ATP binding facilitates target search of SWR1 chromatin remodeler by promoting one-dimensional diffusion on DNA One-dimensional (1D) target search is a well characterized phenomenon for many DNA binding proteins but is poorly understood for chromatin remodelers. Herein, we characterize the 1D scanning properties of SWR1, a yeast chromatin remodeler that performs histone exchange on +1 nucleosomes adjacent to a nucleosome depleted region (NDR) at promoters. We demonstrate that SWR1 has a kinetic binding preference for DNA of NDR length as opposed to gene-body linker length DNA. Using single and dual color single particle tracking on DNA stretched with optical tweezers, we directly observe SWR1 diffusion on DNA. We found that various factors impact SWR1 scanning, including ATP which promotes diffusion through nucleotide binding rather than ATP hydrolysis. A DNA binding subunit, Swc2, plays an important role in the overall diffusive behavior of the complex, as the subunit in isolation retains similar, although faster, scanning properties as the whole remodeler. ATP-bound SWR1 slides until it encounters a protein roadblock, of which we tested dCas9 and nucleosomes. The median diffusion coefficient, 0.024 m2/sec, in the regime of helical sliding, would mediate rapid encounter of NDR-flanking nucleosomes at length scales found in cells.
Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate Ribonucleoprotein bodies are exemplars of membraneless biomolecular condensates that can form via spontaneous or driven phase transitions. The fungal protein Whi3 forms ribonucleoprotein condensates with different RNA molecules, and these condensates are implicated in key processes such as cell-cycle control and generating cell polarity. Whi3 has a modular architecture that includes a Q-rich intrinsically disordered region (IDR) and a tandem RNA recognition module. Here, we demonstrate that a 21-residue stretch within the Q-rich IDR has a weak intrinsic preference for forming alpha-helical conformations. Through mutagenesis, we find that increased alpha helicity enhances oligomerization in the dilute phase. One consequence of enhanced oligomerization is a dilution of Whi3 in the dense phase. The opposite behavior is observed when helicity within the 21-residue stretch of the Q-rich region is abrogated. Thus, the formation of dilute phase oligomers, driven by a specific sequence motif and potential synergies with the rest of the IDR, opposes incorporation of the Whi3 protein into the dense phase, thereby altering the dense phase stoichiometry of protein to RNA. Our findings, which stand in contrast to other systems where oligomerization has been shown to enhance the drive for phase separation, point to a novel mechanism that might be operative for influencing compositions of condensates. Our work also points to routes for designing synthetic ribonucleoprotein condensates whereby modulation of protein oligomerization via homotypic interactions can impact dense phase concentrations, stoichiometries, and material properties.

SignificanceA large sub-class of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.
Caveolae respond to acute oxidative stress through membrane lipid peroxidation, cytosolic release of CAVIN1, and downstream regulation of NRF2 Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to lipid peroxidation-induced ferroptosis. Thus, caveolae, via lipid peroxidation and CAVIN1 release, maintain cellular susceptibility to oxidative stress-induced cell death demonstrating a crucial role for this enigmatic organelle in cellular homeostasis and wound response. Fluorescence-Activated Droplet Sorting of Polyethylene terephthalate-degrading Enzymes Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. However, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PET-degrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new PET-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of PETases using synthetic biology. Artificial seawater based long-term culture of colonial ascidians Tunicates are highly diverse marine invertebrate filter-feeders that are vertebrates closest relatives. These organisms, despite a drastically different body plan during their adulthood, have a tissue complexity related to that of vertebrates. Ascidians, which compose most of the Tunicata, are benthic sessile hermaphrodites that reproduce sexually through a motile tadpole larval stage. Over half of the known ascidians species are able to reproduce asexually by budding, typically leading to the formation of colonies where animals, called zooids, are interconnected through an external vascular system. In addition, colonial ascidians are established models for important biological processes including allorecognition, immunobiology, aging, angiogenesis and whole-body regeneration. However, the current paucity in breeding infrastructures limits the study of these animals to coastal regions.

To promote a wider scientific spreading and popularity of colonial ascidians, we have developed a flexible recirculating husbandry setup for their long-term in-lab culture. Our system is inspired both by the flow-through aquariums used by coastal ascidian labs, as well as by the recirculating in-lab systems used for zebrafish research. Our hybrid system thus combines colony breeding, water filtering and food culturing in a semi-automated system where specimens develop on hanging microscopy glass slides. Temperature, light/dark cycles, flow speed and feeding rates can be controlled independently in four different breeding environments to provide room for species-specific optimization as well as for running experiments. This setup is complemented with a quarantine for the acclimatization of wild isolates.

Herein we present our success in breeding Botrylloides diegensis, a species of colonial ascidians, for more than 3 years in recirculating artificial seawater over 600 km away from their natural habitat. We show that colonies adapt well to in-lab culturing provided that a suitable marine microbiome is present, and that a specific strain can be isolated, propagated and efficiently used for research over prolonged periods of time. The flexible and modular structure of our system can be scaled and adapted to the needs of specific species, such as Botryllus schlosseri, as well as of particular laboratory spaces. Overall, we show that Botrylloides diegensis can be proficiently bred in-land and suggest that our results can be extended to other species of colonial ascidians to promote research on these fascinating animals.

HighlightsO_LIFirst artificial seawater based recirculating aquaculture for colonial ascidians
C_LIO_LIOver 3 years of continuous breeding
C_LIO_LISemi-automated setup with minimized maintenance
C_LIO_LIGood biomass production for strain propagation
C_LIO_LI4 different culture conditions for optimized breeding for species of interest
C_LI Affordable Caenorhabditis elegans tracking system for classroom use For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system. Potential geographic distribution and ecological niche of New World dobsonflies (Megaloptera, Corydalidae): the case of the Neartic-Neotropical transition zone The dobsonflies (Megaloptera, Corydalidae) are an interesting group of insects, but among the New World dobsonflies, it is not known the effect of the Neartic-Neotropical transition zone on their biogeographic distribution. Here we studied at the species level, the records on the geographic range of the dobsonflies that occurred in and near the transition zone. We presented potential geographic distributions based on ecological niche models for several species of dobsonflies. Results suggested that the geographic range of dobsonflies in the transition zone is associated to mountainous formations and that most species favor warm climates with higher precipitation rates. Climate types tend to be important for species that show narrow geographic ranges, but precipitation tends to be the most important variable to explain species dispersion. Overall, our models support the dispersion of dobsonflies from the Neotropics to North America and explain the two endemic species in Mexico as the result of the formation of the transition zone. Circular RNA's competing endogenous gene pair as motif in serous ovarian cancer The non-coding RNA (ncRNA) regulation apprears to be associated to the diagnosis and targeted therapy of complex diseases. Motifs of non-coding RNAs and genes in the competing endogenous RNA (ceRNA) network would probably contribute to the accurate prediction of serous ovarian carcinoma (SOC). Hence, we profiled a whole transcriptome of eight human SOCs and eight controls and constructed a ceRNA network including mRNAs, long ncRNAs, and circular RNAs (circRNAs). We identified the mRNA-ncRNA-mRNA motifs in the ceRNA network named the non-coding RNAs competing endogenous gene pairs (ceGPs), through the denoised individualized pair analysis of gene expression (deiPAGE) proposed in this study. 18 cricRNAs ceGPs (cceGPs) were identified from multiple cohorts and were fused as an indicator (SOC index) for SOC discrimination, which carried a high predictive capacity in independent cohorts. It was found that the index was negatively correlated with the CD8+/CD4+ ratio in tumour-infiltration, reflecting the migration and growth of tumour cells in ovarian cancer progression. Characterising ontogeny of quantity discrimination in zebrafish A sense of non-symbolic numerical magnitudes is widespread in the animal kingdom and has been documented in adult zebrafish. Here we investigated the ontogeny of this ability using a group size preference task in juvenile zebrafish. Fish showed group size preference from 21 days post fertilization (dpf) and reliably chose the larger group when presented with discriminations of between 1 vs. 3, 2 vs. 5 and 2 vs. 3 conspecifics but not 2 vs. 4 conspecifics. When the ratio between the number of conspecifics in each group was maintained at 1:2, fish could discriminate between 1 vs. 2 individuals and 3 vs. 6, but again, not when given a choice between 2 vs. 4 individuals. These findings are in agreement with studies in other species suggesting the systems involved in quantity representation do not operate separately from other cognitive mechanisms. Rather they suggest quantity processing in fish may be the result of an interplay between attentional, cognitive and memory-related mechanisms as in humans and other animals. Our results emphasise the potential of the use of zebrafish to explore the genetic and neural processes underlying the ontogeny and function of number cognition. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Force profile of the two-handed hardstyle kettlebell swing in novice older adults: an exploratory profile BackgroundUnderstanding the force profile of an exercise increases clinical confidence when assessing the benefits and potential risks of a prescribed exercise. This exploratory study presents the force profile of the hardstyle kettlebell swing in novice older adults and compares peak force with kettlebell deadlifts. These data will help inform healthcare providers and coaches who are considering prescribing kettlebell exercises for older adults.

MethodsThirty-five community-dwelling males and females (59-79 years) were recruited, from applicants to participate in the BELL trial. Two-handed hardstyle swings were performed with 8-16 kg. Deadlifts were performed with 8-24 kg and 8-32 kg for females and males, respectively. Ground reaction force was obtained from a floor-mounted force platform. Pairwise comparisons of peak force, forward force, rate of force development, swing cadence, sex, and kettlebell mass, were investigated for the kettlebell swing, with representative force-time curves described. Pairwise comparisons of peak force, sex and kettlebell mass were investigated for the deadlift, with comparisons of peak force, kettlebell mass, and sex, between swings and deadlifts.

ResultsFor kettlebells up to 16 kg, paired samples T-tests show a large exercise effect ({delta} > 1.4) with peak force higher for swings than deadlifts. Data shows: (i) higher peak force during swings than deadlifts ({delta} = 1.77), reaching 4.5 (1.0) N.kg-1, (ii) peak force during an 8 kg swing was greater than a 32kg deadlift, (iii) negligible difference in normalised peak force between males and females performing kettlebell swings, but a moderately large effect size during deadlifts (males > females, {delta} = 0.69), (iv) mean rate of force development of 19.9 (4.7) N.s-1.kg-1 with a very weak, positive correlation with kettlebell mass (y = 14.4 + 0.32x), and trivial effect of sex, (v) mean forward force equal to 5.5% of vertical force during swings, increasing from 3.8 (1.6) % with 8 kg to 7.1 (2.6) % with 16 kg.

ConclusionDuring kettlebell swings, there is negligible difference in normalised net peak force between novice males and females using the same absolute loads. Where ground reaction force is a therapeutic target, kettlebell swings with an 8 kg kettlebell could have similar effects to much heavier deadlifts (>24 kg). Kettlebell swings performed with lighter loads, could provider similar therapeutic value to much heavier deadlifts, and may be a more appealing, affordable, and convenient option for older adults. Model sharing in the human medial temporal lobe Effective planning involves knowing where different actions will take us. However natural environments are rich and complex, leading to an exponential increase in memory demand as a plan grows in depth. One potential solution to this problem is to share the neural state transition functions used for planning between similar contexts. Here, we asked human participants to perform a sequential decision making task designed so that knowledge could be shared between some contexts but not others. Computational modelling showed that participants shared a model of state transitions between contexts where appropriate. fMRI data identified the medial temporal lobe as a locus for learning of state transitions, and within the same region, correlated BOLD patterns were observed in contexts where state transition information were shared. Finally, we show that the transition model is updated more strongly following the receipt of positive compared to negative outcomes, a finding that challenges conventional theories of planning which assume knowledge about our environment is updated independently of outcomes received. Together, these findings propose a computational and neural account of how information relevant for planning can be shared between contexts. SUMM4 biochemically complex couples insulator function and DNA replication timingcontrol The mechanisms that establish DNA replication timing programs in eukaryotes remain incompletely understood. Drosophila SNF2-related factor SUUR imparts under-replication (UR) of late-replicating intercalary heterochromatin (IH) in polytene chromosomes. We developed a proteomics technique termed MERCI to isolate a native complex SUMM4 comprising SUUR and chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates the ATPase activity of SUUR and is required for its normal spatiotemporal distribution in vivo. Both SUMM4 subunits mediate the activities of gypsy insulator disrupting enhancer-promoter interactions and establishing chromatin barriers. Furthermore, SuUR or mod(mdg4) mutations reverse UR of IH. Our findings uncover a critical role for architectural proteins in attenuating replication fork progression and suggest an alternative mechanism for DNA replication timing that does not depend on an asynchronous firing of replication origins.

One-Sentence SummaryA stable protein complex comprising an insulator factor and a SNF2-like ATPase imparts late replication of heterochromatin. Elevation of major constitutive heat shock proteins is heat shock factor independent and essential for establishment and growth of Lgl loss and Yorkie gain-mediated tumors in Drosophila Cancer cells overexpress heat shock proteins (Hsps), the major components of cellular stress response, to overcome the diversity of stresses and to survive. However, the specific roles of Hsps in initiation and establishment of cancers remain unclear. Using loss of Lgl function-mediated epithelial tumorigenesis in Drosophila, we induced tumorigenic MARCM clones of different genetic backgrounds in wing imaginal discs to examine the temporal and spatial expression and roles of major heat shock proteins in tumor growth. The constitutively expressed Hsp83, Hsc70 (heat shock cognate), Hsp60 and Hsp27 show elevated levels in all cells of the tumorigenic clone since early stages, which persists till their transformation. However, the stress-inducible Hsp70 is expressd only in a few cells at later stage of established tumorous clones that show high F-actin aggregation. Intriguingly, levels of heat shock factor (HSF), the master regulator of Hsps, remain unaltered in these tumorous cells and its down-regulation does not affect tumorigenic growth of lgl- clones overexpressing Yorkie, although down-regulation of Hsp83 prevents their survival and growth. Interestingly, overexpression of HSF or Hsp83 in lgl- cells makes them competitively successful in establishing tumorous clones. These results show that the major constitutively expressed Hsps, but not the stress-inducible Hsp70, are involved in early as well as late stages of epithelial tumors and their elevated expression in lgl- clones co-overexpressing Yorkie is independent of HSF. Cold stress upregulates the expression of heat shock and Frost genes, but the evolution of cold stress resistance is apparently not mediated through either heat shock or Frost genes in the cold stress selected populations of Drosophila melanogaster The ability to thermal adaptation can alter insects reproductive traits and physiologic response. A number of studies in Drosophila documented the higher expression levels of heat shock proteins (Hsps) and Frost (Fst) genes during the recovery phase of cold shock treatment and suggested that higher expression levels of these genes and lipids can protect from cold stress. However, these genes expression and cold adaptation have not been well studied. Therefore, understanding the molecular and biochemical basis of cold adaptation, we examined the levels of lipid and expression patterns of Hsp22, Hsp23, Hsp40, Hsp68, Hsp70, Hsp 83, and Fst genes under the cold stress or benign condition in the populations of Drosophila melanogaster selected for increased resistance to cold shock.

We observed the significant up-regulation of Hsp22, Hsp23, Hsp40, Hsp68, and Fst genes in the FSB and FCB populations during the recovery phase post cold shock compared to the no shock condition. However, there was not a significant change in the transcript levels of these genes between FSB and FCB under both the cold stress or no shock condition. Additionally, we noticed higher total lipid levels in the females from cold shock treatment than those from no shock treatment. This finding suggests that cold selected populations have different mechanisms to sustain cold stress. Genome-wide association study suggests an independent genetic basis of zinc and cadmium concentrations in fresh sweet corn kernels Despite being one of the most consumed vegetables in the United States, the elemental profile of sweet corn (Zea mays L.) is limited in its dietary contributions. To address this through genetic improvement, a genome-wide association study was conducted for the concentrations of 15 elements in fresh kernels of a sweet corn association panel. In concordance with mapping results from mature maize kernels, we detected a probable pleiotropic association of zinc and iron concentrations with nicotianamine synthase5 (nas5), which purportedly encodes an enzyme involved in synthesis of the metal chelator nicotianamine. Additionally, a pervasive association signal was identified for cadmium concentration within a recombination suppressed region on chromosome 2. The likely causal gene underlying this signal was heavy metal ATPase3 (hma3), whose counterpart in rice, OsHMA3, mediates vacuolar sequestration of cadmium and zinc in roots, whereby regulating zinc homeostasis and cadmium accumulation in grains. In our association panel, hma3 associated with cadmium but not zinc accumulation in fresh kernels. This finding implies that selection for low cadmium will not affect zinc levels in fresh kernels. Although less resolved association signals were detected for boron, nickel, and calcium, all 15 elements were shown to have moderate predictive abilities via whole-genome prediction. Collectively, these results help enhance our genomics-assisted breeding efforts centered on improving the elemental profile of fresh sweet corn kernels. Similarities Between Bacterial GAD and Human GAD65: Implications in Gut Mediated Autoimmune Type 1 Diabetes A variety of islet autoantibodies (AAbs) can predict and possibly dictate eventual type 1 diabetes (T1D) diagnosis. Upwards of 75% of those with T1D are positive for AAbs against glutamic acid decarboxylase (GAD65), a producer of gamma-aminobutyric acid (GABA) in human pancreatic beta cells. Interestingly, bacterial populations within the human gut also express GAD65 and produce GABA. Evidence suggests that dysbiosis of the microbiome may correlate with T1D pathogenesis and physiology. Therefore, autoimmune linkages between the gut microbiome and islets susceptible to autoimmune attack need to be further elucidated. Utilizing silico analyses, we show here that 25 GAD sequences from different human gut bacterial sources show sequence and motif similarities to human beta cell GAD65. Our motif analyses determined that a majority of gut GAD sequences contain the pyroxical dependent decarboxylase domain of human GAD65 which is important for its enzymatic activity. Additionally, we showed overlap with known human GAD65 T-cell receptor epitopes which may implicate the immune destruction of beta cells. Thus, we propose a physiological hypothesis in which changes in the gut microbiome in those with T1D result in a release of bacterial GAD, thus causing miseducation of the host immune system. Due to the notable similarities, we found between humans and bacterial GAD, these deputized immune cells may then go on to target human beta cells leading to the development of T1D. Epitope-tagging of the endogenous murine BiP/GRP78/Hspa5 locus allows direct analysis of the BiP interactome and protein misfolding in vivo BiP/GRP78, encoded by the Hspa5 gene, is the major HSP70 family member in the endoplasmic reticulum (ER) lumen, and controls ER protein folding. The essential functions of BiP in facilitating proper protein folding are mainly mediated through its dynamic interaction with unfolded or misfolded client proteins, and by serving as a negative regulator of the Unfolded Protein Response. A mechanistic understanding of the dynamics of BiP interaction with its protein partners is essential to understand ER biology, and therefore, we have sought to develop a tractable model to study misfolded protein interaction with BiP. For this purpose, we have used homologous recombination to insert a 3xFLAG epitope tag into the endogenous murine Hspa5 gene, just upstream from the essential KDEL signal necessary for ER localization of BiP. Tagging BiP in this way did not alter Hspa5 expression under basal or ER-stress induced conditions in hepatocytes ex vivo or in fibroblasts. Furthermore, the tag did not alter the cellular localization of BiP or its functionality. All of these findings in primary tissue culture were also confirmed in vivo in livers of heterozygous mice harboring one WT and one FLAG-tagged Hspa5 allele. Hepatocyte-specific BiP-FLAG modification did not alter liver function or UPR signaling. Importantly, immunoprecipitation with anti-FLAG antibody completely pulled down FLAG-tagged BiP from lysates of BiP-FLAG expressing livers. Affinity purification-mass spectrometry (AP-MS) of BiP-FLAG protein complexes isolated from the BiP-FLAG-expressing livers of tunicamycin (TM)- and vehicle-treated mice revealed a marked increase in interaction of glycoproteins with BiP-FLAG in response to inhibition of N-glycosylation due to TM-treatment, validating utility of our BiP-Flag mice as a tool to identify ER misfolded proteins in vivo. Significantly, our AP-MS analysis also provided in vivo evidence demonstrating that BiP-FLAG binds to UPR transducers IRE1 and PERK under basal conditions but is released upon TM-treatment to activate UPR. We have also employed this mouse model to demonstrate that proinsulin in pancreatic {beta} cells misfolds and interacts with BiP-FLAG in healthy mice. In summary, we generated a novel model that can be used to investigate the BiP interactome in vivo under physiological and pathophysiological conditions in a cell type-specific manner. This tool provides, for the first time, an unbiased approach to identify unfolded and misfolded BiP-client proteins, and a new approach to study ER protein misfolding in a cell-type and temporal manner to uncover the role of BiP in many essential ER processes. Algal photosystem I dimer and high resolution model of PSI:plastocyanin complex Photosystem I (PSI) enables photo-electron transfer and regulates photosynthesis in the bioenergetic membranes of cyanobacteria and chloroplasts. Being a multi-subunit complex, its macromolecular organization affects the dynamics of photosynthetic membranes. Here, we reveal a chloroplast PSI from the green alga Chlamydomonas reinhardtii that is organized as a homodimer, comprising 40 protein subunits with 118 transmembrane helices that provide scaffold for 568 pigments. Our cryo-EM structure identifies that the absence of PsaH and Lhca2 gives rise to a head-to-head relative orientation of the PSI-LHCI monomers in a way that is essentially different from the oligomer formation in cyanobacteria. The light-harvesting protein Lhca9 is the key element for mediating this dimerization. The interface between the monomers is lacking PsaH, and thus partially overlaps with the surface area that would bind one of the LHCII complexes in state transitions. We also define the most accurate available PSI-LHCI model at 2.3 [A] resolution, including a flexibly bound electron donor plastocyanin, and assign correct identities and orientations of all the pigments, as well as 621 water molecules that affect energy transfer pathways. GPI-anchored FGF directs cytoneme-mediated bidirectional signaling to self-regulate tissue-specific dispersion How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. FGF-source and recipient ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source cell membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling. Biophysical characterization of the oligomeric states of recombinant Immunoglobulins type-M and their C1q binding kinetics by Biolayer Interferometry The Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs but due to their high oligomeric complexity, in vitro production, biochemical and biophysical characterizations are challenging. In the present study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (AUC, SEC-MALLS, Mass Photometry and Transmission Electron Microscopy). Each IgM oligomer has individual specific expression pattern and yield with different protein quality likely due to intrinsic IgM properties and patterning. Nevertheless, the purified recombinant IgMs retain their ability to activate complement in a C1q dependent manner. And more importantly, a new method to evaluate their functional quality attribute by characterizing the kinetics of C1q binding to recombinant IgM has been developed using BioLayer Interferometry (BLI). We show that recombinant IgMs possess similar C1q binding properties as IgMs purified from human plasma. Quantifying the Morphology and Mechanisms of Cancer Progression in 3D in-vitro environments: Integrating Experiments and Multiscale Models Mathematical models of cancer growth have become increasingly more accurate both in the space and time domains. However, the limited amount of data typically available has resulted in a larger number of qualitative rather than quantitative studies. In the present study, we provide an integrated experimental-computational framework for the quantification of the morphological characteristics and the mechanistic modelling of cancer progression in 3D environments. The proposed framework allows for the calibration of multiscale, spatiotemporal models of cancer growth using state-of-the-art 3D cell culture data, and their validation based on the resulting experimental morphological patterns. Its implementation enabled us to pursue two goals; first, the quantitative description of the morphology of cancer progression in 3D cultures, and second, the relation of tumour morphology with underlying biophysical mechanisms that govern cancer growth and migration. We applied this framework to the study of the spatiotemporal progression of Triple Negative Breast Cancer cells cultured in Matrigel scaffolds, and validated the hypothesis of chemotactic migration using a multiscale Keller-Segel model. The results revealed transient, non-random spatial distributions of cancer cells that consist of clustered, and dispersion patterns. The proposed model was able to describe the general characteristics of the experimental observations and suggests that cancer cells exhibited chemotactic migration and accumulation, as well as random motion during the examined time period of development. To our knowledge, this is the first time that a multiscale model is used to quantify the relationship between the spatial patterns and the underlying mechanisms of cancer growth in 3D environments. Polycystins are upregulated in fibrotic kidneys and promote renal fibrosis Autosomal polycystic kidney disease (ADPKD) is caused by mutations in PKD1or PKD2 gene, which encodes polycystin-1 or polycystin-2 respectively. Paradoxically, polycystins are up-regulated in human ADPKD kidneys and overexpression of polycystin-1 even induced renal cyst formation in mice, suggesting that up-regulation of polycystin is pathogenic. In this study, we determined the role of polycystin in renal fibrosis and explored its underlying mechanisms. We found that the expression of polycystin-1 or polycystin-2 was positively correlated with the expression of collagen-I in the kidney of three different fibrotic mouse models. Pharmaceutical inhibition or genetic knockout of polycystin-2 reduced the expression of pro-fibrotic markers in fibrotic kidneys of two animal models. Moreover, conditional knockout of Pkd1 gene attenuated renal fibrosis in two mouse models. Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase which plays important role in renal fibrosis. We found that EZH2 was positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of the Ezh2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in fibrotic kidneys.

In conclusion, polycystins are up-regulated in injured kidneys and polycystins promote renal fibrosis through up-regulation of EZH2 in fibrotic kidneys, suggesting that renal fibrosis in polycystic kidneys is not the direct effect of PKD gene mutations.

SignificanceMutations of polycystins cause autosomal polycystic kidney disease (ADPKD) which is associated with increased renal fibrosis in cystic kidneys. However, the role of polycystin in renal fibrosis remains unclear. Here, we showed that the expression of polycystins is positively correlated with the development of renal fibrosis in injured kidneys. Pharmaceutical inhibition or genetic deletion of polycystin-2 attenuated renal fibrosis in fibrotic kidneys. Moreover, genetic deletion of polycystin-1 inhibited renal fibrosis in two different animal models. Finally, we showed that ablation of the methyltransferase EZH2 mitigated the anti-fibrotic effect of polycystin-1 deletion in fibrotic kidneys. We conclude that polycystin promotes through EZH2 and renal fibrosis in cystic kidney is not the direct effect of PKD gene mutations. Common anti-haemostatic medications increase the severity of uropathogenic Escherichia coli sepsis Uropathogenic Escherichia coli (UPEC) causes urinary tract infections that can result in sepsis. The haemostatic system is protective in the pyelonephritis stage of ascending UPEC infection, but the role of the haemostatic system has not been investigated during sepsis. Here we utilize a zebrafish-UPEC systemic infection model to visualize infection-induced coagulation and examine the effects of commonly prescribed anti-haemostatic medications on the infection severity. Treatment of systemically infected zebrafish with warfarin, aspirin, or ticagrelor reduced host survival, while stabilization of clots with aminocaproic acid increased host survival. Anti-haemostatic drug treatment increased UPEC burden. Our findings provide evidence that commonly prescribed anti-haemostatic medications may worsen the outcome of severe UPEC infection. Unevolved proteins from modern and prebiotic amino acids manifest distinct structural profiles Natural proteins represent numerous but tiny structure/function islands in a vast ocean of possible protein sequences, most of which has not been explored by either biological evolution or research. Recent studies have suggested this uncharted sequence space possesses surprisingly high structural propensity, but development of an understanding of this phenomenon has been awaiting a systematic high-throughput approach.

Here, we designed, prepared, and characterized two combinatorial protein libraries consisting of randomized proteins, each 105 residues in length. The first library constructed proteins from the entire canonical alphabet of 20 amino acids. The second library used a subset of only 10 residues (A,S,D,G,L,I,P,T,E,V) that represent a consensus view of plausibly available amino acids through prebiotic chemistry. Our study shows that compact conformations resistant to proteolysis are (i) abundant (up to 40%) in random sequence space, (ii) independent of general Hsp70 chaperone system activity, and (iii) not granted solely by "late" and complex amino acid additions. The Hsp70 chaperone system effectively increases solubility and refoldability of the canonical alphabet but has only a minor impact on the "early" library. The early alphabet proteins are inherently more soluble and refoldable, possibly assisted by the cell-like environment in which these assays were performed.

Our work indicates that both early and modern amino acids are predisposed to supporting protein structure (either in forms of oligomers or globular/molten globule structures) and that protein structure may not be a unique outcome of evolution.