Machine learning-assisted image label-free smartphone platform for rapid segmentation and robust multi-urinalysis.

This study presents a groundbreaking approach for the early detection of chronic kidney disease (CKD) and other urological disorders through an image-label-free, multi-dipstick identification method, eliminating the need for complex machinery, label libraries, or preset coordinates. Our research successfully identified reaction pads on 187 multi-dipsticks, each with 11 pads, leveraging machine learning algorithms trained on human urine data. This technique aims to surpass traditional colourimetric methods and concentration-colour curve fitting, offering more robust and precise community screening and home monitoring capabilities. The developed algorithms enhance the generalizability of machine learning models by extracting primary colours and correcting urine colours on each reaction pad. This method's cost-effectiveness and portability are significant, as it requires no additional equipment beyond a standard smartphone. The system's performance rivals professional medical equipment without auxiliary lighting or flash under regular indoor light conditions, effectively managing false positives and negatives across various categories with remarkable accuracy. In a controlled experimental setting, we found that random forest algorithms, based on a Bagging strategy and applied in the HSV colour space, showed optimal results in smartphone-assisted urinalysis. This study also introduces a novel urine colour correction method, significantly improving machine learning model performance. Additionally, ISO parameters were identified as crucial factors influencing the accuracy of smartphone-based urinalysis in the absence of additional lighting or optical configurations, highlighting the potential of this technology in low-resource settings.

Liquid-liquid phase separation of amyloid-ß oligomers modulates amyloid fibrils formation.

Soluble amyloid-ß oligomers (AßOs) are proposed to instigate and mediate the pathology of Alzheimer's disease, but the mechanisms involved are not clear. In this study, we reported that AßOs can undergo liquid-liquid phase separation (LLPS) to form liquid-like droplets in vitro. We determined that AßOs exhibited an α-helix conformation in a membrane-mimicking environment of SDS. Importantly, SDS is capable of reconfiguring the assembly of different AßOs to induce their LLPS. Moreover, we found that the droplet formation of AßOs was promoted by strong hydrated anions and weak hydrated cations, suggesting that hydrophobic interactions play a key role in mediating phase separation of AßOs. Finally, we observed that LLPS of AßOs can further promote Aß to form amyloid fibrils, which can be modulated by (-)-epigallocatechin gallate. Our study highlights amyloid oligomers as an important entity involved in protein liquid-to-solid phase transition and reveals the regulatory role of LLPS underlying amyloid protein aggregation, which may be relevant to the pathological process of Alzheimer's disease.

Liquid-liquid phase separation of RBGD2/4 is required for heat stress resistance in Arabidopsis.

As sessile organisms, plants are highly sensitive to environmental stresses. In response to stresses, globally repressed translation initiation leads to stress granule (SG) formation. Protein liquid-liquid phase separation (LLPS) contributes to SG formation, but a direct link between protein LLPS and stress resistance has not yet been found in plants. Here, we report that two RNA-binding proteins, RBGD2 and RBGD4, function redundantly to improve heat resistance in Arabidopsis. RBGD2 and RBGD4 undergo LLPS in vitro and condense into heat-induced SGs in vivo via tyrosine residue array (TRA). Importantly, disrupting LLPS by mutating TRA abolishes RBGD2/4 condensation in SGs and impairs their protective function against heat stress (HS). Further study found that upon HS, the RBGD2/4 interaction network expands with additional SG proteins and heat-responsive mRNA. Our work shows a mechanistic basis that underlies protein LLPS in HS response in plants and suggests manipulation of protein LLPS as a general strategy to improve plant stress resistance.

Study on the Control of Dichloroacetonitrile Generation by Two-Point Influent Activated Carbon-Quartz Sand Biofilter.

Aiming at the problem of highly toxic Nitrogenous disinfection by-products (N-DBPs) produced by disinfection in the process of drinking water, two-point influent activated carbon-quartz sand biofilter, activated carbon-quartz sand biofilter, and quartz sand biofilter are selected. This study takes typical N-DBPs Dichloroacetonitrile (DCAN) as the research object and aromatic amino acid Tyrosine (Tyr), an important precursor of DCAN, as the model precursor. By measuring the changes of conventional pollutants in different biofilters, and the changes of Tyr, the output DCAN formation potential of the biofilters, this article investigates the control of DCAN generation of the two-point influent activated carbon-quartz sand biofilter. The results show that the average Tyr removal rate of the three biofilters during steady operation is 73%, 50%, and 20%, respectively, while the average effluent DCAN generation potential removal rate is 78%, 52%, and 23%, respectively. The two-point influent activated carbon-sand biofilter features the highest removal rate. The two-point water intake improves the hypoxia problem of the lower filter material of the activated carbon-quartz sand biofilter, and at the same time, the soluble microbial products produced by microbial metabolism can be reduced by an appropriate carbon sand ratio, which is better than traditional quartz sand filters and activated carbon-quartz sand biofilters in the performance of controlling the precursors of N-DBPs.

The nuclear localization sequence mediates hnRNPA1 amyloid fibril formation revealed by cryoEM structure.

Human heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) serves as a key regulating protein in RNA metabolism. Malfunction of hnRNPA1 in nucleo-cytoplasmic transport or dynamic phase separation leads to abnormal amyloid aggregation and neurodegeneration. The low complexity (LC) domain of hnRNPA1 drives both dynamic phase separation and amyloid aggregation. Here, we use cryo-electron microscopy to determine the amyloid fibril structure formed by hnRNPA1 LC domain. Remarkably, the structure reveals that the nuclear localization sequence of hnRNPA1 (termed PY-NLS), which is initially known to mediate the nucleo-cytoplamic transport of hnRNPA1 through binding with karyopherin-ß2 (Kapß2), represents the major component of the fibril core. The residues that contribute to the binding of PY-NLS with Kapß2 also exert key molecular interactions to stabilize the fibril structure. Notably, hnRNPA1 mutations found in familial amyotrophic lateral sclerosis (ALS) and multisystem proteinopathoy (MSP) are all involved in the fibril core and contribute to fibril stability. Our work illuminates structural understandings of the pathological amyloid aggregation of hnRNPA1 and the amyloid disaggregase activity of Kapß2, and highlights the multiple roles of PY-NLS in hnRNPA1 homeostasis.

Hsp27 chaperones FUS phase separation under the modulation of stress-induced phosphorylation.

Protein phase separation drives the assembly of membraneless organelles, but little is known about how these membraneless organelles are maintained in a metastable liquid- or gel-like phase rather than proceeding to solid aggregation. Here, we find that human small heat-shock protein 27 (Hsp27), a canonical chaperone that localizes to stress granules (SGs), prevents FUS from undergoing liquid-liquid phase separation (LLPS) via weak interactions with the FUS low complexity (LC) domain. Remarkably, stress-induced phosphorylation of Hsp27 alters its activity, leading Hsp27 to partition with FUS LC to preserve the liquid phase against amyloid fibril formation. NMR spectroscopy demonstrates that Hsp27 uses distinct structural mechanisms for both functions. Our work reveals a fine-tuned regulation of Hsp27 for chaperoning FUS into either a polydispersed state or a LLPS state and suggests an essential role for Hsp27 in stabilizing the dynamic phase of stress granules.

Exploiting mammalian low-complexity domains for liquid-liquid phase separation-driven underwater adhesive coatings.

Many biological materials form via liquid-liquid phase separation (LLPS), followed by maturation into a solid-like state. Here, using a biologically inspired assembly mechanism designed to recapitulate these sequential assemblies, we develop ultrastrong underwater adhesives made from engineered proteins containing mammalian low-complexity (LC) domains. We show that LC domain-mediated LLPS and maturation substantially promotes the wetting, adsorption, priming, and formation of dense, uniform amyloid nanofiber coatings on diverse surfaces (e.g., Teflon), and even penetrating difficult-to-access locations such as the interiors of microfluidic devices. Notably, these coatings can be deposited on substrates over a broad range of pH values (3 to 11) and salt concentrations (up to 1 M NaCl) and exhibit strong underwater adhesion performance. Beyond demonstrating the utility of mammalian LC domains for driving LLPS in soft materials applications, our study illustrates a powerful example of how combining LLPS with subsequent maturation steps can be harnessed for engineering protein-based materials.

Structural basis for reversible amyloids of hnRNPA1 elucidates their role in stress granule assembly.

Subcellular membrane-less organelles consist of proteins with low complexity domains. Many of them, such as hnRNPA1, can assemble into both a polydisperse liquid phase and an ordered solid phase of amyloid fibril. The former mirrors biological granule assembly, while the latter is usually associated with neurodegenerative disease. Here, we observe a reversible amyloid formation of hnRNPA1 that synchronizes with liquid-liquid phase separation, regulates the fluidity and mobility of the liquid-like droplets, and facilitates the recruitment of hnRNPA1 into stress granules. We identify the reversible amyloid-forming cores of hnRNPA1 (named hnRACs). The atomic structures of hnRACs reveal a distinct feature of stacking Asp residues, which contributes to fibril reversibility and explains the irreversible pathological fibril formation caused by the Asp mutations identified in familial ALS. Our work characterizes the structural diversity and heterogeneity of reversible amyloid fibrils and illuminates the biological function of reversible amyloid formation in protein phase separation.

Modular genetic design of multi-domain functional amyloids: insights into self-assembly and functional properties.

Engineering functional amyloids through a modular genetic strategy represents new opportunities for creating multifunctional molecular materials with tailored structures and performance. Despite important advances, how fusion modules affect the self-assembly and functional properties of amyloids remains elusive. Here, using Escherichia coli curli as a model system, we systematically studied the effect of flanking domains on the structures, assembly kinetics and functions of amyloids. The designed amyloids were composed of E. coli biofilm protein CsgA (as amyloidogenic cores) and one or two flanking domains, consisting of chitin-binding domains (CBDs) from Bacillus circulans chitinase, and/or mussel foot proteins (Mfps). Incorporation of fusion domains did not disrupt the typical ß-sheet structures, but indeed affected assembly rate, morphology, and stiffness of resultant fibrils. Consequently, the CsgA-fusion fibrils, particularly those containing three domains, were much shorter than the CsgA-only fibrils. Furthermore, the stiffness of the resultant fibrils was heavily affected by the structural feature of fusion domains, with ß-sheet-containing domains tending to increase the Young's modulus while random coil domains decreasing the Young's modulus. In addition, fibrils containing CBD domains showed higher chitin-binding activity compared to their CBD-free counterparts. The CBD-CsgA-Mfp3 construct exhibited significantly lower binding activity than Mfp5-CsgA-CBD due to inappropriate folding of the CBD domain in the former construct, in agreement with results based upon molecular dynamics modeling. Our study provides new insights into the assembly and functional properties of designer amyloid proteins with increasing complex domain structures and lays the foundation for the future design of functional amyloid-based structures and molecular materials.

Fibril Self-Assembly of Amyloid-Spider Silk Block Polypeptides.

Because of their association with debilitating diseases and their potential applications in developing novel bionanomaterials, highly ordered amyloid fibrils have recently received considerable attention. While many studies have thus far focused on amyloid fibrils made with short peptides containing just one steric zipper-forming segment of native amyloid proteins, the self-assembly of proteins containing multiple steric zipper-forming segments has been rarely explored. Here we develop a strategy to create four block polypeptides, each containing 16 repeats of a zipper-forming segment from four different amyloid morphological classes. All four block polypeptides self-assemble into fibrils that display the cross-ß structure characteristic of amyloids. These amyloid-spider silk block polypeptides displayed fast self-assembly kinetics, and their fibrils exhibited high thermal stability. These novel synthetic amyloids provide insights into the self-assembly of proteins containing multiple zipper-forming segments, and our approach of creating block polypeptide fibrils could be used to expand the capability of amyloid-based bionanomaterials.

PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins.

Mutations in RNA-binding proteins (RBPs) localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregation, which is a pathological hallmark of various neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In this study, we investigate the function of poly(ADP-ribosyl)ation (PARylation), an important PTM involved in DNA damage repair and cell death, in RNP granule-related neurodegeneration. We reveal that PARylation levels are a major regulator of the assembly-disassembly dynamics of RNP granules containing disease-related RBPs, hnRNP A1 and TDP-43. We find that hnRNP A1 can both be PARylated and bind to PARylated proteins or poly(ADP-ribose) (PAR). We further uncover that PARylation of hnRNP A1 at K298 controls its nucleocytoplasmic transport, whereas PAR-binding via the PAR-binding motif (PBM) of hnRNP A1 regulates its association with stress granules. Moreover, we reveal that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, both genetic and pharmacological inhibition of PARP mitigates hnRNP A1- and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, our findings suggest a novel and crucial role for PARylation in regulating the dynamics of RNP granules, and that dysregulation in PARylation and PAR levels may contribute to ALS disease pathogenesis by promoting protein aggregation.

Amyloid fibril structure of α-synuclein determined by cryo-electron microscopy.

α-Synuclein (α-syn) amyloid fibrils are the major component of Lewy bodies, which are the pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. High-resolution structure of α-syn fibril is important for understanding its assembly and pathological mechanism. Here, we determined a fibril structure of full-length α-syn (1-140) at the resolution of 3.07 Å by cryo-electron microscopy (cryo-EM). The fibrils are cytotoxic, and transmissible to induce endogenous α-syn aggregation in primary neurons. Based on the reconstructed cryo-EM density map, we were able to unambiguously build the fibril structure comprising residues 37-99. The α-syn amyloid fibril structure shows two protofilaments intertwining along an approximate 21 screw axis into a left-handed helix. Each protofilament features a Greek key-like topology. Remarkably, five out of the six early-onset PD familial mutations are located at the dimer interface of the fibril (H50Q, G51D, and A53T/E) or involved in the stabilization of the protofilament (E46K). Furthermore, these PD mutations lead to the formation of fibrils with polymorphic structures distinct from that of the wild-type. Our study provides molecular insight into the fibrillar assembly of α-syn at the atomic level and sheds light on the molecular pathogenesis caused by familial PD mutations of α-syn.

Atomic structures of FUS LC domain segments reveal bases for reversible amyloid fibril formation.

Thermostable cross-ß structures are characteristic of pathological amyloid fibrils, but these structures cannot explain the reversible nature of fibrils formed by RNA-binding proteins such as fused in sarcoma (FUS), involved in RNA granule assembly. Here, we find that two tandem (S/G)Y(S/G) motifs of the human FUS low-complexity domain (FUS LC) form reversible fibrils in a temperature- and phosphorylation-dependent manner. We named these motifs reversible amyloid cores, or RAC1 and RAC2, and determined their atomic structures in fibrillar forms, using microelectron and X-ray diffraction techniques. The RAC1 structure features an ordered-coil fibril spine rather than the extended ß-strand typical of amyloids. Ser42, a phosphorylation site of FUS, is critical in the maintenance of the ordered-coil structure, which explains how phosphorylation controls fibril formation. The RAC2 structure shows a labile fibril spine with a wet interface. These structures illuminate the mechanism of reversible fibril formation and dynamic assembly of RNA granules.

Diverse Supramolecular Nanofiber Networks Assembled by Functional Low-Complexity Domains.

Self-assembling supramolecular nanofibers, common in the natural world, are of fundamental interest and technical importance to both nanotechnology and materials science. Despite important advances, synthetic nanofibers still lack the structural and functional diversity of biological molecules, and the controlled assembly of one type of molecule into a variety of fibrous structures with wide-ranging functional attributes remains challenging. Here, we harness the low-complexity (LC) sequence domain of fused in sarcoma (FUS) protein, an essential cellular nuclear protein with slow kinetics of amyloid fiber assembly, to construct random copolymer-like, multiblock, and self-sorted supramolecular fibrous networks with distinct structural features and fluorescent functionalities. We demonstrate the utilities of these networks in the templated, spatially controlled assembly of ligand-decorated gold nanoparticles, quantum dots, nanorods, DNA origami, and hybrid structures. Owing to the distinguishable nanoarchitectures of these nanofibers, this assembly is structure-dependent. By coupling a modular genetic strategy with kinetically controlled complex supramolecular self-assembly, we demonstrate that a single type of protein molecule can be used to engineer diverse one-dimensional supramolecular nanostructures with distinct functionalities.

[Preparation and evaluation of a novel liposome-based fluorescent probe for tumor imaging].

In this study, a fluorescent nanoprobe based on liposome was synthesized by the hydrophobic interaction of phosphatidyl ethanolamine and indocyanine green(ICG).The nanoprobe was called LipoICG. In order to enhance the stability of liposome, we made a new LipoICG by coating it with human serum albumin (HSA). The new fluorescent nanoprobe, H-LipoICG, was produced for tumor imaging. The LipoICG and H-LipoICG were observed as spherical shape with uniform size distribution. The particle size of LipoICG was 94.47 nm, zeta potential was-43.5 m V and encapsulation efficiency (EE) was 81.5%.The particle size of H-LipoICG was 121.5 nm, zeta potential was-32.3 m V and EE was 98.2%.The coating of HSA could enhance the stability of liposome and increase the EE of ICG. Studies on drug release demonstrated that the release of ICG in H-LipoICG was slower than LipoICG, which suggests that HSA may reduce the ICG leakage from liposome, the fluorescence intensity could be enhanced in the nanoprobe. The Cell Counting Kit-8 assay demonstrated that LipoICG and H-LipoICG was not toxic for MCF-7 cells with good biocompatibility. In the study of biodistribution in mice, our experiments demonstrated that H-LipoICG had better tumor targeting ability and exhibited an enhanced fluorescence intensity than LipoICG. An optimize tumor contrast was observed after 8 h intravenous administration, the tumor margins could be clearly detected for up to 24 h after injection. So, H-LipoICG was an effective fluorescent probe for tumor imaging.