Single-molecule imaging of von Willebrand factor reveals tension-dependent self-association.

von Willebrand factor (VWF) is an ultralong concatemeric protein important in hemostasis and thrombosis. VWF molecules can associate with other VWF molecules, but little is known about the mechanism. Hydrodynamic drag exerts tensile force on surface-tethered VWF that extends it and is maximal at the tether point and declines linearly to 0 at the downstream free end. Using single-molecule fluorescence microscopy, we directly visualized the kinetics of binding of free VWF in flow to surface-tethered single VWF molecules. We showed that self-association requires elongation of tethered VWF and that association increases with tension in tethered VWF, reaches half maximum at a characteristic tension of ∼10 pN, and plateaus above ∼25 pN. Association is reversible and hence noncovalent; a sharp decrease in shear flow results in rapid dissociation of bound VWF. Tethered primary VWF molecules can recruit more than their own mass of secondary VWF molecules from the flow stream. Kinetics show that instead of accelerating, the rate of accumulation decreases with time, revealing an inherently self-limiting self-association mechanism. We propose that this may occur because multiple tether points between secondary and primary VWF result in lower tension on the secondary VWF, which shields more highly tensioned primary VWF from further association. Glycoprotein Ibα (GPIbα) binding and VWF self-association occur in the same region of high tension in tethered VWF concatemers; however, the half-maximal tension required for activation of GPIbα is higher, suggesting differences in molecular mechanisms. These results have important implications for the mechanism of platelet plug formation in hemostasis and thrombosis.

CHIP control degradation of mutant ETF:QO through ubiquitylation in late-onset multiple acyl-CoA dehydrogenase deficiency.

Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common form of lipid storage myopathy. The disease is mainly caused by mutations in electron-transfer flavoprotein dehydrogenase gene (ETFDH), which leads to decreased levels of ETF:QO in skeletal muscle. However, the specific underlying mechanisms triggering such degradation remain unknown. We constructed expression plasmids containing wild type ETF:QO and mutants ETF:QO-A84T, R175H, A215T, Y333C, and cultured patient-derived fibroblasts containing the following mutations in ETFDH: c.250G>A (p.A84T), c.998A>G (p.Y333C), c.770A>G (p.Y257C), c.1254_1257delAACT (p. L418TfsX10), c.524G>A (p.R175H), c.380T>A (p.L127P), and c.892C>T (p.P298S). We used in vitro expression systems and patient-derived fibroblasts to detect stability of ETF:QO mutants then evaluated their interaction with Hsp70 interacting protein CHIP with active/inactive ubiquitin E3 ligase carboxyl terminus using western blot and immunofluorescence staining. This interaction was confirmed in vitro and in vivo by co-immunoprecipitation and immunofluorescence staining. We confirmed the existence two ubiquitination sites in mutant ETF:QO using mass spectrometry (MS) analysis. We found that mutant ETF:QO proteins were unstable and easily degraded in patient fibroblasts and in vitro expression systems by ubiquitin-proteasome pathway, and identified the specific ubiquitin E3 ligase as CHIP, which forms complex to control mutant ETF:QO degradation through poly-ubiquitination. CHIP-dependent degradation of mutant ETF:QO proteins was confirmed by MS and site-directed mutagenesis of ubiquitination sites. Hsp70 is directly involved in this process as molecular chaperone of CHIP. CHIP plays an important role in ubiquitin-proteasome pathway dependent degradation of mutant ETF:QO by working as a chaperone-assisted E3 ligase, which reveals CHIP's potential role in pathological mechanisms of late-onset MADD.

Spectrum of SLC20A2, PDGFRB, PDGFB, and XPR1 mutations in a large cohort of patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder with four causative genes (SLC20A2, PDGFRB, PDGFB, and XPR1) that have been identified. Here, we aim to describe the mutational spectrum of four causative genes in a series of 226 unrelated Chinese PFBC patients. Mutations in four causative genes were detected in 16.8% (38/226) of PFBC patients. SLC20A2 mutations accounted for 14.2% (32/226) of all patients. Mutations in the other three genes were relatively rare, accounting for 0.9% (2/226) of all patients, respectively. Clinically, 44.8% of genetically confirmed patients (probands and relatives) were considered symptomatic. The most frequent symptoms were chronic headache, followed by movement disorders and vertigo. Moreover, the total calcification score was significantly higher in the symptomatic group compared to the asymptomatic group. Functionally, we observed impaired phosphate transport induced by seven novel missense mutations in SLC20A2 and two novel mutations in XPR1. The mutation p.D164Y in XPR1 might result in low protein expression through an enhanced proteasome pathway. In conclusion, our study further confirms that mutations in SLC20A2 are the major cause of PFBC and provides additional evidence for the crucial roles of phosphate transport impairment in the pathogenies of PFBC.

High prevalence of Pfdhfr-Pfdhps quadruple mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea.

BACKGROUND: Sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment of malaria in Africa. However, increasing SP resistance (SPR) affects the therapeutic efficacy of the SP. As molecular markers, Pfdhfr (dihydrofolate reductase) and Pfdhps (dihydropteroate synthase) genes are widely used for SPR surveillance. This study aimed to assess the prevalence of Pfdhfr and Pfdhps genes mutations and haplotypes in Plasmodium falciparum isolates collected from Bioko Island, Equatorial Guinea (EG). METHODS: In total, 180 samples were collected in 2013-2014. The single nucleotide polymorphisms (SNPs) of the Pfdhfr and Pfdhps genes were identified with nested PCR and Sanger sequencing. The genotypes and linkage disequilibrium (LD) tests were also analysed. RESULTS: Sequences of Pfdhfr and Pfdhps genes were obtained from 92.78% (167/180) and 87.78% (158/180) of the samples, respectively. For Pfdhfr, 97.60% (163/167), 87.43% (146/167) and 97.01% (162/167) of the samples carried N51I, C59R and S108N mutant alleles, respectively. The prevalence of the Pfdhps S436A, A437G, K540E, A581G, and A613S mutations were observed in 20.25% (32/158), 90.51% (143/158), 5.06% (8/158), 0.63% (1/158), and 3.16% (5/158) of the samples, respectively. In total, 3 unique haplotypes at the Pfdhfr locus and 8 haplotypes at the Pfdhps locus were identified. A triple mutation (CIRNI) in Pfdhfr was the most prevalent haplotype (86.83%), and a single mutant haplotype (SGKAA; 62.66%) was predominant in Pfdhps. A total of 130 isolates with 12 unique haplotypes were found in the Pfdhfr and Pfdhps combined haplotypes, 65.38% (85/130) of them carried quadruple allele combinations (CIRNI-SGKAA), whereas only one isolate (0.77%, 1/130) was found to carry the wild-type (CNCSI-SAKAA). For LD analysis, the Pfdhfr N51I was significantly associated with the Pfdhps A437G (P < 0.05). CONCLUSION: Bioko Island possesses a high prevalence of the Pfdhfr triple mutation (CIRNI) and Pfdhps single mutation (SGKAA), which will undermine the pharmaceutical effect of SP for malaria treatment strategies. To avoid an increase in SPR, continuous molecular monitoring and additional control efforts are urgently needed in Bioko Island, Equatorial Guinea.

Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease.

Polycystic kidney disease (PKD) is a life-threatening disorder, commonly caused by defects in polycystin-1 (PC1) or polycystin-2 (PC2), in which tubular epithelia form fluid-filled cysts. A major barrier to understanding PKD is the absence of human cellular models that accurately and efficiently recapitulate cystogenesis. Previously, we have generated a genetic model of PKD using human pluripotent stem cells and derived kidney organoids. Here we show that systematic substitution of physical components can dramatically increase or decrease cyst formation, unveiling a critical role for microenvironment in PKD. Removal of adherent cues increases cystogenesis 10-fold, producing cysts phenotypically resembling PKD that expand massively to 1-centimetre diameters. Removal of stroma enables outgrowth of PKD cell lines, which exhibit defects in PC1 expression and collagen compaction. Cyclic adenosine monophosphate (cAMP), when added, induces cysts in both PKD organoids and controls. These biomaterials establish a highly efficient model of PKD cystogenesis that directly implicates the microenvironment at the earliest stages of the disease.

Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development.

A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378.

A Facile Preparation Process of Magnetic Aldehyde-Functionalized Ni(0.5)Zn(0.5)Fe2O4@SiO2 Nanocomposites for Immobilization of Penicillin G Acylase (PGA).

A facile sol combustion and gel calcination process has been reported for the preparation of core­ shell magnetic Zn0.5Fe2O4@SiOnanocomposites. The morphology, chemical composition, structure and magnetic property of as-prepared nanocomposites were investigated by XRD, VSM, BET, SEM, and TEM, and the magnetic Ni0.5Zn0.5Fe2O4@SiO2 nanocomposites were characterized with average size of about 25 nm, saturation magnetization of 90.8 Am²/kg and the specific surface area of 67.1 m2/g. The surface of Ni0.5Zn0.5Fe2O4@SiO2 nanocomposites was functionalized with glutaraldehyde to form the aldehyde-functionalized magnetic Ni0.5Zn0.5Fe2O4@SiO2 nanocomposites, and penicillin G acylase (PGA) was successfully immobilized onto them. And the immobilized PGA exhibited high effective activity, good stability of enzyme catalyst and good reusability, and could retain 63.5% of initial activity after 12 consecutive operations. The kinetic parameters were determined, and the value of K m for the immobilized PGA (161.7 mmol/L) is higher than that of the free PGA (3.5 mmol/L), while v max (1.626 mmol/min) is also larger than that of the free PGA (0.838 mmol/min), which revealed that the immobilization of PGA onto Ni0.5Zn0.5Fe2O4@SiO2 nanocomposites was an efficient and simple way for preparation of stable PGA.

Rapid Recognition and Isolation of Live Colon Cancer Stem Cells by Using Metabolic Labeling of Azido Sugar and Magnetic Beads.

New approach for colon cancer stem cells (CSCs) recognition and isolation is reported. Colon CSCs are responsible for colonic tumor growth, metastasis, and resistance for radio-/chemotherapies. An accurate identification and isolation method is critical for understanding and characterization of these cells. In our work, we recognized CSCs' population from colon cancer cells by using metabolic labeling of azido sugar based on the quiescent nature of these cells, which differed fundamentally from previously described methods by using specific cellular markers to recognize and isolate CSCs. Later the putative CSCs were isolated by using commercially available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and an mRNA level of colon stem cells marker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests.

Significant clinical heterogeneity with similar ETFDH genotype in three Chinese patients with late-onset multiple acyl-CoA dehydrogenase deficiency.

Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) with electron transfer flavoprotein dehydrogenase (ETFDH) gene mutations is the most common lipid storage myopathy (LSM) in China. Its clinical features vary widely and pose a challenge for diagnosis. We presented the significant clinical heterogeneity among three Chinese late-onset MADD patients with similar ETFDH genotype by collecting clinical information, muscle histology, and genetic analysis. Three novel compound heterozygous variants of ETFDH gene were identified: c.892C > T (p.Pro298Ser), c.453delA (p.Glu152ArgfsTer15), and c.449_453delTAACA (p.Leu150Ter). Moreover, all patients carried a hotspot mutation c.250G > A (p.Ala84Thr). Western blot analysis of the patients' muscular tissue showed a significantly reduced ETFDH expression, and normal electron transfer flavoprotein A (ETFA) and electron transfer flavoprotein B (ETFB) expression. Two patients with similar genotypes (c.453delA and c.449_453delTAACA) presented a significant clinical heterogeneity. Among them, one exhibited muscle weakness and exercise intolerance as initial and major symptoms, and the other showed episodic recurrent gastrointestinal symptoms before a serious muscle weakness appeared in later life. The novel variants in ETFDH and the corresponding clinical features enrich the variant spectrum of late-onset MADD and provide a new insight into the genotype-phenotype relationship. Late-onset MADD should be included in differential diagnosis for adult myopathy along with chronic digestive disease.

Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules.

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein.

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.

Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements.

Double-stranded DNA is a dynamic molecule whose structure can change depending on conditions. While there is consensus in the literature about many structures DNA can have, the state of highly-stretched DNA is still not clear. Several groups have shown that DNA in the torsion-unconstrained B-form undergoes an "overstretching" transition at a stretching force of around 65 pN, which leads to approximately 1.7-fold elongation of the DNA contour length. Recent experiments have revealed that two distinct structural transitions are involved in the overstretching process: (i) a hysteretic "peeling" off one strand from its complementary strand, and (ii) a nonhysteretic transition that leads to an undetermined DNA structure. We report the first simultaneous determination of the entropy (ΔS) and enthalpy changes (ΔH) pertaining to these respective transitions. For the hysteretic peeling transition, we determined ΔS âˆ¼ 20 cal/(K.mol) and ΔH âˆ¼ 7 kcal/mol. In the case of the nonhysteretic transition, ΔS âˆ¼ -3 cal/(K.mol) and ΔH âˆ¼ 1 kcal/mol. Furthermore, the response of the transition force to salt concentration implies that the two DNA strands are spatially separated after the hysteretic peeling transition. In contrast, the corresponding response after the nonhysteretic transition indicated that the strands remained in close proximity. The selection between the two transitions depends on DNA base-pair stability, and it can be illustrated by a multidimensional phase diagram. Our results provide important insights into the thermodynamics of DNA overstretching and conformational structures of overstretched DNA that may play an important role in vivo.

Sparse representation for infrared Dim target detection via a discriminative over-complete dictionary learned online.

It is difficult for structural over-complete dictionaries such as the Gabor function and discriminative over-complete dictionary, which are learned offline and classified manually, to represent natural images with the goal of ideal sparseness and to enhance the difference between background clutter and target signals. This paper proposes an infrared dim target detection approach based on sparse representation on a discriminative over-complete dictionary. An adaptive morphological over-complete dictionary is trained and constructed online according to the content of infrared image by K-singular value decomposition (K-SVD) algorithm. Then the adaptive morphological over-complete dictionary is divided automatically into a target over-complete dictionary describing target signals, and a background over-complete dictionary embedding background by the criteria that the atoms in the target over-complete dictionary could be decomposed more sparsely based on a Gaussian over-complete dictionary than the one in the background over-complete dictionary. This discriminative over-complete dictionary can not only capture significant features of background clutter and dim targets better than a structural over-complete dictionary, but also strengthens the sparse feature difference between background and target more efficiently than a discriminative over-complete dictionary learned offline and classified manually. The target and background clutter can be sparsely decomposed over their corresponding over-complete dictionaries, yet couldn't be sparsely decomposed based on their opposite over-complete dictionary, so their residuals after reconstruction by the prescribed number of target and background atoms differ very visibly. Some experiments are included and the results show that this proposed approach could not only improve the sparsity more efficiently, but also enhance the performance of small target detection more effectively.

Liraglutide Improves PCOS Symptoms in Rats by Targeting FDX1.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a gynecological endocrine disorder characterized by ovulatory disorders, hyperandrogenemia, and polycystic changes in the ovaries. FDX1 is a ferredoxin-reducing protein on human mitochondria that plays an important role in steroid anabolism. Liraglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA), has recently emerged as a potential therapeutic agent for PCOS. Recent studies have suggested that FDX1 may be associated with the development of PCOS. This study aims to explore the pivotal role of FDX1 in the amelioration of PCOS through liraglutide intervention. MATERIALS AND METHODS: A PCOS rat model was induced via subcutaneous DHEA injections. Following successful model establishment, the rats were treated with liraglutide combined with metformin, or with each drug individually, over a six-week period. After 6 weeks of treatment, we assessed changes in body weight, fasting blood glucose, sex hormone levels, estrous cycle regularity, ovarian morphology, FDX1 expression in ovarian tissue, and ovarian ROS levels. RESULTS: PCOS rats exhibited significant increases in body weight and fasting blood glucose levels, disrupted estrous cycles, and polycystic ovarian morphology. FDX1 expression was notably reduced in the ovarian tissues of PCOS rats. Treatment with liraglutide, both alone and in combination with metformin, led to improvements in body weight, fasting blood glucose, sex hormone balance, estrous cycle regularity, ovarian morphology, and ovarian ROS levels. Notably, FDX1 expression was significantly restored in all treatment groups, with the most substantial increase observed in the liraglutide-treated group. CONCLUSION: This study suggests that FDX1 could serve as a potential biomarker for elucidating the underlying mechanisms of liraglutide's therapeutic effects in PCOS management.

Interferon-γ induces combined pyroptotic angiopathy and APOL1 expression in human kidney disease.

Elevated interferon (IFN) signaling is associated with kidney diseases including COVID-19, HIV, and apolipoprotein-L1 (APOL1) nephropathy, but whether IFNs directly contribute to nephrotoxicity remains unclear. Using human kidney organoids, primary endothelial cells, and patient samples, we demonstrate that IFN-γ induces pyroptotic angiopathy in combination with APOL1 expression. Single-cell RNA sequencing, immunoblotting, and quantitative fluorescence-based assays reveal that IFN-γ-mediated expression of APOL1 is accompanied by pyroptotic endothelial network degradation in organoids. Pharmacological blockade of IFN-γ signaling inhibits APOL1 expression, prevents upregulation of pyroptosis-associated genes, and rescues vascular networks. Multiomic analyses in patients with COVID-19, proteinuric kidney disease, and collapsing glomerulopathy similarly demonstrate increased IFN signaling and pyroptosis-associated gene expression correlating with accelerated renal disease progression. Our results reveal that IFN-γ signaling simultaneously induces endothelial injury and primes renal cells for pyroptosis, suggesting a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy, which can be targeted therapeutically.

Transition dynamics and selection of the distinct S-DNA and strand unpeeling modes of double helix overstretching.

Recent studies have revealed two distinct pathways for the DNA overstretching transition near 65 pN: 'unpeeling' of one strand from the other, and a transition from B-DNA to an elongated double-stranded 'S-DNA' form. However, basic questions concerning the dynamics of these transitions, relative stability of the two competing overstretched states, and effects of nicks and free DNA ends on overstretching, remain open. In this study we report that: (i) stepwise extension changes caused by sequence-defined barriers occur during the strand-unpeeling transition, whereas rapid, sequence-independent extension fluctuations occur during the B to S transition; (ii) the secondary transition that often occurs following the overstretching transition is strand-unpeeling, during which the extension increases by 0.01-0.02 nm per base pair of S-DNA converted to single-stranded DNA at forces between 75 and 110 pN; (iii) even in the presence of nicks or free ends, S-DNA can be stable under physiological solution conditions; (iv) distribution of small GC-rich islands in a large DNA plays a key role in determining the transition pathways; and (v) in the absence of nicks or free ends, torsion-unconstrained DNA undergoes the overstretching transition via creation of S-DNA. Our study provides a new, high-resolution understanding of the competition between unpeeling and formation of S-DNA.

Loss-of-function variant in the LRR domain of SLITRK2 implicated in a neurodevelopmental disorder.

Background: Neurodevelopmental disorders are characterized by different combinations of intellectual disability (ID), communication and social skills deficits, and delays in achieving motor or language milestones. SLITRK2 is a postsynaptic cell-adhesion molecule that promotes neurite outgrowth and excitatory synapse development. Methods and Results: In the present study, we investigated a single patient segregating Neurodevelopmental disorder. SLITRK2 associated significant neuropsychological issues inherited in a rare X-linked fashion have recently been reported. Whole-exome sequencing and data analysis revealed a novel nonsense variant [c.789T>A; p.(Cys263*); NM_032539.5; NP_115928.1] in exon 5 of the SLITRK2 gene (MIM# 300561). Three-dimensional protein modeling revealed substantial changes in the mutated SLITRK2 protein, which might lead to nonsense-medicated decay. Conclusion: This study confirms the role of SLITRK2 in neuronal development and highlights the importance of including the SLITRK2 gene in the screening of individuals presenting neurodevelopmental disorders.

Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract.

Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.

Two distinct overstretched DNA states.

The DNA double helix undergoes an 'overstretching' transition in a narrow force range near 65 pN. Despite numerous studies the basic question of whether the strands are separated or not remains controversial. Here we show that overstretching in fact involves two distinct types of double-helix reorganization: slow hysteretic 'unpeeling' of one strand off the other; and a fast, non-hysteretic transition to an elongated double-stranded form. We demonstrate that the relative fraction of these two overstretched forms is sensitive to factors that affect DNA base pair stability, including DNA sequence, salt concentration and temperature. The balance between the two forms shifts near physiological solution conditions. This result, in addition to establishing the existence of an overstretched double-stranded state, also shows that double helix physical properties are tuned so that either unpeeling or overextension can be selected via small changes in molecule environment.

Modelling ciliopathy phenotypes in human tissues derived from pluripotent stem cells with genetically ablated cilia.

The functions of cilia-antenna-like organelles associated with a spectrum of disease states-are poorly understood, particularly in human cells. Here we show that human pluripotent stem cells (hPSCs) edited via CRISPR to knock out the kinesin-2 subunits KIF3A or KIF3B can be used to model ciliopathy phenotypes and to reveal ciliary functions at the tissue scale. KIF3A-/- and KIF3B-/- hPSCs lacked cilia, yet remained robustly self-renewing and pluripotent. Tissues and organoids derived from these hPSCs displayed phenotypes that recapitulated defective neurogenesis and nephrogenesis, polycystic kidney disease (PKD) and other features of the ciliopathy spectrum. We also show that human cilia mediate a critical switch in hedgehog signalling during organoid differentiation, and that they constitutively release extracellular vesicles containing signalling molecules associated with ciliopathy phenotypes. The capacity of KIF3A-/- and KIF3B-/- hPSCs to reveal endogenous mechanisms underlying complex ciliary phenotypes may facilitate the discovery of candidate therapeutics.