Organized Assemblies Research Articles

Inorganic nanoparticles modify the phase behavior and viscoelastic properties of non-lamellar lipid mesophases

The inclusion of inorganic nanoparticles (NPs) within organized lipid assemblies combines the rich polymorphism of lipid phases with advanced functional properties provided by the NPs, expanding the applicative spectrum of these materials. In spite of the relevance of these hybrid systems, fundamental knowledge on the effects of NPs on the structure and physicochemical properties of lipid mesophases is still limited. This contribution combines Small-Angle X-ray Scattering (SAXS) and Rheology to connect the structural properties with the viscoelastic behavior of liquid crystalline mesophases of Phytantriol (Phyt) containing two kinds of hydrophobic NPs of similar size, i.e., gold NPs (AuNPs) and Superparamagnetic Iron Oxide NPs (SPIONs). Both types of NPs spontaneously embed in the hydrophobic domains of the liquid crystalline mesophase, deeply affecting its phase behavior, as SAXS results disclose. We propose a general model to interpret and predict the structure of cubic mesophases doped with hydrophobic NPs, where the effects on lipid phase behavior depend only on NPs’ size and volume fraction but not on chemical identity. The rheological measurements reveal that NPs increase the solid-like behavior of the hybrid and, surprisingly, this effect depends on the chemical nature of the NPs. We interpret these results by suggesting that the long-range dipolar interactions of SPIONs affect the viscoelastic response of the material and provide an additional control parameter on mechanical properties. Overall, this study discloses new fundamental insights into hybrid liquid crystalline mesophases doped with hydrophobic NPs, highly relevant for future applications, e.g. in the biomedical field as smart materials for drug delivery.

Supercharging enables organized assembly of synthetic biomolecules

Symmetrical protein oligomers are ubiquitous in biological systems and perform key structural and regulatory functions. However, there are few methods for constructing such oligomers. Here we have engineered completely synthetic, symmetrical oligomers by combining pairs of oppositely supercharged variants of a normally monomeric model protein through a strategy we term 'supercharged protein assembly' (SuPrA). We show that supercharged variants of green fluorescent protein can assemble into a variety of architectures including a well-defined symmetrical 16-mer structure that we solved using cryo-electron microscopy at 3.47 Å resolution. The 16-mer is composed of two stacked rings of octamers, in which the octamers contain supercharged proteins of alternating charges, and interactions within and between the rings are mediated by a variety of specific electrostatic contacts. The ready assembly of this structure suggests that combining oppositely supercharged pairs of protein variants may provide broad opportunities for generating novel architectures via otherwise unprogrammed interactions.

Nanoparticles and organized lipid assemblies: from interaction to design of hybrid soft devices.

This contribution reviews the state of art on hybrid soft matter assemblies composed of inorganic nanoparticles (NP) and lamellar or non-lamellar lipid bilayers. After a short outline of the relevant energetic contributions, we address the interaction of NPs with synthetic lamellar bilayers, meant as cell membrane mimics. We then review the design of hybrid nanostructured materials composed of lipid bilayers and some classes of inorganic NPs, with particular emphasis on the effects on the amphiphilic phase diagram and on the additional properties contributed by the NPs. Then, we present the latest developments on the use of lipid bilayers as coating agents for inorganic NPs. Finally, we remark on the main achievements of the last years and our vision for the development of the field.

Natural rubber-SiO2 nanohybrids: interface structures and dynamics.

Homogeneous dispersion of silica nanoparticles (SiO2 NPs) in natural rubber (NR) is a key challenge for engineering high-performance nanocomposites and elucidation of their structure on a molecular basis. Towards this, the present work devised a novel route for obtaining 3D self-assembled SiO2 NP-NR nanocomposites under aqueous conditions and in the presence of Mg2+, by establishing a molecular bridge that clamped the negatively charged NR and SiO2 colloidal particles with a favoured NR-SiO2 NP hetero-aggregation. The characteristic NR-SiO2 NP hetero-aggregates displayed a decreased heat capacity with increase in the SiO2 mass-fraction, implying a restricted NR chain mobility. Such changes in the interfacial layers were tapped by 29Si NMR, DFT calculations and molecular dynamics simulations towards a mechanistic understanding of the structure and dynamics of the NR/SiO2 NP hybrid. Simple models were used to illustrate basic ideas; specific electrostatic interactions such as ion-dipole and H-bonding interactions proved to be the driving forces for the organized assembly leading to the NR-SiO2 hetero-aggregate over the NR-NR or SiO2 NP-SiO2 NP homo-aggregate. Molecular dynamics simulation of the aqueous canonical ensemble of the hybrid showed the stable molecular conformation to reveal a SiO2 NP spherical core encapsulated by a hydrophobically interconnected NR polymer layer as the outer shell, as a unique structural model. Specifically, the lipid end of the NR was involved electrostatically while the lysine end (the protein part of NR) H-bonded to the core silica cluster thereby restricting random aggregation. The calculated negative free energy changes for the hetero-aggregate composites via their vibrational and rotational spectra proved the spontaneity of composite formation.

Inhibition of protein kinase D disrupts spindle formation and actin assembly during porcine oocyte maturation.

Protein kinase D (PKD) subfamily which includes PKD1, PKD2 and PKD3 is a novel family of serine/threonine kinases. PKD has been widely implicated in the regulation of multiple physiological effects including immune responses, apoptosis and cell proliferation. However, the roles of PKD in oocytes have not been fully clarified. In this study we investigated the regulatory functions of PKD during porcine oocyte maturation. Our results indicated that PKD expressed in porcine oocytes and the inhibition of PKD family activity led to the failure of meiosis resumption and the first polar body extrusion. Further analysis indicated that the spindle assembly and chromosome alignment were disrupted after PKD family inhibition, and this might be through its regulatory role on MAPK phosphorylation. We also found that PKD phosphorylated cofilin for actin assembly, which further affected cortical actin distribution, indicating the roles of PKD family on cytoskeleton. In addition, a decreased expression of PKD in postovulatory aging porcine oocytes was observed, which might connect PKD with cytoskeleton defects in aged oocytes. Taken together, these results suggest that PKD possesses important functions in porcine oocyte maturation by regulating spindle organization and actin assembly.

A Fibrous Hybrid Patch Couples Cell-Derived Matrix and Poly(l-lactide-co-caprolactone) for Endothelial Cells Delivery and Skin Wound Repair.

Skin wound healing is an intricate orchestration that involves different cell types, an extracellular matrix (ECM), cytokines, and growth factors. On the basis of the great benefits of cell-derived ECM in regenerative applications, here we propose an electrospun fibrous membrane that combines poly(l-lactide-co-caprolactone) (PLCL) and human fibroblast-derived ECM (hFDM). hFDM-deposited PLCL (hFDM-PLCL) was obtained via decellularization of a confluent layer of fibroblasts cultivated on PLCL. An organized assembly of fibrillar structure on hFDM-PLCL was notable via immunostaining. As human umbilical vein endothelial cells (HUVECs) were seeded on the hFDM-PLCL, they proliferated faster and exhibited more elongated, capillary-like morphology than those on PLCL or fibronectin-coated PLCL (Fn-PLCL). HUVECs have a relatively large aspect ratio, spreading area, and vinculin-positive area per cell on the fibrillary structure of hFDM-PLCL. In addition, transwell cell migration assay showed the chemoattractant effect of hFDM for HUVECs and human dermal fibroblasts. Furthermore, HUVECs-loaded hFDM-PLCL membranes showed the most promising therapeutic effects on a mouse skin wound model as proved via the wound closure rate, neovascularization effect, regenerated epidermis, and skin appendage. This study shows that biodegradable PLCL fibers not only support the weak mechanical properties of hFDM but also allow hFDM to reserve ECM macromolecules and to maintain structural integrity. Current results also demonstrate the critical role of hFDM with biochemical and biophysical cues on HUVECs adhesion, proliferation, and vascular morphogenesis in vitro and even on the wound healing process in vivo. Taken together, hFDM-functionalized PLCL patch should be a promising platform for cell delivery and regenerative applications.

Bacteriophages: A Therapy Concept against Multi-Drug-Resistant Bacteria.

Bacteriophages (phages) are viruses that kill bacteria specifically but cannot infect other kinds of organisms. They have attracted new attention since the increasing antibiotic resistance developed into a global crisis. Phage therapy, a 100-year-old form of antibacterial treatment in medicine, is gaining momentum because phages represent a therapy concept without such negative side effects as toxicity; phages are the only therapeutic agent that regulates itself at the sites of infection and decays when the infectious bacteria have been killed. Nature is an almost infinite phage resource: New ones can be isolated for most kinds of problem bacteria as needed; bacteria and their phages constantly co-evolve. This is important as new pathogenic bacterial variants evolve and new challenging situations arise. In human therapy, “cocktails” of multiple phages may reduce the probability of selecting bacteria that developed resistance to a certain phage. Antibiotic agents can be applied together with phages in many circumstances; the two often function synergistically. Phages cannot be expected to replace antibiotic agents in our medical arsenal, but can be used where antibiotic agents fail. The selected phages, however, must be obligately virulent, well-characterized, and highly purified before application. Countless patients and their physicians are waiting for re-establishing phage therapy as a flexible, tailored medicine; infrastructures should be built in all countries urgently: The 2015 World Health Organization assembly resolution 68.7.3. called for national action plans by May 2017 to combat the antimicrobial drug resistance crisis. This article discusses the therapeutic potential of phages and describes challenges and recent developments.

GuttaFlow Bioseal promotes spontaneous differentiation of human periodontal ligament stem cells into cementoblast-like cells

ObjectivesTo evaluate in vitro the cementogenic potential and the biological effects of GuttaFlow Bioseal, GuttaFlow 2, MTA Fillapex and AH Plus on human periodontal ligament stem cells (hPDLSCs). MethodsCell viability, cell migration and cell morphology assays were performed using eluates of each material. To evaluate cell attachment, hPDLSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy (SEM). The effects of endodontic sealers on cementum protein 1 (CEMP1), cementum-derived attachment protein (CAP), bone sialoprotein (BSP), ameloblastin (AMBN), amelogenin (AMELX) and alkaline phosphatase (ALP) gene expression on hPDLSCs were investigated by qPCR and immunofluorescence (IF). Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α<0.05). ResultsMore than 90% of viable cells were obtained using extracts of GuttaFlow Bioseal and GuttaFlow2 after 72h of culture. By contrast, AH Plus and MTA Fillapex induced significantly lower levels of cell viability. GuttaFlow2 and GuttaFlow Bioseal promoted wound closure in a concentration-dependent manner, comparable to that observed with control extracts (*p<0.05). However, with AH Plus and MTA Fillapex, cell migration was significantly lower than in the control (***p<0.0001). SEM analysis pointed to an organized stress fiber assembly and high degree of cell adhesion on GuttaFlow Bioseal disks but low rates on GuttaFlow2, MTA Fillapex and AH Plus. When hPDLSCs were cultured with GuttaFlow Bioseal-conditioned media, qPCR assays and IF showed a higher level of AMELX, AMBN, CEMP1 and CAP expression than the control (*p<0.05)), whereas no such expression was observed in the other sealers. SignificanceOur results showed that GuttaFlow sealers were more cytocompatible than AH Plus and MTA Fillapex, while GuttaFlow Bioseal favored cementoblast differentiation of hPDLSCs in the absence of any growth factors.

Nonequilibrium Spatiotemporal Sensing within Acoustically Patterned Two-Dimensional Protocell Arrays.

Acoustically trapped periodic arrays of horseradish peroxidase (HRP)-loaded poly(diallydimethylammonium chloride) / adenosine 5′-triphosphate coacervate microdroplet-based protocells exhibit a spatiotemporal biochemical response when exposed to a codiffusing mixture of substrate molecules (o-phenylenediamine (o-PD) and hydrogen peroxide (H2O2)) under nonequilibrium conditions. Unidirectional propagation of the chemical concentration gradients gives rise to time- and position-dependent fluorescence signal outputs from individual coacervate microdroplets, indicating that the organized protocell assembly can dynamically sense encoded information in the advancing reaction-diffusion front. The methodology is extended to arrays comprising spatially separated binary populations of HRP- or glucose oxidase-containing coacervate microdroplets to internally generate a H2O2 signal that chemically connects the two protocell communities via a concerted biochemical cascade reaction. Our results provide a step toward establishing a systematic approach to study dynamic interactions between organized protocell consortia and propagating reaction-diffusion gradients, and offer a new methodology for exploring the complexity of protocellular communication networks operating under nonequilibrium conditions.

Ccdc61 controls centrosomal localization of Cep170 and is required for spindle assembly and symmetry.

Microtubule nucleation was uncovered as a key principle of spindle assembly. However, the mechanistic details about microtubule nucleation and the organization of spindle formation and symmetry are currently being revealed. Here we describe the function of coiled-coil domain containing 61 (Ccdc61), a so far uncharacterized centrosomal protein, in spindle assembly and symmetry. Our data describe that Ccdc61 is required for spindle assembly and precise chromosome alignments in mitosis. Microtubule tip-tracking experiments in the absence of Ccdc61 reveal a clear loss of the intrinsic symmetry of microtubule tracks within the spindle. Furthermore, we show that Ccdc61 controls the centrosomal localization of centrosomal protein of 170 kDa (Cep170), a protein that was shown previously to localize to centrosomes as well as spindle microtubules and promotes microtubule organization and microtubule assembly. Interestingly, selective disruption of Ccdc61 impairs the binding between Cep170 and TANK binding kinase 1, an interaction that is required for microtubule stability. In summary, we have discovered Ccdc61 as a centrosomal protein with an important function in mitotic microtubule organization.

Amyloid self‐assembling peptides: Potential applications in nanovaccine engineering and biosensing

AbstractLiving organisms are an inestimable source of inspiration for the design of biomaterials and nanostructures for medical and technological applications. Amyloids, which were historically associated with diseases, have recently been recognized as a biological structure that performs vital physiological functions in host organisms, highlighting their potential as life‐inspired assemblies. Amyloids are highly organized proteinaceous assemblies characterized by a cross‐β‐sheet quaternary conformation. The mechanical, physical, and biological properties of amyloids suggest that these nanostructures hold great potential as soft materials, nanoparticles, and biomatrices. This potential is associated with many characteristics, including the spontaneous self‐assembly of many polypeptide sequences, high mechanical resistance, biocompatibility, biodegradability as well as thermal, chemical, and enzymatic stability. Moreover, peptide‐based amyloid assemblies can efficiently be obtained by standard solid phase peptide synthesis and orthogonally functionalized with a wide range of biomolecules, such as large proteins and DNA. In this review, after briefly introducing the amyloid structure and the mechanisms of self‐assembly, we describe approaches to identify and design short self‐assembling amyloidogenic peptides. Afterward, we introduce strategies used to functionalize amyloid materials and we highlight some relevant examples in the development of nanovaccines and biosensors.

Serine protease PRSS55 is crucial for male mouse fertility via affecting sperm migration and sperm\u2013egg binding

Testis-specific PRSS55 is a highly conserved chymotrypsin-like serine protease among mammalian species. So far, the physiological function of PRSS55 remains unknown. Here, we show that PRSS55 is a GPI-anchored membrane protein, specifically expressed in adult mouse testis and mainly observed in the luminal side of seminiferous tubules and sperm acrosome. Mice deficient for Prss55 develop male infertile with normal reproduction-related parameters observed. Interestingly, in vivo fertilization rate of Prss55−/− males is dramatically decreased, possibly due to incapable migration of Prss55−/− sperm from uterus into oviduct. However, in vitro fertilization rate has no difference between two genotypes although Prss55−/− sperm presents defective recognition/binding to zona-intact or zona-free oocytes. Further study reveals that mature ADAM3 is almost undetectable in Prss55−/− sperm, while precursor ADAM3 remains unchanged in the testis. However, it is shown that ADAM3 has no interaction with PRSS55 by immunoprecipitation with anti-PRSS55 antibody. The expression levels of several proteins known to be related to the observed phenotypes remain comparable between wt and Prss55−/− mice. Moreover, we found that Prss55 deficiency has no effect on PRSS37 or vice versa albeit two mutant males share almost the same phenotypes. Microarray analysis reveals a total of 72 differentially expressed genes in Prss55−/− testis, most of which are associated with cellular membrane and organelle organization, protein transport and complex assembly, and response to stimulus and signaling. In conclusion, we have demonstrated that PRSS55 plays vital roles in regulating male fertility of mice, including in vivo sperm migration and in vitro sperm–egg interaction, possibly by affecting the maturation of ADAM3 in sperm and the expression of multiple genes in testis.

Disease burden of hepatitis C in the Austrian state of Tyrol - Epidemiological data and model analysis to achieve elimination by 2030.

BackgroundIn 2016, the World Health Organization (WHO) and 69th World Health Assembly approved the first global health sector strategy (GHSS) on viral hepatitis with the goal to eliminate hepatitis C virus (HCV) infections worldwide. The aim is a 90% reduction of new infections and 65% reduction of HCV-related deaths by 2030.AimThis study reports on the epidemiology of HCV infections in the Austrian state of Tyrol (total population 750,000) and uses a predictive model to identify how the WHO strategy for elimination of HCV can be achieved.MethodsWe developed a regional disease burden model based on observed local diagnosis data from 2001 to 2016. Scenarios were developed to evaluate the impact of diagnosis and treatment on HCV-related outcomes (viremic prevalence, decompensated cirrhosis, hepatocellular carcinoma, and liver-related deaths) from 2015 through 2030.ResultsIn the last 15 years, 1,721 patients living in Tyrol have been diagnosed with chronic HCV infection. When ageing, mortality and treatment were factored in, there were an estimated 2,043 viremic HCV infections in 2016, of which 1,136 cases had been diagnosed. A baseline model predicts a decrease of 588 HCV cases from 2015 to 2030, which would not translate into the significant reduction of infections needed to achieve WHO global health recommendations. A total of 1,843 infected individuals need to be identified and treated to achieve the WHO goals by 2030 (1,254 averted cases as compared to baseline model). Implementation of this strategy would avoid 523 new HCV infections and decreases HCV-related mortality by 73%.ConclusionHCV elimination and >65% reduction of associated mortality are possible for Tyrol, but requires a significant increase in new diagnoses and treatment rate. The model presented in this study could serve as an example for other regions to reliably predict regional disease burden and estimate how WHO goals can be met in the future.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

SummaryWe show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes’ expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

Thermally Responsive Hyperbranched Poly(ionic liquid)s: Assembly and Phase Transformations

A library of linear and branched amphiphilic poly(ionic liquid)s based on hydrophobic cores and peripheral thermally sensitive shells was synthesized and studied with regard to their ability to form stimuli-responsive, organized assemblies in aqueous media. The thermally responsive derivatives of poly(ionic liquid)s were synthesized by neutralizing 32 terminal carboxyl groups of functionalized polyester cores by amine-terminated poly(N-isopropylacrylamide)s (PNIPAM) (50% and 100%). We observed that these hyperbranched poly(ionic liquid)s possessed a narrow low critical solution transition (LCST) window with LCST for hyperbranched compounds being consistently lower than that for linear PNIPAM containing counterparts. We found that the poly(ionic liquid)s form spherical micellar assemblies with diverse morphologies, such as micelles and their aggregates, depending on the terminal compositions with reduced sizes for hyperbranched poly(ionic liquid)s. Increasing temperature above LCST promoted formation of ne...

The phenomenon of collaboration in a life science network

PurposeThe purpose of this paper is to outline the operative meaning of collaboration in a life science network.Design/methodology/approachData were collected through participatory observations and interviews between 2014 and 2015. The data presented were derived from field notes from participant observations, interviews and documents within a life science network in the Öresund region (southern part of Sweden and the Copenhagen area).FindingsThe findings suggest that collaboration within the life science network should be viewed as a lively, organizational assembly in the process of becoming, in contrast to the idea that it is operating on the basis of organic principles. Collaboration thus could be viewed as consisting of self-subsistent parts (participants and organizations) that are detached and plugged into different collaborative networks.Originality/valueIn the context of the emerging idea of re-building the state welfare system with the economic support of producing and selling knowledge, there seems to be a growing interest, especially from the point of view of policymakers, in the phenomenon of collaboration. This paper offers exclusive ethnographic illustrations into the heterogeneity of collaboration.

De novo transcriptome assembly and positive selection analysis of an individual deep-sea fish

BackgroundHigh hydrostatic pressure and low temperatures make the deep sea a harsh environment for life forms. Actin organization and microtubules assembly, which are essential for intracellular transport and cell motility, can be disrupted by high hydrostatic pressure. High hydrostatic pressure can also damage DNA. Nucleic acids exposed to low temperatures can form secondary structures that hinder genetic information processing. To study how deep-sea creatures adapt to such a hostile environment, one of the most straightforward ways is to sequence and compare their genes with those of their shallow-water relatives.ResultsWe captured an individual of the fish species Aldrovandia affinis, which is a typical deep-sea inhabitant, from the Okinawa Trough at a depth of 1550 m using a remotely operated vehicle (ROV). We sequenced its transcriptome and analyzed its molecular adaptation. We obtained 27,633 protein coding sequences using an Illumina platform and compared them with those of several shallow-water fish species. Analysis of 4918 single-copy orthologs identified 138 positively selected genes in A. affinis, including genes involved in microtubule regulation. Particularly, functional domains related to cold shock as well as DNA repair are exposed to positive selection pressure in both deep-sea fish and hadal amphipod.ConclusionsOverall, we have identified a set of positively selected genes related to cytoskeleton structures, DNA repair and genetic information processing, which shed light on molecular adaptation to the deep sea. These results suggest that amino acid substitutions of these positively selected genes may contribute crucially to the adaptation of deep-sea animals. Additionally, we provide a high-quality transcriptome of a deep-sea fish for future deep-sea studies.

Study on the Conformation of Entrapped Protein inside the Reverse Micellar Confinement Based on the Amino Acid Derived Ionic Liquid

AbstractOwing to superior surface‐activity and versatility in functionalization compared to conventional surfactants, surface‐active ionic liquids (SAILs) gained immense interest in recent years. Toxicity and biodegradation remain central issues while dealing with the SAILs and thus, the quest for synthesis of greener SAILs is increasing day by day. Keeping in view of the importance of SAIL's performance, we undertook the present study for the formulation of reverse micelles (RMs) using biodegradable L‐proline propyl ester lauryl sulfate ([ProC3][LS]) in cyclohexane (Cy). The formation of RMs was confirmed from the phase behavior and dynamic light scattering (DLS) studies. Fourier‐transform infrared spectroscopy (FTIR) study revealed the solvation of anionic head group through H‐bonding by added water. An increased micropolarity and reduced microviscosity were evidenced inside the RM droplets as a function of hydration level. Finally, the encapsulation of BSA protein in RMs was investigated through the fluorescence, circular dichroism and DLS studies, which showed conformation with higher degree of secondary structural content than the native state inside the droplet core at higher hydration. Our results signify the importance of the role of hydration in the function of enzyme or protein molecules in molecular crowding environments. These facts certainly prove the versatility of this kind of organized assemblies to alter their inherent properties simply by changing water content.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane

The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E.coli surface, with a nearest neighbor distance of ∼200nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.

Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture.

Chromosome conformation capture (3C) technologies can be used to investigate 3D genomic structures. However, high background noise, high costs, and a lack of straightforward noise evaluation in current methods impede the advancement of 3D genomic research. Here we developed a simple digestion-ligation-only Hi-C (DLO Hi-C) technology to explore the 3D landscape of the genome. This method requires only two rounds of digestion and ligation, without the need for biotin labeling and pulldown. Non-ligated DNA was efficiently removed in a cost-effective step by purifying specific linker-ligated DNA fragments. Notably, random ligation could be quickly evaluated in an early quality-control step before sequencing. Moreover, an in situ version of DLO Hi-C using a four-cutter restriction enzyme has been developed. We applied DLO Hi-C to delineate the genomic architecture of THP-1 and K562 cells and uncovered chromosomal translocations. This technology may facilitate investigation of genomic organization, gene regulation, and (meta)genome assembly.