive and proangiogenic chemokines, as well as anti-angiogenic factors, were detectable in low abundance. Pronounced pro-inflammatory, chemotactic or phagocytic trends were not observed, however, the expression of certain receptors, signaling and ECM proteins indicated the presence of such capabilities.ConclusionsComprehensive proteomic profiling of HMC3 cells stimulated with anti-inflammatory cytokines revealed a spectrum of microglia phenotypes supportive of cancer development in the brain via microenvironment-dependent biological mechanisms.

Molecular investigation of proteinase inhibitor (PI) gene in tomato plants induced by Meloidogyne species

BackgroundThe plant parasitic nematode genus Meloidogyne parasitize almost all flowering crops. Plants respond with a variety of morphological and molecular mechanisms to reduce the effects of pathogens. Proteinase inhibitors (PI), a special group of plant proteins which are small proteins, involve in protective role in the plants attacked by microorganisms. Still, the plant response using PI against nematodes has not been well understood. Therefore, this study was aimed to determine the expression of proteinase inhibitor I (PI-I) gene subsequent the infection of M. incognita, M. javanica, and M. chitwoodi in tomato plants post nematode infections. Molecular methods were used to determine the PI gene expressions at different days post nematode infections in host tissues. ResultsResults revealed that the population of M. incognita species reached the highest level of nematode population followed by M. javanica and M. chitwoodi, respectively. All Meloidogyne species induced expression of PI-I gene reached at the utmost level at 3 days post infection (dpi) in host tissues. Relative gene expression level was sharply dropped at 7 dpi, 14 dpi, and 21 dpi in M. incognita induced gene expression in host tissues. Similar results were observed in host tissues after infection of M. javanica and M. chitwoodi. ConclusionsThe commonalities of plant response across a diverse Meloidogyne species interaction and the expression of PI gene may be related to plant defense system. Increased level of PI gene expressions in early infection days in host tissues induced by parasitic nematodes may share resemblances to the mechanisms of resistance on biotrophic interactions.

Erratum to: Gene Expression of Protease Inhibitors in Tomato Plants with Invasion by Root-Knot Nematode Meloidogyne incognita and Modulation of Their Activity with Salicylic and Jasmonic Acids

An Erratum to this paper has been published: https://doi.org/10.1134/S1062359021440011

Water Deficiency and Induced Defense Against a Generalist Insect Herbivore in Desert and Mediterranean Populations of Eruca sativa.

In natural and agricultural ecosystems, plants are often simultaneously or sequentially exposed to combinations of stressors. Here we tested whether limited water availability (LWA) affects plant response to insect herbivory using two populations of Eruca sativa from desert and Mediterranean habitats that differ in their induced defenses. Considering that such differences evolved as responses to biotic and possibly abiotic stress factors, the two populations offered an opportunity to study ecological aspects in plant response to combined stresses. Analysis of chemical defense mechanisms showed that LWA significantly induced total glucosinolate concentrations in the Mediterranean plants, but their concentrations were reduced in the desert plants. However, LWA, with and without subsequent jasmonate elicitation, significantly induced the expression of proteinase inhibitor in the desert plants. Results of a no-choice feeding experiment showed that LWA significantly increased desert plant resistance to Spodoptera littoralis larvae, whereas it did not affect the relatively strong basal resistance of the Mediterranean plants. LWA and subsequent jasmonate elicitation increased resistance against the generalist insect in Mediterranean plants, possibly due to both increased proteinase inhibitor expression and glucosinolate accumulation. The effect of LWA on the expression of genes involved in phytohormone signaling, abscisic acid (ABA-1) and jasmonic acid (AOC1), and the jasmonate responsive PDF1.2, suggested the involvement of abscisic acid in the regulation of defense mechanisms in the two populations. Our results indicate that specific genotypic responses should be considered when estimating general patterns in plant response to herbivory under water deficiency conditions.