Proteinase Inhibitor Research Articles

Cocculus hirsutus trypsin inhibitor (ChTI) as a biocontrol agent for post harvest bacterial disease in tomato

The widespread distribution of proteinase inhibitors (PIs) in various plant parts has made them the most abundant class of defensive proteins. PIs restrict microbial developmental processes by attenuating respective proteases, making them a highly promising area of research. In our current investigation, we evaluated the bactericidal potential of a serine proteinase inhibitor (SPI), specifically the Cocculus hirsutus trypsin inhibitor (ChTI). Recombinant ChTI was expressed, purified using a Sepharose-4B affinity column, and utilized in bioassays against both gram-negative and gram-positive bacteria. Pre-incubation of bacterial trypsin-like proteinase (TLP) with 400 TIU/mg of ChTI resulted in TLP inhibition rates of 64%, 61.7%, and 56.4%, with IC50 values of 1.67 µg/ml, 1.74 µg/ml, and 1.90 µg/ml for Xanthomonas axonopodis, Escherichia coli, and Ralstonia solanacearum, respectively. Further evaluation against bacterial pathogens Leuconostoc citreum and Lactobacillius fructivorans, associated with postharvest tomato loss, indicated ChTI's potential in extending tomato shelf life. ChTI-treated tomatoes remained viable for over 20 days compared to 11 days for control fruits. Bacterial populations on treated fruits were significantly reduced, with ChTI inhibiting up to 49% and 36% of TLP for L. citreum and L. fructivorans, respectively. The inhibition pattern of bacterial proteinase was further confirmed through in-gel activity assays and bacterial cell mortality assays conducted by incubating live cells with ChTI. The study confirmed ChTI’s strong affinity for bacterial cell wall proteins, as demonstrated by affinity column purification, SDS-PAGE, and western blotting. Overall, the inhibitory properties of ChTI, demonstrated through bacterial bioassays, indicate its significant antibacterial activity and potential for post-harvest preservation.

Effect of Endophytic Bacteria Bacillus subtilis 26D and Bacillus velezensis M66 on Resistance of Potato Plants to the Causative Agent of Early Blight Alternaria solani

The effect of Bacillus velezensis M66 and Bacillus subtilis 26D bacteria on the resistance of potato plants to the necrotrophic fungus Alternaria solani (the causative agent of early blight) was studied. The accumulation of viable bacterial cells of these strains in the internal tissues of the potato stem, roots, and tubers over a long period of time was demonstrated for the first time. A significant reduction in the area affected by the early blight on leaves inoculated with plant endophytes, as well as inhibition of the pathogen growth under the action of bacterial strains, was detected, which can be explained by the synthesis of lipopeptide antibiotics (the genes responsible for the synthesis of which were found by the PCR method) and proteolytic enzymes (the activity of which was demonstrated in vitro). The formation of plant resistance under the influence of inoculation with B. subtilis 26D and B. velezensis M66 was accompanied by an accumulation of hydrogen peroxide in the first hours after plant infection with A. solani spores and by a decrease in this index at the late stages of pathogenesis due to an increase in the activity of catalase and peroxidases. Limitation of the fungus spread was accompanied by an increase in the activity of proteinase inhibitors in plants, which probably decreased the negative effect of proteolytic enzymes of the necrotrophic pathogen A. solani. It can be assumed that the inoculation of plants with bacterial cells of the B. velezensis M66 strain contributed to the formation of resistance of potato plants to the early blight by efficient priming of the phytoimmune potential, which is comparable to the B. subtilis 26D strain (successfully used in the field conditions), an active component of the Fitosporin-M biopreparation.

Exploring the protective role of jelly capsules in jelly‐nest tree frog eggs based on proteomic and peptidomic studies

AbstractTypically, frog eggs are aquatic, being deposited and undergoing development within freshwater habitats. However, certain species within the Rhacophoridae have evolved reproductive modes that are non‐aquatic. The jelly‐nest tree frog (Feihyla palpebralis) has terrestrial eggs that are enclosed in gel, with embryos detaching from the aquatic milieu during the embryonic phase. The jelly enveloping these embryos performs a critical role in defense against biotic and abiotic threats in the terrestrial environment. To investigate the protective function of the jelly, we characterized its proteome and peptidome. In total, 83 proteins were identified and categorized into eight main groups: protease inhibitors, glycoproteins, cytoskeleton proteins, nucleotide‐binding proteins, regulatory proteins, metabolic enzyme, glycoprotein hormones, and oxidative stress‐related proteins. These proteins are believed to play a role in protecting the eggs by creating biochemical and physical barriers, maintaining moisture and structural integrity, and preventing oxidative damage. In addition, 1642 peptides were isolated from tree frog jelly envelopes. Among these, RPPGFSPFR showed predator deterrent properties, LPF and AVPYPQ performed anti‐inflammatory and antioxidant activities, and IYSP functioned as a proteinase inhibitor. These findings help to clarify the protective mechanisms involved in the egg development process of the jelly‐nest tree frog.

A mutation in the Bombyx mori BmSPI51 gene results in alterations protein abundance in cocoon and unveils compensatory mechanism of silk gland proteinase inhibitors

Proteinase inhibitors constitute a significant component of Lepidoptera cocoon. BmSPI51 is the most abundant protease inhibitors in silkworm cocoons. In this study, we aimed to elucidate the biological function of BmSPI51 in both the silkworm silk gland and cocoon formation. To achieve this, we utilized CRISPR/Cas9 gene editing to target the BmSPI51 gene, resulting in premature termination of translation at the 33rd amino acid residue. The mutation of BmSPI51 did not affect the growth and development. Nevertheless, it led to a substantial decrease in the economic traits of silkworm cocoons. Proteomic analysis indicated the abundance of fibroin heavy chain (Fib-H), fibroin light chain (Fib-L), and fibrohexamerin (P25) decreased significantly in the homozygous mutants. Further analysis of cocoon proteins found that the mutants significantly increased the secretion of other protease inhibitors in order to deal with the increased environmental stress resulting from the absence of BmSPI51. Surprisingly, homozygous mutant cocoons exhibited an enhanced inhibitory ability against Saccharomyces cerevisiae compared to the WT cocoons. In conclusion, our study provides a valuable insight into the biological function of protease inhibitors and revealed their roles in cocoon formation and potential applications in biotechnology.

Suppressing Expression of SERPINE1/PAI1 Through Activation of GPER1 Reduces Progression of Vulvar Carcinoma.

The serine proteinase inhibitor 1 (SERPINE1) gene codes for the plasminogen activator inhibitor 1 (PAI1) protein and is thought to play a tumor supportive role in various cancers. In this work we aimed to uncover the role PAI1 plays in the proliferation, migration, and invasion of vulvar cancer (VC), and define the protein's function as an oncogene or tumor suppressor. Through treatment with an agonist (G1) and antagonist (G36) of G-coupled estrogen receptor 1 (GPER1), an upstream regulator of SERPINE1 expression, and a forward transfection knockdown protocol, the expression of SERPINE1/PAI1 in VC cells was altered. The effects these altered SERPINE1/PAI1 levels had on tumor cell functions were then examined. Proliferation was analyzed using the resazurin assay, while migration was studied via the gap closure assay. Through colony- and tumor sphere- formation assays clonogenicity was tested, and western blots showed protein expression. In A431 VC cells, when the levels of PAI1 were reduced via knockdown or treatment with G1, migration, proliferation, and colony growth was reduced. Treatment with G36 increased expression of PAI1 and increased migration and colony size in CAL39 cells. Based on the findings in this study, suppressing PAI1 expression in VC cells appears to reduce their progression and tumorigenic potential. Therefore, PAI1 could possibly function as an oncogene in VC. GPER1 appears to be a suitable target for suppressing PAI1 in VC.

Profiling of Key Hub Genes Using a Two-State Weighted Gene Co-Expression Network of 'Jao Khao' Rice under Soil Salinity Stress Based on Time-Series Transcriptome Data.

RNA-sequencing enables the comprehensive detection of gene expression levels at specific time points and facilitates the identification of stress-related genes through co-expression network analysis. Understanding the molecular mechanisms and identifying key genes associated with salt tolerance is crucial for developing rice varieties that can thrive in saline environments, particularly in regions affected by soil salinization. In this study, we conducted an RNA-sequencing-based time-course transcriptome analysis of 'Jao Khao', a salt-tolerant Thai rice variety, grown under normal or saline (160 mM NaCl) soil conditions. Leaf samples were collected at 0, 3, 6, 12, 24, and 48 h. In total, 36 RNA libraries were sequenced. 'Jao Khao' was found to be highly salt-tolerant, as indicated by the non-significant differences in relative water content, cell membrane stability, leaf greenness, and chlorophyll fluorescence over a 9-day period under saline conditions. Plant growth was slightly retarded during days 3-6 but recovered by day 9. Based on time-series transcriptome data, we conducted differential gene expression and weighted gene co-expression network analyses. Through centrality change from normal to salinity network, 111 key hub genes were identified among 1,950 highly variable genes. Enriched genes were involved in ATP-driven transport, light reactions and response to light, ATP synthesis and carbon fixation, disease resistance and proteinase inhibitor activity. These genes were upregulated early during salt stress and RT-qPCR showed that 'Jao Khao' exhibited an early upregulation trend of two important genes in energy metabolism: RuBisCo (LOC_Os10g21268) and ATP synthase (LOC_Os10g21264). Our findings highlight the importance of managing energy requirements in the initial phase of the plant salt-stress response. Therefore, manipulation of the energy metabolism should be the focus in plant resistance breeding and the genes identified in this work can serve as potentially effective candidates.

Food as a factor determining the physiological state of populations of the phytopagous pests of agricultural crops

Relevance. Trophic relationships along with competition and mutualism are the most basic and significant interactions in ecosystems. To develop, survive, and multiply, insects need to consume a certain amount of nutrients at a certain ratio. Food products are complex mixes of nutrients and non-nutritive substances (sometimes toxic ones): macronutrients (proteins, carbohydrates, and lipids), micronutrients (vitamins and minerals), and water. Some nutrients are essential; insects lack the ability to synthesize them in their bodies and must obtain them from their diet or through symbiosis with beneficial organisms. Identification mechanisms being well developed in the system “phytophagous insect – plant” allow insects to successfully spread, multiply, and feed on certain plant species. The complex of hydrolytic enzymes in the insect intestine is one of the main targets for plant defense responses because these enzymes determine the availability of structural compounds for phytophagous insects. For this reason, enzymes in the insect intestine play a key role in the adaptation of insects to the pest resistance of fodder plants. For instance, when proteinase inhibitors are synthesized in a fodder plant as the result of induced insect resistance the complex of enzymes in an insect intestine might change negating the effect of these inhibitors. The development of co-adaptations due to interactions among species in food chains depends on a complicated constellation of conflicting relationships between consumers and food objects. The mechanisms of this influence may be rooted in the allelochemical interactions in the system “phytophagous insect – plant recipient”. Allelopathic interactions are among the most complex interactions because they are constituted by direct and indirect effects. Plants when damaged by phytophagous insects activate defense responses, which incorporate several mechanisms, including an increase in the concentration of secondary metabolites, many of which are phenolic compounds.The aim of the work is to describe the mechanisms of relationships in the system “phytophage-plant”. Conclusion. Management of processes of intra-water divergence of insect-phytophages in agrobiocoenoses in order to prevent the emergence of races and populations of pests adapted to live on initially resistant to them plant forms is possible in compliance with the transition to a targeted selection of agricultural crops for resistance to a complex of pests.

Engineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

Antithrombin regulates neutrophil activities through the stimulation of C-type lectin family 1A

AbstractIt has been suggested that a serine proteinase inhibitor, antithrombin (AT), exerts anti-inflammatory effects on different types of cells, independent of thrombin inhibition. In this study, we aimed to identify a specific receptor for AT by a screening method using a transmembrane–tethered AT ligand expressed on HEK293T cells together with the coexpression of candidate receptors, followed by the immunoprecipitation of a complex of AT ligand with a receptor. We identified C-type lectin family 1A (CLEC1A) as a receptor for AT. We confirmed the binding of AT to the extracellular domain of CLEC1A using surface plasmon resonance. Recombinant as well as native AT concentration-dependently induced the rounding of purified human neutrophils in shape, associated with the suppression of spontaneous reactive oxygen species production in vitro, but argatroban did not, indicating the independence of AT effects on thrombin inhibition. Native AT maintained the passage of neutrophils through the artificial microcapillaries. Both AT enhanced the phagocytosis of pHrodo-labeled Escherichia coli and prolonged the viability of the neutrophils. The cellular effects of AT were similar to those of histidine-rich glycoprotein, which has the same CLEC1A as a receptor, and were partially inhibited by the addition of anti-CLEC1A antibody to the incubation media. These results suggested that CLEC1A is a novel receptor for AT, and the stimulation of CLEC1A by AT at least in part mediates the important functional changes of human neutrophils.

Lung antimicrobial proteins and peptides: from host defense to therapeutic strategies.

Representing severe morbidity and mortality globally, respiratory infections associated with chronic respiratory diseases, including complicated pneumonia, asthma, interstitial lung disease, and chronic obstructive pulmonary disease, are a major public health concern. Lung health and the prevention of pulmonary disease rely on the mechanisms of airway surface fluid secretion, mucociliary clearance, and adequate immune response to eradicate inhaled pathogens and particulate matter from the environment. The antimicrobial proteins and peptides contribute to maintaining an antimicrobial milieu in human lungs to eliminate pathogens and prevent them from causing pulmonary diseases. The predominant antimicrobial molecules of the lung environment include human α- and β-defensins and cathelicidins, among numerous other host defense molecules with antimicrobial and antibiofilm activity such as PLUNC (palate, lung, and nasal epithelium clone) family proteins, elafin, collectins, lactoferrin, lysozymes, mucins, secretory leukocyte proteinase inhibitor, surfactant proteins SP-A and SP-D, and RNases. It has been demonstrated that changes in antimicrobial molecule expression levels are associated with regulating inflammation, potentiating exacerbations, pathological changes, and modifications in chronic lung disease severity. Antimicrobial molecules also display roles in both anticancer and tumorigenic effects. Lung antimicrobial proteins and peptides are promising alternative therapeutics for treating and preventing multidrug-resistant bacterial infections and anticancer therapies.

Regulation of corticosteroid-binding globulin release in murine leydig tumor cell line mLTC-1 by luteinizing hormone and interleukin-6

Exogenous assaults interfere with homeostatic processes in the body by inducing stress responses. Corticosteroid-binding globulin (CBG) binds to stress hormone glucocorticoids to transport and dynamically control their availability to target tissues. In our previous study, we confirmed that CBG is locally produced by Leydig cells in the testes. Here, we explored the potential regulators of CBG using a murine Leydig tumor cell line (mLTC-1). Results indicated that luteinizing hormone (LH) and interleukin-6 (IL-6) were important factors stimulating the release of CBG from mLTC-1 cells. In addition, IL-6 stimulated mLTC-1 cells to release alpha-1 antitrypsin (AAT), a serine proteinase inhibitor (serpin) that affects CBG conformation. The results implied that any challenge that altered LH or IL-6 levels also changed the release and binding status of CBG with steroid hormones in the testicular microenvironment and modulated cellular responses to these stress hormones. In addition, secretory proteomic analysis indicated that the extracellular matrix (ECM), cytoskeleton, and proteasomes were essentially produced by the mLTC-1 cells, and LH evoked the secretion of proteins involved in binding and metabolism. These results emphasize that Leydig cells may undertake more functions than just steroidogenesis, and the regulation of Leydig cells by LH is versatile.

Combinatorial protection of cochlear hair cells: not too little but not too much.

A number of drugs are toxic to the cochlear sensory cells known as hair cells (HCs), resulting in hearing loss. Treatment with survival-promoting growth factors, antioxidants, and inhibitors of cell death pathways or proteinases have been shown to reduce HC damage in in vivo and/or in vitro animal models. Conversely, translation to humans has often been disappointing. This may be due to the complexity of intracellular damage processes. We hypothesized that combining treatments targeting different cellular processes would be more effective. Using an in vitro model of gentamicin ototoxicity for murine cochlear hair cells, we screened all 56 possible combinations of inhibitors targeting five different cell damage mechanisms, plus the activator of one cell survival pathway, each of which have been shown to be singly effective in preventing HC loss in experimental studies. A high dose of gentamicin (200 μM) was used over three days in culture. All compounds were added at a dosage below that required for significant protection in the assay, and only this single dose was then employed. This was done so that we could more easily detect interactive, as opposed to additive, effects. Increasing protection of hair cells was observed as combinations of compounds were increased from two to four factors, although not all combinations were equally protective. The optimal combination of four compounds consisted of an anti-oxidant, an apoptosis inhibitor, an autophagy inhibitor and a protective growth factor. Increasing the number of factors to five or six resulted in decreased protection. The results support the hypothesis that targeting multiple cellular damage or survival pathways provides more an effective hair cell protection approach. The results help to identify critical interactions among the cellular processes that operate in gentamicin ototoxicity. They also suggest that inhibiting too many biological processes impairs functions critical to HC survival, resulting in decreased protection.

LOCAL IMMUNE STATUS OF PATIENTS AFTER HYSTEROSCOPIC MYOMECTOMY AND METHODS OF ITS CORRECTION

Aim. To evaluate changes in the local immune status of women after hysteroscopic myomectomy and the effect of the proposed treatment complex on them. Materials and methods. 221 patients with submucous uterine myoma who underwent hysteroscopic myomectomy were examined. 121 patients of the main group in the semi-operative period received complex rehabilitation therapy, 100 patients of the comparison group did not receive additional treatment after surgery. 80 women who applied for a routine preventive examination formed the control group. In all patients, the content of factors of local immunity – IL-6, IL-10, lactoferrin and secretory inhibitor of leukocyte proteinase in cervical mucus was studied by enzyme-linked immunosorbent assay (ELISA) using Atiscare reagents in the laboratory conditions of the Center. The indicators were studied before surgery, 3 and 6 months after surgery. Results. In patients with submucosal uterine myoma, an increased content of pro-inflammatory IL-6 in cervical mucus was found (357.9 pg/ml and 345.3 pg/ml), in the control group 134.7 pg/ml and a decrease in the concentration of anti-inflammatory IL-10 (37 .6 pg/ml and 34.3 pg/ml) while the value of the control group was 78.5 pg/ml. The increase in the cervical content of the secretory inhibitor of leukocyte proteinase in women with submucous uterine myoma (274.2 pg/ml and 284.9 pg/ml against 114.5 pg/ml in the control group) is evidence of an active reaction of the mucous membrane to the inflammatory process. The role of the factor in the implementation of antisperm immunity is a factor of impaired fertility in such women. Elevated lactoferrin (27.4 ng/ml and 28.4 ng/ml vs. 3.5 ng/ml in healthy women) is also associated with infertility. Performing hysteroscopic myomectomy leads to a gradual, but incomplete normalization of the content of the specified immune factors in the cervical mucus, and the improvement of the treatment complex leads to the full achievement of the indicator of the control group after 6 months, which is important for the implementation of reproductive function. Conclusions. 1. Patients with submucous uterine myoma are characterized by an increased content of pro-inflammatory factors of local immunity – IL-6, lactoferrin and secretory inhibitor of leukocyte proteinase, as well as a reduced concentration of anti-inflammatory IL-10. 2. After hysteroscopic myomectomy for 6 months, there is a decrease in the content of pro-inflammatory factors and an increase in the concentration of IL-10 in the cervical mucus, but the indicators do not reach the values of healthy women. 3. The use of the proposed complex of treatment after hysteroscopic myomectomy allows to achieve faster changes in the indicators of local cervical immunity, and after 6 months to achieve the values obtained in healthy women.

Alpha-2-Macroglobulin Attenuates Posttraumatic Osteoarthritis Cartilage Damage by Inhibiting Inflammatory Pathways With Modified Intra-articular Drilling in a Yucatan Minipig Model

Background: Posttraumatic osteoarthritis (PTOA) arises secondarily to joint trauma and is driven by catabolic inflammatory pathways. Alpha-2-macroglobulin (α2M) is a naturally occurring proteinase inhibitor found in human serum and synovial fluid that binds proteases as well as proinflammatory cytokines involved in the pathogenesis of PTOA. Purpose: (1) To investigate the therapeutic potential of intra-articular α2M injections during the acute stages of PTOA by inhibiting inflammatory pathways driven by the cytokines expressed by the synovium in a large preclinical Yucatan minipig model and (2) to determine if 3 intra-articular α2M injections have greater chondroprotective effects compared with 1 intra-articular injection. Study Design: Controlled laboratory study. Methods: A total of 48 Yucatan minipigs were randomized into 4 groups (n = 12 each): (1) modified intra-articular drilling (mIAD) and saline (mIAD + saline), (2) mIAD and 1 intra-articular α2M injection (mIAD +α2M-1), (3) mIAD and 3 α2M injections (mIAD +α2M-3), and (4) sham control. Surgical hindlimbs were harvested at 15 weeks after surgery. Cartilage degeneration, synovial changes, inflammatory gene expression, and matrix metalloproteinase levels were evaluated. Gait asymmetry was measured before and after surgery using a pressure-sensing walkway system. Results: Macroscopic lesion areas and microscopic cartilage degeneration scores were lower in the mIAD +α2M-1 and mIAD +α2M-3 groups compared with the mIAD + saline group (P < .05) and similar to those in the sham group (P > .05). Synovial membrane scores of the mIAD +α2M-1 and mIAD +α2M-3 groups were lower than that of the mIAD + saline group (P < .05) and higher than that of the sham group (P < .05). Interleukin-1 beta, nuclear factor kappa B, and tumor necrosis factor alpha mRNA expression in the synovium and matrix metalloproteinase-1 levels in synovial fluid were significantly lower in the mIAD +α2M-1 and mIAD +α2M-3 groups compared with the mIAD + saline group (P < .05). No significant differences were observed between the mIAD +α2M-1 and mIAD +α2M-3 groups for all measured outcomes. There were early changes in gait (P < .05) between preoperative and postoperative time points for the mIAD + saline, mIAD +α2M-1, and mIAD +α2M-3 groups that normalized by 15 weeks. Conclusion: Animals receiving early α2M treatment exhibited less cartilage damage, milder synovitis, and lower inflammation compared with animals with no α2M treatment. These results exemplify the early anti-inflammatory effects of α2M and provide evidence that intra-articular α2M injections may slow the progression of PTOA. Clinical Relevance: In patients presenting with an acute joint injury, an early intervention with α2M may have the potential to reduce cartilage degeneration from catabolic pathways and delay the development of PTOA.

Nucleoside Reverse Transcriptase Inhibitors Are the Major Class of HIV Antiretroviral Therapeutics That Induce Neuropathic Pain in Mice.

The development of combination antiretroviral therapy (cART) has transformed human immunodeficiency virus (HIV) infection from a lethal diagnosis into a chronic disease, and people living with HIV on cART can experience an almost normal life expectancy. However, these individuals often develop various complications that lead to a decreased quality of life, some of the most significant of which are neuropathic pain and the development of painful peripheral sensory neuropathy (PSN). Critically, although cART is thought to induce pain pathogenesis, the relative contribution of different classes of antiretrovirals has not been systematically investigated. In this study, we measured the development of pathological pain and peripheral neuropathy in mice orally treated with distinct antiretrovirals at their translational dosages. Our results show that only nucleoside reverse transcriptase inhibitors (NRTIs), not other types of antiretrovirals such as proteinase inhibitors, non-nucleoside reverse transcriptase inhibitors, integrase strand transfer inhibitors, and CCR5 antagonists, induce pathological pain and PSN. Thus, these findings suggest that NRTIs are the major class of antiretrovirals in cART that promote the development of neuropathic pain. As NRTIs form the essential backbone of multiple different current cART regimens, it is of paramount clinical importance to better understand the underlying mechanism to facilitate the design of less toxic forms of these drugs and/or potential mitigation strategies.

The frequency and chemical phenotype of neighboring plants determine the effects of intraspecific plant diversity.

Associational effects, whereby plants influence the biotic interactions of their neighbors, are an important component of plant-insect interactions. Plant chemistry has been hypothesized to mediate these interactions. The role of chemistry in associational effects, however, has been unclear in part because the diversity of plant chemistry makes it difficult to tease apart the importance and roles of particular classes of compounds. We examined the chemical ecology of associational effects using backcross-bred plants of the Solanum pennellii introgression lines. We used eight genotypes from the introgression line system to establish 14 unique neighborhood treatments that maximized differences in acyl sugars, proteinase inhibitor, and terpene chemical diversity. We found that the chemical traits of the neighboring plant, rather than simply the number of introgression lines within a neighborhood, influenced insect abundance on focal plants. Furthermore, within-chemical class diversity had contrasting effects on herbivore and predator abundances, and depended on the frequency of neighboring plant chemotypes. Notably, we found insect mobility-flying versus crawling-played a key role in insect response to phytochemistry. We highlight that the frequency and chemical phenotype of plant neighbors underlie associational effects and suggest this may be an important mechanism in maintaining intraspecific phytochemical variation within plant populations.

SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis

Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.

Peptide-stimulated T cells bypass immune checkpoint inhibitor resistance and eliminate autologous microsatellite instable colorectal cancer cells

Two hypermutated colon cancer cases with patient-derived cell lines, peripheral and tumor-infiltrating T cells available were selected for detailed investigation of immunological response.T cells co-cultured with autologous tumor cells showed only low levels of pro-inflammatory cytokines and failed at tumor recognition. Similarly, treatment of co-cultures with immune checkpoint inhibitors (ICI) did not boost antitumor immune responses. Since proteinase inhibitor 9 (PI-9) was detected in tumor cells, a specific inhibitor (PI-9i) was used in addition to ICI in T cell cytotoxicity testing. However, only pre-stimulation with tumor-specific peptides (cryptic and neoantigenic) significantly increased recognition and elimination of tumor cells by T cells independently of ICI or PI-9i.We showed, that ICI resistant tumor cells can be targeted by tumor-primed T cells and also demonstrated the superiority of tumor-naïve peripheral blood T cells compared to highly exhausted tumor-infiltrating T cells. Future precision immunotherapeutic approaches should include multimodal strategies to successfully induce durable anti-tumor immune responses.

Ovomucoid gene polymorphism and its influence on quality changes at various storage timepoint of eggs from two strains of Japanese quail

During storage, irreversible changes occur in eggs, resulting in a decline in their quality, predominantly affecting the albumen. Ovomucoid, a major protein found in egg white, belongs to the Kazal-type serine proteinase inhibitors and serves to protect the embryo from microorganisms. Notably, in chicken eggs, it is a significant allergen. There is a possibility that its polymorphism also influences the quality and stability of table eggs. Hence, this study aimed to evaluate the potential effect of polymorphism in the ovomucoid gene and protein on quality changes during the storage of eggs derived from 2 strains of Japanese quail, encompassing various utility types. Eggs from selected females of laying and meat-type breeds were stored for 14 wk, with egg quality traits assessed 10 times during this duration. DNA was isolated from each female, and sequencing was conducted on all exons of the ovomucoid gene. In total, 5 SNPs were identified in exons and adjacent intronic sequences, with SNP1 (13:12355585), SNP4 (13:12356594), and SNP5 (13:12358538) leading to amino acid substitutions in the ovomucoid protein. Notably, all SNPs except SNP5 were identified in the ovomucoid gene of Japanese quail for the first time. The results demonstrated that in the F33 strain, SNP1, SNP3, and SNP4 exhibited significant associations with egg weight, whereas in the S22 strain, SNP5 significantly affected yolk color and various eggshell quality traits, including eggshell weight, eggshell thickness, and breaking strength, throughout the storage period. Furthermore, a haplotype block containing 2 SNPs (3 and 4) was identified, exhibiting 2 distinct haplotypes that significantly affected egg weight, eggshell weight, and breaking strength at various storage time points during egg quality analyses. These findings provide novel insights into the genetic basis of egg quality during storage and have the potential to be integrated into breeding programs for these strains.

Drought stress enhances plastid-mediated RNA interference for efficient the willow leaf beetle management

Plastid-mediated RNA interference has emerged as a promising and effective approach for pest management. By expressing high levels of double-stranded RNAs (dsRNAs) in plastid that target essential pest genes, it has been demonstrated to effectively control certain herbivorous beetles and spider mites. However, as plants are sessile organisms, they frequently experience a combination of biotic and abiotic stresses. It remains unclear whether abiotic stress, such as drought stress, influences the accumulation of dsRNAs produced in plastids and its effectiveness in controlling pests. In this study, we aimed to investigate the effects of drought stress on dsACT expression in transplastomic poplar plants and its control efficiency against the willow leaf beetle (Plagiodera versicolora). Our findings revealed that drought stress did not significantly affect the dsRNA contents in transplastomic poplar plants, but it did lead to higher mortality of insect larvae. This increased mortality may be attributed to increased levels of jasmonic acid and cysteine proteinase inhibitor induced by water deficit. These results contribute to understanding of the mechanisms linking water deficit in plants to insect performance and provide valuable insights for implementing appropriate pest control strategies under drought stress conditions.