Crystallographic molecular replacement using an in silico‐generated search model of SARS‐CoV‐2 ORF8

The majority of crystal structures are determined by the method of molecular replacement (MR). The range of application of MR is limited mainly by the need for an accurate search model. In most cases, pre‐existing experimentally determined structures are used as search models. In favorable cases, ab initio predicted structures have yielded search models adequate for MR. The ORF8 protein of SARS‐CoV‐2 represents a challenging case for MR using an ab initio prediction because ORF8 has an all β‐sheet fold and few orthologs. We previously determined experimentally the structure of ORF8 using the single anomalous dispersion (SAD) phasing method, having been unable to find an MR solution to the crystallographic phase problem. Following a report of an accurate prediction of the ORF8 structure, we assessed whether the predicted model would have succeeded as an MR search model. A phase problem solution was found, and the resulting structure was refined, yielding structural parameters equivalent to the original experimental solution.     

Sex‐ and strain‐specific effects of mitochondrial uncoupling on age‐related metabolic diseases in high‐fat diet‐fed mice

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti‐aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled‐release mitochondrial protonophore (CRMP) that is functionally liver‐directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver‐directed fashion could reduce oxidative damage and improve age‐related metabolic disease and lifespan in diet‐induced obese mice. Oral administration of CRMP (20 mg/[kg‐day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74‐week‐old) high‐fat diet (HFD)‐fed C57BL/6J male mice, independently of changes in body weight, whole‐body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long‐term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94–104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex‐specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof‐of‐concept data to support further studies investigating the use of liver‐directed mitochondrial uncouplers to promote healthy aging in humans.     

Genetic diversity and sex‐biased dispersal in the brown spotted pitviper (Protobothrops mucrosquamatus): Evidence from microsatellite markers

Dispersal plays a vital role in the geographical distribution, population genetic structure, quantity dynamics, and evolution of a species. Sex‐biased dispersal is common among vertebrates and many studies have documented a tendency toward male‐biased dispersal in mammals and female‐biased dispersal in birds. However, dispersal patterns in reptiles remain poorly understood. In this study, we explored the genetic diversity and dispersal patterns of the widely distributed Asian pitviper Protobothrops mucrosquamatus. In total, 16 polymorphic microsatellite loci were screened in 150 snakes (48 males, 44 females, 58 samples without sex information) covering most of their distribution. Microsatellite analysis revealed high genetic diversity in P. mucrosquamatus. Bayesian clustering of population assignment identified two major clusters for all populations, somewhat inconsistent with the mitochondrial DNA phylogeny of P. mucrosquamatus reported in previous research. Analyses based on 92 sex‐determined and 37 samples of P. mucrosquamatus from three small sites in Sichuan, China (Mingshan, Yibin, and Zizhong) consistently suggested female‐biased dispersal in P. mucrosquamatus, which is the first example of this pattern in snakes. The female‐biased dispersal patterns in P. mucrosquamatus may be explained by local resource competition.     

Contrasting definitive hosts as determinants of the genetic structure in a parasite with complex life cycle along the south‐eastern Pacific

The spatial genetic structure (and gene flow) of parasites with complex life cycles, such as digeneans, has been attributed mainly to the dispersion ability of the most mobile host, which most often corresponds to the definitive host (DH). In this study, we compared the genetic structure and diversity of adult Neolebouria georgenascimentoi in two fish species (DHs) that are extensively distributed along the south‐eastern Pacific (SEP). The analysis was based on the cytochrome oxidase subunit I gene sequences of parasites collected between 23°S and 45°S. In total, 202 sequences of N. georgenascimentoi in Pinguipes chilensis isolated from nine sites and 136 sequences of Prolatilus jugularis from five sites were analysed. Our results showed that N. georgenascimentoi is a species complex that includes three different parasite species; however, in this study, only lineage 1 and 2 found in P. chilensis and P. jugularis, respectively, were studied because they are widely distributed along the coastline. Lineage 1 parasites had two common haplotypes with wide distribution and unique haplotypes in northern sites. Lineage 2 had only one common haplotype with wide distribution and a large number of unique haplotypes with greater genetic diversity. Both lineages have experienced recent population expansion. Only lineage 1 exhibited a genetic structure that was mainly associated with a biogeographical break at approximately 30°S along the SEP. Our finding suggests that host access to different prey (=intermediate hosts) could affect the genetic structure of the parasite complex discovered here. Consequently, difference between these patterns suggests that factors other than DH dispersal are involved in the genetic structure of autogenic parasites.     

Analysis of microbial diversity and succession during Xiaoqu Baijiu fermentation using high‐throughput sequencing technology

In this study, high‐throughput sequencing (HTS) was used to compare and analyze the microbial diversity and succession during the brewing process of xiaoqu Baijiu. A total of 34 phyla and 378 genera of bacteria, as well as four phyla, 32 genera of fungi were detected. At the phylum level, Firmicutes, Proteobacteria, Ascomycota, and Mucoromycota were the dominant groups. During the brewing process of xiaoqu Baijiu, the dominant bacteria were Weissella and unidentified Rickettsiales within the first 2 days of brewing, followed by Lactobacillus at 3 days until to the end of brewing. The dominant fungi were Rhizopus, Saccharomyces, and Issatchenkia. The relative abundance of Rhizopus decreased with the extension of brewing time, while the relative abundance of Saccharomyces increased, and Saccharomyces became the dominant species at the second day of brewing. This study revealed the diversity and changes of the microbial community during the brewing process of xiaoqu Baijiu, providing theoretical support and laying a foundation for future study on the contribution of microbial metabolism during brewing of xiaoqu Baijiu, thereby promoting the development of xiaoqu Baijiu industry.     

High diversity in Delta variant across countries revealed by genome‐wide analysis of SARS‐CoV‐2 beyond the Spike protein

The highly contagious Delta variant of SARS‐CoV‐2 has become a prevalent strain globally and poses a public health challenge around the world. While there has been extensive focus on understanding the amino acid mutations in the Delta variant’s Spike protein, the mutational landscape of the rest of the SARS‐CoV‐2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS‐CoV‐2 proteins from nearly 2 million SARS‐CoV‐2 genomes from 176 countries/territories. Six highly prevalent missense mutations in the viral life cycle‐associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, and Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant. Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of amino acid mutations in the Delta variant’s proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.     

Assessing the diversity and distribution of Cephalothrix species (Nemertea: Palaeonemertea) in European waters by comparing different species delimitation methods

Soft‐bodied marine taxa, like ribbon worms (Nemertea), often lack clear diagnostic morphological characters impeding traditional species delimitation. Therefore, recent studies concentrated on molecular genetic methods to solve taxonomic issues. Different delimitation methods were employed to explore species boundaries and the presence of cryptic species. However, the performance of the different delimitation methods needs to be tested. A particularly promising nemertean genus in this regard is the palaeonemertean genus Cephalothrix that is commonly found in European waters. In order to gain information on the number and distribution of European cephalotrichids and to test different tree‐based and non‐tree‐based delimitation methods, we analyzed a dataset comprising the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) of 215 European Cephalothrix specimens, of which 78 were collected for this study. Our results show the presence of 12–13 European lineages of which several can be assigned to known European species. Analyzing a second dataset comprising 74 additional sequences from the Pacific and the Atlantic Oceans helped identify some of the unassigned European specimens. One resulting clade seems to represent a non‐native introduced Cephalothrix species, while another has never been recorded from Europe before. In our analysis, especially the tree‐based methods and the phylogenetic analysis proved to be a useful tool when delimiting species. It remains unclear whether the different identified clades result from cryptic speciation or from a high genetic variability of the COI gene.     

A genotyping‐in‐thousands by sequencing panel to inform invasive deer management using noninvasive fecal and hair samples

Studies in ecology, evolution, and conservation often rely on noninvasive samples, making it challenging to generate large amounts of high‐quality genetic data for many elusive and at‐risk species. We developed and optimized a Genotyping‐in‐Thousands by sequencing (GT‐seq) panel using noninvasive samples to inform the management of invasive Sitka black‐tailed deer (Odocoileus hemionus sitkensis) in Haida Gwaii (Canada). We validated our panel using paired high‐quality tissue and noninvasive fecal and hair samples to simultaneously distinguish individuals, identify sex, and reconstruct kinship among deer sampled across the archipelago, then provided a proof‐of‐concept application using field‐collected feces on SGang Gwaay, an island of high ecological and cultural value. Genotyping success across 244 loci was high (90.3%) and comparable to that of high‐quality tissue samples genotyped using restriction‐site associated DNA sequencing (92.4%), while genotyping discordance between paired high‐quality tissue and noninvasive samples was low (0.50%). The panel will be used to inform future invasive species operations in Haida Gwaii by providing individual and population information to inform management. More broadly, our GT‐seq workflow that includes quality control analyses for targeted SNP selection and a modified protocol may be of wider utility for other studies and systems where noninvasive genetic sampling is employed.     

Structural investigation of CDCA3‐Cdh1 protein–protein interactions using in vitro studies and molecular dynamics simulation

The anaphase‐promoting complex/cyclosome (APC/C) ubiquitin ligase and its cofactor, Cdh1, regulate the expression of several cell‐cycle proteins and their functions during mitosis. Levels of the protein cell division cycle‐associated protein 3 (CDCA3), which is functionally required for mitotic entry, are regulated by APC/C^Cdh1. CDCA3 is an intrinsically disordered protein and contains both C‐terminal KEN box and D‐box recognition motifs, enabling binding to Cdh1. Our previous findings demonstrate that CDCA3 has a phosphorylation‐dependent non‐canonical ABBA‐like motif within the linker region bridging these two recognition motifs and is required for efficient binding to Cdh1. Here, we sought to identify and further characterize additional residues that participate within this ABBA‐like motif using detailed in vitro experiments and in silico modeling studies. We identified the role of H‐bonds, hydrophobic and ionic interactions across the CDCA3 ABBA‐like motif in the linker region between KEN and D‐box motifs. This linker region adopts a well‐defined structure when bound to Cdh1 in the presence of phosphorylation. Upon alanine mutation, the structure of this region is lost, leading to higher flexibility, and alteration in affinities due to binding to alternate sites on Cdh1. Our findings identify roles for the anchoring residues in the non‐canonical ABBA‐like motif to promote binding to the APC/C^Cdh1 and regulation of CDCA3 protein levels.     

Restoration of species‐rich grasslands by transfer of local plant material and its impact on species diversity and genetic variation—Findings of a practical restoration project in southeastern Germany

Restoration of species‐rich grasslands is a key issue of conservation. The transfer of seed‐containing local plant material is a proven technique to restore species‐rich grassland, since it potentially allows to establish genetically variable and locally adapted populations. In our study, we tested how the transfer of local plant material affected the species diversity and composition of restored grasslands and the genetic variation of the typical grassland plant species Knautia arvensis and Plantago lanceolata.For our study, we selected fifteen study sites in southeastern Germany. We analyzed species diversity and composition and used molecular markers to investigate genetic variation within and among populations of the study species from grasslands that served as source sites for restoration and grasslands, which were restored by transfer of green hay and threshed local plant material.The results revealed no significant differences in species diversity and composition between grasslands at source and restoration sites. Levels of genetic variation within populations of the study species Knautia arvensis and Plantago lanceolata were comparable at source and restoration sites and genetic variation among populations at source and their corresponding restoration sites were only marginal different.Our study suggests that the transfer of local plant material is a restoration approach highly suited to preserve the composition of species‐rich grasslands and the natural genetic pattern of typical grassland plant species.     

Genetic prediction of ICU hospitalization and mortality in COVID‐19 patients using artificial neural networks

There is an unmet need of models for early prediction of morbidity and mortality of Coronavirus disease‐19 (COVID‐19). We aimed to a) identify complement‐related genetic variants associated with the clinical outcomes of ICU hospitalization and death, b) develop an artificial neural network (ANN) predicting these outcomes and c) validate whether complement‐related variants are associated with an impaired complement phenotype. We prospectively recruited consecutive adult patients of Caucasian origin, hospitalized due to COVID‐19. Through targeted next‐generation sequencing, we identified variants in complement factor H/CFH, CFB, CFH‐related, CFD, CD55, C3, C5, CFI, CD46, thrombomodulin/THBD, and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS13). Among 381 variants in 133 patients, we identified 5 critical variants associated with severe COVID‐19: rs2547438 (C3), rs2250656 (C3), rs1042580 (THBD), rs800292 (CFH) and rs414628 (CFHR1). Using age, gender and presence or absence of each variant, we developed an ANN predicting morbidity and mortality in 89.47% of the examined population. Furthermore, THBD and C3a levels were significantly increased in severe COVID‐19 patients and those harbouring relevant variants. Thus, we reveal for the first time an ANN accurately predicting ICU hospitalization and death in COVID‐19 patients, based on genetic variants in complement genes, age and gender. Importantly, we confirm that genetic dysregulation is associated with impaired complement phenotype.     

Reversion of a resistance‐breaking mutation shows reversion costs and high virus diversity at necrotic local lesions

An instance of host range evolution relevant to plant virus disease control is resistance breaking. Resistance breaking can be hindered by across‐host fitness trade‐offs generated by negative effects of resistance‐breaking mutations on the virus fitness in susceptible hosts. Different mutations in pepper mild mottle virus (PMMoV) coat protein result in the breaking in pepper plants of the resistance determined by the L ³ resistance allele. Of these, mutation M138N is widespread in PMMoV populations, despite associated fitness penalties in within‐host multiplication and survival. The stability of mutation M138N was analysed by serial passaging in L ³ resistant plants. Appearance on passaging of necrotic local lesions (NLL), indicating an effective L ³ resistance, showed reversion to nonresistance‐breaking phenotypes was common. Most revertant genotypes had the mutation N138K, which affects the properties of the virus particle, introducing a penalty of reversion. Hence, the costs of reversion may determine the evolution of resistance‐breaking in addition to resistance‐breaking costs. The genetic diversity of the virus population in NLL was much higher than in systemically infected tissues, and included mutations reported to break L ³ resistance other than M138N. Infectivity assays on pepper genotypes with different L alleles showed high phenotypic diversity in respect to L alleles in NLL, including phenotypes not reported in nature. Thus, high diversity at NLL may potentiate the appearance of genotypes that enable the colonization of new host genotypes or species. Collectively, the results of this study contribute to better understanding the evolutionary dynamics of resistance breaking and host‐range expansions.     

Dissecting molecular details and functional effects of the high‐affinity copper binding site in plasminogen activator Inhibitor‐1

Plasminogen activator inhibitor‐1 (PAI‐1) is the primary inhibitor for plasminogen activators, tissue‐type plasminogen activator (tPA) and urokinase‐type plasminogen activator (uPA). As a unique member in the serine protease inhibitor (serpin) family, PAI‐1 is metastable and converts to an inactive, latent structure with a half‐life of 1–2 hr under physiological conditions. Unusual effects of metals on the rate of the latency conversion are incompletely understood. Previous work has identified two residues near the N‐terminus, H2 and H3, which reside in a high‐affinity copper‐binding site in PAI‐1 [Bucci JC, McClintock CS, Chu Y, Ware GL, McConnell KD, Emerson JP, Peterson CB (2017) J Biol Inorg Chem 22:1123–1,135]. In this study, neighboring residues, H10, E81, and H364, were tested as possible sites that participate in Cu(II) coordination at the high‐affinity site. Kinetic methods, gel sensitivity assays, and isothermal titration calorimetry (ITC) revealed that E81 and H364 have different roles in coordinating metal and mediating the stability of PAI‐1. H364 provides a third histidine in the metal‐coordination sphere with H2 and H3. In contrast, E81 does not appear to be required for metal ligation along with histidines; contacts made by the side‐chain carboxylate upon metal binding are perturbed and, in turn, influence dynamic fluctuations within the region encompassing helices D, E, and F and the W86 loop that are important in the pathway for the PAI‐1 latency conversion. This investigation underscores a prominent role of protein dynamics, noncovalent bonding networks and ligand binding in controlling the stability of the active form of PAI‐1.     

Benefits and limitations of a new genome‐based PCR‐RFLP genotyping assay (GB‐RFLP): A SNP‐based detection method for identification of species in extremely young adaptive radiations

High‐throughput DNA sequencing technologies make it possible now to sequence entire genomes relatively easily. Complete genomic information obtained by whole‐genome resequencing (WGS) can aid in identifying and delineating species even if they are extremely young, cryptic, or morphologically difficult to discern and closely related. Yet, for taxonomic or conservation biology purposes, WGS can remain cost‐prohibitive, too time‐consuming, and often constitute a “data overkill.” Rapid and reliable identification of species (and populations) that is also cost‐effective is made possible by species‐specific markers that can be discovered by WGS. Based on WGS data, we designed a PCR restriction fragment length polymorphism (PCR‐RFLP) assay for 19 Neotropical Midas cichlid populations (Amphilophus cf. citrinellus), that includes all 13 described species of this species complex. Our work illustrates that identification of species and populations (i.e., fish from different lakes) can be greatly improved by designing genetic markers using available “high resolution” genomic information. Yet, our work also shows that even in the best‐case scenario, when whole‐genome resequencing information is available, unequivocal assignments remain challenging when species or populations diverged very recently, or gene flow persists. In summary, we provide a comprehensive workflow on how to design RFPL markers based on genome resequencing data, how to test and evaluate their reliability, and discuss the benefits and pitfalls of our approach.     

Molecular evidence suggesting the persistence of residual SARS‐CoV‐2 and immune responses in the placentas of pregnant patients recovered from COVID‐19

ObjectivesRecent studies have shown the presence of SARS‐CoV‐2 in the tissues of clinically recovered patients and persistent immune symptoms in discharged patients for up to several months. Pregnant patients were shown to be a high‐risk group for COVID‐19. Based on these findings, we assessed SARS‐CoV‐2 nucleic acid and protein retention in the placentas of pregnant women who had fully recovered from COVID‐19 and cytokine fluctuations in maternal and foetal tissues.Materials and MethodsRemnant SARS‐CoV‐2 in the term placenta was detected using nucleic acid amplification and immunohistochemical staining of the SARS‐CoV‐2 protein. The infiltration of CD14+ macrophages into the placental villi was detected by immunostaining. The cytokines in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens at delivery were profiled using the Luminex assay.ResultsResidual SARS‐CoV‐2 nucleic acid and protein were detected in the term placentas of recovered pregnant women. The infiltration of CD14+ macrophages into the placental villi of the recovered pregnant women was higher than that in the controls. Furthermore, the cytokine levels in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens fluctuated significantly.ConclusionsOur study showed that SARS‐CoV‐2 nucleic acid (in one patient) and protein (in five patients) were present in the placentas of clinically recovered pregnant patients for more than 3 months after diagnosis. The immune responses induced by the virus may lead to prolonged and persistent symptoms in the maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid.     

Ecological theory of mutualism: Robust patterns of stability and thresholds in two‐species population models

Mutualisms are ubiquitous in nature, provide important ecosystem services, and involve many species of interest for conservation. Theoretical progress on the population dynamics of mutualistic interactions, however, comparatively lagged behind that of trophic and competitive interactions, leading to the impression that ecologists still lack a generalized framework to investigate the population dynamics of mutualisms. Yet, over the last 90 years, abundant theoretical work has accumulated, ranging from abstract to detailed. Here, we review and synthesize historical models of two‐species mutualisms. We find that population dynamics of mutualisms are qualitatively robust across derivations, including levels of detail, types of benefit, and inspiring systems. Specifically, mutualisms tend to exhibit stable coexistence at high density and destabilizing thresholds at low density. These dynamics emerge when benefits of mutualism saturate, whether due to intrinsic or extrinsic density dependence in intraspecific processes, interspecific processes, or both. We distinguish between thresholds resulting from Allee effects, low partner density, and high partner density, and their mathematical and conceptual causes. Our synthesis suggests that there exists a robust population dynamic theory of mutualism that can make general predictions.     

Population genetic structure and evolution of Batesian mimicry in Papilio polytes from the Ryukyu Islands,Japan, analyzed by genotyping‐by‐sequencing

Batesian mimicry is a striking example of Darwinian evolution, in which a mimetic species resembles toxic or unpalatable model species, thereby receiving protection from predators. In some species exhibiting Batesian mimicry, nonmimetic individuals coexist as polymorphism in the same population despite the benefits of mimicry. In a previous study, we proposed that the abundance of mimics is limited by that of the models, leading to polymorphic Batesian mimicry in the swallowtail butterfly, Papilio polytes, on the Ryukyu Islands in Japan. We found that their mimic ratios (MRs), which varied among the Islands, were explained by the model abundance of each habitat, rather than isolation by distance or phylogenetic constraint based on the mitochondrial DNA (mtDNA) analysis. In the present study, this possibility was reexamined based on hundreds of nuclear single nucleotide polymorphisms (SNPs) of 93 P. polytes individuals from five Islands of the Ryukyus. We found that the population genetic and phylogenetic structures of P. polytes largely corresponded to the geographic arrangement of the habitat Islands, and the genetic distances among island populations show significant correlation with the geographic distances, which was not evident by the mtDNA‐based analysis. A partial Mantel test controlling for the present SNP‐based genetic distances revealed that the MRs of P. polytes were strongly correlated with the model abundance of each island, implying that negative frequency‐dependent selection interacting with model species shaped and maintained the mimetic polymorphism. Taken together, our results support the possibility that predation pressure, not isolation by distance or other neutral factors, is a major driving force of evolution of the Batesian mimicry in P. polytes from the Ryukyus.     

N‐terminal acetylation modestly enhances phase separation and reduces aggregation of the low‐complexity domain of RNA‐binding protein fused in sarcoma

The RNA‐binding protein fused in sarcoma (FUS) assembles via liquid–liquid phase separation (LLPS) into functional RNA granules and aggregates in amyotrophic lateral sclerosis associated neuronal inclusions. Several studies have demonstrated that posttranslational modification (PTM) can significantly alter FUS phase separation and aggregation, particularly charge‐altering phosphorylation of the nearly uncharged N‐terminal low complexity domain of FUS (FUS LC). However, the occurrence and impact of N‐terminal acetylation on FUS phase separation remains unexplored, even though N‐terminal acetylation is the most common PTM in mammals and changes the charge at the N‐terminus. First, we find that FUS is predominantly acetylated in two human cell types and stress conditions. Next, we show that recombinant FUS LC can be acetylated when co‐expressed with the NatA complex in Escherichia coli. Using NMR spectroscopy, we find that N‐terminal acetylated FUS LC (FUS LC Nt‐Ac) does not notably alter monomeric FUS LC structure or motions. Despite no difference in structure, Nt‐Ac‐FUS LC phase separates more avidly than unmodified FUS LC. More importantly, N‐terminal acetylation of FUS LC reduces aggregation. Our findings highlight the importance of N‐terminal acetylation of proteins that undergo physiological LLPS and pathological aggregation.