Search

Fatal pathogenic organisms have been detected by various point‐of‐care tests (PoCTs) in clinical samples. PoCTs based on portable electrical devices are able to detect organisms in simple and rapid method. Although electrical PoCT devices show ultra sensitivity when detecting pathogens in standard samples, their industrialization has been limited due to low reproducibility, sensitivity, and specificity, resulting from nonspecific binding issues by media. In this research, a perpendicular N‐heterocyclic carbene self‐assembled monolayer (NHC‐SAM) conjugated on graphene micropattern field‐effect transistors (NGMFETs) is first demonstrated for monitoring pathogens. Two single bonds of NHCSAM on GM are investigated via density functional theory (DFT) and surface analyses. Bioreceptors are conjugated on the side gate‐modulated NGMFET for ultra‐stable on‐site detection. The side‐gated BNGMFETs system displayed remarkable performance in the detection of the following: i) SARS‐CoV‐2 spike protein S1 (limit of detection (LOD): ≈10 pg mL−1) in CTM, ii) O. tsutsugamushi Ab (LOD: ≈1 pg mL−1), and iii) E. coli (LOD: 10° CFU mL−1) in serum. Compared to commercial methodology, the platform presents 102 times higher LODs towards those pathogens in clinical samples with higher reproducibility. Based on these results, side‐gated BNGMFETs can be utilized as universal PoCT during pandemics. [ABSTRACT FROM AUTHOR]

A serological immunoassay based on enzyme‐linked immunosorbent assay (ELISA) is a crucial tool for screening and identification of human SARS‐CoV‐2 seroconversion. Various immunoassays are developed to detect the spike 1 (S1) and nucleocapsid (NP) proteins of SARS‐CoV‐2; however, these serological tests have low sensitivity. Here, a novel microplate (MP) is developed on which a ZnO nanowire (NW) is fabricated by a modified hydrothermal synthesis method. This plate is coated with SARS‐CoV‐2 NP and used as a fluorescent immunoassay (FIA) to detect antibodies specific for SARS‐CoV‐2 NP. Compared with the bare MP, the ZnO‐NW MP binds high levels (up to 5 µg mL−1) of SARS‐CoV‐2 NP tagged to histidine without any surface treatment. A novel serological assay based on the ZnO‐NW MP is more sensitive than a commercial immunoassay, enabling early detection (within <5 days of a reverse transcription polymerase chain reaction‐confirmed COVID‐19 infection) of anti‐SARS‐CoV‐2 NP IgG antibodies in asymptomatic patients with COVID‐19. This is the first assay to detect early antibody responses to SARS‐CoV‐2 in asymptomatic patients. Therefore, this serological assay will facilitate accurate diagnosis of COVID‐19, as well as estimation of COVID‐19 prevalence and incidence. [ABSTRACT FROM AUTHOR]

We solved the crystal structure of a functionally uncharacterized protein, HP0902, from Helicobacter pylori. Its structure demonstrated an all-β cupin fold that cannot bind metal ions due to the absence of a metal-binding histidine that is conserved in many metallo-cupins. In contrast, isothermal titration calorimetry and NMR titration demonstrated that HP0902 is able to bind bacterial endotoxin lipopolysaccharides ( LPS) through its surface-exposed loops, where metal-binding sites are usually found in other metallo-cupins. This report constitutes the first identification of an LPS-interacting protein, both in the cupin family and in H. pylori. Furthermore, identification of the ability of HP0902 to bind LPS uncovers a putative role for this protein in H. pylori pathogenicity. [ABSTRACT FROM AUTHOR]

3,6-Anhydro- l-galactonate cycloisomerase (ACI), which is found in the marine bacterium Vibrio sp. strain EJY3, converts 3,6-anhydro- l-galactonate into 2-keto-3-deoxygalactonate. ACI is a key enzyme in the metabolic pathway of 3,6-anhydro- l-galactose (AHG). Study of AHG metabolism is important for the efficient fermentation of agar and biofuel production, because AHG is a sugar that is non-fermentable by commercial microorganisms. The aci gene from Vibrio sp. strain EJY3 was cloned, and the recombinant protein was overexpressed and crystallized in order to determine the structure and understand the function of the protein. The crystals diffracted to 2.2 Å resolution and belonged to space group P41212 or P43212, with unit-cell parameters a = b = 87.9, c = 143.5 Å. The Matthews coefficient was 2.3 Å3 Da−1, with a solvent content of 47%. [ABSTRACT FROM AUTHOR]

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1-RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1-RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432-Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1-RASSF5 complex showed a longer helical structure (Ser438-Lys480) than that in the MST1 homodimer (Val441-Lys480). Moreover, extensive polar and nonpolar contacts in the MST1-RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST-RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1-RASSF5 SARAH domain in apoptosis signalling. [ABSTRACT FROM AUTHOR]

Objective: Although the levonorgestrel-releasing intrauterine system (LNG-IUS) is effective in reducing the recurrence of endometriosis-associated pain, its efficacy in preventing endometrioma recurrence is questionable. We compared the efficacy of postoperative use of LNG-IUS with oral contraceptives (OC) for preventing endometrioma recurrence.Design: A retrospective cohort study.Setting: Medical university hospital.Population: Ninety-nine women with endometriomas.Methods: A chart review was performed of women of reproductive age who had undergone laparoscopic surgery for endometrioma followed by three cycles of gonadotropin-releasing hormone agonist (leuprolide acetate) treatment. Women were categorized into two groups: a group that had postoperative LNG-IUS placement (n = 42) and a group that received postoperative, cyclic, low-dose, monophasic, OCs (n = 57). Main outcome measures. Endometrioma recurrence was analyzed according to several clinical variables and postoperative treatment modalities.Results: During the follow-up period (median 17 months), recurrent endometriomas were detected in eight women (8.1%). Patients with LNG-IUS had a recurrence rate of 4.8% (2/42), whereas women receiving OC had a recurrence rate of 10.5% (6/57). Cumulative recurrence-free survival assessment revealed that mean disease-free survival times for both groups were similar, but that for LNG-IUS was slightly longer than that for OC, with statistical significance (34.4 ± 1.0 months, 95% confidence interval 32.3–36.5, vs. 33.4 ± 1.3 months, 95% confidence interval 30.8–36.0, p = 0.045). Univariate analysis revealed a hazard ratio of 0.178 (95% confidence interval 0.029–1.075) (p = 0.060) for postoperative LNG-IUS use and endometrioma recurrence. However, for the multivariate regression analysis, only postoperative serum CA 125 levels were significantly associated with endometrioma recurrence (hazard ratio 1.012, p = 0.010).Conclusions: Postoperative LNG-IUS use seemed to be comparable to the use of cyclic OC in preventing endometrioma recurrence. [ABSTRACT FROM AUTHOR]

Problem We investigated the association of COX -2 gene-765G/C polymorphism and risk of advanced-stage endometriosis in Korean women. Method of study This study consisted of 268 women with advanced-stage endometriosis and 242 control women without endometriosis in Korea. Subjects were genotyped for the -765 G/ C polymorphism of the COX -2 gene by RFLP- PCR analysis. Results There were significant differences in the genotype distributions of the -765 G/ C polymorphism between patients with advanced-stage endometriosis and control subjects. The C allele for -765 G/ C was associated with significantly lower risk of advanced-stage endometriosis ( OR, 0.14; 95% CI, 0.06-0.30). Conclusions We have demonstrated a significant genetic association between the -765 G/ C polymorphism and advanced-stage endometriosis in Korean women. The -765 C allele may be protective against the development of the disease in Korean women. [ABSTRACT FROM AUTHOR]

Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpFℓSTR® MiniFiler™ kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs ( n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpFℓSTR® MiniFiler™ and Identifiler™ kits. Whereas only 21.87% of larger-sized loci profiles could be obtained with the Identifiler™ kit, 75% of the same loci profiles were determined by MiniFiler™ kit analysis. This very successful amplification of large-sized STR markers from highly degraded aDNA suggests that the MiniFiler™ kit could be a useful complement to conventional STR kit analysis of ancient samples. [ABSTRACT FROM AUTHOR]

HP0902 from Helicobacter pylori 26695 belongs to the cupin superfamily of proteins, which encompasses proteins with a great diversity in function. In this work, two types of recombinant HP0902 protein were crystallized: one with an N-terminal His6 tag (H6HP0902) and the other with a C-terminal His6 tag (HP0902H6). The H6HP0902 crystal diffracted to 1.40 Å resolution and belonged to space group P21, with unit-cell parameters a = 33.5, b = 78.6, c = 41.4 Å. The HP0902H6 crystal belonged to space group P43212 or P41212 and diffracted to 2.5 Å resolution, with unit-cell parameters a = b = 50.4, c = 142.0 Å. [ABSTRACT FROM AUTHOR]

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C-terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I-CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75-fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90-fold. [ABSTRACT FROM AUTHOR]

Obtaining well-diffracting crystals of macromolecules remains a significant barrier to structure determination. Here we propose and test a new approach to crystallization, in which the crystallization target is fused to a polymerizing protein module, so that polymer formation drives crystallization of the target. We test the approach using a polymerization module called 2TEL, which consists of two tandem sterile alpha motif (SAM) domains from the protein translocation Ets leukemia (TEL). The 2TEL module is engineered to polymerize as the pH is lowered, which allows the subtle modulation of polymerization needed for crystal formation. We show that the 2TEL module can drive the crystallization of 11 soluble proteins, including three that resisted prior crystallization attempts. In addition, the 2TEL module crystallizes in the presence of various detergents, suggesting that it might facilitate membrane protein crystallization. The crystal structures of two fusion proteins show that the TELSAM polymer is responsible for the majority of contacts in the crystal lattice. The results suggest that biological polymers could be designed as crystallization modules. [ABSTRACT FROM AUTHOR]

If unpaid family workers are considered (they represent 10.3% of all workers and 6.1% of all non-farm workers), the rate of self-employment is about 40% of all workers and 31% of non-farm workers (National Statistical Office of Korea, 1999). In a labor market where workers are differentiated by various abilities and characteristics, self-employment would be the form of employment for workers who were not successful in obtaining employment by others. This type of self-employed worker stood in contrast to those who became self-employed because they were less capable workers, making it difficult for them to get salary jobs or because their reservation wage was too high relative to the available wages and salaries. Those who had fewer employees were more willing to leave self-employment, a finding that implies that businesses that employed more workers were more successful and that business owners may have felt a greater responsibility to keep the business running. 3. For reference, in 1988, average annual household income for wage and salary workers was 25,596,000 won ($26,000) and household expenditures for wage and salary workers were 18,432,000 won ($18,000) in all cities. [Extracted from the article]

Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P21, with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å3 Da−1. [ABSTRACT FROM AUTHOR]

The meso isomer of diaminopimelate (meso-DAP) is a biosynthetic precursor of L-lysine in bacteria and plants, and is a key component of the peptidoglycan layer in the cell walls of Gram-negative and some Gram-positive bacteria. Diaminopimelate epimerase (DapF) is a pyridoxal-5'-phosphate-independent racemase which catalyses the interconversion of (6S,2S)-2,6-diaminopimelic acid (LL-DAP) and meso-DAP. In this study, DapF from Acinetobacter baumannii was overexpressed in Escherichia coli strain SoluBL21, purified and crystallized using a vapour-diffusion method. A native crystal diffracted to a resolution of 1.9 Å and belonged to space group P3(1) or P3(2), with unit-cell parameters a = b = 74.91, c = 113.35 Å, α = β = 90, γ = 120°. There were two molecules in the asymmetric unit.

Peptidoglycan-associated lipoprotein (Pal) is one component of the Tol-Pal system that is involved in maintaining the integrity and stability of the outer membrane. The C-terminal OmpA-like domain of Pal interacts noncovalently with peptidoglycan. In this study, the OmpA-like domain of Pal from Acinetobacter baumannii was overexpressed in Escherichia coli strain BL21 (DE3), purified and crystallized using the vapour-diffusion method. A native crystal diffracted to 1.4 Å resolution and belonged to space group P6(1) or P6(5), with unit-cell parameters a=b=72.58, c=44.65 Å, a calculated Matthews coefficient of 2.64 Å3 Da(-1) and one molecule per asymmetric unit.

We investigated whether a combination of recently introduced methods, total demineralization and ion-exchange columns, would increase DNA recovery from old bone. Ten bone samples taken after a burial period of approximately 60 years were used in this study. Bone powder was digested using total or incomplete demineralization. DNA was extracted by the standard organic method. The DNA extract was purified with ion-exchange columns or QIAquick spin columns. The efficiency of different DNA extraction methods was compared in terms of DNA concentration, inhibitors generated by real-time PCR, and conventional STR typing results. The mean DNA concentration using the total demineralization method is approximately 3 times higher than that using the incomplete demineralization method. For DNA purification, the method using QIAquick spin columns appeared to yield approximately double the DNA than the method using ion-exchange columns. Furthermore, 2 out of 10 samples showed higher levels of inhibition with C(T) values of IPC > or =30 cycles when using only ion-exchange columns. In STR results, total demineralization yielded more locus profiles by 4.2 loci than incomplete demineralization, and QIAquick spin columns also yielded more locus profiles by 3.5 loci than ion-exchange columns. Total demineralization of bone powder significantly increased DNA yield and improved STR typing results. However, the use of ion-exchange columns was not efficient when compared with the method using QIAquick spin columns. It is suggested that the combination of total demineralization and QIAquick spin columns lead to greatly improved STR typing results.

Problem: The aim of this study was to evaluate osteopontin (OPN) mRNA expression in eutopic endometrium and plasma OPN levels in patients with endometriosis., Method of Study: A total of 79 patients with histologically confirmed endometriosis and 43 patients without endometriosis participated in this study. OPN mRNA expression in endometrial tissues was measured by real-time quantitative polymerase chain reaction (PCR) and plasma concentrations of OPN were quantified using a specific commercial sandwich enzyme-linked immunosorbent assays (ELISA)., Results: Osteopontin mRNA expression in endometrial tissue was significantly higher in women with endometriosis than in controls (P = 0.010). The mean plasma levels of OPN (mean +/- S.E.M.) in patients with endometriosis and controls were 407.31 +/- 37.80 ng/mL and 165.84 +/- 19.29 ng/mL, respectively (P < 0.001). Receiver operating characteristic (ROC) analysis for plasma OPN revealed an area under the curve (AUC) of 0.894, with a sensitivity of 93.0%, specificity of 72.4%, positive likelihood ratio of 3.37, and negative likelihood ratio of 0.1 using a cut-off value of 167.68 ng/mL., Conclusion: Osteopontin may be involved in the pathogenesis of endometriosis and plasma OPN may be a useful non-invasive marker for the diagnosis of endometriosis.

Problem: The aim of this study was to evaluate serum and urinary levels of vascular endothelial growth factors, tumor necrosis factor-alpha (TNF-alpha), and soluble fms-like tyrosine kinase (sFlt-1) in patients with endometriosis., Method of Study: During surgery for pelvic pain, pelvic mass or infertility, serum and urine were collected. Of 70 patients, 46 had histology-proven endometriosis and 24 patients without endometriosis participated as controls., Results: Serum TNF-alpha levels and urinary sFlt-1 levels corrected for creatinine excretion were significantly increased in the endometriosis group (P=0.001 and P=0.011 respectively). Serum sFlt-1 levels and urinary sFlt-1 levels corrected for creatinine were significantly higher in patients with minimal-to-mild disease (P=0.014 and P=0.015 respectively), where serum TNF-alpha levels were increased in moderate-to-severe endometriosis (P<0.001)., Conclusion: The pathogenesis of minimal-to-mild endometriosis and moderate-to-severe endometriosis seems to be different. Increased sFlt-1 levels in serum and urine of minimal-to-mild disease indicate that sFlt-1 may have an important role in inhibiting angiogenic process of the disease.