The prevalence of obstructive sleep apnea syndrome after COVID-19 infection.

Obstructive sleep apnea is a well-known risk factor regarding the severity of COVID-19 infection. However, to date, relatively little research performed on the prevalence of obstructive sleep apnea in COVID-19 survivors. The purpose of this study was to investigate the risk of obstructive sleep apnea after COVID-19 infection. This study was based on data collected from the US Collaborative Network in TriNetX. From January 1, 2020 to June 30, 2022, participants who underwent the SARS-CoV-2 test were included in the study. Based on their positive or negative results of the COVID-19 test results (the polymerase chain reaction [PCR] test), we divided the study population into two groups. The duration of follow-up began when the PCR test was administered and continued for 12 months. Hazard ratios (HRs) and 95% confidence intervals (CIs) for newly recorded COVID-19 positive subjects for obstructive sleep apnea were calculated using the Cox proportional hazards model and compared to those without COVID-19 infection. Subgroup analyses were performed for the age, sex, and race, groups. The COVID-19 group was associated with an increased risk of obstructive sleep apnea, at both 3 months of follow-up (HR: 1.51, 95% CI: 1.48-1.54), and 1 year of follow-up (HR: 1.57, 95% CI: 1.55-1.60). Kaplan-Meier curves regarding the risk of obstructive sleep apnea revealed a significant difference of probability between the two cohorts in the follow-up periods of 3 months and 1 year (Log-Rank test, p < 0.001). The risks of obstructive sleep apnea among COVID-19 patients were significant in the less than 65 year of age group (HR: 1.50, 95% CI: 1.47-1.52), as well as in the group older than or equal to 65 years (HR:1.69, 95% CI: 1.64-1.73). Furthermore, the risks of obstructive sleep apnea were evident in both the male and female COVID-19 groups. Compared to the control group, the risks of obstructive sleep apnea in the COVID-19 participants increased in the subgroups of White (HR: 1.62, 95% CI: 1.59-1.64), Blacks/African Americans (HR: 1.50, 95% CI: 1.45-1.55), Asian (HR: 1.46, 95% CI: 1.32-1.62) and American Indian/Alaska Native (HR: 1.36, 95% CI: 1.07-1.74). In conclusion, the incidence of new diagnosis obstructive sleep apnea could be substantially higher after COVID-19 infection than non-COVID-19 comparison group. Physicians should evaluate obstructive sleep apnea in patients after COVID-19 infection to help prevent future long-term adverse effects from occurring in the future, including cardiovascular and neurovascular disease.

Lipofundin mediates major inhibition of intravenous propofol on phorbol myristate acetate and Escherichia coli-induced neutrophil extracellular traps.

BACKGROUND: Neutrophil extracellular traps (NETs) consist of chromatin DNA networks that are studded with cytosolic and granular antimicrobial proteins to trap or kill an infected microorganism. A lipid emulsion, the solvent of pure propofol for intravenous application, is given to clinical patients who require intravenous feeding of fatty acids and fat for energy. Intravenous propofol is widely used to sedate critically ill patients. Both intravenous propofol and its lipid emulsion have immunomodulatory activity. However, the role of lipid emulsion of intravenous propofol on NET induction remains unclear. METHODS: In this study, neutrophils were stimulated with phorbol myristate acetate (PMA) or Escherichia coli (E. coli) in the absence or presence of intravenous propofol (Propofol-Lipuro®), its solvent lipid emulsion (Lipofundin) or pure propofol, and NETs were stained with SYTOX Green for visualization and quantification. Total HOCl was determined by measuring the taurine-chloramine complex, and intracellular HOCl was evaluated with BioTracker™ TP-HOCl 1 dye. RESULTS: PMA-induced NETs were not efficiently inhibited when Propofol-Lipuro® was added after PMA stimulation. Clinically relevant concentrations of Lipofundin exerted a significant reduction in PMA-induced NETs and total reactive oxidative species (ROS), which was comparable to that observed for Propofol-Lipuro®. Lipofundin transiently reduced intracellular HOCl production and the phosphorylation level of extracellular regulated kinase (p-ERK) but did not scavenge HOCl. Moreover, Lipofundin decreased E. coli-induced NETs in a ROS-independent pathway, similar to Propofol-Lipuro®. CONCLUSIONS: All data agree that Lipofundin, the major component of Propofol-Lipuro®, inhibits intracellular HOCl and p-ERK to suppress PMA-induced NET formation but reduces E.coli-induced NETs in a ROS-independent pathway.

Development of Bactericidal Peptides against Multidrug-Resistant Acinetobacter baumannii with Enhanced Stability and Low Toxicity.

Pathogenic superbugs are the root cause of untreatable complex infections with limited or no treatment options. These infections are becoming more common as clinical antibiotics have lost their effectiveness over time. Therefore, the development of novel antibacterial agents is urgently needed to counter these microbes. Antimicrobial peptides (AMPs) are a viable treatment option due to their bactericidal potency against multiple microbial classes. AMPs are naturally selected physiological microbicidal agents that are found in all forms of organisms. In the present study, we developed two tilapia piscidin 2 (TP2)-based AMPs for antimicrobial application. Unlike the parent peptide, the redesigned peptides showed significant antimicrobial activity against multidrug-resistant bacterial species. These peptides also showed minimal cytotoxicity. In addition, they were significantly active in the presence of physiological salts, 50% human serum and elevated temperature. The designed peptides also showed synergistic activity when combined with clinical antibiotics. The current approach demonstrates a fruitful strategy for developing potential AMPs for antimicrobial application. Such AMPs have potential for progression to further trials and drug development investigations.

Flavopereirine Inhibits Autophagy via the AKT/p38 MAPK Signaling Pathway in MDA-MB-231 Cells.

Autophagy is a potential target for the treatment of triple negative breast cancer (TNBC). Because of a lack of targeted therapies for TNBC, it is vital to find optimal agents that avoid chemoresistance and metastasis. Flavopereirine has anti-proliferation ability in cancer cells, but whether it regulates autophagy in breast cancer cells remains unclear. A Premo™ Tandem Autophagy Sensor Kit was used to image the stage at which flavopereirine affects autophagy by confocal microscopy. A plasmid that constitutively expresses p-AKT and siRNA targeting p38 mitogen-activated protein kinase (MAPK) was used to confirm the related signaling pathways by Western blot. We found that flavopereirine induced microtubule-associated protein 1 light chain 3 (LC3)-II accumulation in a dose- and time-dependent manner in MDA-MB-231 cells. Confocal florescent images showed that flavopereirine blocked autophagosome fusion with lysosomes. Western blotting showed that flavopereirine directly suppressed p-AKT levels and mammalian target of rapamycin (mTOR) translation. Recovery of AKT phosphorylation decreased the level of p-p38 MAPK and LC3-II, but not mTOR. Moreover, flavopereirine-induced LC3-II accumulation was partially reduced in MDA-MB-231 cells that were transfected with p38 MAPK siRNA. Overall, flavopereirine blocked autophagy via LC3-II accumulation in autophagosomes, which was mediated by the AKT/p38 MAPK signaling pathway.

miR-338-5p inhibits cell proliferation, colony formation, migration and cisplatin resistance in esophageal squamous cancer cells by targeting FERMT2.

Esophageal cancer is one of the leading causes of cancer death in the male population of Eastern Asia. In addition, esophageal squamous cell carcinoma (ESCC) is the major type of esophageal cancer among the world. Owing to the poor overall 5-year survival rate, novel effective treatment strategies are needed. MicroRNAs are important gene regulators that are dysregulated in many cancer types. In our previous study, we applied next-generation sequencing to demonstrate that miR-338-5p was downregulated in the tumor tissue of patients with versus without recurrence. In this study, we further studied the roles of miR-338-5p in ESCC. The expression of endogenous miR-338-5p was at lower levels in ESCC cells compared with normal cells. Functional assays showed that miR-338-5p reduced cell proliferation, colony formation, migration and cisplatin resistance in an ESCC cell line, CE-81T. Potential target genes of miR-338-5p were identified by microarray and prediction tools, and 31 genes were selected. Among these, Fermitin family homolog 2 (FERMT2) plays an oncogenic role in ESCC, so it was chosen for further study. Luciferase assays showed the direct binding between miR-338-5p and the 3' untranslated region of FERMT2. Silencing of FERMT2 inhibited cell proliferation, colony formation, migration and cisplatin resistance. Pathway analysis revealed that the integrin-linked protein kinase signaling pathway, in which FERMT2 participates, was significantly affected by a miR-338-5p mimic. Our results suggest that miR-338-5p may play an antioncogenic role in ESCC via repressing FERMT2.

Propofol specifically suppresses IL-1ß secretion but increases bacterial survival in Staphylococcus aureus-infected RAW264.7 cells.

Anesthetics have immunomodulatory effects, but the use of different assay systems has contributed to inconsistent results in the literature. IL-1ß and reactive oxygen species (ROS) secreted by phagocytes are important factors that protect against Staphylococcus aureus infection. In this study, the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on IL-1ß secretion, ROS, and bacterial survival in S. aureus-infected RAW264.7 cells were evaluated. S. aureus-infected RAW264.7 cells with or without intravenous anesthetic treatment were established as the experimental model. Cell supernatants were subjected to ELISAs to measure secreted IL-1ß. Cell pellets were subjected to qPCR and western blot analyses to analyze IL-1ß mRNA and protein levels. Luminol chemiluminescence assays were used to detect ROS, and bacterial survival was determined by counting the colony forming units at the beginning and end of the infection. Compared with the levels after treatment with the other intravenous anesthetics, secreted IL-1ß levels were lowest in the supernatant of S. aureus-infected RAW264.7 cell cultures after propofol treatment, but propofol did not decrease IL-1ß mRNA or protein expression. However, thiamylal sodium and midazolam decreased IL-1ß mRNA and protein expression in a dose-dependent manner. Additionally, propofol substantially decreased S. aureus-stimulated ROS and phagocytosis. Bacterial survival was strongly increased by propofol treatment. Of the four intravenous anesthetics, propofol was the most potent inhibitor of IL-1ß secretion and ROS level in S. aureus-infected RAW264.7 cells; moreover, propofol resulted in an increase in bacterial survival by inhibiting ROS and phagocytosis.

Recombinant Epinephelus lanceolatus serum amyloid A as a feed additive: Effects on immune gene expression and resistance to Vibrio alginolyticus infection in Epinephelus lanceolatus.

Recombinant Epinephelus lanceolatus serum amyloid A (rElSAA) exhibits strong immunostimulant activity and enhances phagocytic clearance of bacteria by macrophages. However, the effects of dietary rElSAA supplementation on growth performance, immunomodulation and disease resistance in giant grouper have not been previously evaluated. To test whether oral administration of rElSAA affects growth, fish were fed with 0, 0.88, 4.4 or 22 mg/kg rElSAA-containing diet for 28 days. No statistically significant differences in body weight were observed between groups. Next, we tested whether oral administration of rElSAA may enhance disease resistance. Fish were fed with 0, 0.88, 4.4 or 22 mg/kg rElSAA-containing diet for 3, 7, 14, 21 or 28 days, followed by challenge with Vibrio alginolyticus. Survival was then monitored for 4 days. Fish that were fed with rElSAA-containing diet for 28 days showed significantly improved survival after infection. In addition, the expression levels of immune defense-associated genes in hepatic tissue were assessed by quantitative real-time polymerase chain reaction before and after V. alginolyticus infection. Oral administration of rElSAA increased the expression level of toll-like receptor 5, whereas the expression levels of CC chemokine 1, SAA and C reactive protein were decreased. Thus, the data suggest that rElSAA may enhance host immunity by attenuating regulatory T cell-mediated suppression of inflammation. Together, our results demonstrate that rElSAA is a promising candidate as a feed additive for giant grouper, which may effectively enhance disease resistance after being administered for several weeks.

Recombinant expression of Epinephelus lanceolatus serum amyloid A (ElSAA) and analysis of its macrophage modulatory activities.

Serum amyloid A (SAA) is an acute-phase protein that plays a crucial role in the inflammatory response. In this study, we identified an SAA homolog from Epinephelus lanceolatus (ElSAA). Molecular characterization revealed that ElSAA contains a fibronectin-like motif that is typical of SAAs. Recombinant ElSAA protein (rElSAA) was produced in E. coli BL21 (DE3) cells and purified as a soluble protein. To analyze its biological activity, mouse Raw264.7 macrophage cells were treated with various concentrations of rElSAA. Expression of several inflammation-related cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, IL-6, and IL-10, was induced by rElSAA. This protein also triggered macrophage differentiation, as evidenced by increases in cell size and complexity. To determine whether rElSAA regulates macrophage polarization, we assessed gene expression of M1 and M2 markers. The results demonstrated that rElSAA induced the expression of both M1 and M2 markers, suggesting that it promotes the differentiation of macrophages into a mixed M1/M2 phenotype. To evaluate whether rElSAA enhances phagocytosis via an opsonization-dependent mechanism, GFP-labeled E. coli cells were pretreated with rElSAA, followed by incubation with Raw264.7 cells. Flow cytometry was used to monitor the phagocytic uptake of GFP-labeled E. coli by macrophages. Surprisingly, incubating E. coli with rElSAA did not enhance bacterial uptake by macrophages. However, preincubating Raw264.7 cells with various concentrations of rElSAA, followed by infection with E. coli (multiplicity of infection = 20 or 40), resulted in a clear enhancement of macrophage phagocytic capacity. In conclusion, we have identified SAA from E. lanceolatus and have demonstrated that rElSAA promotes inflammatory cytokine production and macrophage differentiation. In addition, rElSAA enhances phagocytosis of bacteria by macrophages via an opsonization-independent mechanism.

Electrotransfer of the tilapia piscidin 3 and tilapia piscidin 4 genes into skeletal muscle enhances the antibacterial and immunomodulatory functions of Oreochromis niloticus.

Tilapia piscidin 3 (TP3) and tilapia piscidin 4 (TP4) are antimicrobial peptides recently isolated from Oreochromis niloticus. We previously showed that synthetic TP3 and TP4 possessed antimicrobial activities. Here, we analyzed the bactericidal abilities and immunomodulatory properties of these AMPs following the electroporation of pCMV-GFP-TP3 or pCMV-GFP-TP4 plasmid into tilapia (O. niloticus) muscle and subsequent infection with Vibrio vulnificus or Streptococcus agalactiae. Prior overexpression of TP3 or TP4 in tilapia muscle tissues efficiently reduced bacterial numbers at 24 and 48 h after V. vulnificus infection and reduced bacterial numbers at 24 h after S. agalactiae infection compared to numbers in controls expressing pCMV-GFP (EGFP). Electroporation of pCMV-EGFP-TP3 (TP3) or pCMV-EGFP-TP4 (TP4) significantly increased expression of several immune-related genes in muscle (IL-1ß (12 h, TP3), IL-8 (12 h, TP3), TGFß (3 h, TP4), and IκB (48 h, TP3, TP4)) and decreased the expression of TLR5 (12 h and 24 h, TP3) after V. vulnificus infection. Following S. agalactiae infection, expression of the following genes was significantly decreased in muscle: IL-1ß (12 h, TP3), IL-8 (12 h, TP3, TP4), TLR5 (3 h-24 h, TP3, TP4), TGFß (3 h, TP4; 24 h, TP3, TP4), and IκB (3 h, TP3). These data suggest that TP3 and TP4 exert antimicrobial effects after overexpression in the O. niloticus muscle, and also play important roles in the regulation of immune-related gene expression.

Deregulated microRNAs in triple-negative breast cancer revealed by deep sequencing.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNA molecules that play critical roles in human malignancy. However, the regulatory characteristics of miRNAs in triple-negative breast cancer, a phenotype of breast cancer that does not express the genes for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, are still poorly understood. METHODS: In this study, miRNA expression profiles of 24 triple-negative breast cancers and 14 adjacent normal tissues were analyzed using deep sequencing technology. Expression levels of miRNA reads were normalized with the quantile-quantile scaling method. Deregulated miRNAs in triple-negative breast cancer were identified from the sequencing data using the Student's t-test. Quantitative reverse transcription PCR validations were carried out to examine miRNA expression levels. Potential target candidates of a miRNA were predicted using published target prediction algorithms. Luciferase reporter assay experiments were performed to verify a putative miRNA-target relationship. Validated molecular targets of the deregulated miRNAs were retrieved from curated databases and their associations with cancer progression were discussed. RESULTS: A novel 25-miRNA expression signature was found to effectively distinguish triple-negative breast cancers from surrounding normal tissues in a hierarchical clustering analysis. We documented the evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNAs in triple-negative breast cancer. Two of these miRNA clusters (miR-143-145 at 5q32 and miR-497-195 at 17p13.1) were markedly down-regulated in triple-negative breast cancer, while the other five miRNA clusters (miR-17-92 at 13q31.3, miR-183-182 at 7q32.2, miR-200-429 at 1p36.33, miR-301b-130b at 22q11.21, and miR-532-502 at Xp11.23) were up-regulated in triple-negative breast cancer. Moreover, miR-130b-5p from the miR-301b-130b cluster was shown to directly repress the cyclin G2 (CCNG2) gene, a crucial cell cycle regulator, in triple-negative breast cancer cells. Luciferase reporter assays showed that miR-130b-5p-mediated repression of CCNG2 was dependent on the sequence of the 3'-untranslated region. The findings described in this study implicate a miR-130b-5p-CCNG2 axis that may be involved in the malignant progression of triple-negative breast cancer. CONCLUSIONS: Our work delivers a clear picture of the global miRNA regulatory characteristics in triple-negative breast cancer and extends the current knowledge of microRNA regulatory network.

Germinal center kinase-like kinase overexpression in T cells as a novel biomarker in rheumatoid arthritis.

OBJECTIVE: Germinal center kinase-like kinase (GLK; also called MAPKKKK-3) activates protein kinase Cθ (PKCθ) during T cell activation and controls autoimmunity in lupus patients. Intracellular kinases are involved in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the role of GLK in RA. METHODS: The severity of collagen-induced arthritis (CIA) was studied in GLK-deficient mice. Expression levels of GLK from RA patients were determined by Western blotting, flow cytometry, real-time polymerase chain reaction, and immunohistochemical staining. Localization of GLK in T cells was identified by confocal microscopy. RA disease activity was assessed using the Disease Activity Score in 28 joints. RESULTS: GLK-deficient mice displayed impaired CIA development and decreased inflammatory cytokine levels. Local T cell infiltration and collagen restimulation responses were impaired by GLK deficiency. RA patients showed significantly higher GLK protein and messenger RNA levels in peripheral blood T cells than did healthy controls. GLK-overexpressing T cells in synovial fluid and synovial tissue samples from RA patients were increased compared with those from osteoarthritis patients. Confocal microscopy and flow cytometry showed that GLK colocalized and coexisted with phosphorylated PKCθ in T cells from RA patients. Frequencies of GLK-expressing T cells were significantly correlated with RA disease activity. CONCLUSION: GLK overexpression in T cells contributes to the pathogenesis of RA, indicating that GLK is a novel biomarker for autoimmune disease severity and a potential therapeutic target for RA.

Use of natural peptide TP4 as a food preservative prevents contamination by fungal pathogens.

Molecules of natural origin often possess useful biological activities. For instance, the natural peptide Tilapia Piscidin 4 (TP4) exhibits potent antimicrobial activity against a broad spectrum of pathogens. In this study, we explored the potential application of TP4 as a food preservative, asking whether it can prevent spoilage due to microbial contamination. A preliminary in silico analysis indicated that TP4 should interact strongly with fungal cell membrane components. Hence, we tested the activity of TP4 toward Candida albicans within fruit juice and found that the addition of TP4 could abolish fungal growth. We further determined that the peptide acts via a membranolytic mechanism and displays concentration-dependent killing efficiency. In addition, we showed that TP4 inhibited growth of Rhizopus oryzae in whole fruit (tomato) samples. Based on these findings, we conclude that TP4 should be further evaluated as a potentially safe and green solution to prevent food spoilage.

ClinOleic Impairs ROS Production and Phagocytosis in M1 Macrophages Without Affecting M1 Differentiation.

Lipid emulsions are the primary source of calories and fatty acids that are used to provide essential energy and nutrients to patients suffering from severe intestinal failure and critical illness. However, their use has been linked to adverse effects on patient outcomes, notably affecting immune defenses and inflammatory responses. ClinOleic is a lipid emulsion containing a mixture of olive oil and soybean oil (80:20). The effect of ClinOleic on the differentiation of M1 macrophages remains unclear. In this study, we isolated human monocytes and added ClinOleic to differentiation culture media to investigate whether it affects monocyte polarization into M1 macrophages and macrophage functions, such as reactive oxygen species (ROS) production and phagocytosis. ROS production was stimulated by live S. aureus and detected with L-012, a chemiluminescence emission agent. Phagocytic capacity was assayed using pHrodo™ Green S. aureus Bioparticles® Conjugate. We found that M1 cell morphology, surface markers (CD80 and CD86), and M1-associated cytokines (TNF-α and IL-6) did not significantly change upon incubation with ClinOleic during M1 polarization. However, S. aureus-triggered ROS production was significantly lower in M1 macrophages differentiated with ClinOleic than in those not treated with ClinOleic. The inhibitory effect of ClinOleic on macrophage function also appeared in the phagocytosis assay. Taken together, these findings reveal that ClinOleic has a limited impact on the M1 differentiation phenotype but obviously reduces ROS production and phagocytosis.

Optimization of the Black Garlic Processing Method and Development of Black Garlic Jam Using High-Pressure Processing.

Black garlic has many beneficial effects, and it has a less spicy flavor. However, its aging conditions and related products still need to be further investigated. The present study aims to analyze the beneficial effects under different processing conditions and utilize high-pressure processing (HPP) in the production of black garlic jam. The highest antioxidant activities, including the DPPH scavenging, total antioxidant capacity, and reducing power (86.23%, 88.44%, and A700 = 2.48, respectively), were observed in black garlic that had been aged for 30 days. Similarly, the highest total phenols and flavonoids were observed in black garlic that had been aged for 30 days (76.86 GAE/g dw and 13.28 mg RE/g dw, respectively). The reducing sugar in black garlic was significantly increased to about 380 (mg GE/g dw) after 20 days of aging. The free amino acids in black garlic were decreased time-dependently to about 0.2 mg leucine/g dw after 30 days of aging. For the browning indexes of black garlic, the uncolored intermediate and browning products were increased in a time-dependent manner and reached a plateau at day 30. Another intermediate product in the Maillard reaction, 5-hydroxymethylfurfural (5-HMF), was observed in concentrations that increased to 1.81 and 3.04 (mg/g dw) at day 30 and 40, respectively. Furthermore, the black garlic jam made by HPP was analyzed for its texture and sensory acceptance, showing that a 1:1.5:2 ratio of black garlic/water/sugar was the most preferred and was classified as "still acceptable". Our study suggests suitable processing conditions for black garlic and outlines the prominent beneficial effects after 30 days of aging. These results could be further applied in HPP jam production and increase the diversity of black garlic products.

Haematopoietic cell-derived exosomes in cancer development and therapeutics: From basic science to clinical practice.

BACKGROUND: The tumour microenvironment (TME) is a specialised niche involving intercellular communication among cancer cells and various host cells. Among the host cells, the quantity and quality of immune cells within the TME play essential roles in cancer development and management. The immunologically suppressive, so-called 'cold' TME established by a series of tumour-host interactions, including generating immunosuppressive cytokines and recruiting regulatory host immune cells, is associated with resistance to therapies and worse clinical outcomes. MAIN BODY: Various therapeutic approaches have been used to target the cold TME, including immune checkpoint blockade therapy and adoptive T-cell transfer. A promising, less explored therapeutic strategy involves targeting TME-associated exosomes. Exosomes are nanometer-sized, extracellular vesicles that transfer material from donor to recipient cells. These particles can reprogram the recipient cells and modulate the TME. In particular, exosomes from haematopoietic cells are known to promote or suppress cancer progression under specific conditions. Understanding the effects of haematopoietic cell-secreted exosomes may foster the development of therapeutic exosomes (tExos) for personalised cancer treatment. However, the development of exosome-based therapies has unique challenges, including scalable production, purification, storage and delivery of exosomes and controlling batch variations. Clinical trials are being conducted to verify the safety, feasibility, availability and efficacy of tExos. CONCLUSION: This review summarises our understanding of how haematopoietic cell-secreted exosomes regulate the TME and antitumour immunity and highlights present challenges and solutions for haematopoietic cell-derived exosome-based therapies.

Rational Design of Stapled Antimicrobial Peptides to Enhance Stability and In Vivo Potency against Polymicrobial Sepsis.

In this work, we sought to develop a TP4-based stapled peptide that can be used to counter polymicrobial sepsis. First, we segregated the TP4 sequence into hydrophobic and cationic/hydrophilic zones and substituted the preferred residue, lysine, as the sole cationic amino acid. These modifications minimized the intensity of cationic or hydrophobic characteristics within small segments. Then, we incorporated single or multiple staples into the peptide chain, bracketing the cationic/hydrophilic segments to improve pharmacological suitability. Using this approach, we were able to develop an AMP with low toxicity and notable in vivo efficacy. IMPORTANCE In our in vitro studies, one dual stapled peptide out of the series of candidates (TP4-3: FIIXKKSXGLFKKKAGAXKKKXIKK) showed significant activity, low toxicity, and high stability (in 50% human serum). When tested in cecal ligation and puncture (CLP) mouse models of polymicrobial sepsis, TP4-3 improved survival (87.5% on day 7). Furthermore, TP4-3 enhanced the activity of meropenem against polymicrobial sepsis (100% survival on day 7) compared to meropenem alone (37.5% survival on day 7). Molecules such as TP4-3 may be well suited for a wide variety of clinical applications.

Octanoic acid promotes clearance of antibiotic-tolerant cells and eradicates biofilms of Staphylococcus aureus isolated from recurrent bovine mastitis.

Antibiotic therapy is the primary treatment for bovine mastitis, but the drawbacks of this strategy include poor cure rate and economic losses from the need to discard milk with antibiotic residues. Unfortunately, few other treatment options are currently available for mastitis. Failure of antibiotic treatments is often attributed to formation of bacterial biofilms and abscesses in the mammary gland tissue, which lead to chronic infections that are difficult to eradicate and drive recurrent disease. A major mastitis-causing pathogen (MCP) associated with biofilms in bovine mastitis is Staphylococcus aureus. In this study, we demonstrate that octanoic acid has broad-spectrum microbicidal activity against MCPs and effectively inhibits S. aureus biofilm formation in milk (>50% inhibition at 3.13 mM). Octanoic acid effectively clears biofilms (95% eradication at 1X minimum bactericidal concentration, MBC) and infrequently induces S. aureus small colony variants (SCVs) that may cause recurrent mastitis. Additionally, octanoic acid rapidly kills persistent biofilm cells and cells with antibiotic tolerance (within 4 h). In contrast, antibiotics treated at >100X MBC cannot eradicate biofilms but do induce SCVs and antibiotic-tolerant cells. These effects may accelerate the transition from biofilm to chronic infection. Thus, octanoic acid exhibits bactericidal action against S. aureus biofilms, and it is less likely than antibiotic therapy to induce persistent cells and pathogen tolerance. Moreover, octanoic acid acts additively with antibiotics against S. aureus, and it attenuates tetracycline-induced virulence factor gene expression in S. aureus cells. According to these data, octanoic acid may prevent the pathological progression of bovine mastitis and offer a new strategy for treating the condition.

Strategic modification of low-activity natural antimicrobial peptides confers antibacterial potential in vitro and in vivo.

Antimicrobial peptides (AMPs) show great promise for clinical applications, but the utility of naturally occurring AMPs is often limited by their stability. Here, we used a rational design approach to improve the characteristics of a pair of inactive peptides, tilapia piscidin 1 and 2 (TP1 and TP2). From each starting peptide, we generated a series of novel derivatives by substituting residues to adjust cationic charge density, percent hydrophobicity and hydrophilicity/hydrophobicity coefficients. This approach yielded a novel peptide, TP2-5 (KKCIAKAILKKAKKLLKKLVNP), that exhibits significant bactericidal potency, low cytotoxicity and high stability. The designed peptide further showed antibiofilm activity, rapid antibacterial action and a low capacity to induce bacterial resistance. Importantly, we also demonstrated that TP2-5 can protect mice in a Vibrio vulnificus-infected wound model. Therefore, our peptide modification strategy successfully generated a novel AMP with high potential for future clinical application.

The cellular responses and antibacterial activities of silver nanoparticles stabilized by different polymers.

Silver nanoparticles (AgNPs) are known for their excellent antibacterial activities. The possible toxicity, however, is a major concern for their applications. Three types of AgNPs were prepared in this study by chemical processes. Each was stabilized by a polymer surfactant, which was expected to reduce the exposure of cells to AgNPs and therefore their cytotoxicity. The polymer stabilizers included poly(oxyethylene)-segmented imide (POEM), poly(styrene-co-maleic anhydride)-grafting poly(oxyalkylene) (SMA) and poly(vinyl alcohol) (PVA). The cytotoxicity of these chemically produced AgNPs to mouse skin fibroblasts (L929), human hepatocarcinoma cells (HepG2), and mouse monocyte macrophages (J774A1) was compared to that of physically produced AgNPs and gold nanoparticles (AuNPs) as well as the standard reference material RM8011 AuNPs. Results showed that SMA-AgNPs were the least cytotoxic among all materials, but cytotoxicity was still observed at higher silver concentrations (>30 ppm). Macrophages demonstrated the inflammatory response with cell size increase and viability decrease upon exposure to 10 ppm of the chemically produced AgNPs. SMA-AgNPs did not induce hemolysis at a silver concentration below 1.5 ppm. Regarding the antibacterial activity, POEM-AgNPs and SMA-AgNPs at 1 ppm silver content showed 99.9% and 99.3% growth inhibition against E. coli, while PVA-AgNPs at the same silver concentration displayed 79.1% inhibition. Overall, SMA-AgNPs demonstrated better safety in vitro and greater antibacterial effects than POEM-AgNPs and PVA-AgNPs. This study suggested that polymer stabilizers may play an important role in determining the toxicity of AgNPs.