Multipronged regulation of autophagy and apoptosis: emerging role of TRIM proteins.

TRIM proteins are characterized by their conserved N-terminal RING, B-box, and coiled-coil domains. These proteins are efficient regulators of autophagy, apoptosis, and innate immune responses and confer immunity against viruses and bacteria. TRIMs function as receptors or scaffold proteins that target substrates for autophagy-mediated degradation. Most TRIMs interact with the BECN1-ULK1 complex to form TRIMosomes, thereby efficiently targeting substrates to autophagosomes. They regulate the functions of ATG proteins through physical interactions or ubiquitination. TRIMs affect the lipidation of MAP1LC3B1 to form MAP1LC3B2, which is a prerequisite for phagophore and autophagosome formation. In addition, they regulate MTOR kinase and TFEB, thereby regulating the expression of ATG genes. TRIM proteins are efficient regulators of apoptosis and are crucial for regulating cell proliferation and tumor formation. Many TRIM proteins regulate intrinsic and extrinsic apoptosis via the cell surface receptors TGFBR2, TNFRSF1A, and FAS. Mitochondria modulate the anti- and proapoptotic functions of BCL2, BAX, BAK1, and CYCS. These proteins use a multipronged approach to regulate the intrinsic and extrinsic apoptotic pathways, culminating in coordinated activation or inhibition of the initiator and executor CASPs. Furthermore, TRIMs can have a dual effect in determining cell fate and are therefore crucial for cellular homeostasis. In this review, we discuss mechanistic insights into the role of TRIM proteins in regulating autophagy and apoptosis, which can be used to better understand cellular physiology. These findings can be used to develop therapeutic interventions to prevent or treat multiple genetic and infectious diseases.

PRKAA2, MTOR, and TFEB in the regulation of lysosomal damage response and autophagy.

Lysosomes function as critical signaling hubs that govern essential enzyme complexes. LGALS proteins (LGALS3, LGALS8, and LGALS9) are integral to the endomembrane damage response. If ESCRT fails to rectify damage, LGALS-mediated ubiquitination occurs, recruiting autophagy receptors (CALCOCO2, TRIM16, and SQSTM1) and VCP/p97 complex containing UBXN6, PLAA, and YOD1, initiating selective autophagy. Lysosome replenishment through biogenesis is regulated by TFEB. LGALS3 interacts with TFRC and TRIM16, aiding ESCRT-mediated repair and autophagy-mediated removal of damaged lysosomes. LGALS8 inhibits MTOR and activates TFEB for ATG and lysosomal gene transcription. LGALS9 inhibits USP9X, activates PRKAA2, MAP3K7, ubiquitination, and autophagy. Conjugation of ATG8 to single membranes (CASM) initiates damage repair mediated by ATP6V1A, ATG16L1, ATG12, ATG5, ATG3, and TECPR1. ATG8ylation or CASM activates the MERIT system (ESCRT-mediated repair, autophagy-mediated clearance, MCOLN1 activation, Ca2+ release, RRAG-GTPase regulation, MTOR modulation, TFEB activation, and activation of GTPase IRGM). Annexins ANAX1 and ANAX2 aid damage repair. Stress granules stabilize damaged membranes, recruiting FLCN-FNIP1/2, G3BP1, and NUFIP1 to inhibit MTOR and activate TFEB. Lysosomes coordinate the synergistic response to endomembrane damage and are vital for innate and adaptive immunity. Future research should unveil the collaborative actions of ATG proteins, LGALSs, TRIMs, autophagy receptors, and lysosomal proteins in lysosomal damage response.

An injectable hydrogel of porcine cholecyst extracellular matrix for accelerated wound healing.

Hydrogel formulations of xenogeneic extracellular matrices have been widely used for topical wound care because of their exceptional tunability over other formulations like lyophilized sheets, powders, non-injectable gels, pastes, and ointments. This is important in the treatment of wounds with irregular shapes and depth. This study identified an injectable hydrogel formulation of porcine cholecyst extracellular matrix (60%) in medical-grade carboxymethyl cellulose (40%) as vehicle and evaluated its biomaterial properties. Further, an in-depth evaluation of in vivo wound healing efficacy was conducted in a rat full-thickness skin excision wound healing model, which revealed that the hydrogel formulation accelerated wound healing process compared to wounds treated with a commercial formulation and untreated wounds. The hydrogel appeared to have promoted a desirable pro-regenerative tissue reaction predominated by Th2 helper lymphocytes and M2 macrophages as well as an effective collagen remodeling indicative of diminished scarring. In conclusion, the porcine cholecyst extracellular matrix injectable hydrogel formulation appeared to be a promising candidate formulation as an advanced wound care biomaterial for faster healing of skin wounds with minimal scarring.

CRISPR­based diagnostic approaches: Implications for rapid management of future pandemics (Review).

Sudden viral outbreaks have increased in the early part of the 21st century, such as those of severe acute respiratory syndrome coronavirus (SARS­CoV), Middle East respiratory syndrome corona virus, and SARS­CoV­2, owing to increased human access to wildlife habitats. Therefore, the likelihood of zoonotic transmission of human­associated viruses has increased. The emergence of severe acute respiratory syndrome coronavirus 2 in China and its spread worldwide within months have highlighted the need to be ready with advanced diagnostic and antiviral approaches to treat newly emerging diseases with minimal harm to human health. The gold­standard molecular diagnostic approaches currently used are time­consuming, require trained personnel and sophisticated equipment, and therefore cannot be used as point­of­care devices for widespread monitoring and surveillance. Clustered regularly interspaced short palindromic repeats (CRISPR)­associated (Cas) systems are widespread and have been reported in bacteria, archaea and bacteriophages. CRISPR­Cas systems are organized into CRISPR arrays and adjacent Cas proteins. The detection and in­depth biochemical characterization of class 2 type V and VI CRISPR­Cas systems and orthologous proteins such as Cas12 and Cas13 have led to the development of CRISPR­based diagnostic approaches, which have been used to detect viral diseases and distinguish between serotypes and subtypes. CRISPR­based diagnostic approaches detect human single nucleotide polymorphisms in samples from patients with cancer and are used as antiviral agents to detect and destroy viruses that contain RNA as a genome. CRISPR­based diagnostic approaches are likely to improve disease detection methods in the 21st century owing to their ease of development, low cost, reduced turnaround time, multiplexing and ease of deployment. The present review discusses the biochemical properties of Cas12 and Cas13 orthologs in viral disease detection and other applications. The present review expands the scope of CRISPR­based diagnostic approaches to detect diseases and fight viruses as antivirals.

Point-of-care optical devices in clinical imaging and screening: A review on the state of the art.

Integration of optical technologies in biomedical sciences permitted light manipulation at smaller time-length scales for specific detection and imaging of biological entities. Similarly, advances in consumer electronics and wireless telecommunications strengthened the development of affordable and portable point-of-care (POC) optical devices, circumventing the necessity of conventional clinical analyses by trained personnel. However, many of the POC optical technologies translated from bench to bedside require industrial support for their commercialization and dissemination to the population. This review aims to demonstrate the intriguing progress and challenges of emerging POC devices utilizing optics for clinical imaging (depth-resolved and perfusion imaging) and screening (infections, cancer, cardiac health, and haematologic disorders) with a focus on research studies over the previous 3 years. Special attention is given to POC optical devices that can be utilized in resource-constrained environments.

A porcine cholecystic extracellular matrix conductive scaffold for cardiac tissue repair.

Cardiac tissue engineering using cells, scaffolds or signaling molecules is a promising approach for replacement or repair of damaged myocardium. This study addressed the contemporary need for a conductive biomimetic nanocomposite scaffold for cardiac tissue engineering by examining the use of a gold nanoparticle-incorporated porcine cholecystic extracellular matrix for the same. The scaffold had an electrical conductivity (0.74 ± 0.03 S/m) within the range of native myocardium. It was a suitable substrate for the growth and differentiation of cardiomyoblast (H9c2) as well as rat mesenchymal stem cells to cardiomyocyte-like cells. Moreover, as an epicardial patch, the scaffold promoted neovascularisation and cell proliferation in infarcted myocardium of rats. It was concluded that the gold nanoparticle coated cholecystic extracellular matrix is a prospective biomaterial for cardiac tissue engineering.

Gelatin-Modified Cholecyst-Derived Scaffold Promotes Angiogenesis and Faster Healing of Diabetic Wounds.

Compromised angiogenesis is a major factor contributing delayed wound healing in diabetic patients. Graft-assisted healing using synthetic and natural scaffolds supplemented with micromolecules for stimulating angiogenesis is the contemporary tissue engineering strategy for treating diabetic wounds. This study deployed the carbodiimide chemical reaction for coupling gelatin with a porcine cholecyst-derived scaffold (CDS) for enhancing angiogenesis. The modification was confirmed by the trinitrobenzene sulfonic acid assay and scanning electron microscopy. The gelatin-coupled CDS was more stable than the bare CDS in an in vitro proteolytic environment and allowed survival of keratinocytes (HaCaT), indicating its suitability for chronic skin wound application. The gelatin coupling brought significant improvement in the in vitro angiogenic potential of the CDS as evident from the enhanced viability of endothelial cells. An in ovo chorioallantoic membrane assay also demonstrated the angiogenic potential of the modified scaffold. Further, the modified scaffold promoted angiogenesis and aided faster healing of full-thickness excision wounds in streptozotocin-induced diabetic rats. It is concluded that the gelatin-coupled CDS is a potential advanced wound care material for treating diabetic wounds.

Surface Modification of Polypropylene Mesh with a Porcine Cholecystic Extracellular Matrix Hydrogel for Mitigating Host Tissue Reaction.

Polypropylene (PP) meshes are widely used for repairing skeletal muscle defects like abdominal hernia despite the chances of undesirable pro-inflammatory tissue reactions that demand revision surgeries in about 45% of cases. Attempts have been made to address the problem by modifying the mesh surface and architecture. These procedures have yielded only incremental improvements in the management of overall postoperative complications, and the search for a clinically viable therapeutic strategy continues. This study deployed a tissue engineering approach for mitigating PP-induced adverse tissue reaction by dip-coating the mesh with a hydrogel formulation of the porcine cholecystic extracellular matrix (CECM). The biomaterial properties of the CECM hydrogel-coated PP (C-PP) meshes were studied and their biocompatibility was evaluated by in vitro and in vivo tests based on ISO standards. Further, the nature of tissue reactions induced by the hydrogel-coated mesh and a commercial PP hernia repair graft was compared in a rat model of partial-thickness abdominal wall defect. Histomorphologically, in comparison with the PP graft-induced tissue reaction, C-PP caused a favorable graft-acceptance response characterized by reduced numbers of pro-inflammatory M1 macrophages and cytotoxic lymphocytes. Remarkably, the differential inflammatory response of the C-PP graft-assisted healing was associated with a fibrotic reaction predominated by deposition of type I collagen rather than type III collagen, as desired during skeletal muscle repair. It was concluded that the CECM hydrogel is a potential biomaterial for surface modification of polymeric biomedical devices.

A cholecystic extracellular matrix-based hybrid hydrogel for skeletal muscle tissue engineering.

Tailoring the properties of extracellular matrix (ECM) based hydrogels by conjugating with synthetic polymers is an emerging method for designing hybridhydrogels for a wide range of tissue engineering applications. In this study, poly(ethylene glycol) diacrylate (PEGDA), a synthetic polymer at variable concentrations (ranging from 0.2 to 2% wt/vol) was conjugated with porcine cholecyst derived ECM (C-ECM) (1% wt/vol) and prepared a biosynthetic hydrogel having enhanced physico-mechanical properties, as required for skeletal muscle tissue engineering. The C-ECM was functionalized with acrylate groups using activated N-hydroxysuccinimide ester-based chemistry and then conjugated with PEGDA via free-radical polymerization in presence of ammonium persulfate and ascorbic acid. The physicochemical characteristics of the hydrogels were evaluated by Fourier transform infrared spectroscopy and environmental scanning electron microscopy. Further, the hydrogel properties were studied by evaluating rheology, swelling, gelation time, percentage gel fraction, in vitro degradation, and mechanical strength. Biocompatibility of the gel formulations were assessed using the C2C12 skeletal myoblast cells. The hydrogel formulations containing 0.2 and 0.5% wt/vol of PEGDA were non-cytotoxic and found suitable for growth and proliferation of skeletal myoblasts. The study demonstrated a method for modulating the properties of ECM hydrogels through conjugation with bio-inert polymers for skeletal muscle tissue engineering applications.