Outer membrane proteins (Omps) of Gram-negative bacteria represent porins involved in a wide range of virulence- and pathogenesis-related cellular processes, including transport, adhesion, penetration, and the colonization of host tissues. Most outer membrane porins share a specific spatial structure called the β-barrel that provides their structural integrity within the membrane lipid bilayer. Recent data suggest that outer membrane proteins from several bacterial species are able to adopt the amyloid state alternative to their β-barrel structure. Amyloids are protein fibrils with a specific spatial structure called the cross-β that gives them an unusual resistance to different physicochemical influences. Various bacterial amyloids are known to be involved in host-pathogen and host-symbiont interactions and contribute to colonization of host tissues. Such an ability of outer membrane porins to adopt amyloid state might represent an important mechanism of bacterial virulence. In this work, we investigated the amyloid properties of the OmpC and OmpF porins from two species belonging to Enterobacteriaceae family, Escherichia coli, and Salmonella enterica. We demonstrated that OmpC and OmpF of E. coli and S. enterica form toxic fibrillar aggregates in vitro. These aggregates exhibit birefringence upon binding Congo Red dye and show characteristic reflections under X-ray diffraction. Thus, we confirmed amyloid properties for OmpC of E. coli and demonstrated bona fide amyloid properties for three novel proteins: OmpC of S. enterica and OmpF of E. coli and S. enterica in vitro. All four studied porins were shown to form amyloid fibrils at the surface of E. coli cells in the curli-dependent amyloid generator system. Moreover, we found that overexpression of recombinant OmpC and OmpF in the E. coli BL21 strain leads to the formation of detergent- and protease-resistant amyloid-like aggregates and enhances the birefringence of bacterial cultures stained with Congo Red. We also detected detergent- and protease-resistant aggregates comprising OmpC and OmpF in S. enterica culture. These data are important in the context of understanding the structural dualism of Omps and its relation to pathogenesis.

Hereditary neurodegenerative diseases (hNDDs) such as Alzheimer’s, Parkinson’s, Huntington’s disease, and others are primarily characterized by their progressive nature, severely compromising both the cognitive and motor abilities of patients. The underlying genetic component in hNDDs contributes to disease risk, creating a complex genetic landscape. Considering the fact that growth factors play crucial roles in regulating cellular processes, such as proliferation, differentiation, and survival, they could have therapeutic potential for hNDDs, provided appropriate dosing and safe delivery approaches are ensured. This article presents a detailed overview of growth factors, and explores their therapeutic potential in treating hNDDs, emphasizing their roles in neuronal survival, growth, and synaptic plasticity. However, challenges such as proper dosing, delivery methods, and patient variability can hinder their clinical application.

The development of gene therapies for rare hereditary disorders is hindered by small patient cohorts, incomplete characterization of natural disease history, and the impracticality of conducting long-term clinical trials. Surrogate biomarkers—quantifiable indicators predictive of clinical outcomes—represent a promising strategy to accelerate the evaluation of therapeutic efficacy. This review examines the role of surrogate endpoints in gene therapy, outlining essential validation criteria, including biological plausibility, analytical reproducibility, and clinical predictive value. Regulatory frameworks governing surrogate markers in the United States, European Union, Russia, Japan, China, and Canada are compared, with emphasis on mechanisms for expedited or conditional approval. Challenges associated with biomarker validation and extrapolation in the context of rare diseases are discussed, alongside future perspectives that integrate multi-omics technologies and artificial intelligence to enhance biomarker discovery and facilitate regulatory acceptance.

Amyloids are protein fibrils with a characteristic cross-β structure that is responsible for the unusual resistance of amyloids to various physical and chemical factors, as well as numerous pathogenic and functional consequences of amyloidogenesis. The greatest diversity of functional amyloids was identified in bacteria. The majority of bacterial amyloids are involved in virulence and pathogenesis either via facilitating formation of biofilms and adaptation of bacteria to colonization of a host organism or through direct regulation of toxicity. Recent studies have shown that, beside their commonly known activity, amyloids may be involved in the spatial regulation of proteome by modulating aggregation of other amyloidogenic proteins with multiple functional or pathological effects. Although the studies on the role of microbiome-produced amyloids in the development of amyloidoses in humans and animals have only been started, it is clear that humans as holobionts contain amyloids encoded not only by the host genome, but also by microorganisms that constitute the microbiome. Amyloids acquired from external sources (e.g., food) can interact with holobiont amyloids and modulate the effects of bacterial and host amyloids, thus adding another level of complexity to the holobiont-associated amyloid network. In this review, we described bacterial amyloids directly or indirectly involved in disease pathogenesis in humans and discussed the significance of bacterial amyloids in the three-component network of holobiont-associated amyloids.

SARS-CoV-2 main protease (M^pro) is essential for viral polyprotein processing and represents a prime target for antiviral drug discovery. However, most available screening strategies rely on computational predictions or cell-free biochemical approaches that provide limited functional context and often require specialized instrumentation, while mammalian cell-based models remain costly and require high biosafety levels. Accordingly, there remains a shortage of simple, rapid, and biosafe functional screening tools suitable for early-stage prioritization of potential M^pro inhibitors, particularly those derived from natural sources and in urgent situations such as the COVID-19 pandemic. In this study, a bacterial colorimetric reporter assay was developed that directly links SARS-CoV-2 M^pro activity to β-galactosidase function in Escherichia coli. To the best of our knowledge, the developed assay represents the first bacterial colorimetric model for functional detection of SARS-CoV-2 M^pro inhibition based on a phenotypic readout. The assay enables the rapid visual detection of protease inhibition on X-gal-containing medium and provides a cost-effective and biosafe platform for prioritizing candidate inhibitors, under standard laboratory conditions, prior to further validation. Due to its bacterial expression context, this assay is intended for functional screening to provide the most promising candidate compounds and/or extracts for subsequent biochemical or mammalian cell-based validation; it is not intended to determine quantitative potency or to replace further validation approaches. It should be noted that the selective compound uptake in E. coli restricts the range of chemical compositions that can be evaluated using this method. Therefore, proof-of-concept application was demonstrated using pomegranate juice, a representative natural inhibitor source, rather than most currently known specific M^pro inhibitors. In addition, other plant-derived preparations, including rhubarb, grape, and red/black currant juices, were tested demonstrating the assay’s applicability to diverse natural matrices.

Crystal (Cry) toxins produced by Bacillus thuringiensis are highly specific insecticidal proteins that are widely used as biological insecticides and function through proteolytic activation in insect midguts, which generates a three-domain toxic core responsible for membrane disruption as well as cleaved N- and C-terminal domains. While the structure and function of this core have been extensively characterized, the fate of the cleaved C-terminal region remains poorly understood. Amyloids are protein fibrils with a characteristic cross-β structure that participate in a wide range of functions in bacteria. Here, using bioinformatic approaches, we demonstrated that the C-terminal domains of Cry are rich in potentially amyloidogenic regions. We investigated the aggregation behavior of C-terminal domains from Cry1Ea11 and Cry1Ab12 proteins using a combination of biochemical and structural approaches. We found that the C-terminal domains of Cry1Ea11 and Cry1Ab12 undergo self-assembly in vitro, forming aggregates with fibrillar morphology. These assemblies bind amyloid-associated dyes, exhibit partial resistance to ionic detergents and proteolytic digestion, and display X-ray diffraction patterns confirming their amyloid properties. Functional assessment in insect cells showed that although these amyloids do not induce detectable cytotoxicity, amyloids of the C-terminal domains of Cry1Ea11 alter the readouts of the MTT assay in a manner consistent with previously described amyloid-associated effects on cellular formazan handling. Together, our findings demonstrate that the C-terminal domains of Cry toxins, which are released in the insect midgut as a result of proteolytic cleavage, are able to form bona fide amyloids that affect the metabolism of insect cells.