Introducing a comprehensive data reduction algorithm for high-precision U-Th geochronology with isotope dilution MC-ICP-MS

Multi collector inductively coupled plasma mass spectrometry (MC-ICP-MS) is being increasingly utilized for U-Th geochronology of carbonate deposits with comparable precision to thermal ionization mass spectrometry (TIMS) [1, 2]. While attention has been paid to propagation of uncertainties for U-Th-Pb analysis by TIMS and the isochron technique [3,4], a comprehensive data processing scheme is lacking for MC-ICP-MS. To address this need, we have developed an algorithm in Mathematica application to allow for step-by-step monitoring of the data reduction process. The program is flexible and affords the user easy control over input variables. Adjustments for background and spike isotope contributions, abundance sensitivity and instrumental mass bias are implemented through the code, followed by age calculation and propagation of uncertainties with Monte Carlo simulation. A rigorous standard bracketing procedure was adopted using Uranium (CRM-112A) and Th (IRMM-035) standard solutions, doped with IRMM-3636a ^(233)U/^(236)U “double-spike”, to account for deviations of isotope ratios from certificate values and improve accuracy. Following a single U/TEVA extraction chromatography step to separate U from Th, ten replicate ages from a speleothem in Cathedral Cave (CC), Utah showed excellent agreement (R^2 = 0.999) with results previously measured at the University of Minnesota by single collection ICP-MS [5]. The external reproducibility of our analytical technique was evaluated by analyzing six aliquots of an in-house standard, prepared by homogenizing a piece of the CC speleothem, which returned a mean age of 21468±120 y (2SD). A limited amount of the standard powder is available upon request for interlaboratory calibration. We have successfully dated 36 samples from caves in the Bahamas, the Dominican Republic and Iran

Introducing a comprehensive data reduction algorithm for high-precision U-Th geochronology with isotope dilution MC-ICP-MS

Multi collector inductively coupled plasma mass spectrometry (MC-ICP-MS) is being increasingly utilized for U-Th geochronology of carbonate deposits with comparable precision to thermal ionization mass spectrometry (TIMS) [1, 2]. While attention has been paid to propagation of uncertainties for U-Th-Pb analysis by TIMS and the isochron technique [3,4], a comprehensive data processing scheme is lacking for MC-ICP-MS. To address this need, we have developed an algorithm in Mathematica application to allow for step-by-step monitoring of the data reduction process. The program is flexible and affords the user easy control over input variables. Adjustments for background and spike isotope contributions, abundance sensitivity and instrumental mass bias are implemented through the code, followed by age calculation and propagation of uncertainties with Monte Carlo simulation. A rigorous standard bracketing procedure was adopted using Uranium (CRM-112A) and Th (IRMM-035) standard solutions, doped with IRMM-3636a ^(233)U/^(236)U “double-spike”, to account for deviations of isotope ratios from certificate values and improve accuracy. Following a single U/TEVA extraction chromatography step to separate U from Th, ten replicate ages from a speleothem in Cathedral Cave (CC), Utah showed excellent agreement (R^2 = 0.999) with results previously measured at the University of Minnesota by single collection ICP-MS [5]. The external reproducibility of our analytical technique was evaluated by analyzing six aliquots of an in-house standard, prepared by homogenizing a piece of the CC speleothem, which returned a mean age of 21468±120 y (2SD). A limited amount of the standard powder is available upon request for interlaboratory calibration. We have successfully dated 36 samples from caves in the Bahamas, the Dominican Republic and Iran

Carbapenem-resistant Acinetobacter baumannii recovered from burn patients

Purpose. Emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) and their prolonged presence in burn units increases the risk of acquisition of CRAB. Methods. From November 2012 to September 2013, 1474 burn patients were screened for CRAB isolates through testing susceptibility to imipenem and its comparators meropenem, and doripenem. Furthermore, the in vitro activity of other antibiotics against CRAB was investigated. Results. Three patients were infected with carbapenem-susceptible A. baumannii (CSAB) and 168 were infected with CRAB. Approximately one-fifth (n=32) of CRAB isolates were obtained from patients hospitalized in Burn Intensive Care Unit (BICU). Most of CRABs were isolated from wound. The mean length of stay (LOS) in hospital prior to A. baumannii isolation was significantly higher for CRAB compared to CSAB cases (P=0.04). Amongst the independent variables, percentage of total burn surface area (TBSA) significantly increased the mortality rate using multivariate logistic regression (P=0.001, OR= 16.5; 95 CI: 4.72-57.7). The majority of tested isolates were resistant to imipenem (94.8), and to its comparators, doripenem (97.7), and meropenem (97.7). The susceptibility of CRAB isolates was less than 10 to all tested antibiotics except for colistin (100), doxycycline (61.9), gentamicin (18.5), and tigecycline (11.9). Conclusion. Resistance to carbapenem reduces the number of effective antibiotics. The coordinated and intensive efforts of healthcare personnel are required to meet the challenge of dissemination of CRA

Optimization and efficient purification in production of Brucella melitensis recombinant HSP and TF proteins with low endotoxin contents

Background: The development of an effective subunit vaccine against brucellosis is a research area of intense. But optimization of recombinant proteins production in Escherichia coli and content of endotoxins associated with final recombinant proteins are very important. Objectives: In the present study, expression and purification of Brucella melitensis rHSP and rTF were optimized to reduce endotoxin contaminants. Materials and Methods: pDEST-tf and pDEST-hsp were transformed into E. coli BL21 (DE3), and then B. melitensis recombinant HSPA and TF proteins were overexpressed. Purification of these proteins was optimized to remove most of endotoxin contaminants from the end product using 0.1 Triton X-114 in washing buffers. Results: An endotoxin reduction of less than 0.05 EUmg/1 was achieved with protein recovery close to an 80 yield. Conclusions: As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense. © 2013, Ahvaz Jundishapur University of Medical Sciences; Licensee Kowsar Ltd

Multiplex SNP typing by bioluminometric assay coupled with terminator incorporation (BATI)

A multiplex single-nucleotide polymorphism (SNP) typing platform using ‘bioluminometric assay coupled with terminator [2′,3′-dideoxynucleoside triphosphates (ddNTPs)] incorporation’ (named ‘BATI’ for short) was developed. All of the reactions are carried out in a single reaction chamber containing target DNAs, DNA polymerase, reagents necessary for converting PPi into ATP and reagents for luciferase reaction. Each of the four ddNTPs is dispensed into the reaction chamber in turn. PPi is released by a nucleotide incorporation reaction and is used to produce ATP when the ddNTP dispensed is complementary to the base in a template. The ATP is used in a luciferase reaction to release visible light. Only 1 nt is incorporated into a template at a time because ddNTPs do not have a 3′ hydroxyl group. This feature greatly simplifies a sequencing spectrum. The luminescence is proportional to the amount of template incorporated. Only one peak appears in the spectrum of a homozygote sample, and two peaks at the same intensity appear for a heterozygote sample. In comparison with pyrosequencing using dNTP, the spectrum obtained by BATI is very simple, and it is very easy to determine SNPs accurately from it. As only one base is extended at a time and the extension signals are quantitative, the observed spectrum pattern is uniquely determined even for a sample containing multiplex SNPs. We have successfully used BATI to type various samples containing plural target sequence areas. The measurements can be carried out with an inexpensive and small luminometer using a photodiode array as the detector. It takes only a few minutes to determine multiplex SNPs. These results indicate that this novel multiplexed approach can significantly decrease the cost of SNP typing and increase the typing throughput with an inexpensive and small luminometer

Molecular detection of prostate specific antigen in patients with prostate cancer or benign prostate hyperplasia the first investigation from Iran

Prostate cancer is the second common form of cancer in men. Detection of circulating Prostate Specific Antigen (PSA) transcripts has effectively been used for early diagnosis of prostate cancer cells. This investigation employed a reverse transcriptase polymerase chain reaction (RT-PCR) technique to distinguish the patients with either localized or metastatic prostate cancer (CaP) vs. Benign Prostate Hyperplasia (BPH) and control subjects, as compared with clinical and pathological records. With reservation of ethical issues, blood samples were collected from 60 cases. Based on pathological and clinical findings, 25 patients (20 with localized cancer, 5 with metastatic), 22 with BPH, and 13 healthy (including 3 females) subjects as negative controls, were selected from Shariati, Mehrad, Sina,, Khatam and Atie Hospitals in Tehran, Iran. RT-PCR for a 260 bp PSA transcript was then performed. Clinical and pathological records were used for the assessment and comparison of PSA RT-PCR results. None of the control subjects and BPH (with 7 exceptions) were found positive by RT-PCR (Relative specificity= 72.7). In patients with prostate cancer, 21 out of 25 were found PSA positive (Relative sensitivity= 83.4) and the remaining 3 have been shown to be PSA negative (Positive predictive value= 83.4). All of 5 metastatic patients (100) revealed PSA positive results. Our data reflects the clinical relevance and significance of RT-PCR results as assessed with clinical and pathological examinations. PSA RT-PCR might be used as a powerful means for diagnosis, even when either pathological or clinical findings are negative, and could be employed for further molecular epidemiology surveys

Vaccine potential of lena and lcpa proteins of leptospira interrogans in combination with escherichia coli heat-labile enterotoxin, b subunit (LTB)

Background and Objectives: Leptospirosis is a zooanthroponosis caused by the genus of Leptospira. It is an emerging public health problem due to its increasing incidence. The achievement to a vaccine that prevent from entrance of Leptospira interrogans to the deeper tissues of the host is needed. This study aimed to investigate the immunogenicity of LcpA (rLcpA) and LenA (rLenA) recombinant proteins in combination with LTB (rLTB) recombinant protein as an adjuvant against leptospiral infection in hamsters. Materials and Methods: The genes encoding these proteins were cloned into pGH cloning vector and then lenA, lcpA and ltb genes subcloned into pET-15b and pET-28a expression vectors, respectively. The hamsters were immunized with the purified recombinant proteins and challenged with Leptospira interrogans for evaluation of their survival. The antibody responses to the recombinant proteins were determined by ELISA. Then, data entered into SPSS software. Statistical Kruskal-Wallis test was used to compare the significant differences among different groups. The groups with significant differences were further analyzed by post hoc tests. The p value < 0.05 statistically was considered significant. Results: Immunized hamsters with rLenA-plus-rLTB, rLcpA-plus-rLTB and rLenA-plus-rLcpA-plus-rLTB proteins showed 60, 74, and 80 survival rates, respectively. A significant amount of interleukin-17 (IL-17), interleukin-4 (IL-4) and gamma interferon (IFNγ) cytokines were produced in immunized hamsters. Conclusion: Based on our findings, rLcpA and rLenA proteins in combination with rLTB can protect the hamsters against L. interrogans and effectively induce a protective antibody response. Thus, these proteins can be used as an additional prophylactic tool against leptospira. © 2018, Tehran University of Medical Science. All rights reserved

Analysis of Short Tandem Repeats by Parallel DNA Threading

The majority of studies employing short tandem repeats (STRs) require investigation of several of these genetic markers. As such, we demonstrate the feasibility of the trinucleotide threading (TnT) approach for scalable analysis of STRs. The TnT method represents a parallel amplification alternative that addresses the obstacles associated with multiplex PCR. In this study, analysis of the STR fragments was performed with capillary gel electrophoresis; however, it should be possible to combine our approach with the massive 454 sequencing platform to considerably increase the number of targeted STRs

The risk factors of prostate cancer: A multicentric case-control study in Iran

Prostate cancer (PC), in Iran, is the third most frequently diagnosed visceral cancer among men and the seventh most common underlying cause of cancer mortality. We evaluated the relation between speculated factors and PC risk using data from a multicentric case-control study conducted in Iran from 2005 to 2007 on 130 cases of incident, clinicopathologically confirmed PC, and 75 controls admitted to the same network of hospitals without any malignant disease. Odds ratios(OR) and corresponding 95 confidence intervals (CIs) were estimated using conditional logistic regression models. The risk of PC was increased with aging (OR: 5.35, 95 CI: 2.17-13.19; P<0.0001), and with the number of sexual intercourse �2 times/week (OR: 3.14, 95 CI: 1.2-8.2; P=0.02). One unit elevation in serum estradiol and testosterone concentration was related to increase (OR: 1.04, 95 CI: 1.01-1.06; P=0.006) and decrease (OR: 0.79; 95 CI: 0.64-0.96; P=0.02) of PC risk, respectively. Cases were less likely to have a history of diabetes (OR: 0.34, 95 CI: 0.12-0.98; P=0.04). Increasing in dietary consumption of lycopene and fat was associated with declined (OR: 0.45, 95 CI: 0.09-2.12) and increased (OR: 2.38, 95 CI: 0.29-19.4) PC development, respectively. Other factors including educational level, marriage status, dietary meat consumption, vasectomy and smoking have not been shown to affect PC risk in the Iranian population. Ourstudy adds further information on the potential risk factors of PC and is the first epidemiologic report from Iran. However, justification of these results requires more well-designed studies with a larger number of participants