BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation.

Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.

Relationship between lower urinary tract symptoms and cardiovascular risk scores including Framingham risk score and ACC/AHA risk score.

AIMS: This study attempted to investigate the association between lower urinary tract symptoms (LUTS) and cardiovascular disease (CVD) risk using International Prostate Symptom Score (IPSS) and CVD risk scores and to overcome the limitations of previous relevant studies. METHODS: A total of 2994 ostensibly healthy males, who participated in a voluntary health check in a health promotion center from January 2010 to December 2014, were reviewed. CVD risk scores were calculated using Framingham risk score and American College of Cardiology (ACC)/American Heart Association (AHA) score. Correlation and multivariate logistic regression analysis to predict the CVD risk severity were performed. RESULTS: Correlation between total IPSS with CVD risk scores demonstrated significant positive associations, which showed higher correlation with ACC/AHA score than the Framingham score (r = 0.18 vs 0.09, respectively). For ACC/AHA score, the partial correlation after adjustment of body mass index (BMI) showed significant positive correlations between all LUTS parameters and PSA. For the Framingham score, all variables, except IPSS Q2 and IPSS Q6, showed significant positive correlations. After adjustment of BMI, prostate volume and PSA, only the severe LUTS group showed significant relationship with intermediate-high CVD risk severity, as compared with normal LUTS group (OR = 2.97, 95%CI (1.35-6.99)). CONCLUSION: Using two validated CVD risk calculators, we observed that LUTS is closely associated with future CVD risk. To predict the intermediate-high CVD risk severity, severe LUTS was a sentinel sign, the presence of which warrants the importance of an earlier screening for CVD.

The nucleus is an intracellular propagator of tensile forces in NIH 3T3 fibroblasts.

Nuclear positioning is a crucial cell function, but how a migrating cell positions its nucleus is not understood. Using traction-force microscopy, we found that the position of the nucleus in migrating fibroblasts closely coincided with the center point of the traction-force balance, called the point of maximum tension (PMT). Positioning of the nucleus close to the PMT required nucleus-cytoskeleton connections through linker of nucleoskeleton-to-cytoskeleton (LINC) complexes. Although the nucleus briefly lagged behind the PMT following spontaneous detachment of the uropod during migration, the nucleus quickly repositioned to the PMT within a few minutes. Moreover, traction-generating spontaneous protrusions deformed the nearby nucleus surface to pull the nuclear centroid toward the new PMT, and subsequent retraction of these protrusions relaxed the nuclear deformation and restored the nucleus to its original position. We propose that the protruding or retracting cell boundary transmits a force to the surface of the nucleus through the intervening cytoskeletal network connected by the LINC complexes, and that these forces help to position the nucleus centrally and allow the nucleus to efficiently propagate traction forces across the length of the cell during migration.

Kordia zosterae sp. nov., isolated from the seaweed, Zostera marina.

A Gram-stain-negative, gliding and rod shaped bacterium, designated strain ZO2-23T was isolated from a seaweed sample collected from the West Sea, Republic of Korea. Cells are catalase-negative and oxidase-positive. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain ZO2-23T forms an independent lineage within the genus Kordia. Strain ZO2-23T was related to Kordia ulvae SC2T (98.0 %, 16S rRNA gene sequence similarity) and K. antarctica IMCC3317T (97.9 %). The major fatty acids of strain ZO2-23T were iso-C15 : 0, iso-C17 : 0 3-OH, summed feature 3 and iso-C15 : 0 3-OH. The only isoprenoid quinone of the isolate was menaquinone-6. The DNA G+C content of strain ZO2-23T was 31.7 mol%. Phenotypic characteristics distinguished strain ZO2-23T from the related species of the genus Kordia. On the basis of the evidences presented in this study, novel species, Kordia zosterae sp. nov., is proposed for strain ZO2-23T (=KCTC 52268T=JCM 31799T).

Sphingomonas limnosediminicola sp. nov. and Sphingomonas palustris sp. nov., isolated from freshwater environments.

Two aerobic, Gram-stain-negative, gliding and yellow-pigmented bacteria, designated strains 03SUJ6T and WM95T were isolated from freshwater sediment of Juam reservoir and freshwater of Woopo wetland, Republic of Korea, respectively. Cells of the two strains are motile by gliding and catalase- and oxidase-positive. The 16S rRNA gene sequence similarity between 03SUJ6T and WM95T was 97.7 %, but their DNA-DNA relatedness was 55.1 %. A maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences showed that 03SUJ6T and WM95T each form independent lineages within the genus Sphingomonas. 03SUJ6T was related distantly to Sphingomonas daechungensis CH15-11T (97.4 % 16S rRNA gene sequence similarity), Sphingomonas ginsengisoliGsoil 634T (97.3 %) and Sphingomonas astaxanthinifaciens DMS 22298T (97.1 %). Closest relatives of strain WM95T were S. daechungensis CH15-11T (98.2 %), Sphingomonasjaspsi DSM 18422T (97.6 %), Sphingomonas sediminicolaDae 20T (97.5 %), Sphingomonaslutea JS5T (97.4 %) and S. ginsengisoliGsoil 634T (97.2 %). The major fatty acids of the two isolates were summed feature 8 and C16 : 0. The predominant isoprenoid quinone was ubiquinone-10. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid. sym-Homospermidine was the major polyamine of the isolates. Phenotypic characteristics distinguished 03SUJ6T and WM95T from the related species of the genus Sphingomonas. On the basis of the evidence presented in this study, the novel species, Sphingomonas limnosediminicola sp. nov. and Sphingomonas palustris sp. nov. are proposed for strain 03SUJ6T (=KCTC 23331T=JCM 17543T) and strain WM95T (=KACC 18738T=JCM 31399T), respectively.

Probing nuclear pore complex architecture with proximity-dependent biotinylation.

Proximity-dependent biotin identification (BioID) is a method for identifying protein associations that occur in vivo. By fusing a promiscuous biotin ligase to a protein of interest expressed in living cells, BioID permits the labeling of proximate proteins during a defined labeling period. In this study we used BioID to study the human nuclear pore complex (NPC), one of the largest macromolecular assemblies in eukaryotes. Anchored within the nuclear envelope, NPCs mediate the nucleocytoplasmic trafficking of numerous cellular components. We applied BioID to constituents of the Nup107-160 complex and the Nup93 complex, two conserved NPC subcomplexes. A strikingly different set of NPC constituents was detected depending on the position of these BioID-fusion proteins within the NPC. By applying BioID to several constituents located throughout the extremely stable Nup107-160 subcomplex, we refined our understanding of this highly conserved subcomplex, in part by demonstrating a direct interaction of Nup43 with Nup85. Furthermore, by using the extremely stable Nup107-160 structure as a molecular ruler, we defined the practical labeling radius of BioID. These studies further our understanding of human NPC organization and demonstrate that BioID is a valuable tool for exploring the constituency and organization of large protein assemblies in living cells.

Sphingomonas lutea sp. nov., isolated from freshwater of an artificial reservoir.

An aerobic, Gram-stain-negative, gliding and yellow-pigmented bacterium, designated strain JS5T, was isolated from freshwater of Juam reservoir, Republic of Korea. Cells were catalase-positive and oxidase-negative. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain JS5T forms an independent lineage within the genus Sphingomonas. Strain JS5T was related distantly to 'Sphingomonas parvus' GP20-2 (98.2 % 16S rRNA gene sequence similarity), Sphingomonas sediminicola Dae 20T (96.8 %) and Sphingomonas daechungensis CH15-11T (96.7 %). The major fatty acids of strain JS5T were C16 : 0, summed feature 3 comprising C16 : 1ω7c and/or C16 : 1ω6c and summed feature 8 comprising C18 : 1ω7c and/or C18 : 1ω6c. The predominant isoprenoid quinone of the isolate was ubiquinone-10. The DNA G+C content of strain JS5T was 65 mol%. Phenotypic characteristics distinguished strain JS5T from related species of the genus Sphingomonas. On the basis of the evidence presented in this study, a novel species, Sphingomonaslutea sp. nov., is proposed to accommodate strain JS5T (=KCTC 23642T=JCM 18309T).

Pontibacter rugosus sp. nov., isolated from seawater.

A motile and rod-shaped bacterium, designated strain KYW1030T, was isolated from seawater collected from the Gwangyang bay (Republic of Korea). Cells were Gram-reaction-negative, aerobic, catalase-positive and oxidase-negative. The major fatty acids were summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B), iso-C15 : 0 and iso-C17 : 0 3-OH. The strain contained MK-7 as the major isoprenoid quinone, phosphatidylethanolamine and diphosphatidylglycerol as the major polar lipids and sym-homospermidine as the major polyamine. The DNA G+C content was 46 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain KYW1030T forms an evolutionary lineage within the radiation enclosing the members of the genus Pontibacter, with Pontibacterakesuensis AKS 1T (97.9 % 16S rRNA gene sequence similarity) as its nearest neighbour. A number of phenotypic characteristics distinguished strain KYW1030T from the related members of the genus Pontibacter. On the basis of the evidence presented in this study, a novel species, Pontibacter rugosus sp. nov., is proposed for strain KYW1030T (=KACC 18739T=JCM 31319T).

Making the LINC: SUN and KASH protein interactions.

Cell nuclei are physically integrated with the cytoskeleton through the linker of nucleoskeleton and cytoskeleton (LINC) complex, a structure that spans the nuclear envelope to link the nucleoskeleton and cytoskeleton. Outer nuclear membrane KASH domain proteins and inner nuclear membrane SUN domain proteins interact to form the core of the LINC complex. In this review, we provide a comprehensive analysis of the reported protein-protein interactions for KASH and SUN domain proteins. This critical structure, directly connecting the genome with the rest of the cell, contributes to a myriad of cellular functions and, when perturbed, is associated with human disease.

The efficacy of detecting arrhythmia is higher with 7-day continuous electrocardiographic patch monitoring than with 24-h Holter monitoring.

Background: Detecting high-risk arrhythmia is important in diagnosing patients with palpitations. We compared the diagnostic accuracies of 7-day patch-type electrocardiographic (ECG) monitoring and 24-h Holter monitoring for detecting significant arrhythmias in patients with palpitations. Methods: This was a single-center prospective trial with 58 participants who presented with palpitations, chest pain or syncope. Outcomes were defined as the detection of any one of six arrhythmias, including supraventricular tachycardia (SVT), atrial fibrillation or atrial flutter lasting more than 30 s, pauses of more than 3 s, high-degree atrioventricular block, ventricular tachycardia (VT) >3 beats, or polymorphic VT/ventricular fibrillation. The McNemar test for paired proportions was used to compare arrhythmia detection rates. Results: The overall arrhythmia detection rate was higher with 7-day ECG patch monitoring than with 24-h Holter monitoring (34.5% vs. 19.0%, p = .008). Compared with the use of 24-h Holter monitors, the use of 7-day ECG patch monitors was associated with higher detection of SVT (29.3% vs. 13.8%, p = .042). No serious adverse skin reactions were reported among the ECG patch-monitored participants. Conclusions: The results suggest that a 7-day patch-type continuous ECG monitor is more effective for the detection of supraventricular tachycardia than is a 24-h Holter monitor. However, the clinical significance of device detected arrhythmia should be consolidated.

The establishment of ecological conservation for herpetofauna species in hotspot areas of South Korea.

Understanding the geographic distribution of species is crucial for establishing protected areas. This study aimed to identify the preferred habitat environment of South Korean herpetofauna using distribution point information, providing the information necessary to protect their habitat by establishing a species distribution model. We found that climate variables in the region where 19 amphibians and 20 reptiles were distributed correlated with the altitude, suggesting that altitude had a major influence on their distribution. The species distribution modeling indicated that 10-12 amphibian and 13-16 reptile species inhabit the Gangwon-do region, forming hotspot areas in the eastern and western regions around the Taebaek Mountains. Some of these hotspot areas occurred in the Demilitarized Zone and national parks, which are government-managed ecological conservation areas. However, some hotspot areas are vulnerable to habitat destruction due to development and deforestation as they are not designated conservation areas. Therefore, it is necessary to establish new conservation areas with a focus on herpetofauna after confirming the actual inhabitation of species through precise monitoring in predicted hotspot areas and designating them as protected areas. Our results can serve as important basic data for establishing protection measures and designating protected areas for herpetofauna species.

Gene flow from herbicide-tolerant GM rice and the heterosis of GM rice-weed F2 progeny.

Gene flow from genetically modified (GM) crops to non-GM cultivars or weedy relatives may lead to the development of more aggressive weeds. We quantified the amount of gene flow from herbicide-tolerant GM rice (Protox GM, derived from the cultivar Dongjin) to three cultivars (Dongjin, Aranghyangchal and Hwaseong) and a weedy rice line. Gene flow frequency generally decreased with increasing distance from the pollen donor. At the shortest distance (0.5 m), we observed a maximum frequency (0.039%) of gene flow. We found that the cultivar Dongjin received the greatest amount of gene flow, with the second being weedy rice. Heterosis of F2 inbred progeny was also examined between Protox GM and weedy rice. We compared growth and reproduction between F2 progeny (homozygous or hemizygous for the Protox gene) and parental rice lines (GM and weedy rice). Here, transgene-homozygous F2 progeny was significantly taller and produced more seeds than the transgene-hemizygous F2 progeny and parental lines. Although the gene flow frequency was generally low, our results suggest that F2 progeny between GM and weedy relatives may exhibit heterosis.

Making Connections: Integrative Signaling Mechanisms Coordinate DNA Break Repair in Chromatin.

DNA double-strand breaks (DSBs) are hazardous to genome integrity and can promote mutations and disease if not handled correctly. Cells respond to these dangers by engaging DNA damage response (DDR) pathways that are able to identify DNA breaks within chromatin leading ultimately to their repair. The recognition and repair of DSBs by the DDR is largely dependent on the ability of DNA damage sensing factors to bind to and interact with nucleic acids, nucleosomes and their modified forms to target these activities to the break site. These contacts orientate and localize factors to lesions within chromatin, allowing signaling and faithful repair of the break to occur. Coordinating these events requires the integration of several signaling and binding events. Studies are revealing an enormously complex array of interactions that contribute to DNA lesion recognition and repair including binding events on DNA, as well as RNA, RNA:DNA hybrids, nucleosomes, histone and non-histone protein post-translational modifications and protein-protein interactions. Here we examine several DDR pathways that highlight and provide prime examples of these emerging concepts. A combination of approaches including genetic, cellular, and structural biology have begun to reveal new insights into the molecular interactions that govern the DDR within chromatin. While many questions remain, a clearer picture has started to emerge for how DNA-templated processes including transcription, replication and DSB repair are coordinated. Multivalent interactions with several biomolecules serve as key signals to recruit and orientate proteins at DNA lesions, which is essential to integrate signaling events and coordinate the DDR within the milieu of the nucleus where competing genome functions take place. Genome architecture, chromatin structure and phase separation have emerged as additional vital regulatory mechanisms that also influence genome integrity pathways including DSB repair. Collectively, recent advancements in the field have not only provided a deeper understanding of these fundamental processes that maintain genome integrity and cellular homeostasis but have also started to identify new strategies to target deficiencies in these pathways that are prevalent in human diseases including cancer.

DNMT1 reads heterochromatic H4K20me3 to reinforce LINE-1 DNA methylation.

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1BAH1). Engagement of DNMT1BAH1-H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

Unmet Healthcare Needs and Associated Factors in Rural and Suburban Vietnam: A Cross-Sectional Study.

The purpose of this study was to examine the current utilization of healthcare services, exploring unmet healthcare needs and the associated factors among people living in rural Vietnam. This cross-sectional study was conducted with 233 participants in a rural area. The methods included face-to-face interviews using a structured questionnaire, and anthropometric and blood pressure measurements. We considered participants to have unmet health needs if they had any kind of health problem during the past 12 months for which they were unable to see a healthcare provider. Multivariate logistic regression analysis was performed to determine the factors associated with unmet healthcare needs. Of the participants, 18% (n = 43) had unmet healthcare needs, for reasons like transportation (30%), a lack of available doctors or medicine (47%), and communication issues with healthcare providers (16%). The multivariate logistic regression showed that living in a rural area, having stage 2 hypertension, and having insurance were associated with unmet healthcare needs. To better meet the healthcare needs in rural or suburban areas of Vietnam, allocation of adequate healthcare resources should be distributed in rural areas and insurance coverage for personalized healthcare needs might be required. Efforts should focus on availability of medicine, improvement of transportation systems, and communication skills of healthcare providers to improve access to healthcare services.

Signature Fragment Ions of Biotinylated Peptides.

The use of biotin or biotin-containing reagents is an essential component of many protein purification and labeling technologies. Owing to its small size and high affinity to the avidin family of proteins, biotin is a versatile molecular handle that permits both enrichment and purity that is not easily achieved by other reagents. Traditionally, the use of biotinylation to enrich for proteins has not required the detection of the site of biotinylation. However, newer technologies for discovery of protein-protein interactions, such as APEX and BioID, as well as some of the click chemistry-based labeling approaches have underscored the importance of determining the exact residue that is modified by biotin. Anti-biotin antibody-based enrichment of biotinylated peptides (e.g., BioSITe) coupled to LC-MS/MS permit large-scale detection and localization of sites of biotinylation. As with any chemical modification of peptides, understanding the fragmentation patterns that result from biotin modification is essential to improving its detection by LC-MS/MS. Tandem mass spectra of biotinylated peptides has not yet been studied systematically. Here, we describe the various signature fragment ions generated with collision-induced dissociation of biotinylated peptides. We focused on biotin adducts attached to peptides generated by BioID and APEX experiments, including biotin, isotopically heavy biotin, and biotin-XX-phenol, a nonpermeable variant of biotin-phenol. We also highlight how the detection of biotinylated peptides in high-throughput studies poses certain computational challenges for accurate quantitation which need to be addressed. Our findings about signature fragment ions of biotinylated peptides should be helpful in the confirmation of biotinylation sites.

Spring and summer microhabitat use by Schlegel's Japanese gecko, Gekko japonicus (Reptilia: Squamata: Gekkonidae), in urban areas.

Differential microhabitat use may be beneficial to achieving fitness in seasonally variable environmental conditions. To explore whether the microhabitat use of the nocturnal Schlegel's Japanese gecko, Gekko japonicus, varies seasonally and depends on juvenile, male, and female reproductive groups, we investigated five categorical and five quantitative measure variables of microhabitat use in a wild population both in spring and summer. Most geckos were found on white, vertical planes of concrete and plastered brick walls. None of the categorical variables (type of location, substrate, substrate color, light source, and refuge) significantly differed according to season or group, while substrate temperature and irradiance at the location where geckos were observed and the distance from the nearest potential refuge were significantly greater in summer than in spring. The quantitative measure variables did not differ among the reproductive groups. These results suggest that G. japonicus seasonally adjusts its microhabitat use mainly in terms of quantitative measure variables rather than categorical variables.

BioID: A Screen for Protein-Protein Interactions.

BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. © 2018 by John Wiley & Sons, Inc.

HPV prevalence in the foreskins of asymptomatic healthy infants and children: Systematic review and meta-analysis.

The true HPV prevalence in the foreskins of infants and children has been little documented, but reporting on this prevalence is of great importance given its impact on the rationale for treating asymptomatic boys. We searched multiple databases from 1960 to 2016 for observational or prospective studies that reported on HPV prevalence in foreskins. We conducted a meta-analysis using a random-effects model to pool for HPV prevalence in the foreskins of infants and children. Eight studies, with a total of 556 infants and children with phimosis, were eligible for the meta-analysis. The pooled overall prevalence of general HPV, high-risk HPV, low-risk HPV, HPV 16/18, HPV 16, and HPV 18 were 17.3 (95%CI: 0.8-46.3), 12.1 (95% CI: 0.9-31.5), 2.4 (95% CI: 0.0-11.2), 4.8 (95% CI: 0.0-16.8), 1.7 (95% CI: 0.0-5.1), and 0 (95% CI: 0-0.5), respectively. The estimated HPV prevalence in foreskins was not zero among infants and children, which implies HPV transmission other than by sexual contact. Considering that high-risk HPV is detected in asymptomatic infants and children, future studies are warranted to determine whether preventive treatments in asymptomatic infants and children could be effective in preventing persistence or transmission of high-risk HPV.

VRK2A is an A-type lamin-dependent nuclear envelope kinase that phosphorylates BAF.

The nuclear envelope (NE) is critical for numerous fundamental cellular functions, and mutations in several NE constituents can lead to a heterogeneous spectrum of diseases. We used proximity biotinylation to uncover new constituents of the inner nuclear membrane (INM) by comparative BioID analysis of lamin A, Sun2 and a minimal INM-targeting motif. These studies identify vaccinia-related kinase-2 (VRK2) as a candidate constituent of the INM. The transmembrane VRK2A isoform is retained at the NE by association with A-type lamins. Furthermore, VRK2A physically interacts with A-type, but not B-type, lamins. Finally, we show that VRK2 phosphorylates barrier to autointegration factor (BAF), a small and highly dynamic chromatin-binding protein, which has roles including NE reassembly, cell cycle, and chromatin organization in cells, and subtly alters its nuclear mobility. Together these findings support the value of using BioID to identify unrecognized constituents of distinct subcellular compartments refractory to biochemical isolation and reveal VRK2A as a transmembrane kinase in the NE that regulates BAF.