Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

PARP1 condensates differentially partition DNA repair proteins and enhance DNA ligation.

Poly(ADP-ribose) polymerase 1 (PARP1) is one of the first responders to DNA damage and plays crucial roles in recruiting DNA repair proteins through its activity - poly(ADP-ribosyl)ation (PARylation). The enrichment of DNA repair proteins at sites of DNA damage has been described as the formation of a biomolecular condensate. However, it is not understood how PARP1 and PARylation contribute to the formation and organization of DNA repair condensates. Using recombinant human PARP1 in vitro, we find that PARP1 readily forms viscous biomolecular condensates in a DNA-dependent manner and that this depends on its three zinc finger (ZnF) domains. PARylation enhances PARP1 condensation in a PAR chain-length dependent manner and increases the internal dynamics of PARP1 condensates. DNA and single-strand break repair proteins XRCC1, LigIII, Polß, and FUS partition in PARP1 condensates, although in different patterns. While Polß and FUS are both homogeneously mixed within PARP1 condensates, FUS enrichment is greatly enhanced upon PARylation whereas Polß partitioning is not. XRCC1 and LigIII display an inhomogeneous organization within PARP1 condensates; their enrichment in these multiphase condensates is enhanced by PARylation. Functionally, PARP1 condensates concentrate short DNA fragments and facilitate compaction of long DNA and bridge DNA ends. Furthermore, the presence of PARP1 condensates significantly promotes DNA ligation upon PARylation. These findings provide insight into how PARP1 condensation and PARylation regulate the assembly and biochemical activities in DNA repair foci, which may inform on how PARPs function in other PAR-driven condensates.

Regulation of EZH2 protein stability: new mechanisms, roles in tumorigenesis, and roads to the clinic.

The importance of EZH2 as a key methyltransferase has been well documented theoretically. Practically, the first EZH2 inhibitor Tazemetostat (EPZ6438), was approved by FDA in 2020 and is used in clinic. However, for most solid tumors it is not as effective as desired and the scope of clinical indications is limited, suggesting that targeting its enzymatic activity may not be sufficient. Recent technologies focusing on the degradation of EZH2 protein have drawn attention due to their potential robust effects. This review focuses on the molecular mechanisms that regulate EZH2 protein stability via post-translational modifications (PTMs), mainly including ubiquitination, phosphorylation, and acetylation. In addition, we discuss recent advancements of multiple proteolysis targeting chimeras (PROTACs) strategies and the latest degraders that can downregulate EZH2 protein. We aim to highlight future directions to expand the application of novel EZH2 inhibitors by targeting both EZH2 enzymatic activity and protein stability.

High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL.

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.

CTCF and R-loops are boundaries of cohesin-mediated DNA looping.

Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.

Mass spectrometry imaging and single-cell transcriptional profiling reveal the tissue-specific regulation of bioactive ingredient biosynthesis in Taxus leaves.

Taxus leaves provide the raw industrial materials for taxol, a natural antineoplastic drug widely used in the treatment of various cancers. However, the precise distribution, biosynthesis, and transcriptional regulation of taxoids and other active components in Taxus leaves remain unknown. Matrix-assisted laser desorption/ionization-mass spectrometry imaging analysis was used to visualize various secondary metabolites in leaf sections of Taxus mairei, confirming the tissue-specific accumulation of different active metabolites. Single-cell sequencing was used to produce expression profiles of 8846 cells, with a median of 2352 genes per cell. Based on a series of cluster-specific markers, cells were grouped into 15 clusters, suggesting a high degree of cell heterogeneity in T. mairei leaves. Our data were used to create the first Taxus leaf metabolic single-cell atlas and to reveal spatial and temporal expression patterns of several secondary metabolic pathways. According to the cell-type annotation, most taxol biosynthesis genes are expressed mainly in leaf mesophyll cells; phenolic acid and flavonoid biosynthesis genes are highly expressed in leaf epidermal cells (including the stomatal complex and guard cells); and terpenoid and steroid biosynthesis genes are expressed specifically in leaf mesophyll cells. A number of novel and cell-specific transcription factors involved in secondary metabolite biosynthesis were identified, including MYB17, WRKY12, WRKY31, ERF13, GT_2, and bHLH46. Our research establishes the transcriptional landscape of major cell types in T. mairei leaves at a single-cell resolution and provides valuable resources for studying the basic principles of cell-type-specific regulation of secondary metabolism.

Genome-wide identification and expression analysis of TCP family genes in Catharanthus roseus.

Introduction: The anti-tumor vindoline and catharanthine alkaloids are naturally existed in Catharanthus roseus (C. roseus), an ornamental plant in many tropical countries. Plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play important roles in various plant developmental processes. However, the roles of C. roseus TCPs (CrTCPs) in terpenoid indole alkaloid (TIA) biosynthesis are largely unknown. Methods: Here, a total of 15 CrTCP genes were identified in the newly updated C. roseus genome and were grouped into three major classes (P-type, C-type and CYC/TB1). Results: Gene structure and protein motif analyses showed that CrTCPs have diverse intron-exon patterns and protein motif distributions. A number of stress responsive cis-elements were identified in promoter regions of CrTCPs. Expression analysis showed that three CrTCP genes (CrTCP2, CrTCP4, and CrTCP7) were expressed specifically in leaves and four CrTCP genes (CrTCP13, CrTCP8, CrTCP6, and CrTCP10) were expressed specifically in flowers. HPLC analysis showed that the contents of three classic TIAs, vindoline, catharanthine and ajmalicine, were significantly increased by ultraviolet-B (UV-B) and methyl jasmonate (MeJA) in leaves. By analyzing the expression patterns under UV-B radiation and MeJA application with qRT-PCR, a number of CrTCP and TIA biosynthesis-related genes were identified to be responsive to UV-B and MeJA treatments. Interestingly, two TCP binding elements (GGNCCCAC and GTGGNCCC) were identified in several TIA biosynthesis-related genes, suggesting that they were potential target genes of CrTCPs. Discussion: These results suggest that CrTCPs are involved in the regulation of the biosynthesis of TIAs, and provide a basis for further functional identification of CrTCPs.

Integrated mass spectrometry imaging and single-cell transcriptome atlas strategies provide novel insights into taxoid biosynthesis and transport in Taxus mairei stems.

Taxol, which is a widely used important chemotherapeutic agent, was originally isolated from Taxus stem barks. However, little is known about the precise distribution of taxoids and the transcriptional regulation of taxoid biosynthesis across Taxus stems. Here, we used MALDI-IMS analysis to visualize the taxoid distribution across Taxus mairei stems and single-cell RNA sequencing to generate expression profiles. A single-cell T. mairei stem atlas was created, providing a spatial distribution pattern of Taxus stem cells. Cells were reordered using a main developmental pseudotime trajectory which provided temporal distribution patterns in Taxus stem cells. Most known taxol biosynthesis-related genes were primarily expressed in epidermal, endodermal, and xylem parenchyma cells, which caused an uneven taxoid distribution across T. mairei stems. We developed a single-cell strategy to screen novel transcription factors (TFs) involved in taxol biosynthesis regulation. Several TF genes, such as endodermal cell-specific MYB47 and xylem parenchyma cell-specific NAC2 and bHLH68, were implicated as potential regulators of taxol biosynthesis. Furthermore, an ATP-binding cassette family transporter gene, ABCG2, was proposed as a potential taxoid transporter candidate. In summary, we generated a single-cell Taxus stem metabolic atlas and identified molecular mechanisms underpinning the cell-specific transcriptional regulation of the taxol biosynthesis pathway.

Investigation of the role of TmMYB16/123 and their targets (TmMTP1/11) in the tolerance of Taxus media to cadmium.

The toxicity and stress caused by heavy metal contamination has become an important constraint to the growth and flourishing of trees. In particular, species belonging to the genus Taxus, which are the only natural source for the anti-tumor medicine paclitaxel, are known to be highly sensitive to environmental changes. To investigate the response of Taxus spp. to heavy metal stress, we analyzed the transcriptomic profiles of Taxus media trees exposed to cadmium (Cd2+). In total, six putative genes from the metal tolerance protein (MTP) family were identified in T. media, including two Cd2+ stress inducible TMP genes (TmMTP1, TmMTP11 and Taxus media). Secondary structure analyses predicted that TmMTP1 and TmMTP11, which are members of the Zn-CDF and Mn-CDF subfamily proteins, respectively, contained six and four classic transmembrane domains, respectively. The introduction of TmMTP1/11 into the ∆ycf1 yeast cadmium-sensitive mutant strain showed that TmMTP1/11 might regulate the accumulation of Cd2+ to yeast cells. To screen the upstream regulators, partial promoter sequences of the TmMTP1/11 genes were isolated using the chromosome walking method. Several myeloblastosis (MYB) recognition elements were identified in the promoters of these genes. Furthermore, two Cd2+-induced R2R3-MYB TFs, TmMYB16 and TmMYB123, were identified. Both in vitro and in vivo assays confirmed that TmMTB16/123 play a role in Cd2+ tolerance by activating and repressing the expression of TmMTP1/11 genes. The present study elucidated new regulatory mechanisms underlying the response to Cd stress and can contribute to the breeding of Taxus species with high environmental adaptability.

Identification and functional analysis of calcium sensor calmodulins from heavy metal hyperaccumulator Noccaea caerulescens.

Noccaea caerulescens (J. Presl & C. Presl) F. K. Mey. is a heavy metal hyperaccumulator exhibiting extreme tolerance to various environmental stresses. To date, the functional role of Ca2+ -binding protein in this plant is largely unknown. To investigate the function of calmodulins (CaMs) in N. caerulescens , CaM2 , a Ca2+ sensor encoding gene, was identified and functionally characterised. Protein structure analysis showed that NcCaM2 contains four classic exchange factor (EF)-hand motifs with high sequence similarity to the CaM proteins from model plant Arabidopsis thaliana L. Tissue specific expression analysis showed that NcCaM2 is constitutively expressed in stems, leaves, and roots. Expression level of NcCaM2 was significantly upregulated under various environmental stimulus, indicating a potential involvement of NcCaM2 in the tolerance to abiotic stresses. The heterologous expression of NcCaM2 in a yeast mutant strain increased the heavy metal tolerance in yeast cells. Furthermore, the constitutive expression of NcCaM2 enhanced the heavy metal tolerance capability of transgenic tobacco (Nicotiana tabacum L.) plants. Our data suggested an important role of NcCaM2 in the responses to environmental stresses and provided a potential target gene to enhance of the ability to hyperaccumulate metals.

MATE transporter GFD1 cooperates with sugar transporters, mediates carbohydrate partitioning and controls grain-filling duration, grain size and number in rice.

More than half of the world's food is provided by cereals, as humans obtain >60% of daily calories from grains. Producing more carbohydrates is always the final target of crop cultivation. The carbohydrate partitioning pathway directly affects grain yield, but the molecular mechanisms and biological functions are poorly understood, including rice (Oryza sativa L.), one of the most important food sources. Here, we reported a prolonged grain filling duration mutant 1 (gfd1), exhibiting a long grain-filling duration, less grain number per panicle and bigger grain size without changing grain weight. Map-based cloning and molecular biological analyses revealed that GFD1 encoded a MATE transporter and expressed high in vascular tissues of the stem, spikelet hulls and rachilla, but low in the leaf, controlling carbohydrate partitioning in the stem and grain but not in the leaf. GFD1 protein was partially localized on the plasma membrane and in the Golgi apparatus, and was finally verified to interact with two sugar transporters, OsSWEET4 and OsSUT2. Genetic analyses showed that GFD1 might control grain-filling duration through OsSWEET4, adjust grain size with OsSUT2 and synergistically modulate grain number per panicle with both OsSUT2 and OsSWEET4. Together, our work proved that the three transporters, which are all initially classified in the major facilitator superfamily family, could control starch storage in both the primary sink (grain) and temporary sink (stem), and affect carbohydrate partitioning in the whole plant through physical interaction, giving a new vision of sugar transporter interactome and providing a tool for rice yield improvement.

DRB2 Modulates Leaf Rolling by Regulating Accumulation of MicroRNAs Related to Leaf Development in Rice.

As an important agronomic trait in rice (Oryza sativa), moderate leaf rolling helps to maintain the erectness of leaves and minimize shadowing between leaves, leading to improved photosynthetic efficiency and grain yield. However, the molecular mechanisms underlying rice leaf rolling still need to be elucidated. Here, we isolated a rice mutant, rl89, showing adaxially rolled leaf phenotype due to decreased number and size of bulliform cells. We confirmed that the rl89 phenotypes were caused by a single nucleotide substitution in OsDRB2 (LOC_Os10g33970) gene encoding DOUBLE-STRANDED RNA-BINDING2. This gene was constitutively expressed, and its encoded protein was localized to both nucleus and cytoplasm. Yeast two-hybrid assay showed that OsDRB2 could interact with DICER-LIKE1 (DCL1) and OsDRB1-2 respectively. qRT-PCR analysis of 29 related genes suggested that defects of the OsDRB2-miR166-OsHBs pathway could play an important role in formation of the rolled leaf phenotype of rl89, in which OsDRB2 mutation reduced miR166 accumulation, resulting in elevated expressions of the class III homeodomain-leucine zipper genes (such as OsHB1, 3 and 5) involved in leaf polarity and/or morphology development. Moreover, OsDRB2 mutation also reduced accumulation of miR160, miR319, miR390, and miR396, which could cause the abnormal leaf development in rl89 by regulating expressions of their target genes related to leaf development.

Mutation of Protoporphyrinogen IX Oxidase Gene Causes Spotted and Rolled Leaf and Its Overexpression Generates Herbicide Resistance in Rice.

Protoporphyrinogen IX (Protogen IX) oxidase (PPO) catalyzes the oxidation of Protogen IX to Proto IX. PPO is also the target site for diphenyl ether-type herbicides. In plants, there are two PPO encoding genes, PPO1 and PPO2. To date, no PPO gene or mutant has been characterized in monocotyledonous plants. In this study, we isolated a spotted and rolled leaf (sprl1) mutant in rice (Oryza sativa). The spotted leaf phenotype was sensitive to high light intensity and low temperature, but the rolled leaf phenotype was insensitive. We confirmed that the sprl1 phenotypes were caused by a single nucleotide substitution in the OsPPO1 (LOC_Os01g18320) gene. This gene is constitutively expressed, and its encoded product is localized to the chloroplast. The sprl1 mutant accumulated excess Proto(gen) IX and reactive oxygen species (ROS), resulting in necrotic lesions. The expressions of 26 genes associated with tetrapyrrole biosynthesis, photosynthesis, ROS accumulation, and rolled leaf were significantly altered in sprl1, demonstrating that these expression changes were coincident with the mutant phenotypes. Importantly, OsPPO1-overexpression transgenic plants were resistant to the herbicides oxyfluorfen and acifluorfen under field conditions, while having no distinct influence on plant growth and grain yield. These finding indicate that the OsPPO1 gene has the potential to engineer herbicide resistance in rice.

Identification and functional analysis of cation-efflux transporter 1 from Brassica juncea L.

BACKGROUND: Brassica juncea behaves as a moderate-level accumulator of various heavy metal ions and is frequently used for remediation. To investigate the roles of metal ion transporters in B. juncea, a cation-efflux family gene, BjCET1, was cloned and functionally characterized. RESULTS: BjCET1 contains 382 amino acid residues, including a signature motif of the cation diffusion facilitator protein family, six classic trans-membrane-spanning structures and a cation-efflux domain. A phylogenetic analysis showed that BjCET1 has a high similarity level with metal tolerance proteins from other Brassica plants, indicating that this protein family is highly conserved in Brassica. BjCET1 expression significantly increased at very early stages during both cadmium and zinc treatments. Green fluorescence detection in transgenic tobacco leaves revealed that BjCET1 is a plasma membrane-localized protein. The heterologous expression of BjCET1 in a yeast mutant increased the heavy-metal tolerance and decreased the cadmium or zinc accumulations in yeast cells, suggesting that BjCET1 is a metal ion transporter. The constitutive expression of BjCET1 rescued the heavy-metal tolerance capability of transgenic tobacco plants. CONCLUSIONS: The data suggest that BjCET1 is a membrane-localized efflux transporter that plays essential roles in heavy metal ion homeostasis and hyper-accumulation.

How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein.

E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid-liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL, promoting amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.

On the Machinability Evolution in Asymmetric Milling of TC25 Ti Alloy Aiming at High Performance Machining.

In this paper, the evolutions of cutting force, cutting temperature, and surface roughness, and the corresponding machinability in asymmetric up-milling of TC25 alloy are investigated. The results indicated that radial depth of cut generated opposite influence on the cutting force/cutting temperature versus surface roughness. The reason can be accounted as the intertwining of feed marks at low radial depth of cut, and the mechanism of hard cutting at a high radial depth of cut. Moreover, the asymmetry has a significant effect on the machinability in asymmetry up-milling TC25 alloy. Changing the asymmetry, i.e., the radial depth of cut, can alter the machinability while maintain the balanced development of various indexes. The machinability reaches the best when the radial depth of cut is ae = 8 mm. The axial depth of cut and feed per tooth should be selected as large as possible to avoid work hardening and to improve machining efficiency in asymmetric up-milling TC25 alloy. The cutting speed should be controlled within Vc = 100-120 m/min to obtain better machinability. On the basis of this research, it is expected to find optimized milling parameters to realize high efficiency milling of TC25 alloy.

LPS1, Encoding Iron-Sulfur Subunit SDH2-1 of Succinate Dehydrogenase, Affects Leaf Senescence and Grain Yield in Rice.

The iron-sulfur subunit (SDH2) of succinate dehydrogenase plays a key role in electron transport in plant mitochondria. However, it is yet unknown whether SDH2 genes are involved in leaf senescence and yield formation. In this study, we isolated a late premature senescence mutant, lps1, in rice (Oryza sativa). The mutant leaves exhibited brown spots at late tillering stage and wilted at the late grain-filling stage and mature stage. In its premature senescence leaves, photosynthetic pigment contents and net photosynthetic rate were reduced; chloroplasts and mitochondria were degraded. Meanwhile, lps1 displayed small panicles, low seed-setting rate and dramatically reduced grain yield. Gene cloning and complementation analysis suggested that the causal gene for the mutant phenotype was OsSDH2-1 (LOC_Os08g02640), in which single nucleotide mutation resulted in an amino acid substitution in the encoded protein. OsSDH2-1 gene was expressed in all organs tested, with higher expression in leaves, root tips, ovary and anthers. OsSDH2-1 protein was targeted to mitochondria. Furthermore, reactive oxygen species (ROS), mainly H2O2, was excessively accumulated in leaves and young panicles of lps1, which could cause premature leaf senescence and affect panicle development and pollen function. Taken together, OsSDH2-1 plays a crucial role in leaf senescence and yield formation in rice.

RADX condenses single-stranded DNA to antagonize RAD51 loading.

RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using single-molecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

Human cohesin compacts DNA by loop extrusion.

Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.

Prevalence and distribution of HPV types in genital warts in Xi'an, China: a prospective study.

OBJECTIVES: To characterise the prevalence and distribution of human papillomavirus (HPV) types in genital warts in Xi'an, China. METHODS: This prospective study was conducted in Shaanxi Provincial Institute for Skin Disease and STD Control (SPISSC) between September 2014 and April 2017. Genital wart samples were obtained from 879 patients, including 512 men and 367 women. HPV genotyping was performed by using an automatic nucleic acid hybridisation system. RESULTS: Of the 879 patients with genital warts, the detectable rates of low-risk, high-risk and total HPV types were 45.4%, 34.5% and 57.8%, respectively. The detectable rate of low-risk HPV types (45.4%) was significantly higher than that of high-risk HPV types (34.5%) (χ2=21.85, p<0.01). The detectable rate of low-risk HPV types of men (52.3%) was significantly higher than that of women (35.7%) (χ2=23.90, p<0.01). The detectable rates of one HPV type infection and two and three or more HPV type coinfections were 26.1%, 17.5% and 14.2%, respectively. HPV6 (24.9%), HPV11 (17.9%), HPV52 (9.9%) and HPV16 (7.3%) were the four most common HPV types. CONCLUSIONS: The results of this study suggest that low-risk HPV types are major pathogens of genital warts, but high-risk HPV type infections and multiple HPV type coinfections are also common in genital warts. HPV6, 11, 52 and 16 are the four most common HPV types in genital wart in Xi'an, China.