CD Assay Research Articles

Discovery of a potent Guanidine derivative that selectively binds and stabilizes the human BCL-2 G-quadruplex DNA and downregulates the transcription

G-quadruplex also known as G4 (GQ) structures, are a non-canonical kind of DNA or RNA secondary structure that may develop inside guanine-rich nucleic acid sequences. They may be found in a variety of locations in the human genome, such as gene promoters, 5ˈ untranslated region, and telomeres, among others. Because of their significance in biology, G4 structures are recognized as promising pharmacological targets, particularly for therapeutics against cancer. This has led to the discovery of small molecules that can stabilize G4 structures. Small molecules that interact with quadruplexes offer a wide range of potential applications, including not just as medications but also as sensors for quadruplexes structures. The BCL-2 is a proto-oncogene that often gets mutated in lethal cancer and could be an interesting target for developing an anti-cancer drug. In the present study, we have employed various biophysical techniques such as fluorescence, CD, Isothermal calorimetry, gel retardation, and PCR stop assay, indicating that Guanidine derivatives GD-1 and GD-2 selectively interact with high affinity with BCL-2 GQ over other G-quadruplex DNA and duplex DNA. The most promising small molecule GD-1 increases the thermostability of the BCL-2 GQ structure by 12°C. Our biological experiments such as ROS generation, qRT-PCR, western blot, TFP based reporter assay, show that the GD-1 ligand causes a synthetic lethal interaction by suppressing the expression of BCL-2 genes via interaction and stabilization of its promoter GQ strucure in HeLa cells and act as a potential anti-cancer agent.

Cholic acid inhibits amyloid fibrillation: Interplay of protonation and deprotonation

Amyloidopathies are the consequence of misfolding with subsequent aggregation affecting people worldwide. Irrespective of speedy advancement in the field of therapeutics no agent for treating amyloidopathies has been discovered and thus targeting amyloid fibrillation process via repositioning of small molecules can be fruitful. According to previous reports potential amyloid inhibitors possess unique features like, hydrophobicity, aromaticity, charge etc. Herein, we have explored the effect of Cholic acid (CA) on amyloid fibrillation irrespective of the charge (determined by Zetasizer) using four proteins Human Serum Albumin, Bovine Serum Albumin, Human Insulin and Beta-lactoglobulin (HSA, BSA, HI and BLG) employing biophysical, imaging and computational techniques. ThT results revealed that CA in both protonated and deprotonated form is potent to curb HSA, BSA, BLG aggregation ~50% and HI aggregation ~96% in a dose dependent manner (in accord with CD, ANS and Congo red assay). Interestingly, CA treated samples displayed reduced cytotoxicity (Hemolytic assay) with altered morphology (TEM) and mechanism behind inhibition may be the interaction of CA with proteins via hydrophobic interactions and hydrogen bonding (supported by molecular docking results). This study proved CA (irrespective of the pH) a potential inhibitor of amyloidosis thus can be helpful in generalizing and repurposing the related drugs/compounds for their anti-aggregation behavior as an implication towards treating amyloidopathies.

766. Readmissions of Hospitalized Patients with Clostridioides difficile Infection (CDI) for Recurrent CDI Is Common

Abstract Background Hospital acquired infections (HAI) and hospital readmissions are of particular focus by Centers for Medicare and Medicaid Services. Clostridioides difficile infection (CDI) is an HAI notorious for causing recurrent illness and potentially resulting in re-hospitalizations. The purpose of our study was to identify the frequency of follow-up appointments in patients with CDI and determine the rate of re-hospitalization for recurrent CDI (rCDI). Methods This was a single-center, retrospective, chart review at a tertiary medical center. Through the electronic medical record, we queried all hospitalized patients with a positive stool test for C. difficile (GI panel PCR, FilmArray, Biofire, or C. difficile PCR, Xpert CD assay, Cepheid) with or without an ICD-10 code Enterocolitis due to C. difficile (A04.7, A04.71, A04.72) from January 2018 through April 2018. Demographic and clinical data at the time of diagnosis and up to 90 days after were collected from patient records. Results One-hundred and eighty-five patient episodes were evaluated. Of these, 147 (79.5%) were primary CDI, 13 (7.0%) were rCDI, and 25 (13.5%) were determined to be colonization. Twenty-two (11.9%) patients from the total cohort attended a follow-up appointment for CDI within 30 days, most often with a primary care provider or infectious disease physician. Twenty-three (12.4%) patients, 18 of whom were hospitalized for primary CDI episodes, developed a recurrent episode within 90 days of their initial CDI episode. Of these 23 patients with rCDI, 10 (43.5%) patients were re-hospitalized for their rCDI. Only 4 (17.4%) patients with rCDI had a follow-up appointment after their primary episode and among the 10 patients re-hospitalized for rCDI, only 2 (20.0%) patient had been seen for follow up for their previous CDI episode. Conclusion In our study, few patients had a follow-up appointment for CDI. Also, more than one third of the patients who had rCDI had to be re-hospitalized for the recurrent episode. Our study highlights a concern that the majority of patients re-hospitalized with rCDI did not have a follow-up appointment within 30 days of their initial diagnosis. Further study is necessary to determine if a dedicated follow-up appointment for CDI would result in decreased re-hospitalizations associated with rCDI. Disclosures Stuart Johnson, MD, Acurx Pharmaceuticals (Advisor or Review Panel member)Bio-K+ (Advisor or Review Panel member)Ferring Pharmaceutical (Advisor or Review Panel member)

Evaluation of the Cepheid Xpert C. difficile diagnostic assay: an update meta-analysis

BackgroundAccurate and rapid diagnosis of Clostridium difficile infection (CDI) is critical for effective patient management and implementation of infection control measures to prevent transmission.ObjectivesWe updated our previous meta-analysis to provide a more reliable evidence base for the clinical diagnosis of Xpert C. difficile (Xpert C. difficile) assay.MethodsWe searched PubMed, EMBASE, Cochrane Library, Chinese National Knowledge Infrastructure (CNKI), and the Chinese Biomedical Literature Database (CBM) databases to identify studies according to predetermined criteria. STATA 13.0 software was used to analyze the tests for sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the summary receiver operating characteristic curves (AUC). QUADAS-2 was used to assess the quality of included studies with RevMan 5.2. Heterogeneity in accuracy measures was tested with Spearman correlation coefficient and chi-square. Meta-regressions and subgroup analyses were performed to figure out the potential sources of heterogeneity. Model diagnostics were used to evaluate the veracity of the data.ResultsA total of 26 studies were included in the meta-analysis. The pooled sensitivity (95% confidence intervals [CI]) for diagnosis was 0.97(0.95–0.98), and specificity was 0.96(0.95–0.97). The AUC was 0.99 (0.98–1.00). Model diagnostics confirmed the robustness of our meta-analysis’s results. Significant heterogeneity was still observed when we pooled most of the accuracy measures of selected studies. Meta-regression and subgroup analyses showed that the sample size and type, ethnicity, and disease prevalence might be the conspicuous sources of heterogeneity.ConclusionsThe up-to-date meta-analysis showed the Xpert CD assay had good accuracy for detecting CDI. However, the diagnosis of CDI must combine clinical presentation with diagnostic testing to better answer the question of whether the patient actually has CDI in the future, and inclusion of preanalytical parameters and clinical outcomes in study design would provide a more objective evidence base.

An in-vitro elucidation of inhibitory potential of carminic acid: Possible therapeutic approach for neurodegenerative diseases

Protein misfolding leads to several human pathologies. So far, there has not been much success on the remediations of these protein conformational disorders. Since these diseases arise as a result of protein aggregation, hence inhibition of aggregation could be a promising approach for designing new therapeutics. For which; polyphenols, antibiotics and other small compounds have been tested by the researchers in the recent past, but so far there has not been much success. Here we have investigated the effect of carminic acid over Human serum albumin in an in vitro manner. It was observed that there was severe resistance in the increment of fluorescence intensity in the presence of carminic acid when observed through ANS, ThT and RLS, which was further supported by CD and Congo red assay. Microscopic analysis also confirmed that there was aggregation inhibition in the presence of180μM carminic acid. ROS and SDS-PAGE results also established the same. Furthermore, molecular docking was performed to understand the interacting residues which were found to be Arg, Leu, Glu. It is noteworthy that inhibitory effect was concentration dependent and maximum inhibition was found to be in presence of 180 μM of carminic acid. These results shall be helpful in designing the new therapeutics against amyloidosis.

Bifunctional carbon dots for cell imaging and inhibition of human insulin fibrillation in the whole aggregation process

Due to the favorable stability, water solubility and good biocompatibility, carbon dots have attracted much attention. Herein, a novel nitrogen-doping bifunctional carbon dots (N-BCDs) with ultra-highly quantum yield (QYabs = 70.4%) is prepared through microwave-assisted method. 50 μg/mL of N-BCDs emit intense fluorescence in HeLa and GES-1 cells with negligible cytotoxicity. In addition, effective inhibition of N-BCDs to human insulin (HI) fibrillation is observed even at 10:1 (mass ratio of HI: N-BCDs) by ThT fluorescence, CD assay and TEM. N-BCDs prevent HI from fibrillation with prolonged lag time and reduced fluorescent intensity at equilibrium, regardless of the addition time of N-BCDs (HI: N-BCDs = 1:1, mass ratio), which has been rarely reported before. Furthermore, the morphology of final HI fibrils is shorter and thinner in the presence of N-BCDs. Mechanism studies reveal that the enhanced hydrogen bond between HI monomers and N-BCDs inhibits nucleation during the lag stage (Ka: 1.54 × 104 L·mol−1, 298 K), while the accumulation of N-BCDs blocks the growth of profibrils in the elongation stage. To the best of our knowledge, it's the first time to observe the accumulation of N-BCDs around HI profibrils with TEM. Our study provides a new strategy for developing efficient nanoparticle inhibitors for protein fibrillation.

2349. C. diff-erent results: Eliminating Inappropriate Stool PCR Tests Through an Interdisciplinary Infection Prevention and Mirobiology Collaboration

BackgroundDiagnosis of Clostridium difficile infection (CDI) is based on symptoms and laboratory results. Distinguishing between CDI vs colonization or antibiotic-associated diarrhea is challenging. Widespread adoption of a highly sensitive real-time nucleic-acid amplification assay for toxin B DNA (Xpert CD assay, Cepheid) by US hospital-based microbiology labs has increased the challenge, resulting in overreporting of healthcare facility-onset CDI. Excess testing is inevitable in hospitals with EPIC electronic medical record (EMR) that incorporate best practice alerts (BPAs) prompting for C. difficile testing (CDT) when loose stools are charted by nursing staff.MethodsBeginning October 1, 2018 microbiology and infection prevention (IP) staff at our 650 bed teaching hospital in central IL agreed to partner on a diagnostic stewardship effort to engage providers on potentially unnecessary or inappropriate CDTs. All stool samples sent to lab for CDT are held pending IP review. The IP review tool provides a guide for EMR review of contributing factors in diarrhea, including patient history, medications (e.g., laxatives, stool softeners), nutrition (e.g., tube feeds), symptoms (abdominal pain), and labs (e.g., serum creatinine, WBC count). Inappropriate CDT was defined as test of patients receiving pro-motility treatment, without signs of infection.ResultsBetween October 1, 2018 and December 31, 2018, 383 CDT were ordered on inpatients. 196 were requested within 3 days of admission, and 187 were requested on beyond day 4. 56.6% (107/187) HO-CDTs were deemed inappropriate and canceled; 80 HO-CDT were deemed appropriate. 18.8% (15/80) of HO-CDTs were positive. Compared with October 1 - December 31, 2017, HO-CDI cases decreased by 66.7% (1.0 - 15/45) over the same period in 2018.ConclusionA 66.7% reduction in HO-CDI (vs. FY2017 cases) was seen in the first 3 months following adoption of a team-based (IPs, microbiology lab staff and MD director), CDT diagnostic stewardship intervention employing EMR review and patient provider engagement (RNs and MDs). Opportunities identified during the first 90 days of CDT stewardship: (a) Lack of provider awareness of pro-motility therapies; (b)inappropriate prompting for CDT generated by the EPIC EMR. Disclosures All authors: No reported disclosures.

Chemical-induced formation of BZ-junction with base extrusion

The crystal structure of BZ-junction reveals that left-handed Z-DNA stabilized by Z-DNA binding domain (Zα) is continuously stacked to right-handed B-DNA with AT bases’ extrusion in the junction site. However, this structure might not fully represent the BZ-junction in solution due to the possibility of the junction formation either by crystal packing or Zα interaction. Therefore, we investigated BZ-junction in solution with chemical Z-DNA inducers using CD and 2-aminopurine base-extrusion assay. We confirmed the formation of Z-DNA and BZ-junction with base-extrusion by chemical Z-DNA inducers. However, neither typical Z-DNA nor base-extrusion could be detected with some inducers such as spermine, suggesting that the energy barrier for the formation of the BZ junction might vary depending on the Z-DNA induction conditions.

A highly sensitive electrochemical sensor for Cd(II) based on protic salt derived nitrogen and sulfur co-doped porous carbon

Lately, an “all-in-one” strategy via direct pyrolysis of protic salt has attracted great attention in the preparation of hetero-doped porous carbons due to its distinctive simplicity. However, protic salt derived carbons usually process large grain sizes thus hamper their applications. Herein, a protic salt derived N, S co-doped porous carbon (N, S-PC) with improved porosity was synthesized by nanocasting and carbonization of protic salt. In the nanocasting silica sphere was used as auxiliary template and an easily obtained protic salt ([pPDA][nHSO4]) was served as C, N and S sources. The obtained N, S-PC contains rich N, S contents and hierarchical textural porosity, thus favors the electrochemical detection of toxic cadmium ions. The N, S-PC based sensor shows excellent sensitivity and selectivity for Cd(II) versus other metals, with a low detection limit of 0.1 μg L−1 (S/N = 3) over a wide concentration range of 4–80 μg L−1. Applications of the N, S-PC based sensor for Cd(II) assay in tap water samples achieved results with good recoveries, indicating that the N, S-PC based electrode is promising in real sample analysis.

Accuracy of Xpert Clostridium difficile assay for the diagnosis of Clostridium difficile infection: A meta analysis.

BackgroundThere is an urgent need for rapid and accurate microbiological diagnostic assay for detection of Clostridium difficile infection (CDI). We assessed the diagnostic accuracy of the Xpert Clostridium difficile assay (Xpert CD) for the diagnosis of CDI.MethodsWe searched PubMed, EMBASE, and Cochrane Library databases to identify studies according to predetermined criteria. STATA 13.0 software was used to analyze the tests for sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the summary receiver operating characteristic curves (AUC). QUADAS-2 was used to assess the quality of included studies with RevMan 5.2. Heterogeneity in accuracy measures was tested with Spearman correlation coefficient and Chi-square.ResultsA total of 22 studies were included in the meta-analysis. The pooled sensitivity (95% confidence intervals [CI]) was 0.97 (0.95–0.99) and specificity was 0.95 (0.94–0.96). The AUC was 0.99 (0.97–0.99). Significant heterogeneity was observed when we pooled most of the accuracy measures of selected studies.ConclusionsThe Xpert CD assay is a useful diagnostic tool with high sensitivity and specificity in diagnosing toxigenic CDI, and this method has excellent usability due to its rapidity and simplicity.

A multifunctional fluorescent aptamer probe for highly sensitive and selective detection of cadmium(II).

We report a highly sensitive and selective strategy for Cd(II) assay using a singly labeled multifunctional probe consisting of a Cd(II)-specific aptamer (CAP), which acted as a recognition element for Cd(II) and a signal reporter. The presence of Cd(II) can induce the conformational switching of the CAP, accompanied by a change in fluorescence intensity. Thereby, a fluorescence strategy for Cd(II) assay was established. The proposed method has a detection limit of 2.15nM, which is much lower than the detection limits reported in related literature. This strategy involves only an aptamer probe, and the use of such a G4-based quencher avoids the dual labeling of the CAP with fluorophore/quencher units. It is obviously more convenient and economical than the other aptamer-based biosensors for Cd(II) detection. The mechanism by which Cd(II) induces the CAP to change from a random coil sequence to a stem-loop structure was studied in a series of control experiments. This strategy would be helpful in the design of a sensitive analytical platform for various target assays in environmental and biomedical fields. Graphical Abstract The presence of Cd2+ leads to the conformational change of CAP from a random coil sequence to a stem-loop structure, resulting in a quenching in the fluorescence.

Functional Basis and Biophysical Approaches to Characterize the C-Terminal Domain of Human—Ribosomal S6 Kinases-3

Ribosomal S6 kinases (RSKs) are the major functional components in mitogen-activated protein kinase (MAPK) pathway, and these are activated by upstream Extracellular signal-regulated kinase. Upon activation, RSKs activate a number of substrate molecules involved in transcription, translation and cell-cycle regulation. But how cellular binding partners are engaged in the MAPK pathways and regulate the molecular mechanisms have not been explored. Considering the importance of protein-protein interactions in cell signalling and folding pattern of native protein, functional C-terminal kinase domain of RSK3 has been characterized using in vitro, in silico and biophysical approaches. RSKs discharge different functions by binding to downstream kinase partners. Hence, depending upon cellular binding partners, RSKs translocate between cytoplasm and nucleus. In our study, it has been observed that the refolded C-terminal Kinase domain (CTKD) of RSK 3 has a compact domain structure which is predominantly α-helical in nature by burying the tryptophans deep into the core, which was confirmed by CD, Fluorescence spectroscopy and limited proteolysis assay. Our study also revealed that RSK 3 CTKD was found to be a homotrimer from DLS experiments. A model was also built for RSK 3 CTKD and was further validated using PROCHECK and ProSA webservers.

A flow cytometry assay that measures cellular sensitivity to DNA-damaging agents, customized for clinical routine laboratories

ObjectivesThe clonogenic assay examines cell sensitivity to toxic agents and has been shown to correlate with normal tissue sensitivity to radiotherapy in cancer patients. The clonogenic assay is not clinically applicable due to its intra-individual variability and the time frame of the protocol. We aimed to develop a clinically applicable assay that correlated with the clonogenic assay. Design and methodsWe have developed a faster and less labor-intensive cell division assay (CD assay) using flow cytometry and incorporation of a fluorescent thymidine analogue. The CD assay was calibrated to the clonogenic assay and optimized for peripheral blood lymphocytes. ResultsFollowing ionizing radiation of primary human skin fibroblasts, the four-day CD assay gave similar results as the 14-day clonogenic survival assay. In lymphocytes isolated from patient blood samples, the CD assay was able to detect increased radiosensitivity in ataxia telangiectasia patients and increased radiosensitivity after in vitro treatment with DNA-PK and ATM inhibitors. The CD assay found a variation in the intrinsic radiosensitivity of lymphocytes isolated from healthy control samples. The CD assay was able to measure the anti-proliferation effect of different chemotherapeutic drugs in lymphocytes. ConclusionsOur results indicate that the CD assay is a fast and reliable method to measure the anti-proliferation effect of DNA-damaging agents with a potential to find the most sensitive patients in the work-up before cancer treatment.

Heterologous expression, purification and characterization of L-type lectin homologue from Leishmania donovani

Leishmaniasis, a disease of the developing world affects about 12 million people and has limited therapeutic interventions available. L-type lectins, Endoplasmic Reticulum Golgi Intermediate Compartment/Vesicular Integral Proteins (ERGIC-53/VIP36) are involved in protein sorting in luminal compartments of animal cells and are important for parasite biology. A lectin homologue was identified through a bioinformatics analysis of Leishmania genome and it was found to have N-terminal conserved carbohydrate recognition domain (CRD) and a unique C-terminal region rich in repetitive amino acids and a poly glutamine tract. The N-terminal CRD region was cloned and expressed in Escherichia coli, but gave an insoluble expression which was re-solubilized by on column refolding. The fold integrity was checked through CD, fluorescence and functional assay of hemagglutination activity using rabbit erythrocyte. Bioinformatics analysis identified 15 members from Tritryps (Leishmania spp., Trypanosoma spp.) and they separate out as a distinct clade in the global phylogenetic analysis of all ERGIC-53/VIP36 sequences downloaded from Uniprot. Our analysis shows that the extended C-terminal regions with repeats is unique to Tritryps and this repeat pattern is different in sequences from Leishmania spp. and Trypanosoma spp. and all these features make this protein an interesting candidate for further detailed studies.

LB03.08

Objective: hSLC39A8 (human solute carrier family 39 member 8) encodes a transmembrane protein that co-transports divalent heavy metal cations, such as Cd2+, with elusive physiological role. Recent genome-wide association studies have identified a non-synonymous single nucleotide polymorphism rs13107325 to be associated with hypertension. To investigate the functional impact of rs13107325 resulting in an amino acid substitution from Ala to Thr (A391T) in SLC39A8 on Cd2+ transport and the downstream signalling pathways. Design and method: Intracellular Cd2+ uptake was measured in HEK293 cells overexpressing SLC39A8 (Measure-iTTM Pb and Cd assay kit), and in human umbilical vascular endothelial cells (HUVECs) of different genotypes. Cd2+- and genotype-dependence of ERK1/2 and NF-kB pathway's activation were investigated by immunoblotting and dual-luciferase reporter assay. Cytotoxicity was measured by the lactate dehydrogenase assay and MTS assay. Molecular dynamics simulations were performed to predict in silico the effect of A391T on the structure and dynamics of SLC39A8 by using Robetta, TMHMM and etc. Results: Overexpression of Ala variant in HEK293 resulted in higher Cd2+ uptake and higher cytotoxicity as compared with the Thr variant. This is associated with increased phosphorylation of ERK1 and NF-kB activation. Similar trends were observed in HUVECs with endogenous SLC39A8. Bioinformatics tools also suggested a conformational change of the α-helical structural transition (residual 390–392) in the Thr mutant, which potentially attenuates the protein function. Conclusions: Increased Cd2+ uptake by SLC39A8 Ala variant (blood pressure raising allele) is associated with higher cell death in human kidney and endothelial cells. Therefore its altered function due to rs13107325 may indicate a potential therapeutic target in hypertension.

Structural Findings and Molecular Modeling Approach of a TFPI-Like Inhibitor

Specific blood coagulation inhibitors from hematophagous organisms, with different structures and novel mechanism of action, have been described and they represent promising agents for the treatment of a variety of human diseases related to coagulation and cancer. In our lab, the salivary glands transcriptome of the adult Amblyomma cajennense tick was previously characterized by expressed sequence tags (EST). A transcript that codes for a tissue factor pathway inhibitor (TFPI)-like protein with unique structure was found, and the recombinant form of this protein was named Amblyomin-X. This protein was able to inhibit the factor Xa amidolytic activity and the activation of factor X by the extrinsic tenase complex (FVIIa/TF). Herein, it was performed functional and structural evaluation of Amblyomin-X. The CD assay and molecular dynamics simulations revealed that Amblyomin-X is structurally stable and the naturally unfolded regions as well as the presence of three disulfide bridges in its Kunitz-type domain seem to sustain its inhibitory activity. Regarding the electrostatic potential mapping on the Kunitz-type region, the pattern of charged residues was not quite the same in comparison to human TFPI-1 and TFPI-2, pointing out there might be distinct functional and structural features, which are going to be experimentally exploited.

Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum

TroR is a putative metal-dependent regulatory protein that has been linked to the virulence of the human pathogen Treponema pallidum. It shares high homology with the well-known iron-dependent regulatory protein DtxR from Corynebacterium diphtheriae, as well as the manganese-dependent MntR from Bacillus subtilis. However, it has been uncertain whether manganese or zinc is the natural cofactor of TroR to date. Herein, we established an efficient method named "double-fusion tagging" to obtain soluble TroR for the first time. A series of studies, including ICP, CD, fluorescence, ITC, and electrophoresis mobility shift assay (EMSA), were performed to resolve the discrepancies in its metal-binding specificity. In addition, bioinformatic analysis as well as mutation studies were carried out to find the genetic relationships of TroR with its homology proteins. In conclusion, our findings indicate that TroR is a manganese-dependent rather than a zinc-dependent regulatory protein.

Evaluation of the Xpert Clostridium difficile Assay for the Diagnosis of Clostridium difficile Infection

Infection with Clostridium difficile is a growing concern because of the increasing prevalence and spread of nosocomial infections. Emergence of the hypervirulent 027/NAP1/BI strain is also notable. Existing diagnostic methods have low sensitivity or are time-consuming. Therefore, establishing a rapid and accurate microbiological diagnostic assay is needed. We evaluated the Xpert C. difficile assay (Xpert CD assay; Cepheid, USA) to detect toxigenic C. difficile. This assay is a real-time multiplex PCR assay that can be used to detect toxigenic C. difficile strains and differentiate the C. difficile presumptive 027/NAP1/BI strain. A total of 253 loose stool specimens were collected and toxigenic cultures, VIDAS C. difficile A & B assays (VIDAS CDAB assay; bioMérieux, France), and the Xpert CD assay were performed. In comparison to toxigenic cultures, the sensitivity, specificity, and positive and negative predictive values were 100%, 94.6%, 83.1%, and 100%, respectively, for the Xpert CD assay and 40.8%, 98.0%, 100%, and 88.9%, respectively, for VIDAS CDAB assay. Because of the low prevalence of the PCR ribotype 027 in Korea, the evaluation of the usefulness of the Xpert CD assay for screening for the 027 strain was limited. The Xpert CD assay provides great sensitivity in diagnosing toxigenic C. difficile infection. In addition, this method has excellent usability because it is simple and fast.

PH-Induced Molten Globule State of Rhizopus niveus Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, "B" (pH 9.0), native state, "N" (pH 7.4), acid-induced state, "A" (pH 2.2) and molten globule state, "MG" (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This "MG" state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The "R (h)" of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state "B" which is 15-fold higher than state "N." The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the "MG" state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.

Bone-Prognostic Status After Cessation of Cadmium Exposure for One Month in Male Rats

This study investigated bone status after decreased cadmium (Cd) exposure in male rats. Sprague-Dawley male rats were randomly divided into three groups. One group was injected subcutaneously with sodium chloride as control. The others were given CdCl2 by subcutaneous injection at doses of 0.5 mg Cd/kg body weight (bw) for 2 months (Cd+2m) and for 3 months (Cd+3m). For the Cd+2m group, the rats were shifted to cessation of Cd injection for 1 month after 2 months' exposure. At month 3, micro-computed tomography (micro-CT) analyses were performed on the proximal tibia and lumbar spine, and urine was collected from all rats. Rats were then killed and blood collected for metabolic-marker measurement and Cd assay. Bone tissues were also collected for bone-mass assay, biomechanical test, and bone-histology analysis. Cd burdens of rats in the Cd+2m and Cd+3m groups were both significantly greater than those in the control group. Cd burdens of rats were lower in the Cd+2m group compared with the Cd+3m group. Bone damage occurred in the Cd+2m and Cd+3m groups compared with the control group (p<0.05), but no significant improvement was found in the Cd+2m group compared with the Cd+3m group. Cd damage to bone could not be reversed over the short term. More attention should be paid to Cd's toxic effects on bone after decreased exposure.