Evaluation of plant-derived biomaterials for the development of tissue-engineered corneal substitutes.

Corneal blindness affects over 10 million patients worldwide. Due to the limited supply of donor corneas and frequent graft failure, bioengineered alternatives are crucial. To overcome drawbacks associated with corneal substitutes from synthetic biomaterials, fabrication from plant-derived biomaterials is a potential alternative. Herein, soy protein and glutenin in combination with different crosslinkers were evaluated for fabrication of corneal substitutes. Optical, mechanical, and biochemical properties of fabricated constructs and control rabbit corneas were evaluated in vitro. Soy protein crosslinked with peroxidase/H202 possessed transparency and mechanical properties comparable to controls, although their water content and biocompatibility were inferior. In contrast, soy protein crosslinked with tannic acid showed similar water content, tensile strength, and biocompatibility as rabbit corneas; however, these constructs displayed significantly lower transparency and higher strain to failure. Finally, glutenin cross-linked using formaldehyde showed excellent transparency, strain to failure, and biocompatibility, however; they exhibited significantly lower water content and tensile strength than controls. This study is the first to establish CIELAB color values for the rabbit cornea, allowing quantitative optical evaluation of tissue-engineered substitutes. Thus, a crosslinking strategy utilizing plant-derived proteins for fabrication of constructs with properties comparable to rabbit corneas is a promising direction for development of tissue-engineered corneal substitutes.

Immunomodulation by xylan and carrageenan-type polysaccharides from red seaweeds: Anti-inflammatory, wound healing, cytoprotective, and anticoagulant activities.

The immunomodulatory properties of the polysaccharides (carrageenan, xylan) from Chondrus crispus (CC), Ahnfeltiopsis devoniensis (AD), Sarcodiotheca gaudichaudii (SG) and Palmaria palmata (PP) algal species were studied. Using RAW264.7 macrophages, we investigated the proliferation and migration capacity of different extracts along with their immunomodulatory activities, including nitric oxide (NO) production, phagocytosis, and secretion of pro-inflammatory cytokines. Polysaccharides from C. crispus and S. gaudichaudii effectively mitigated inflammation and improved scratch-wound healing. Polysaccharide fractions extracted under cold conditions (25 °C), including CC-1A, SG-1A and SG-1B stimulated cell proliferation, while fractions extracted under hot conditions (95 °C), including CC-3A, CC-2B and A. devoniensis (AD-3A), inhibited cell proliferation after 48 h. Furthermore, RAW264.7 cells treated with the fractions CC-3A, AD-1A, and SG-2A significantly reduced LPS-stimulated NO secretion over 24 h. Phagocytosis was significantly improved by treatment with C. crispus (CC-2B, CC-3B) and A. devoniensis (AD-3A) fractions. RAW264.7 cells treated with the CC-2A and SG-1A fractions showed elevated TGF-ß1 expression without affecting TNF-α expression at 24 h. Polysaccharide fractions of A. devoniensis (ι/κ hybrid carrageenan; AD-2A, AD-3A) showed the highest anti-coagulation activity. CC-2A and SG-1A fractions enhanced various bioactivities, suggesting they are candidates for skin-health applications. The carrageenan fractions (CC-3A: λ-, µ-carrageenan, SG-2A: ν-, ι-carrageenan) tested herein showed great potential for developing anti-inflammatory and upscaled skin-health applications.

A Two Bacteriocinogenic Ligilactobacillus Strain Association Inhibits Growth, Adhesion, and Invasion of Salmonella in a Simulated Chicken Gut Environment.

In this study, we aimed to develop a protective probiotic coculture to inhibit the growth of Salmonella enterica serovar Typhimurium in the simulated chicken gut environment. Bacterial strains were isolated from the digestive mucosa of broilers and screened in vitro against Salmonella Typhimurium ATCC 14028. A biocompatibility coculture test was performed, which identified two biocompatible strains, Ligilactobacillus salivarius UO.C109 and Ligilactobacillus saerimneri UO.C121 with high inhibitory activity against Salmonella. The cell-free supernatant (CFS) of the selected isolates exhibited dose-dependent effects, and the inhibitory agents were confirmed to be proteinaceous by enzymatic and thermal treatments. Proteome and genome analyses revealed the presence of known bacteriocins in the CFS of L. salivarius UO.C109, but unknown for L. saerimneri UO.C121. The addition of these selected probiotic candidates altered the bacterial community structure, increased the diversity of the chicken gut microbiota challenged with Salmonella, and significantly reduced the abundances of Enterobacteriaceae, Parasutterlla, Phascolarctobacterium, Enterococcus, and Megamonas. It also modulated microbiome production of short-chain fatty acids (SCFAs) with increased levels of acetic and propionic acids after 12 and 24 h of incubation compared to the microbiome challenged with S. Typhimurium. Furthermore, the selected probiotic candidates reduced the adhesion and invasion of Salmonella to Caco-2 cells by 37-39% and 51%, respectively, after 3 h of incubation, compared to the control. These results suggest that the developed coculture probiotic strains has protective activity and could be an effective strategy to control Salmonella infections in poultry.

Proteomic Analysis of Chicken Chorioallantoic Membrane (CAM) during Embryonic Development Provides Functional Insight.

In oviparous animals, the egg contains all resources required for embryonic development. The chorioallantoic membrane (CAM) is a placenta-like structure produced by the embryo for acid-base balance, respiration, and calcium solubilization from the eggshell for bone mineralization. The CAM is a valuable in vivo model in cancer research for development of drug delivery systems and has been used to study tissue grafts, tumor metastasis, toxicology, angiogenesis, and assessment of bacterial invasion. However, the protein constituents involved in different CAM functions are poorly understood. Therefore, we have characterized the CAM proteome at two stages of development (ED12 and ED19) and assessed the contribution of the embryonic blood serum (EBS) proteome to identify CAM-unique proteins. LC/MS/MS-based proteomics allowed the identification of 1470, 1445, and 791 proteins in CAM (ED12), CAM (ED19), and EBS, respectively. In total, 1796 unique proteins were identified. Of these, 175 (ED12), 177 (ED19), and 105 (EBS) were specific to these stages/compartments. This study attributed specific CAM protein constituents to functions such as calcium ion transport, gas exchange, vasculature development, and chemical protection against invading pathogens. Defining the complex nature of the CAM proteome provides a crucial basis to expand its biomedical applications for pharmaceutical and cancer research.

Metabolic Influences of Gut Microbiota Dysbiosis on Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBD) are chronic medical disorders characterized by recurrent gastrointestinal inflammation. While the etiology of IBD is still unknown, the pathogenesis of the disease results from perturbations in both gut microbiota and the host immune system. Gut microbiota dysbiosis in IBD is characterized by depleted diversity, reduced abundance of short chain fatty acids (SCFAs) producers and enriched proinflammatory microbes such as adherent/invasive E. coli and H2S producers. This dysbiosis may contribute to the inflammation through affecting either the immune system or a metabolic pathway. The immune responses to gut microbiota in IBD are extensively discussed. In this review, we highlight the main metabolic pathways that regulate the host-microbiota interaction. We also discuss the reported findings indicating that the microbial dysbiosis during IBD has a potential metabolic impact on colonocytes and this may underlie the disease progression. Moreover, we present the host metabolic defectiveness that adds to the impact of symbiont dysbiosis on the disease progression. This will raise the possibility that gut microbiota dysbiosis associated with IBD results in functional perturbations of host-microbiota interactions, and consequently modulates the disease development. Finally, we shed light on the possible therapeutic approaches of IBD through targeting gut microbiome.

Distinct CDK6 complexes determine tumor cell response to CDK4/6 inhibitors and degraders.

CDK4/6 inhibitors (CDK4/6i) are effective in metastatic breast cancer, but they have been only modestly effective in most other tumor types. Here we show that tumors expressing low CDK6 rely on CDK4 function, and are exquisitely sensitive to CDK4/6i. In contrast, tumor cells expressing both CDK4 and CDK6 have increased reliance on CDK6 to ensure cell cycle progression. We discovered that CDK4/6i and CDK4/6 degraders potently bind and inhibit CDK6 selectively in tumors in which CDK6 is highly thermo-unstable and strongly associated with the HSP90/CDC37 complex. In contrast, CDK4/6i and CDK4/6 degraders are ineffective in antagonizing tumor cells expressing thermostable CDK6, due to their weaker binding to CDK6 in these cells. Thus, we uncover a general mechanism of intrinsic resistance to CDK4/6i and CDK4/6i-derived degraders and the need for novel inhibitors targeting the CDK4/6i-resistant, thermostable form of CDK6 for application as cancer therapeutics.

Biotechnological Applications of Eggshell: Recent Advances.

The eggshell (ES) provides protection against pathogenic and physical insults while supplying essential metabolic and nutritional needs for the growing avian embryo. It is constituted mainly of calcium carbonate arranged as calcite crystals. The global chicken egg production in 2018 was over 76.7 million metric tons. In industrialized countries, about 30% of eggs are processed at breaker plants that produce liquid egg products and large quantities of solid ES waste. ES waste is utilized for a variety of low-value applications, or alternatively is disposed in landfill with associated economic and environmental burdens. The number of patents pertaining to ES applications has increased dramatically in recent years; of 673 patents granted in the last century, 536 (80%) were published in the last two decades. This review provides a snapshot of the most recent patents published between 2015 and 2020, with emphasis on different biotechnological applications of ES waste, and summarizes applications for biomedical, chemical, engineering, and environmental technologies. Biomedical technologies include the production of calcium lactate, calcium phosphate, and health-promoting products, while chemical technologies include plant growth promoters, food processing and production, and biodiesel oil catalysis along with active calcium, carbon, soluble proteins, organic calcium, and ultrafine calcium carbonate sources. Engineering technologies address material engineering and nanoparticle production, while environmental technologies pertain to production of biomass, solubilization of sludge as well as production of magnetic ES adsorbents and adsorption of heavy metals, organics, total nitrogen and fluoride, soil pollutants, and radioactive compounds. Although the number of ES-based patents has exponentially increased in the last decade, exploration of innovative top-down approaches and ES development as a physical platform are new endeavors that are expected to further increase the upscaling of ES waste exploitation.

A Survey of Recent Patents in Engineering Technology for the Screening, Separation and Processing of Eggshell.

The chicken egg is a well-known complete food of human daily consumption which serves as a cost-effective, high-quality nutrient resource. About 30% of table eggs are directed to breaker plants in developed countries, leading to the generation of substantial eggshell (ES) waste, which is increasingly explored for potential value-added applications. The number of patents describing ES-based applications has increased dramatically in recent years. This review provides insight into the most recent patents published between 2015 and 2020, with focus on different engineering technologies for the screening, separation, and processing of ES. Screening technologies include detection of ES surface spots and glossiness, ES cracks, and mechanical properties, along with identification of chicken breed and enumeration of surface bacterial count. Collection and separation technologies describe separation strategies of ES from egg white (EW), egg yolk (EY), liquid egg, eggshell membrane (ESM), hatchlings, and cooked egg. Separation of ES from liquid eggs utilizes gravity, rotational forces, or air pressure. Processing of ES involves washing and sterilization along with cutting, crushing, and pulverization technologies that enable the collection of ES suitable for value-added applications. In addition, ES carving (mechanical and laser) opens up the realm of artwork and decoration. Furthermore, intact ES can be utilized for food serving. The exponential increase in innovative screening, separation, collection, and processing technologies reflects industrial interest to upscale low-value ES waste material, and is a first crucial step in the emergence of advanced technologies that exploit the biomedical, chemical, engineering, and environmental applications for ES.

Exploiting Allosteric Properties of RAF and MEK Inhibitors to Target Therapy-Resistant Tumors Driven by Oncogenic BRAF Signaling.

Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF-MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAFV600E therapy-resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAFV600E tumors. SIGNIFICANCE: This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAFV600E tumors.See related commentary by Zhang and Bollag, p. 1620.This article is highlighted in the In This Issue feature, p. 1601.

Changes of Gut-Microbiota-Liver Axis in Hepatitis C Virus Infection.

The gut-liver-axis is a bidirectional coordination between the gut, including microbial residents, the gut microbiota, from one side and the liver on the other side. Any disturbance in this crosstalk may lead to a disease status that impacts the functionality of both the gut and the liver. A major cause of liver disorders is hepatitis C virus (HCV) infection that has been illustrated to be associated with gut microbiota dysbiosis at different stages of the disease progression. This dysbiosis may start a cycle of inflammation and metabolic disturbance that impacts the gut and liver health and contributes to the disease progression. This review discusses the latest literature addressing this interplay between the gut microbiota and the liver in HCV infection from both directions. Additionally, we highlight the contribution of gut microbiota to the metabolism of antivirals used in HCV treatment regimens and the impact of these medications on the microbiota composition. This review shed light on the potential of the gut microbiota manipulation as an alternative therapeutic approach to control the liver complications post HCV infection.

A novel eco-friendly green approach to produce particalized eggshell membrane (PEM) for skin health applications.

The eggshell membrane (ESM) is a natural bioactive material, which is increasingly utilized for various biomedical applications. However, the poor solubility of ESM limits the bioavailability of its constituents and reduces the expression of their potential bioactivity. In this study, we utilized an innovative green strategy to separate ESM from shell, and processed ESM for size reduction by cryo-grinding and homogenization to produce particalized eggshell membrane (PEM) approaching submicron dimensions, with enhanced anti-inflammatory activity and increased antimicrobial activity against skin associated pathogens. Gram-positive Staphylococcus aureus (log10 reduction = 4.5 ± 0.3) was more sensitive to PEM as compared to Gram-negative Pseudomonas aeruginosa (log10 reduction = 2.1 ± 0.3). PEM elicited a dose-dependent reduction in NO accumulation in LPS-induced RAW 264.7 macrophages, suggesting an anti-inflammatory response to ESM particles. These findings suggest that processed PEM possesses great potential as a topical ingredient in skincare applications to maintain skin health by reducing bacterial infections and inflammation.

Experimental datasets on processed eggshell membrane powder for wound healing.

Eggshell (ES) and eggshell membrane (ESM) is a significant byproduct of the egg producing industry (Ahmed et al., 2019). Many studies have been undertaken to utilize ES waste for potential value added applications (Cordeiro and Hincke, 2011). Described here are the datasets from our evaluation of processed eggshell membrane powder (PEP) as a wound healing product using the mouse excisional wound splinting model (Ahmed et al., 2019). PEP biomaterial was characterized by proteomics using various extraction and solubilization strategies including moderate (lithium dodecyl sulphate (LDS) and urea/ammonium bicarbonate) and harsh conditions (3-mercaptopropionic acid (3-MPA) and NaOH/dimethylsulfoxide) in order to progressively overcome its stable, insoluble nature (Ahmed et al., 2019, Ahmed et al., 2017). Analysis of proteomic data allowed the relative abundance of the main PEP protein constituents to be determined. The efficacy of PEP for promotion of wound healing was assessed using the mouse excisional wound splinting model, and well-established semi-quantitative histological scoring. (More details about the PEP biomaterial characterization and its in vivo evaluation can be found in the related research article (Ahmed et al., 2019)).

Recent insights into structure-function relationships of antimicrobial peptides.

The application of antimicrobial peptides (AMPs) in food preservation presents a promising alternative and offers many benefits, such as reducing the use of chemical preservatives, reducing food losses due to spoilage, and development of health-promoting food supplements. The biological activity of AMPs largely dependent on several physicochemical features including charge, the degree of helicity, hydrophobicity, and sequence. The present review provides an overview of the structural classification of AMPs emphasizing the importance of their structural features for biological activity, followed by the description of some antimicrobial mechanism of action. Despite the several hurdles that must be overcome for the exploitation of food-derived AMPs in drug discovery and food systems, the developments discussed in this review offer a taste of future trends in food and pharmaceutical applications of these intriguing molecules. PRACTICAL APPLICATIONS: Numerous AMPs have been reported in recent years as naturally present or released from food proteins upon enzymatic digestion during food processing, fermentation, or gastrointestinal transit. Particularly, food-released AMPs is a promising alternative to satisfy consumer demands for safe, ready-to-eat, extended shelf-life, fresh-tasting, and minimally processed foods, without chemical additives. The potential of several AMPs to inhibit foodborne pathogens is increasingly studied in various food matrices including dairy products, meat, fruits, and beverages. Although extensive progress has been made with respect to our understanding of AMPs structure/function, additional thorough investigation of the factors influencing peptide activity is required. The time has now come for the development of nutraceuticals and pharmaceutical products containing food-derived AMPs. Despite the several hurdles that must be overcome for the exploitation of AMPs, the features and developments discussed in this review offer a taste of future trends in food and pharmaceutical applications of these intriguing molecules.

SHP2 Drives Adaptive Resistance to ERK Signaling Inhibition in Molecularly Defined Subsets of ERK-Dependent Tumors.

Pharmacologic targeting of components of ERK signaling in ERK-dependent tumors is often limited by adaptive resistance, frequently mediated by feedback-activation of RTK signaling and rebound of ERK activity. Here, we show that combinatorial pharmacologic targeting of ERK signaling and the SHP2 phosphatase prevents adaptive resistance in defined subsets of ERK-dependent tumors. In each tumor that was sensitive to combined treatment, p(Y542)SHP2 induction was observed in response to ERK signaling inhibition. The strategy was broadly effective in TNBC models and tumors with RAS mutations at G12, whereas tumors with RAS(G13D) or RAS(Q61X) mutations were resistant. In addition, we identified a subset of BRAF(V600E) tumors that were resistant to the combined treatment, in which FGFR was found to drive feedback-induced RAS activation, independently of SHP2. Thus, we identify molecular determinants of response to combined ERK signaling and SHP2 inhibition in ERK-dependent tumors.

Processed eggshell membrane powder: Bioinspiration for an innovative wound healing product.

Non-healing wounds are a major health problem worldwide and a significant cause of morbidity and mortality. Effective treatments for acute and chronic skin wounds are the focus of intensive research. Eggshell membrane (ESM) is a natural proteinaceous by-product of the food industry and is suitable for biomedical applications. The objective of this study was to evaluate processed eggshell membrane powder (PEP) for the promotion of skin wound healing. PEP was characterized using proteomics and bioinformatics. Proteomic analysis of PEP identified 110 proteins, including structural proteins such as collagen and cysteine-rich eggshell membrane proteins (CREMPs) that together constitute about 40% of PEP. Functional annotation clustering showed various predicted functionalities related to wound healing including response to external stimulus, defense response, inflammatory response, and cell-substrate adhesion. The impact of PEP on wound healing was determined using the mouse excisional wound splinting model with a subsequent assessment by histopathology. PEP was found to significantly accelerate wound closure at days 3, 7, and 10. Histological assessment showed significantly thicker granulation tissue in wounds treated with PEP than non-treated controls at days 10 and 17. In addition, histological scoring showed higher levels of collagen deposition at day 10 in wounds treated with PEP, with limited inflammatory reaction. Therefore, PEP is a biocompatible and non-cytotoxic biomaterial that has great potential for development into a cost-effective wound healing product.

Processed eggshell membrane powder regulates cellular functions and increase MMP-activity important in early wound healing processes.

Avian eggshell membrane (ESM) is a natural biomaterial that has been used as an alternative natural bandage to cure wounds, and is available in large quantities from egg industries. We have previously demonstrated that processed eggshell membrane powder (PEP), aiming to be used in a low cost wound healing product, possesses anti-inflammatory properties. In this study, we further investigated effects of PEP on MMP activities in vitro (a dermal fibroblast cell culture system) and in vivo (a mouse skin wound healing model). Three days incubation with PEP in cell culture led to rearrangement of the actin-cytoskeleton and vinculin in focal adhesions and increased syndecan-4 shedding. In addition, we observed increased matrix metalloproteinase type 2 (MMP-2) enzyme activation, without effects on protein levels of MMP-2 or its regulators (membrane type 1 (MT1)-MMP and tissue inhibitor of matrix metalloproteinase type 2 (TIMP-2). Longer incubation (10 days) led to increased protein levels of MMP-2 and its regulators. We also observed an increased alpha-smooth muscle actin (α-SMA) production, suggesting an effect of PEP on myofibroblast differentiation. In vivo, using the mouse skin wound healing model, PEP treatment (3 days) increased MMP activity at the wound edges, along with increased MMP-2 and MMP-9 protein levels, and increased keratinocyte cell proliferation. Altogether, our data suggest PEP stimulates MMP activity, and with a positive effect on early cellular events during wound healing.

In-depth comparative analysis of the chicken eggshell membrane proteome.

The avian eggshell membrane (ESM) is stabilized by extensive cross-linkages, making the identification of its protein constituents technically challenging. Herein, we applied various extraction/solubilization conditions followed by proteomic analysis to characterize the protein constituents of ESM derived from the unfertilized chicken eggs. The egg white and eggshell proteomes (including previous published work) were determined and compared to ESM to identify proteins that are relatively or highly specific to ESM. Merging the results from different extraction/solubilization conditions with various proteomes allowed the identification of 472, 225, and 488 proteins in the ESM, egg white, and eggshell proteomes, respectively. Of these, 163 and 124 proteins were relatively or highly specific to ESM, respectively. GO term analysis of the common proteins and ESM unique proteins generated 8 and 9 significantly enriched functional groups, respectively. Different families of proteins that were identified as ESM-specific included collagens, CREMPs, histones, AvBDs, lysyl oxidase-like 2 (LOXL2), and ovocalyxin-36 (OCX36). These proteins serve as a foundation for the mechanically stable ESM that rests upon the egg white compartment and is a physical barrier against pathogen invasion. Overall, our results highlight the structural nature of the ESM constituents that are relevant to various biomedical applications, such as wound healing. BIOLOGICAL SIGNIFICANCE: The eggshell membranes (ESM) are a highly resilient double-layered fibrous meshwork that is secreted while the forming egg transits a specialized oviduct segment, the white isthmus. The ESM protects against pathogen invasion and provides a platform for nucleation of the calcitic eggshell (ES). ESM is greatly stabilized by the extensive desmosine, isodesmosine and disulfide cross-linkages which make the identification of its protein constituents by standard proteomic approaches technically challenging. Comparative proteomic analyses of ESM, egg white, and ES proteins showed proteins groups that are relatively or highly specific to ESM. These groups of proteins serve as a foundation for the mechanically stable ESM that rests upon the egg white compartment and is a physical barrier against pathogen invasion. These features are essential for eggshell quality and for the prevention of pathogen invasion which reinforce food safety of the table egg.

An Integrated Model of RAF Inhibitor Action Predicts Inhibitor Activity against Oncogenic BRAF Signaling.

The complex biochemical effects of RAF inhibitors account for both the effectiveness and mechanisms of resistance to these drugs, but a unified mechanistic model has been lacking. Here we show that RAF inhibitors exert their effects via two distinct allosteric mechanisms. Drug resistance due to dimerization is determined by the position of the αC helix stabilized by inhibitor, whereas inhibitor-induced RAF priming and dimerization are the result of inhibitor-induced formation of the RAF/RAS-GTP complex. The biochemical effect of RAF inhibitor in cells is the combined outcome of the two mechanisms. Therapeutic strategies including αC-helix-IN inhibitors are more effective in multiple mutant BRAF-driven tumor models, including colorectal and thyroid BRAF(V600E) cancers, in which first-generation RAF inhibitors have been ineffective.