Affordable Biocidal Ultraviolet Cured Cuprous Oxide Filled Vat Photopolymerization Resin Nanocomposites with Enhanced Mechanical Properties

In this study, Cuprous Oxide (Cu2O), known for its mechanism against bacteria, was used as filler to induce biocidal properties on a common commercial resin stereolithography (SLA) 3D printing resin. The aim was to develop nanocomposites suitable for the SLA process with a low-cost process that mimic host defense peptides (HDPs). Such materials have a huge economic and societal influence on the global technological war on illness and exploiting 3D printing characteristics is an additional asset for these materials. Their mechanical performance was also investigated with tensile, flexural, Charpy’s impact, and Vickers microhardness tests. Morphological analysis was performed through scanning electron microscopy (SEM), atomic force microscopy (AFM), and energy-dispersive X-ray spectroscopy (EDS) analysis, while the thermal behavior was studied through Thermogravimetric Analysis (TGA). The antibacterial activity of the fabricated nanocomposites was investigated using a screening agar well diffusion method, for a gram-negative and a gram-positive bacterium. Three-dimensional printed nanocomposites exhibited antibacterial performance in all loadings studied, while their mechanical enhancement was approximately 20% even at low filler loadings, revealing a multi-functional performance and a potential of Cuprous Oxide implementation in SLA resin matrices for engineering and medical applications.

Investigation of the Biocidal Performance of Multi-Functional Resin/Copper Nanocomposites with Superior Mechanical Response in SLA 3D Printing

Metals, such as silver, gold, and copper are known for their biocidal properties, mimicking the host defense peptides (HDPs) of the immune system. Developing materials with such properties has great importance in medicine, especially when combined with 3D printing technology, which is an additional asset for various applications. In this work, copper nanoparticles were used as filler in stereolithography (SLA) ultraviolet (UV) cured commercial resin to induce such biocidal properties in the material. The nanocomposites developed featured enhanced mechanical responses when compared with the neat material. The prepared nanocomposites were employed to manufacture specimens with the SLA process, to be tested for their mechanical response according to international standards. The process followed was evaluated with Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), energy-dispersive X-ray spectroscopy (EDS), and thermogravimetric analysis (TGA). The antibacterial activity of the fabricated nanocomposites was evaluated using the agar-well diffusion method. Results showed enhanced mechanical performance of approximately 33.7% in the tensile tests for the nanocomposites filled with 1.0 wt.%. ratios, when compared to the neat matrix material, while this loading showed sufficient antibacterial performance when compared to lower filler loadings, providing an added value for the fabrication of effective nanocomposites in medical applications with the SLA process.

Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages

Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.

Gut Microbiota Dysbiosis Aggravates Mycoplasma gallisepticum Colonization in the Chicken Lung

Mycoplasma gallisepticum (MG) is the pathogen that causes chronic respiratory diseases in chickens. Gut microbiota plays an important role in maintaining body health and resisting respiratory infection, but the correlation between gut microbiota and MG infection is poorly defined. Therefore, in this study, the correlation between gut microbiota and MG infection was explored by disturbing gut microbiota in chickens with antibiotic cocktail. The results showed that the gut microbiota dysbiosis impairs pulmonary immune response against MG infection. It has been noted that MG colonization in the lung was significantly increased following gut microbiota dysbiosis, and this could be reversed by intranasally administrated toll-like receptor 2 (TLR2) ligand, recombinant chicken IL-17 protein or recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) protein. In addition, the levels of short-chain fatty acids (SCFAs) and vitamin A were significantly reduced in gut microbiota dysbiosis group, however, butyric acid or vitamin A as feed additives promoted MG clearance in the lung of gut microbiota dysbiosis group via increasing TLR2/IL17/GM-CSF and host defense peptides genes expression. The present study revealed an important role of gut microbiota in the defense against MG colonization in the lung of chicken.

In-Silico Tool for Predicting, Scanning, and Designing Defensins

Defensins are host defense peptides present in nearly all living species, which play a crucial role in innate immunity. These peptides provide protection to the host, either by killing microbes directly or indirectly by activating the immune system. In the era of antibiotic resistance, there is a need to develop a fast and accurate method for predicting defensins. In this study, a systematic attempt has been made to develop models for predicting defensins from available information on defensins. We created a dataset of defensins and non-defensins called the main dataset that contains 1,036 defensins and 1,035 AMPs (antimicrobial peptides, or non-defensins) to understand the difference between defensins and AMPs. Our analysis indicates that certain residues like Cys, Arg, and Tyr are more abundant in defensins in comparison to AMPs. We developed machine learning technique-based models on the main dataset using a wide range of peptide features. Our SVM (support vector machine)-based model discriminates defensins and AMPs with MCC of 0.88 and AUC of 0.98 on the validation set of the main dataset. In addition, we created an alternate dataset that consists of 1,036 defensins and 1,054 non-defensins obtained from Swiss-Prot. Models were also developed on the alternate dataset to predict defensins. Our SVM-based model achieved maximum MCC of 0.96 with AUC of 0.99 on the validation set of the alternate dataset. All models were trained, tested, and validated using standard protocols. Finally, we developed a web-based service “DefPred” to predict defensins, scan defensins in proteins, and design the best defensins from their analogs. The stand-alone software and web server of DefPred are available at https://webs.iiitd.edu.in/raghava/defpred.

SSTP1, a Host Defense Peptide, Exploits the Immunomodulatory IL6 Pathway to Induce Apoptosis in Cancer Cells

While the immunomodulatory pathways initiated in immune cells contribute to therapeutic response, their activation in cancer cells play a role in cancer progression. Also, many of the aberrantly expressed immunomodulators on cancer cells are considered as therapeutic targets. Here, we introduce host defense peptide (HDP), a known immuomodulator, as a therapeutic agent to target them. The cationic host defense peptides (HDPs), an integral part of the innate immune system, possess membranolytic activity, which imparts antimicrobial and antitumor efficacy to it. They act as immunomodulators by activating the immune cells. Though their antimicrobial function has been recently reassigned to immunoregulation, their antitumor activity is still attributed to its membranolytic activity. This membrane pore formation ability, which is proportional to the concentration of the peptide, also leads to side effects like hemolysis, limiting their therapeutic application. So, despite the identification of a variety of anticancer HDPs, their clinical utility is limited. Though HDPs are shown to exert the immunomodulatory activity through specific membrane targets on immune cells, their targets on cancer cells are unknown. We show that SSTP1, a novel HDP identified by shotgun cloning, binds to the active IL6/IL6Rα/gp130 complex on cancer cells, rearranging the active site residues. In contrast to the IL6 blockers inhibiting JAK/STAT activity, SSTP1 shifts the proliferative IL6/JAK/STAT signaling to the apoptotic IL6/JNK/AP1 pathway. In IL6Rα-overexpressing cancer cells, SSTP1 induces apoptosis at low concentration through JNK pathway, without causing significant membrane disruption. We highlight the importance of immunomodulatory pathways in cancer apoptosis, apart from its established role in immune cell regulation and cancer cell proliferation. Our study suggests that identification of the membrane targets for the promising anticancer HDPs might lead to the identification of new drugs for targeted therapy.

Protective Effects of Lactobacillus plantarum Lac16 on Clostridium perfringens Infection-Associated Injury in IPEC-J2 Cells

Clostridium perfringens (C. perfringens) causes intestinal injury through overgrowth and the secretion of multiple toxins, leading to diarrhea and necrotic enteritis in animals, including pigs, chickens, and sheep. This study aimed to investigate the protective effects of Lactobacillus plantarum (L. plantarum) Lac16 on C. perfringens infection-associated injury in intestinal porcine epithelial cell line (IPEC-J2). The results showed that L. plantarum Lac16 significantly inhibited the growth of C. perfringens, which was accompanied by a decrease in pH levels. In addition, L. plantarum Lac16 significantly elevated the mRNA expression levels of host defense peptides (HDPs) in IPEC-J2 cells, decreased the adhesion of C. perfringens to IPEC-J2 cells, and attenuated C. perfringens-induced cellular cytotoxicity and intestinal barrier damage. Furthermore, L. plantarum Lac16 significantly suppressed C. perfringens-induced gene expressions of proinflammatory cytokines and pattern recognition receptors (PRRs) in IPEC-J2 cells. Moreover, L. plantarum Lac16 preincubation effectively inhibited the phosphorylation of p65 caused by C. perfringens infection. Collectively, probiotic L. plantarum Lac16 exerts protective effects against C. perfringens infection-associated injury in IPEC-J2 cells.

Brilacidin, a COVID-19 Drug Candidate, demonstrates broad-spectrum antiviral activity against human coronaviruses OC43, 229E and NL63 through targeting both the virus and the host cell

Brilacidin, a mimetic of host defense peptides (HDPs), is currently in phase 2 clinical trial as an antibiotic drug candidate. A recent study reported that brilacidin has antiviral activity against SARS-CoV-2 by inactivating the virus. In this work, we discovered an additional mechanism of action of brilacidin by targeting heparan sulfate proteoglycans (HSPGs) on host cell surface. Brilacidin, but not acetyl brilacidin, inhibits the entry of SARS-CoV-2 pseudovirus into multiple cell lines, and heparin, a HSPG mimetic, abolishes the inhibitory activity of brilacidin on SARS-CoV-2 pseudovirus cell entry. In addition, we found that brilacidin has broad-spectrum antiviral activity against multiple human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, and HCoV-NL63. Mechanistic studies revealed that brilacidin has a dual antiviral mechanism of action including virucidal activity and binding to coronavirus attachment factor HSPGs on host cell surface. Brilacidin partially loses its antiviral activity when heparin was included in the cell cultures, supporting the host-targeting mechanism. Drug combination therapy showed that brilacidin has a strong synergistic effect with remdesivir against HCoV-OC43 in cell culture. Taken together, this study provides appealing findings for the translational potential of brilacidin as a broad-spectrum antiviral for coronaviruses including SARS-CoV-2.