Peptide Expression Research Articles

Protein Extract of a Probiotic Strain of Hafnia alvei and Bacterial ClpB Protein Improve Glucose Tolerance in Mice

A commercial strain of Hafnia alvei (H. alvei) 4597 bacteria was shown to reduce food intake and promote weight loss, effects possibly induced by the bacterial protein ClpB, an antigen-mimetic of the anorexigenic α-melanocyte-stimulating hormone. A decrease in the basal plasma glucose levels was also observed in overweight fasted humans and mice receiving H. alvei. However, it is not known whether H. alvei influences sweet taste preference and whether its protein extract or ClpB are sufficient to increase glucose tolerance; these are the objectives tested in the present study. C57BL/6J male mice were kept under standard diet and were gavaged daily for 17 days with a suspension of H. alvei (4.5 × 107 CFU/animal) or with H. alvei total protein extract (5 μg/animal) or saline as a control. Sweet taste preference was analyzed via a brief-access licking test with sucrose solution. Glucose tolerance tests (GTT) were performed after the intraperitoneal (IP) or intragastric (IG) glucose administration at the 9th and 15th days of gavage, respectively. The expression of regulatory peptides’ mRNA levels was assayed in the hypothalamus. In another experiment performed in non-treated C57BL/6J male mice, effects of acute IP administration of recombinant ClpB protein on glucose tolerance were studied by both IP- and IG-GTT. Mice treated with the H. alvei protein extract showed an improved glucose tolerance in IP-GTT but not in IG-GTT. Both groups treated with H. alvei bacteria or protein extract showed a reduction of pancreatic tissue weight but without significant changes to basal plasma insulin. No significant effects of H. alvei bacteria or its total protein extract administration were observed on the sweet taste preference, insulin tolerance and expression of regulatory peptides’ mRNA in the hypothalamus. Acute administration of ClpB in non-treated mice increased glucose tolerance during the IP-GTT but not the IG-GTT, and reduced basal plasma glucose levels. We conclude that both the H. alvei protein extract introduced orally and the ClpB protein administered via IP improve glucose tolerance probably by acting at the glucose postabsorptive level. Moreover, H. alvei probiotic does not seem to influence the sweet taste preference. These results justify future testing of both the H. alvei protein extract and ClpB protein in animal models of diabetes.

Understanding Autologous Spliceostatin Transcriptional Regulation to Derive Parts for Heterologous Expression in a Burkholderia Bacterial Host.

Burkholderia β-Proteobacteria are emerging sources of natural products. We are interested in developing Burkholderia sp. FERM BP-3421 into a synthetic biology chassis to facilitate natural product discovery. FERM BP-3421 produces autologous spliceostatins on gram per liter scale. We reasoned that transcription factors and promoters that regulate spliceostatin biosynthesis would provide valuable parts for heterologous expression. Herein we demonstrate that fr9A encodes a pathway-specific transcriptional activator of spliceostatin biosynthesis. In-frame deletion of fr9A abolished spliceostatin production, which was restored by genetic complementation. Using transcriptomics and green fluorescent protein (GFP) reporter assays, we identified four fr9 promoters, three of which are activated by LuxR-type regulator Fr9A. We then constructed an Fr9A-regulated promoter system that was compared to benchmarks and effectively applied for GFP and capistruin lasso peptide expression in an optimized host background. Our findings enrich the genetic toolbox for optimizing heterologous expression and promoting the discovery and development of natural products from Burkholderia bacteria.

Benzenesulfonamide as a novel, pharmaceutical small molecule inhibitor on Aβ gene expression and oxidative stress in Alzheimer's Wistar rats

Alzheimer's disease (AD) is the most prevalent acute neurodegenerative disease described by memory loss and other cognitive functions. Benzenesulfonamide, a novel, potent, and small organic molecule, was synthesized to investigate its effects on the levels of oxidative biomarkers (GPx, ROS, and MDA) and expression of beta-amyloid peptides (Aβ40 and Aβ42) in the pathology of AD. The results were compared with the rivastigmine drug. Applying benzenesulfonamide to Alzheimer's-induced Wistar rats showed a significant increase in the level of oxidative biomarkers (GPx, ROS, and MDA) in both the brain and blood serum as well as amyloid-β40 and 42 gene expressions. Therefore, benzenesulfonamide could be considered a novel therapeutic agent against AD.

Activity of FAAH-Inhibitor JZP327A in an Experimental Rat Model of Migraine.

Increased anandamide levels via fatty acid amide hydrolase (FAAH) inhibition can decrease the pronociceptive responses and inflammatory mediators in animal models of migraine. Here, we profile the pharmacological activity of the FAAH inhibitor JZP327A, a chiral 1,3,4-oxadiazol-2(3H)-one compound, in the mediation of spontaneous and nocifensive behaviour in the animal models of migraine based on nitroglycerin (NTG) administration. JZP327A (0.5 mg/kg, i.p.) or vehicle was administered to male rats 3 h after NTG (10 mg/kg, i.p.) or NTG vehicle injection. The rats were then exposed to the open field test and an orofacial formalin test 1 h later. The levels of endocannabinoids and lipid-related substances, and the expression of pain and inflammatory mediators were evaluated in cranial tissues and serum. The findings show that JZP327A did not affect NTG-induced changes in the spontaneous behaviour of rats, while it inhibited NTG-induced hyperalgesia at the orofacial formalin test. Furthermore, JZP327A dramatically decreased the gene expression of calcitonin gene-related peptide (CGRP), tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in the trigeminal ganglia and medulla-pons, while it did not change endocannabinoids or lipids levels nor CGRP serum levels in the same tissues. These data suggest an anti-hyperalgesic role for JZP327A in the NTG model, which is mediated by the inhibition of the inflammatory cascade of events. This activity does not seem mediated by a change in the levels of endocannabinoids and lipid amides.

A member of the immunoglobulin superfamily lrig-1 might be involved in the immune priming of Scylla paramamosain in response to the infection and re-infection by Vibrio parahaemolyticus

A member of the immunoglobulin superfamily designated leucine-rich repeats and immunoglobulin-like domains protein-1 (lrig-1) encoding a protein with 1109 amino acids with a characteristic IGc2 domain was identified from the transcriptome data of mud crab Scylla paramamosain. Lrig-1 contained: one signaling peptide; one LRR_NT domain; nine LRR domains; three LRR_TYP domains; one LRR_CT domain; three IGc2 regions; one transmembrane region; C-terminal cytoplasmic tail. lrig-1 was widely expressed in all tissues of mud crab and was responsive in hemocytes to first and second Vibrio parahaemolyticus infections. lrig-1 knockdown mediated by RNAi repressed expression of several antimicrobial peptides significantly. Its orthologs in 19 crustacean species were identified and showed high conservation. These results suggest that lrig-1 have a vital role in mud crabs against V. parahaemolyticus infection through expression of multiple antimicrobial peptides. The results obtained in the present study imply the potential roles the lrig-1 played in immune priming in crabs.

Perinatal opioid exposure leads to decreased social play in adolescent male and female rats: Potential role of oxytocin signaling in brain regions associated with social reward

Over the last two decades, the number of infants exposed to opioids in utero has quadrupled in the United States, with some states reporting rates as high as 55 infants per 1000 births. Clinical studies report that children previously exposed to opioids during gestation show significant deficits in social behavior, including an inability to form friendships or other social relationships. To date, the neural mechanisms whereby developmental opioid exposure disrupts social behavior remain unknown. Using a novel paradigm of perinatal opioid administration, we tested the hypothesis that chronic opioid exposure during critical developmental periods would disrupt juvenile play. As oxytocin is a major regulator of sociability, the impact of perinatal morphine exposure on oxytocin peptide expression was also examined. Juvenile play was assessed in vehicle- or morphine-exposed male and female rats at P25, P35, and P45. Classical features of juvenile play were measured, including time spent engaged in social play, time not in contact, number of pins, and number of nape attacks. We report that morphine-exposed males and females spend less time engaged in play behavior than control males and females, with a corresponding increase in time spent alone. Morphine-exposed males and females also initiated fewer pins and nape attacks. Together, these data suggest that male and female rats exposed to morphine during critical developmental periods are less motivated to participate in social play, potentially due to alterations in oxytocin-mediated reward signaling.

Expressing Parasitoid Venom Protein VRF1 in an Entomopathogen Beauveria bassiana Enhances Virulence toward Cotton Bollworm Helicoverpa armigera.

Despite entomopathogenic fungi being used in various insect pest control, it is recognized that they could replace more chemical insecticides if they were more efficient. We have found that cotton bollworm Helicoverpa armigera responded to the infection of entomopathogenic fungus Beauveria bassiana by activating the Toll pathway. Koinobiont wasps also regulate host immunity and development to ensure the survival of their progeny. Previously, venom protein VRF1 was identified in Microplitis mediator. It enters H. armigera hemocytes, suppresses the expression of antimicrobial peptides (AMPs) by inhibiting the Toll pathway, and prevents parasite offspring from being encapsulated. With this in mind, we thought that it might be feasible to increase the virulence of B. bassiana by embedding VRF1 into its genome. Compared with that of wild-type (WT) B. bassiana, the median lethal dose (LD50) of the transformant expressing VRF1 (named BbVRF1) decreased approximately 2.36-fold, and the median time to lethality (LT50) was shortened to 84% when infecting H. armigera (a natural host of M. mediator). The AMP expression level of hemocytes in H. armigera infected with BbVRF1 strain was significantly downregulated compared to that in the control group infected with the WT. In addition, the LD50 of BbVRF1 against the fall armyworm Spodoptera frugiperda (an unnatural host of M. mediator) was decreased 3.45-fold and the LT50 was shortened to 73%, showing a greater virulence. Our research indicated that BbVRF1, an engineered strain of B. bassiana, has greater efficacy against pest insects both within and outside its host range (M. mediator), expanding the utilization of parasitoid wasp virulence effectors. IMPORTANCE Mycoinsecticides are essential for the development of integrated pest management as substitutes to chemical insecticides, but their usage is limited by their inferior virulence. Thus, genetically engineered bioinsecticides, including recombinant entomopathogenic fungi, have been regarded as a breakthrough to rapidly control pests. Deep knowledge of parasitoid wasps allows us to take advantage of this natural enemy of pest insects beyond raising them for field release. Our transformant BbVRF1 (Beauveria bassiana integrated with a venom protein VRF1 from Microplitis mediator) showed a higher virulence in H. armigera and S. frugiperda, demonstrating its potential for managing natural or unnatural hosts of M. mediator. This result provides a new strategy regarding which venom protein of parasitoid wasps can become part of the arsenal with which to equip entomopathogenic fungi. Utilizing parasitoid wasps with this approach could easily overcome the difficulties of artificial culture and enhance the virulence of other biocontrol agents.

Investigating the Correlation Between Clostridioides difficile Infection and Vitamin D Deficiency.

Clostridioides difficile infection (CDI) is the most common healthcare-associated infection in the US. Symptoms include watery diarrhea, nausea, and anorexia and it can present with leukocytosis on laboratory evaluation. Treatment is based on disease severity and recurrence. Despite antibioticusage being the highest risk factor for infection, they are also the first-line treatment for initial CDI. Prevention of CDI mostly involves good hand hygiene, antibiotic stewardship, and appropriate precautions when interacting with infected individuals. Vitamin D deficiency (VDD) has been linked to CDI, however, there is limited insight into the correlation between both states. Our aim was to further investigate the potential link between VDD and CDI. Data were obtained from the National Inpatient Sample (NIS) from 2016 to 2019. Patients with CDI were identified and stratified based on a diagnosis of VDD. Primary outcomes were mortality, CDI recurrence, ileus, toxic megacolon, perforation, and colectomy. Chi-squared and independent t-tests were performed to assess categorical and continuous data, respectively. Multiple logistic regression was used to control for confounders. Patients with VDD had higher rates of CDI recurrence (17.4% versus 14.7%,p<0.05), but lower rates of mortality (3.1% versus 6.1%, p<0.05). Differences in rates of ileus, toxic megacolon, perforation, and colectomy were statistically insignificant. Length of stay was higher in the VDD group (10.38 days versus 9.83 days). Total charges were lower in the VDD group ($93,935.85 versus $102,527.9). CDI patients with comorbid VDD are at higher risk for the recurrence of CDI. This is likely due to the role of vitamin D in the expression of intestinal epithelial antimicrobial peptides, macrophage activation, and maintenance of tight junctions between gut epithelial cells. Furthermore, vitamin D plays a role in maintaining a healthy gut microbiome. Alternatively, deficiency results in poor gut health and detrimental changes to the gut microbiome. In effect, VDD promotes the proliferation of C. difficile within the large colon, resulting in an increased predisposition for CDI.

Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in Chlamydomonas reinhardtii

Anti-lipopolysaccharide factor 3 (ALFPm3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALFPm3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALFPm3 in Chlamydomonas reinhardtii. In this study, signal peptides ARS1 and CAH1 were fused with ALFPm3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALFPm3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALFPm3 could be detected in algal cells and culture medium by immunoblot, meaning that ALFPm3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALFPm3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi, V. alginolyticus, V. anguillarum, and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALFPm3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALFPm3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALFPm3 peptide. Our results provided a new strategy for the secretory production of ALFPm3 with high antibacterial activity in C. reinhardtii, which could improve the application potentiality of ALFPm3 in the aquaculture industry.

A gut-isolated Enterococcus strain (HcM7) triggers the expression of antimicrobial peptides that aid resistance to nucleopolyhedrovirus infection of Hyphantria cunea larvae.

Commensal microorganisms are widely distributed in insect gut tissues and play important roles in to host nutrition, metabolism, reproductive regulation and, especially the immune functioning and tolerance to pathogens. Consequently, gut microbiota represent a promising resource for the development of microbial-based products for pest control and management. However, the interactions among host immunity, entomopathogen infections, and gut microbiota remain poorly understood for many arthropod pests. We previously isolated an Enterococcus strain (HcM7) from Hyphantria cunea larvae guts that increased the survival rates of larvae challenged with nucleopolyhedrovirus (NPV). Here, we further investigated whether this Enterococcus strain stimulates a protective immune response against NPV proliferation. Infection bioassays demonstrated that the re-introduction of the HcM7 strain to germ-free larvae pre-activated the expression of several antimicrobial peptides (particularly H. cunea gloverin 1, HcGlv1), resulting in the significant repression of virus replication in host guts and hemolymph, and consequently improved host survivorship after NPV infection. Furthermore, silencing of the HcGlv1 gene by RNA interference markedly enhanced the deleterious effects of NPV infection, revealing a role of this gut symbiont-induced gene in host defenses against pathogenic infections. These results show that some gut microorganisms can stimulate host immune systems, thereby contributing to resistance to entomopathogens. Furthermore, HcM7, as a functional symbiotic bacteria of H. cunea larvae, may be a potential target for increasing the effectiveness of biocontrol agents against this devastating pest.

Type 2 Diabetes Mellitus, Non-Alcoholic Fatty Liver Disease, and Metabolic Repercussions: The Vicious Cycle and Its Interplay with Inflammation.

The prevalence of metabolic-related disorders, such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (DM2), has been increasing. Therefore, developing improved methods for the prevention, treatment, and detection of these two conditions is also necessary. In this study, our primary focus was on examining the role of chronic inflammation as a potential link in the pathogenesis of these diseases and their interconnections. A comprehensive search of the PubMed database using keywords such as "non-alcoholic fatty liver disease", "type 2 diabetes mellitus", "chronic inflammation", "pathogenesis", and "progression" yielded 177 relevant papers for our analysis. The findings of our study revealed intricate relationships between the pathogenesis of NAFLD and DM2, emphasizing the crucial role of inflammatory processes. These connections involve various molecular functions, including altered signaling pathways, patterns of gene methylation, the expression of related peptides, and up- and downregulation of several genes. Our study is a foundational platform for future research into the intricate relationship between NAFLD and DM2, allowing for a better understanding of the underlying mechanisms and the potential for introducing new treatment standards.

TGF-β Type II Receptor Punt Suppresses Antimicrobial Peptide Expression and Influences Development in Tribolium castaneum.

The transforming growth factor-β (TGF-β) superfamily in insects regulated various physiological events, including immune response, growth and development, and metamorphosis. This complex network of signaling pathways involves conserved cell-surface receptors and signaling co-receptors that allow for precisely coordinated cellular events. However, the roles of TGF-β receptors, particularly the type II receptor Punt, in mediating the innate immunity in insects remains unclear. In this study, we used the red flour beetle, Tribolium castaneum, as a model species to investigate the role of TGF-β type II receptor Punt in mediating antimicrobial peptide (AMP) expression. Developmental and tissue-specific transcript profiles revealed Punt was constitutively expressed throughout development, with the highest transcript level in 1-day female pupae and the lowest transcript level in 18-day larvae. Tissue specific expression profiles showed the highest transcript level of Punt was observed in the Malpighian tubule and ovary in 18-day larvae and 1-day female adults, respectively, suggesting Punt might have distinct functions in larvae and adults. Further results indicated that Punt RNAi in the 18-day larvae led to increased transcript level of AMP genes through transcription factor Relish, leading to inhibition of Escherichia coli proliferation. Knockdown of Punt in larvae also led to splitting of adult elytra and abnormal compound eyes. Furthermore, knockdown of Punt during the female pupal stage resulted in increased transcript levels of AMP genes, as well as abnormal ovary, reduced fecundity, and failure of eggs to hatch. This study deepens our understanding of the biological significance of Punt in insect TGF-β signaling and lays the groundwork for further research of its role in insect immune response, development, and reproduction.

The deubiquitinase OTUD4 inhibits the expression of antimicrobial peptides in Paneth cells to support intestinal inflammation and bacterial infection.

Dysfunction of the intestinal epithelial barrier causes microbial invasion that would lead to inflammation in the gut. Antimicrobial peptides (AMPs) are essential components of the intestinal epithelial barrier, while the regulatory mechanisms of AMPs expression are not fully characterized. Here, we report that the ovarian tumor family deubiquitinase 4 (OTUD4) in Paneth cells restricts the expression of AMPs and thereby promotes experimental colitis and bacterial infection. OTUD4 is upregulated in the inflamed mucosa of ulcerative colitis patients and in the colon of mice treated with dextran sulfate sodium salt (DSS). Knockout of OTUD4 promotes the expression of AMPs in intestinal organoids after stimulation with lipopolysaccharide (LPS) or peptidoglycan (PGN) and in the intestinal epithelial cells (IECs) of mice after DSS treatment or Salmonella typhimurium (S.t.) infection. Consistently, Vil-Cre;Otud4fl/fl mice and Def-Cre;Otud4fl/fl mice exhibit hyper-resistance to DSS-induced colitis and S.t. infection compared to Otud4fl/fl mice. Mechanistically, knockout of OTUD4 results in hyper K63-linked ubiquitination of MyD88 and increases the activation of NF-κB and MAPKs to promote the expression of AMPs. These findings collectively highlight an indispensable role of OTUD4 in Paneth cells to modulate AMPs production and indicate OTUD4 as a potential target for gastrointestinal inflammation and bacterial infection.

Vitamin D modulates expression of antimicrobial peptides and proinflammatory cytokines to restrict Zika virus infection in macrophages

Although the impact of Zika virus (ZIKV) infection on human health has been well documented, we still have no vaccine or effective treatment. This fact highlights the importance of searching for alternative therapy for treating ZIKV. To search for ZIKV antivirals, we examined the effect of vitamin D in monocyte-derived macrophages (MDMs) differentiated in the presence of vitamin D (D3-MDM) and explored the molecular mechanisms by analyzing transcriptional profiles. Our data show the restriction of ZIKV infection in D3-MDMs as compared to MDMs. Transcriptional profiles show that vitamin D alters about 19% of Zika response genes (8.2% diminished and 10.8% potentiated). Among the genes with diminished expression levels, we found proinflammatory cytokines and chemokines such as IL6, TNF, IL1A, IL1B, and IL12B, CCL1, CCL4, CCL7, CXCL3, CXCL6, and CXCL8. On the other hand, genes with potentiated expression were related to degranulation such as Lysozyme, cathelicidin (CAMP), and Serglycin. Since the CAMP gene encodes the antimicrobial peptide LL-37, we treated MDMs with LL-37 and infected them with ZIKV. The results showed a decrease in the proportion of infected cells. Our data provide new insights into the role of vitamin D in restricting ZIKV infection in macrophages that are mediated by induction of cathelicidin/LL-37 expression and downregulation of proinflammatory genes. Results highlight the biological relevance of vitamin D-inducible peptides as an antiviral treatment for Zika fever.

Effects of Starvation and Refeeding on Glucose Metabolism and Immune Responses in Macrobrachium rosenbergii.

Starvation is a common challenge for aquatic animals in both natural and cultured environments. To investigate the effects of starvation and refeeding on glucose metabolism and immunity in Macrobrachium rosenbergii, prawns were starved for 14days and then refed for 7days. Results showed that both glucose and trehalose levels decreased significantly at the beginning of starvation, followed by a significant decrease in glycogen content in the hepatopancreas and muscle. Triglyceride and total protein reserves were also mobilized under starvation, with a slightly quicker response from triglycerides. The mRNA levels of glycolysis (glucokinase) and anabolism-related enzymes (glycogen branching enzyme, diacylglycerol acyltransferase, and transpeptidase) decreased during starvation, while gluconeogenic potential was induced, as indicated by up-regulated transcriptional levels of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase) and catabolism-related enzymes (glycogen debranching enzyme, adipose triglyceride lipase, and cathepsin B). Starvation also stimulated the expression of the crustacean hyperglycemic hormone and inhibited insulin-like peptide expression, indicating their potential role in glucose metabolism regulation. In addition, starvation increased the mRNA levels of superoxide dismutase and prophenoloxidase, indicating an influence on the immune system. After bacterial infection, starved prawns showed enhanced activity of non-specific immunological parameters and reduced mortality. Refeeding for 7days led to a recovery of physiological and biochemical indices and transcriptional levels of metabolism/immune-related genes. Our findings provide a better understanding of the mechanisms underlying energy utilization, metabolic adaptation, and immune response to starvation in M. rosenbergii.

Refinement of the Fusion Tag PagP for Effective Formation of Inclusion Bodies in Escherichia coli.

Methods for efficient insoluble protein production require further exploration. PagP, an Escherichia coli outer membrane protein with high β-sheet content, could function as an efficient fusion partner for inclusion body-targeted expression of recombinant peptides. The primary structure of a given polypeptide determines to a large extent its propensity to aggregate. Herein, aggregation "hot spots" (HSs) in PagP were analyzed using the web-based software AGGRESCAN, leading to identification of a C-terminal region harboring numerous HSs. Moreover, a proline-rich region was found in the β-strands. Substitution of these prolines by residues with high β-sheet propensity and hydrophobicity significantly improved its ability to form aggregates. Consequently, the absolute yields of recombinant antimicrobial peptides Magainin II, Metchnikowin, and Andropin were increased significantly when expressed in fusion with this refined version of PagP. We describe separation of recombinant target proteins expressed in inclusion bodies fused with the tag. An artificial NHT linker peptide with three motifs was implemented for separation and purification of authentic recombinant antimicrobial peptides. IMPORTANCE Fusion tag-induced formation of inclusion bodies provides a powerful means to express unstructured or toxic proteins. For a given fusion tag, how to enhance the formation of inclusion bodies remains to be explored. Our study illustrated that the aggregation HSs in a fusion tag played important roles in mediating its insoluble expression. Efficient production of inclusion bodies could also be implemented by refining its primary structure to form a more stable β-sheet with higher hydrophobicity. This study provides a promising method for improvement of the insoluble expression of recombinant proteins.

LFQ-Based Peptide and Protein Intensity Differential Expression Analysis.

Testing for significant differences in quantities at the protein level is a common goal of many LFQ-based mass spectrometry proteomics experiments. Starting from a table of protein and/or peptide quantities from a given proteomics quantification software, many tools and R packages exist to perform the final tasks of imputation, summarization, normalization, and statistical testing. To evaluate the effects of packages and settings in their substeps on the final list of significant proteins, we studied several packages on three public data sets with known expected protein fold changes. We found that the results between packages and even across different parameters of the same package can vary significantly. In addition to usability aspects and feature/compatibility lists of different packages, this paper highlights sensitivity and specificity trade-offs that come with specific packages and settings.

Localization of relaxin-like gonad-stimulating peptide expression in starfish reveals the gonoducts as a source for its role as a regulator of spawning.

Oocyte maturation and gamete release (spawning) in starfish are triggered by relaxin-like gonad-stimulating peptide (RGP), a neuropeptide that was first isolated from the radial nerve cords of these animals. Hitherto, it has generally been assumed that the radial nerve cords are the source of RGP that triggers spawning physiologically. To investigate other sources of RGP, here we report the first comprehensive anatomical analysis of its expression, using both in situ hybridization and immunohistochemistry to map RGP precursor transcripts and RGP, respectively, in the starfish Asterias rubens. Cells expressing RGP precursor transcripts were revealed in the ectoneural epithelium of the radial nerve cords and circumoral nerve ring, arm tips, tube feet, cardiac stomach, pyloric stomach, and, most notably, gonoducts. Using specific antibodies to A. rubens RGP, immunostaining was revealed in cells and/or fibers in the ectoneural region of the radial nerve cords and circumoral nerve ring, tube feet, terminal tentacle and other arm tip-associated structures, body wall, peristomial membrane, esophagus, cardiac stomach, pyloric stomach, pyloric caeca, and gonoducts. Our discovery that RGP is expressed in the gonoducts of A. rubens proximal to its gonadotropic site of action in the gonads is important because it provides a new perspective on how RGP may act as a gonadotropin in starfish. Thus, we hypothesize that it is the release of RGP from the gonoducts that triggers gamete maturation and spawning in starfish, while RGP produced in other parts of the body may regulate other physiological/behavioral processes.

A frog-derived antimicrobial peptide as a potential anti-biofilm agent in combating Staphylococcus aureus skin infection.

Staphylococcus aureus (S. aureus), one of the most prevalent bacteria found in atopic dermatitis lesions, can induce ongoing infections and inflammation by downregulating the expression of host defence peptides in the skin. In addition, the emergence of the 'superbug' Methicillin-resistant S. aureus (MRSA) has made the treatment of these infections more challenging. Antimicrobial peptides (AMPs), due to their potent antimicrobial activity, limited evidence of resistance development, and potential immunomodulatory effects, have gained increasing attention as potential therapeutic agents for atopic dermatitis. In this study, we report a novel AMP, brevinin-1E-OG9, isolated from the skin secretions of Odorrana grahami, which shows potent antibacterial activity, especially against S. aureus. Based on the characteristics of the 'Rana Box', we designed a set of brevinin-1E-OG9 analogues to explore its structure-activity relationship. Brevinin-1E-OG9c-De-NH2 exhibited the most potent antimicrobial efficacy in both in vitro and ex vivo studies and attenuated inflammatory responses induced by lipoteichoic acid and heat-killed microbes. As a result, brevinin-1E-OG9c-De-NH2 might represent a promising candidate for the treatment of S. aureus skin infections.