Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic but partly depend on conserved, histone-like proteins HUαα and HUαβ and their interactions that span the nanoscale and mesoscale from protein-DNA complexes to the bacterial chromosome and nucleoid structure. We determined the crystal structures of these chromosome-associated proteins in complex with native duplex DNA. Distinct DNA binding modes of HUαα and HUαβ elucidate fundamental features of bacterial chromosome packing that regulate gene transcription. By combining crystal structures with solution x-ray scattering results, we determined architectures of HU-DNA nucleoproteins in solution under near-physiological conditions. These macromolecular conformations and interactions result in contraction at the cellular level based on in vivo imaging of native unlabeled nucleoid by soft x-ray tomography upon HUβ and ectopic HUα38 expression. Structural characterization of charge-altered HUαα-DNA complexes reveals an HU molecular switch that is suitable for condensing nucleoid and reprogramming noninvasive Escherichia coli into an invasive form. Collective findings suggest that shifts between networking and cooperative and noncooperative DNA-dependent HU multimerization control DNA compaction and supercoiling independently of cellular topoisomerase activity. By integrating x-ray crystal structures, x-ray scattering, mutational tests, and x-ray imaging that span from protein-DNA complexes to the bacterial chromosome and nucleoid structure, we show that defined dynamic HU interaction networks can promote nucleoid reorganization and transcriptional regulation as efficient general microbial mechanisms to help synchronize genetic responses to cell cycle, changing environments, and pathogenesis.

Cell-penetrating peptides (CPPs) as small molecular transporters with abilities of cell penetrating, internalization, and endosomal escape have potential prospect in drug delivery systems. However, a bottleneck hampering their application is the poor specificity for cells. By utilizing the function of hydration shell of polyethylene glycol (PEG) and acid sensitivity of hydrazone bond, we constructed a kind of CPP-modified pH-sensitive PEGylated liposomes (CPPL) to improve the selectivity of these peptides for tumor targeting. In CPPL, CPP was directly attached to liposome surfaces via coupling with stearate (STR) to avoid the hindrance of PEG as a linker on the penetrating efficiency of CPP. A PEG derivative by conjugating PEG with STR via acid-degradable hydrazone bond (PEG2000-Hz-STR, PHS) was synthesized. High-performance liquid chromatography and flow cytometry demonstrated that PHS was stable at normal neutral conditions and PEG could be completely cleaved from liposome surface to expose CPP under acidic environments in tumor. An optimal CPP density on liposomes was screened to guaranty a maximum targeting efficiency on tumor cells as well as not being captured by normal cells that consequently lead to a long circulation in blood. In vitro and in vivo studies indicated, in 4 mol% CPP of lipid modified system, that CPP exerted higher efficiency on internalizing the liposomes into targeted subcellular compartments while remaining inactive and free from opsonins at a maximum extent in systemic circulation. The 4% CPPL as a drug delivery system will have great potential in the clinical application of anticancer drugs in future.

The Topo2a-dependent arrest is associated with faithful segregation of sister chromatids and has been identified as dysfunctional in numerous tumour cell lines. This genome-protecting pathway is poorly understood and its characterization is of significant interest, potentially offering interventional opportunities in relation to synthetic lethal behaviours in arrest-defective tumours. Using the catalytic Topo2a inhibitor ICRF193, we have performed a genome-wide siRNA screen in arrest-competent, non-transformed cells, to identify genes essential for this arrest mechanism. In addition, we have counter-screened several DNA-damaging agents and demonstrate that the Topo2a-dependent arrest is genetically distinct from DNA damage checkpoints. We identify the components of the SMC5/6 complex, including the activity of the E3 SUMO ligase NSE2, as non-redundant players that control the timing of the Topo2a-dependent arrest in G2. We have independently verified the NSE2 requirement in fibroblasts from patients with germline mutations that cause severely reduced levels of NSE2. Through imaging Topo2a-dependent G2 arrested cells, an increased interaction between Topo2a and NSE2 is observed at PML bodies, which are known SUMOylation hotspots. We demonstrate that Topo2a is SUMOylated in an ICRF193-dependent manner by NSE2 at a novel non-canonical site (K1520) and that K1520 sumoylation is required for chromosome segregation but not the G2 arrest.

The small GTPase Rab7 controls late endocytic transport by the minus end-directed motor protein complex dynein-dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein-related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP-Rab7-ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150(Glued), which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150(Glued) recruitment by Rab7-RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as betaIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150(Glued) dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7-RILP is transferred by ORP1L to betaIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with betaIII spectrin, which requires the activities of Rab7-RILP and ORP1L.

Epiberberine (EPI) is a novel and potentially effective therapeutic and preventive agent for diabetes and cardiovascular disease. To evaluate its potential value for drug development, a specific, sensitive and robust high-performance liquid chromatography-tandem mass spectrometry assay for the determination of EPI in rat biological samples was established. This assay was used to study the pharmacokinetics, bioavailability and excretion of EPI in rats after oral administration. In addition, a cocktail method was used to compare the inhibition characteristics of EPI on cytochrome P450 (CYP450) isoforms in human liver microsomes (HLMs) and rat liver microsomes (RLMs). The results demonstrated that EPI was rapidly absorbed and metabolized after oral administration (10, 54 or 81 mg/kg) in rats, with Tmax of 0.37-0.42 h and T1/2 of 0.49-2.73 h. The Cmax and area under the curve values for EPI increased proportionally with the dose, and the oral absolute bioavailability was 14.46%. EPI was excreted mainly in bile and feces, and after its oral administration to rats, EPI was eliminated predominantly by the kidneys. A comparison of the current half-maximal inhibitory concentration and Ki values revealed that EPI demonstrated an obvious inhibitory effect on CYP2C9 and CYP2D6. Furthermore, its effect was stronger in HLM than in RLM, more likely to be a result of noncompetitive inhibition.

Diagnosis of syphilis is difficult. Follow-up and therapy evaluation of syphilitic patients are poor. Little is known about positron emission tomography (PET) in syphilis. This review was to systematically review usefulness of PET for diagnosis, disease extent evaluation, follow-up, and treatment response assessment in patients with syphilis.

There are more than 2000 ramie germplasms in the National Ramie Germplasm Nursery affiliated with the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science, China. As it is difficult to perform effective conservation, management, evaluation, and utilization of redundant genetic resources, it is necessary to construct a core collection by using molecular markers. In this study, a core collection of ramie consisting of 22 germplasms was constructed from 108 accessions by heuristic search based on 21 Simple Sequence Repeat (SSR) marker combinations. The results showed that there is a poor relationship between the core collection and the geographic distribution. The number of amplification bands for the core collection was the same as that for the entire collection. Shannon's index for three of the SSR primers (14%) and Nei's index for nine of the SSR primers (19%) were lower in the core collection than in the entire collection. The true core collection had wider genetic diversity compared with the random core collection. Collectively, the core collection constructed in this study is reliable and represents the genetic diversity of all the 108 accessions.

MFN2 encodes mitofusin 2, a membrane-bound mediator of mitochondrial membrane fusion and inter-organelle communication. MFN2 mutations cause axonal neuropathy, with associated lipodystrophy only occasionally noted, however homozygosity for the p.Arg707Trp mutation was recently associated with upper body adipose overgrowth. We describe similar massive adipose overgrowth with suppressed leptin expression in four further patients with biallelic MFN2 mutations and at least one p.Arg707Trp allele. Overgrown tissue was composed of normal-sized, UCP1-negative unilocular adipocytes, with mitochondrial network fragmentation, disorganised cristae, and increased autophagosomes. There was strong transcriptional evidence of mitochondrial stress signalling, increased protein synthesis, and suppression of signatures of cell death in affected tissue, whereas mitochondrial morphology and gene expression were normal in skin fibroblasts. These findings suggest that specific MFN2 mutations cause tissue-selective mitochondrial dysfunction with increased adipocyte proliferation and survival, confirm a novel form of excess adiposity with paradoxical suppression of leptin expression, and suggest potential targeted therapies.

Werner Syndrome (WS) and Bloom Syndrome (BS) are disorders of DNA damage repair caused by biallelic disruption of the WRN or BLM DNA helicases respectively. Both are commonly associated with insulin resistant diabetes, usually accompanied by dyslipidemia and fatty liver, as seen in lipodystrophies. In keeping with this, progressive reduction of subcutaneous adipose tissue is commonly observed. To interrogate the underlying cause of adipose tissue dysfunction in these syndromes, CRISPR/Cas9 genome editing was used to generate human pluripotent stem cell (hPSC) lacking either functional WRN or BLM helicase. No deleterious effects were observed in WRN-/- or BLM-/- embryonic stem cells, however upon their differentiation into adipocyte precursors (AP), premature senescence emerged, impairing later stages of adipogenesis. The resulting adipocytes were also found to be senescent, with increased levels of senescent markers and senescence-associated secretory phenotype (SASP) components. SASP components initiate and reinforce senescence in adjacent cells, which is likely to create a positive feedback loop of cellular senescence within the adipocyte precursor compartment, as demonstrated in normal ageing. Such a scenario could progressively attenuate adipose mass and function, giving rise to "lipodystrophy-like" insulin resistance. Further assessment of pharmacological senolytic strategies are warranted to mitigate this component of Werner and Bloom syndromes.

Inter-organelle interactions are a vital part of normal cellular function; however, these have proven difficult to quantify due to the range of scales encountered in cell biology and the throughput limitations of traditional imaging approaches. Here, we demonstrate that soft X-ray tomography (SXT) can be used to rapidly map ultrastructural reorganization and inter-organelle interactions in intact cells. SXT takes advantage of the naturally occurring, differential X-ray absorption of the carbon-rich compounds in each organelle. Specifically, we use SXT to map the spatiotemporal evolution of insulin vesicles and their co-localization and interaction with mitochondria in pancreatic β cells during insulin secretion and in response to different stimuli. We quantify changes in the morphology, biochemical composition, and relative position of mitochondria and insulin vesicles. These findings highlight the importance of a comprehensive and unbiased mapping at the mesoscale to characterize cell reorganization that would be difficult to detect with other existing methodologies.

Two-photon fluorescence correlation spectroscopy (2P-FCS) within single dendritic spines of living hippocampal pyramidal neurons was used to resolve various subpopulations of mobile F-actin during activity-dependent structural changes such as potentiation induced spine head growth. Two major classes of mobile F-actin were discovered: very dynamic and about a hundred times less dynamic F-actin. Spine head enlargement upon application of Tetraethylammonium (TEA), a protocol previously used for the chemical induction of long-term potentiation (cLTP) strictly correlated to changes in the dynamics and filament numbers in the different actin filament fractions. Our observations suggest that spine enlargement is governed by a mechanism in which longer filaments are first cut into smaller filaments that cooperate with the second, increasingly dynamic shorter actin filament population to quickly reorganize and expand the actin cytoskeleton within the spine head. This process would allow a fast and efficient spine head enlargement using a major fraction of the actin filament population that was already present before spine head growth.

Nearly 350 million persons worldwide are chronically infected with hepatitis B virus (HBV). Ubiquitin (Ub) is a highly conserved small regulatory protein, ubiquitous in eukaryotes, that usually serves as a signal for the target protein that is recognised and degraded in proteasomes . The Ub-mediated processing of antigens is rapid and efficient and stimulates cell-mediated immune responses. Accordingly, Ub-mediated processing of antigens has been widely used in chronic-infection and cancer studies to improve immune response.

Low birth weight and rapid postnatal growth increases the risk of developing insulin resistance and type 2 diabetes in later life. However, underlying mechanisms and potential intervention strategies are poorly defined. Here we demonstrate that male Wistar rats exposed to a low-protein diet in utero that had a low birth weight but then underwent postnatal catch-up growth (recuperated offspring) had reductions in the insulin signaling proteins p110-β (13% ± 6% of controls [P < .001]) and insulin receptor substrate-1 (39% ± 10% of controls [P < .05]) in adipose tissue. These changes were not accompanied by any change in expression of the corresponding mRNAs, suggesting posttranscriptional regulation. Recuperated animals displayed evidence of a proinflammatory phenotype of their adipose tissue with increased IL-6 (139% ± 8% [P < .05]) and IL1-β (154% ± 16% [P < .05]) that may contribute to the insulin signaling protein dysregulation. Postweaning dietary supplementation of recuperated animals with coenzyme Q (CoQ10) (1 mg/kg of body weight per day) prevented the programmed reduction in insulin receptor substrate-1 and p110-β and the programmed increased in IL-6. These findings suggest that postweaning CoQ10 supplementation has antiinflammatory properties and can prevent programmed changes in insulin-signaling protein expression. We conclude that CoQ10 supplementation represents an attractive intervention strategy to prevent the development of insulin resistance that results from suboptimal in utero nutrition.

The small GTPase Rab7 controls fusion and transport of late endocytic compartments. A critical mediator is the Rab7 effector RILP that recruits the minus-end dynein-dynactin motor complex to these compartments. We identified a natural occurring splice variant of RILP (RILPsv) lacking only 27 amino acids encoded by exon VII. Both variants bind Rab7, prolong its GTP-bound state, and induce clustering of late endocytic compartments. However, RILPsv does not recruit the dynein-dynactin complex, implicating exon VII in motor recruitment. Clustering might still occur via dimerization, since both RILP and RILPsv are able to form hetero- and homo-dimers. Moreover, both effectors compete for Rab7 binding but with different outcome for dynein-dynactin recruitment and transport. Hence, RILPsv provides an extra dimension to the control of vesicle fusion and transport by the small GTPase Rab7.

Although domestication has dramatically altered the phenotype, physiology, and life history of ramie (Boehmeria nivea) plants, few studies have investigated the effects of domestication on the structure and expression pattern of genes in this fiber crop. To investigate the selective pattern and genetic relationships among a cultivated variety of ramie (BNZ: B. nivea, ZZ1) and four wild species, BNT (B. nivea var. tenacissima), BNN (B. nivea var. nipononivea), BNW (B. nivea var. nivea), and BAN (B. nivea var. viridula), in the section Tilocnide, we performed an RNA sequencing analysis of these ramie species. The de novo assembly of the "all-ramie" transcriptome yielded 119,114 unigenes with an average length of 633 bp, and a total of 7,084 orthologous gene pairs were identified. The phylogenetic tree showed that the cultivar BNZ clustered with BAN in one group, BNW was closely related to BNT, and BNN formed a separate group. Introgression analysis indicated that gene flow occurred from BNZ to BNN and BAN, and between BAN and BNN. Among these orthologs, 2,425 and 269 genes underwent significant purifying and positive selection, respectively. For these positively selected genes, oxidation-reduction process (GO:0055114) and stress response pathways (GO:0006950) were enriched, indicating that modulation of the cellular redox status was important during both ramie fiber evolution and improvement. Two genes related to the suppression of flowering and one gene annotated as a flowering-promoting factor were subjected to positive selection, probably caused by human manipulation. Additionally, five genes were homologs of those involved in abiotic stress tolerance and disease resistance, with higher expression levels in the cultivar BNZ than in the wild species. Collectively, the results of this study indicated that domestication has resulted in the upregulation of many genes involved in the abiotic and biotic stress responses, fiber yield, and plant growth of ramie.

Accumulated evidence suggests a pathogenic role of reactive oxygen species (ROS) in perpetually rheumatoid joints. Therefore, the application of radical scavengers for reducing the accumulation of ROS is beneficial for patients with rheumatoid arthritis (RA). We synthesized water-soluble fullerenols that could inhibit the production of ROS and applied intra-articular (i.a.) injection in an experimental arthritis model to examine the anti-arthritic effect of the synthesized compound. RAW 264.7 cells were used to examine the activity of the synthesized fullerenol. Collagen-induced arthritis (CIA) was induced in Sprague-Dawley rats by injecting their joints with fullerenol. The therapeutic effects were evaluated using the articular index as well as radiological and histological scores. Dose-dependent suppression of nitric oxide (NO) production caused by the fullerenol was demonstrated in the RAW 264.7 cell culture, thus confirming the ability of fullerenol to reduce ROS production. In the fullerenol-injected joints, articular indexes, synovial expression of ROS, histological and radiological scores, pannus formation, and erosion of cartilage and bone were all reduced. Moreover, interleukin (IL)-1β and vascular endothelial growth factor (VEGF) levels were reduced, and fewer von Willebrand factor (vWF)-stained areas were identified in the fullerenol-treated joints than in control joints. The i.a. injection of fullerenol for reducing ROS production can ameliorate arthritis in joints by suppressing pro-inflammatory cytokine production and the angiogenesis process. Thus, the i.a. injection of fullerenol for reducing the production of ROS can be used as a pharmacological approach for RA patients.

Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85α/p55α/p50α subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85α with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85α overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome.

Most cells exhibit a constant ratio between nuclear and cell volume. The mechanism dictating this constant ratio and the nuclear component(s) that scale with cell size are not known. To address this, we examined the consequences to the size and shape of the budding yeast nucleus when cell expansion is inhibited by down-regulating components of the secretory pathway. We find that under conditions where cell size increase is restrained, the nucleus becomes bilobed, with the bulk of the DNA in one lobe and the nucleolus in the other. The formation of bilobed nuclei is dependent on fatty acid and phospholipid synthesis, suggesting that it is associated with nuclear membrane expansion. Bilobed nuclei appeared predominantly after spindle pole body separation, suggesting that nuclear envelope expansion follows cell-cycle cues rather than cell size. Importantly, cells with bilobed nuclei had the same nuclear:cell volume ratio as cells with round nuclei. Therefore, the bilobed nucleus could be a consequence of continued NE expansion as cells traverse the cell cycle without an accompanying increase in nuclear volume due to the inhibition of cell growth. Our data suggest that nuclear volume is not determined by nuclear envelope availability but by one or more nucleoplasmic factors.

Multidrug-resistant (MDR) bacterial strain is a serious medical problem. Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to many antibiotics and is often associated with several diseases such as arthritis, osteomyelitis, and endocarditis. The development of an alternative treatment for eliminating MDR bacteria such as MRSA has attracted a considerable amount of research attention. Moreover, the development of a material for highly efficient generation of reactive oxygen species (ROS) involving two-photon photodynamic therapy (PDT) is currently desirable.

Alström syndrome (AS), featuring retinal dystrophy, neuronal deafness, cardiomyopathy, metabolic syndrome, and diffuse fibrosis, is caused by biallelic mutations in the centrosomal protein ALMS1. Genotype-phenotype correlation has been suggested without assessment of ALMS1 expression.