Two-step PCR Method Research Articles

Yeast species in the respiratory samples of COVID-19 patients; molecular tracking of Candida auris.

Although the existence of Candida species in the respiratory tract is often considered commensal, it is crucial to recognize the significance of Candida colonization in immunocompromised or COVID-19 patients. The emergence of Candida auris as an emerging pathogen further emphasizes the importance of monitoring yeast infection/colonization, particularly in COVID-19 patients. In this study, respiratory samples mainly from COVID-19 patients, primarily those suspected of having a fungal infection, were cultured on Sabouraud dextrose agar plates and the yeast colonies were identified using a two-step multiplex PCR method. The samples suspected of C. auris underwent specific nested PCR followed by sequence analysis. A total of 199 respiratory samples were collected from 73 women and 126 men, ranging in age from 1.6 to 88 years. Among the patients, 141 had COVID-19, 32 had cancer, 5 were hospitalized in ICU, 2 had chronic obstructive pulmonary disease)COPD(, and others were patients with combination diseases. From these samples, a total of 334 yeast strains were identified. C. albicans (n=132, 39.52%) was the most common species, followed by C. tropicalis (n=67, 20%), C. glabrata (n=56, 16.76%), C. krusei (n=18, 5.4%), C. parapsilosis (n=17, 5.08%), Saccharomyces cerevisiae (n=10, 3%), C. kefyr (n=9, 2.6%), C. dubliniensis (n=7, 2.1%), C. lusitaniae (n=5, 1.5%), C. auris (n=3, 0.9%), C. guilliermondii (n=2, 0.6%), C. rugosa (n=1, 0.3%), C. intermedia (n=1, 0.3%), and Trichosporon spp. (n=1, 0.3%). C. auris was detected in a patient in ICU and two COVID-19 patients. While its presence was confirmed through sequence analysis, our extensive efforts to isolate C. auris were unsuccessful. While C. albicans colonization remains prevalent, our study found no evidence of Candida lung infection. Since the role of Candida colonization in airway secretions remains ambiguous due to limited research, further studies are imperative to shed light on this matter.

A Two-Step Real-Time PCR Method To Identify Mycobacterium tuberculosis Infections and Six Dominant Nontuberculous Mycobacterial Infections from Clinical Specimens.

Tuberculosis (TB) is an ongoing threat to public health, and furthermore, the incidence of infections by nontuberculous mycobacteria (NTM), whose symptoms are not distinguishable from TB, is increasing globally, thus indicating a need for accurate diagnostics for patients with suspected mycobacterial infections. Such diagnostic strategies need to include two steps, (i) detecting the mycobacterial infections and, if the case is an NTM infection, (ii) identifying the causative NTM pathogen. To eliminate a false-positive TB diagnosis for a host vaccinated by the bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis, a new target specific for M. tuberculosis species was selected, together with the species-specific targets for the six dominant NTM species of clinical importance, i.e., M. intracellulare, M. avium, M. kansasii, M. massiliense, M. abscessus, and M. fortuitum. Using sets of primers and probes, a two-step real-time multiplex PCR method was designed. The diagnostic performance was assessed by using a total of 1,772 clinical specimens from patients with suspected TB or NTM infection. A total of 69.4% of M. tuberculosis and 28.8% of NTM infections were positive for the primary step of the real-time PCR corresponding to the culture within 10 weeks, and mycobacterial species of 75.5% of the NTM-positive cases were identified by the secondary step. The two-step method described herein presented promising results and similar diagnostic sensitivity and specificity to commercially available real-time PCR kits for detecting TB and NTM infections. The method also enabled the identification of mycobacterial species in three-quarters of NTM infection cases, thus providing a better treatment strategy. IMPORTANCE Tuberculosis (TB) is an ongoing threat to public health. In addition, infection by nontuberculous mycobacteria (NTM) is a nonnegligible issue for global public health, with increasing incidences. Since the antimicrobial treatment strategy needs to be differed by the causative pathogen, a rapid and accurate diagnostic method is necessary. In this study, we developed a two-step molecular diagnostic method using clinical specimens of TB and NTM infection-suspected patients. The diagnostic power of the new method using the novel target was similar to the widely used TB detection kit, and, among the NTM-positive specimens, three-quarters of the NTM species were able to be identified. This simple and powerful method will be useful as it is, and it could be applied easily to a point-of-care diagnostic apparatus for better application to patients, especially those living in developing countries.

Correction: Temporal and spatially controlled APP transgene expression using Cre-dependent alleles

There were errors in Disease Models & Mechanisms (2022) 15, dmm049330 (doi:10.1242/dmm.049330).The authors recently discovered that one of the Cre-dependent transgenic mouse lines used in the study is not correctly described within the article. The mouse line labelled as R26-APP in the paper was originally described as containing both the Swedish and Iberian mutations, but carries only the Iberian mutation. This was discovered by The Jackson Laboratory when they sequenced through the complete insert to confirm the allele before entering the line into their repository for distribution. The authors also discovered an unintended point mutation located just outside the Aβ domain, M647T by APP695 numbering (M722T by APP770 numbering). They propose that the model should be identified as R26-APPIbe rather than as R26-APP.A diagram of the allele that appears in Fig. 2A has been corrected:In addition, a section describing the cloning methods in the Materials and Methods has been updated. The corrected and original versions are shown below:‘Cloning of R26-LSL-hAPPIbe transgene’ section (corrected). Human APPIbe 695 amino acid cDNA was generated in two steps. The N-terminal 1592 nucleotides of huAPP695 were cut from pcDNA3.1-sAPPα (a gift from Hui Zheng, BCM) with HindIII and SacI and subcloned into the same sites of transfer plasmid L4440 (Addgene, #1654). The remaining fragments were amplified from a human cDNA library using a two-step PCR method. Both PCR products contained the Ibe mutation as well as an overlap region needed to serve as an extended template for the second PCR. The first C-terminal fragment was amplified using hAPP C-terminus forward primer 5′-AACGAAAACCACCGTGGAGCT-3′ and Ibe reverse primer 5′-AAGGTGATGACGAACACTGTCG-3′. The second C-terminal fragment was amplified by Ibe forward primer 5′-CGACAGTGTTCGTCATCACCTT-3′ and hAPP C-terminus reverse primer 5′-GCGCGGATATCCTAGTTCTGCATCTGCTCAAAG-3′. The complete hAPP695Ibe C-terminus was then amplified from a mixture of the two shorter products using hAPP CT forward primer and hAPP CT reverse primer. The purified huAPPIbe C-terminal PCR fragment was digested with EcoRV and SacI and ligated into the EcoRV/SacI sites of L4440-APP(1-1592), resulting in L4440-APPIbe. The complete huAPPIbe sequence was transferred into an intermediary expression plasmid, which was digested with MluI and FseI and cloned into an Ai3-derived ROSA26 targeting vector (Madisen et al., 2010) to generate the final transgene construct pJJ-hAPPIbe. The modified Ai3 targeting vector retained the original Ai3 Cre-dependent CAG-loxP-stop-loxP sequence but removed the PGK-DTA negative selection cassette and shortened the 3′ targeting arm to reduce the overall vector size. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).‘Cloning of R26-LSL-hAPPSwe/Ibe transgene’ section (original). Human APPSwe/Ibe 695 amino acid cDNA was amplified from the pBR322-APPSwe/Ind plasmid used to generate the Tg CRND8 mouse model (Chishti et al., 2001) (a gift from David Westaway, University of Alberta, Canada) with primers that converted the original Indiana mutation V717F into the Iberian mutation I716F (point mutation numbering is for the standard human APP 770 amino acid isoform). The N-terminus of APPSwe/Ibe 695 was amplified using the forward primer 5′-GGCCGGCCGCCACCATGCTGCCCGGTTTGGCACT-3′ and reverse primer 5′-AAGGTGATGACGAACACTGTCG-3′; the C-terminus was amplified using the forward primer 5′-CGACAGTGTTCGTCATCACCTT-3′ and reverse primer 5′-GCGCGACGCGTCTAGTTCTGCATCTGCTCAAAGAA-3′. The resulting hAPPSwe/Ibe fragments were combined by amplification using the forward primer 5′-GGCCGGCCGCCACCATGCTGCCCGGTTTGGCACT-3′ and reverse primer 5′-GCGCGACGCGTCTAGTTCTGCATCTGCTCAAAGAA-3′. The complete hAPPSwe/Ibe 695 cassette was digested with MluI and FseI, and cloned into an Ai3-derived ROSA26 targeting vector (Madisen et al., 2010) that had been cut with the same enzymes to generate the final transgene construct pJJ-hAPPSwe/Ibe. The modified Ai3 targeting vector retained the original Cre-dependent CAGloxP-stop-loxP sequence but removed the PGK-DTA negative selection cassette and shortened the 3′ targeting arm to reduce the overall vector size. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).Throughout the paper, where the text refers to R26-APP, R26-APPSwe/Ibe or ROSA26-CAG-LSL-APPSwe/Ibe, corrections have been made to R26-APPIbe or ROSA26-CAG-LSL-APPIbe. The model will still be distributed by The Jackson Laboratory as stock #037319, but using the line name R26-APPIbe. The study results and conclusions remain unaffected.Both the online full-text and PDF versions have been updated. The authors apologise to readers for these errors and any inconvenience they may have caused.

Screening for gene doping transgenes in horses via the use of massively parallel sequencing.

Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.

Massively parallel sequencing of 25 autosomal STRs including SE33 in four population groups for forensic applications

The introduction of massively parallel sequencing (MPS) in forensic investigation enables sequence-based large-scale multiplexing beyond size-based analysis using capillary electrophoresis (CE). For the practical application of MPS to forensic casework, many population studies have provided sequence data for autosomal short tandem repeats (STRs). However, SE33, a highly polymorphic STR marker, has little sequence-based data because of difficulties in analysis. In this study, 25 autosomal STRs were analyzed, including SE33, using an in-house MPS panel for 350 samples from four populations (African–American, Caucasian, Hispanic, and Korean). The barcoded MPS library was generated using a two-step PCR method and sequenced using a MiSeq System. As a result, 99.88% genotype concordance was obtained between length- and sequence-based analyses. In SE33, the most discordances (eight samples, 0.08%) were observed because of the 4 bp deletion between the CE and MPS primer binding sites. Compared with the length-based CE method, the number of alleles increased from 332 to 725 (2.18-fold) for 25 autosomal STRs in the sequence-based MPS method. Notably, additional 129 unique alleles, a 4.15-fold increase, were detected in SE33 by identifying sequence variations. This population data set provides sequence variations and sequence-based allele frequencies for 25 autosomal STRs.

One-step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kluyveromyces marxianus inulinase.

This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72h. In a 5-l fed-batch fermentation, as high as 148.9g/L of inulin was directly used within 120h, and only 1.2g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9g/L was obtained.

Cell-Free Protein Synthesis of Plant Transcription Factors.

Biotic and abiotic stimuli induce profound transcriptional reprograming in plants through sophisticated regulation of transcription factors (TFs). Recombinant proteins of TFs play an important role in unveiling their molecular functions. Cell-free protein synthesis (CFPS) system from wheat germ has been developed as one of the most efficient protein synthesis platforms. However, preparation of linear DNA templates for in vitro transcription is time-consuming and laborious. Here, we describe a versatile method for in vitro transcription and translation of the wheat germ CFPS system. Our two-step PCR method enables researchers to generate a variety of transcription templates from a single plasmid including fusion proteins of an N- or C-terminal tag and truncated proteins. Thus, this method supports a rapid and high-throughput expression of proteins for a large-scale proteomics analysis.

PCR Assembly of Synthetic Promoters.

In this chapter, we describe a two-step assembly PCR method to construct synthetic promoters. Essentially, this method takes advantage of specific annealing between complimentary DNA sequences to build random TFBS combinations within the assembled PCR products. A DNA polymerase is then employed to fill in the unpaired nucleotides in the generated sequences and also to amplify the assembled PCR products. We have used this method to generate synthetic promoters whereby the orientation of the TFBS can be controlled, the spacing between TFBS can be predetermined, and also the full diversity of the consensus TFBS can be covered through the use of degenerate oligonucleotides .

Evaluating the methylation status of CXCR4 promoter: A cost-effective and sensitive two-step PCR method

The chemokine receptor CXCR4 plays a key role in the bone marrow microenvironment maintenance and in the hematopoietic stem and progenitor cells migration. In addition, CXCR4 is expressed in a broad spectrum of solid tumors where its methylation state has been recently proposed as a biomarker for cancer prognosis. To evaluate methylation status of CXCR4 promoter we developed a sensitive, accurate, specific and cost-effective two-step PCR method that does not require any specific equipment other than a conventional real-time PCR instrument. The principle of the technique relies on a novel normalization strategy which allows the detection and quantification of small methylation differences among pre-amplified DNA samples deriving from low amount of starting material. In addition, the analysis of melting curve profiles of PCR products provides additional information about the methylation status of CpG sites in between the primers. Finally, the principle of this technique can potentially be adapted for the investigation of the methylation status of any other DNA region.

Abstract 857: Diagnostic test system for sensitive, specific and reproducible detection of EML4-ALK RNA fusion transcripts in the blood of patients with NSCLC

Abstract Clinical testing for the detection of RNA fusions in tissue currently include FISH, IHC, PCR and NGS. However, approximately 30% of patients with advanced non-small cell lung cancer (NSCLC) are not candidates for tissue biopsies and in some cases where tissue is obtained, it is not always of sufficient quantity for molecular testing. For these reasons, the detection of nucleic acids in circulation has become increasingly relevant to clinical testing. In this study, we have focused on the development of an EML4-ALK diagnostic test system that includes the prospective collection of whole blood and the reproducible detection of mRNA fusion transcripts by a PCR-based technology. The focus was on the detection of EML4-ALK transcripts from donors with and without previously diagnosed NSCLC. Pre-analytic complexity was reduced by restricting the handling of samples to 72 hours from time of sample receipt to test result. Recovery methods for RNA extraction from donor plasma were then optimized to enrich for RNA recovered from both circulating-free, and RNA within blood vesicles, including platelets and exosomes. A two-step reverse transcription and Droplet Digital™ PCR (RT-ddPCR) method was evaluated in detection testing using a multiplexed EML4-ALK assay that includes variants 1, 2 and 3. Analytic assay specificity and sensitivity was examined for RT-ddPCR efficiency using a cell-line positive for EML4-ALK variant 1 and in vitro RNA designed to mimic variants 1, 2, and 3. Analytic sensitivity was determined to be 0.2% of fusion RNA spiked into a background of normal plasma. Precision studies were conducted with two different amounts of input RNA (high and low), over three consecutive days, three runs in one day and with two operators. The SD of detected fusion transcripts in this study did not exceed 25%. Finally, normal (n = 10) and FISH positive NSCLC donor (n = 9) plasma samples were processed for the recovery of RNA and tested for EML4-ALK fusions. All FISH positive cases were accurately detected by the RT-ddPCR test with EML4-ALK variant copies ranging from 13 to 150 copies. EML4-ALK fusion transcripts were not identified in normal donor plasma. We conclude that the developed test system is highly suited for a 72 hour test to result turnaround, reproducible and sensitive detection of diagnostic fusion RNA variants, including EML4-ALK in blood in the clinical laboratory. Citation Format: Hestia Mellert, Leisa Jackson, Dianna Maar, Dawne Shelton, Gary Pestano. Diagnostic test system for sensitive, specific and reproducible detection of EML4-ALK RNA fusion transcripts in the blood of patients with NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 857.

Seminal vesicle fluid ameliorates autoimmune response within central nervous system.

In 2004, it was demonstrated that changes in female reproductive tissue due to insemination result in immunological priming to paternal antigens and influence pregnancy outcome. During mating, exposure to seminal fluid elicits a tolerant state to paternal allo-antigens by expanding the regulatory T cell (Treg) pool. Notably, mating with males with surgically removed seminal vesicle glands was shown to fail to induce such effects.1 Evidence of antigen-specific Treg responses2,3,4 suggested that the expansion of the Treg pool occurs in response to seminal vesicle fluid.6 Further research has revealed differential recruitment of Tregs into different tissues, including the liver, kidney, heart, spleen, lymph nodes, mucosal tissues, blood, skin, thymus and brain, of which recruitment to the brain was the lowest.1 Owing to these effects on Treg expansion and the lower recruitment to the brain, we administered seminal vesicle fluid via an intra- cerebrospinal fluid (CSF) route in a Lewis rat model of experimental autoimmune encephalomyelitis (EAE) and investigated its effect on Treg expansion and clinical disease course. We induced EAE in Lewis rats and treated them with saline, 0.1 mg/ml chymotrypsin or seminal vesicle fluid (liquefied using chymotrypsin). Then, IFN-γ, IL-4, IL-17 and FoxP3 expression were estimated using a two-step real-time PCR method on samples from the brain, spinal cord and deep cervical lymph node (Supplementary Information). Our EAE Lewis rat model exhibited an acute monophasic disease, with clinical signs beginning on day 7 and reached the peak of the disease on day 14 post-induction. Intra-CSF injection of seminal vesicle fluid, 0.1 mg/ml chymotrypsin or saline was performed on day 7 after EAE induction. Changing trends in clinical signs in seminal vesicle fluid-treated animals were markedly different from those in the animals treated with 0.1 mg/ml chymotrypsin or saline (Figure 1a). The maximum mean clinical scores during the course of study were 1.46, 1.41 and 0.41, in saline-, 0.1 mg/ml chymotrypsin- and seminal vesicle fluid-treated animals, respectively.

Molecular identification and quantification of human rhinoviruses in respiratory samples.

PCR-based molecular assays have become standard diagnostic procedures for the identification and quantification of human rhinoviruses (HRVs) and other respiratory pathogens in most, if not all, clinical microbiology laboratories. Molecular assays are significantly more sensitive than traditional culture-based and serological methods. This advantage has led to the recognition that HRV infections are common causes for not only upper airway symptoms but also more severe lower respiratory illnesses. In addition, molecular assays improve turnaround time, can be performed by technicians with ordinary skills, and can easily be automated. This chapter describes two highly sensitive and specific PCR-based methods for identifying and quantifying HRVs. The first is a two-step PCR method for the detection and typing of HRV. The second is a pan-HRV real-time quantitative (q) PCR method for measuring viral loads in respiratory samples.

Potential clinical significance of ERβ ON promoter methylation in sporadic breast cancer

The aim of the study was to assess how hypermethylation of the ON promoter of the estrogen receptor beta (ERβ) gene affects its expression (at the mRNA and protein level) and to correlate these with some clinical and histopathological parameters. A total of 131 samples of frozen breast cancer tissue was analyzed. A custom-designed, two-step PCR method was used to measure the methylation index of the ERβ gene ON promoter region. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to quantify mRNA of the ERβ1 isoform, while ERβ1 protein was determined using the Western blot method. There was a significant difference in the methylation index of the ERβ gene ON promoter between the groups of patients with negative and positive axillary lymph node status (P = 0.03). In addition, the methylation index of the ON promoter was positively correlated with estrogen receptor alfa (ERα) protein levels (ρ = 0.31, P = 0.02). There was a significant difference in the methylation index of the ON promoter between the progesterone receptor (PR)-negative and PR-positive groups of patients (P = 0.01). ERβ1 protein levels were negatively correlated with ERα protein (ρ = -0.27, P < 0.01). The methylation index of the ON promoter could be a more reliable additional parameter for prediction and/or prognosis in breast cancer than ERβ1-mRNA and/or protein levels.

胆嚢癌および胆嚢異形成における高感度検出法を用いたK-ras codon 12点突然変異の解析

胆嚢癌および胆嚢異形成における高感度検出法を用いたK-ras codon 12点突然変異の解析

High Frequency of CYP3A4*1B among Opiate Dependent Patients in Malaysia

The sharing of injection needles among drug user is a leading cause for the spread of HIV/AIDS. Malaysia introduced methadone as a management of heroin dependents to reduce HIV spread. Methadone has variable pharmacokinetics and CYP3A4 has been implicated in its metabolism. The objective of this study therefore was to determine if polymorphisms exist with CYP3A4 among opiate users in Malaysia. This study was approved by Ethics Committees at University of Malaya and Universiti Sains Malaysia. Control subjects comprised blood donors, students and residents of a village. Opiate-dependents were from methadone clinics and drop-in centers. They signed a written-informed consent to participate and gave blood for DNA CYP3A4 genotyping. DNA was extracted using QIAgen DNA mini kit. A nested two-step allele specific PCR method was developed to detect CYP3A4*1B, CYP3A4*3, CYP3A4*4, CYP3A4*5, CYP3A4*6, CYP3A4*7, CYP3A4*8, CYP3A4*9, CYP3A4*10, CYP3A4*11, CYP3A4*12, CYP3A4*13, CYP3A4*14, CYP3A4*15 and CYP3A4*16. Normal controls comprised Malays, Chinese and Indians but opiate-dependent subjects were majority Malay males. Control subjects all carried the wild-type gene. Mutant CYP3A4*1B allele was found in 2.17% of opiate-dependent subjects. Our results revealed that CYP3A4 was not polymorphic among Malaysian Malays, Chinese and Indians who were not opiate-dependent. To date, we are not aware of any study to associate CYP3A4 polymorphism and heroin addiction. It is conceivable that altered CYP3A4 function may contribute towards addiction liabilities in subsets of individuals. We conclude that CYP3A4 is polymorphic among heroin-dependent individuals. The mutation, CYP3A4*1B is not silent. This may have implications on heroin addiction liability as well as on dose requirements for MMT and HAART.

Quantitative PCR used to Assess HIV-1 Integration and 2-LTR Circle Formation in Human Macrophages, Peripheral Blood Lymphocytes and a CD4+ Cell Line

BackgroundIntegration is an intermediate step in the HIV life cycle and is defined as the insertion of HIV-1 proviral DNA into the host chromosome. If integration does not occur when HIV-1 cDNA enters the nucleus, it circularizes upon itself and forms a 2-LTR circle. Monitoring the level of integrated HIV-1 cDNA in different primary cell subsets is very important, particularly regarding the effect of HAART in HIV-1 infected individuals. Because of limitations of prior HIV-1 integration assays, there is limited data on the level of integration and 2-LTR circle formation in primary cell subsets, particularly in human monocyte-derived macrophages and peripheral blood lymphocytes (PBL).ResultsIn this study, we utilized a well-defined, sensitive two-step quantitative real-time PCR method to detect HIV-1 integration as well as conventional real-time PCR to detect 2-LTR circle formation in human macrophages and PBL isolated from six different healthy donors, as well as U373 CD4+ cells by infecting with HIV-1SX (R5) or dual-tropic isolate HIV-189.6 (R5/X4) virus strains. We used the FDA-approved integrase inhibitor, raltegravir, to determine quantitative differences of integrated HIV viral cDNA in HIV-1 infected cells with and without raltegravir treatment. Our results show that integration and 2-LTR circle formation can be assessed in primary macrophages, PBL, and a CD4+ cell line by this method. Specifically, our results demonstrate that this two-step real-time PCR method can distinguish between HIV-1 integrated viral cDNA and non-integrated nuclear HIV-1 2-LTR circles caused by impaired integration with raltegravir-treatment. This further confirms that only integrated HIV-1 cDNA can be specifically amplified and quantified by two-step PCR without non-specifically detecting non-integrated viral cDNA.ConclusionThese results consistently demonstrate that the well-established real-time PCR assays used are robust, sensitive and quantitative for the detection of HIV-1 integration and 2-LTR circle formation in physiologically relevant human macrophages and PBL using lab-adapted virus strains, instead of pseudovirus. With two-step real-time PCR, we show that unintegrated, nuclear HIV-1 cDNA is not detected in raltegravir-treated cells, while specific for only integrated HIV-1 cDNA in non-treated cells. These methods could be applied as a useful tool in further monitoring specific therapy in HIV-1 infected individuals.

New Mutation in 23S rRNA Gene Associated with High Level of Azithromycin Resistance in Neisseria gonorrhoeae

Although azithromycin (1 g orally in a single dose) is not recommended for treating gonorrhea, it is still used in some parts of the world, and it is widely used either to treat Chlamydia trachomatis infections or, in combination with expanded-spectrum cephalosporins, to treat simultaneous infections with both chlamydia and gonococci (7). A higher dose of 2 g of new formulations of azithromycin with a lower incidence of gastrointestinal intolerance may be more effective (9), but recent descriptions of gonococcal strains showing azithromycin resistance or high-level azithromycin resistance (AzHLR; MIC of azithromycin, >256 mg/liter) would make that treatment untenable if strains of this type were to emerge elsewhere and spread (1, 2, 6). Decreased susceptibility of Neisseria gonorrhoeae to azithromycin is due mainly to the overproduction of an mtrCDE-encoded efflux pump induced by mtrR mutations (10) but also to the presence of the mef gene, whose code for an efflux pump has been associated with resistance of gonococci to macrolides (3). Recently, a gonococcal strain isolated in Argentina for which the MIC of azithromycin was >2,048 mg/liter was described. Neither mutations in the promoter region of the mtrR gene explaining the AzHLR nor the presence of the mef gene was observed in this strain (2). Mutations in the peptidyltransferase loop in domain V of 23S rRNA alleles have also been associated with macrolide resistance in N. gonorrhoeae (5), and now the Argentinian strain with AzHLR has been analyzed for the presence of these mutations within domain V. A two-step PCR method was followed (5) to analyze three gonococcal strains isolated in Argentina showing different levels of azithromycin susceptibility: the strain with AzHLR (azithromycin MIC, >2,048 mg/liter) mentioned above (strain 1782), one isolate (strain 2498) for which the azithromycin MIC was 8 mg/liter (indicating resistance), and one susceptible gonococcal strain (3783; azithromycin MIC, 0.06 mg/liter). The sequences obtained in this study were compared with those (Gene Bank accession numbers {type:entrez-nucleotide-range,attrs:{text:AF450074 to AF450081,start_term:AF450074,end_term:AF450081,start_term_id:22901255,end_term_id:22901262}}AF450074 to AF450081) corresponding to azithromycin-resistant gonococcal strains and associated with mutations at this level (5). The susceptible Argentinian strain did not show mutations in the sequenced fragment from any of the four copies of the 23S rRNA rrl gene. In strain 2498, for which the MIC was 8 mg/liter, all four alleles were found to contain C2599T, which has been associated with this level of resistance (5). The strain showing AzHLR did not have this particular mutation but presented an A2143G mutation (N. gonorrhoeae numbering) in all four alleles. This mutation, which corresponds to A2059G in Escherichia coli, has not been reported to occur in gonococci, but it has been associated with macrolide resistance in Helicobacter pylori, several Mycobacterium species, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Propionibacterium species and more recently in Treponema pallidum (4, 8). Adenosine 2058 or neighbor nucleotides (such as A2059) are the key for macrolide interaction on the ribosome (8), and A2143G is strongly suggested to be responsible for the AzHLR in gonococcal strain 1782, isolated in Argentina. The finding observed for the strain for which the MIC was 8 mg/liter is also interesting: all alleles showed a C2599T mutation, but a dinucleotide (TT) insertion in the promoter region of the mtrR gene, which has also been associated with reduced susceptibility to azithromycin (2), was also found. Ng et al. (5) observed a spontaneous N. gonorrhoeae mutant strain with the same mutation in four rrl alleles and a T insertion in the promoter region of the mtrR gene for which the MIC was 4 mg/liter. As far as we know, Argentinian strain 2498 is the first clinical isolate with both mechanisms of resistance, and it illustrates how gonococcal strains are continuously adapting under antibiotic selection pressure.

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.

Influence of class B scavenger receptors on cholesterol flux across the brush border membrane and intestinal absorption

To learn more about how the step of cholesterol uptake into the brush border membrane (BBM) of enterocytes influences overall cholesterol absorption, we measured cholesterol absorption 4 and 24 h after administration of an intragastric bolus of radioactive cholesterol in mice with scavenger receptor class B, type 1 (SR-BI) and/or cluster determinant 36 (CD36) deleted. The cholesterol absorption efficiency is unaltered by deletion of either one or both of the receptors. In vitro determinations of the cholesterol uptake specific activity of the BBM from the mice reveal that the scavenger receptors facilitate cholesterol uptake into the proximal BBM. It follows that cholesterol uptake into the BBM is not normally rate-limiting for the cholesterol absorption process in vivo; a subsequent step, such as NPC1L1-mediated transfer from the BBM into the interior of the enterocyte, is rate-limiting. The absorption of dietary cholesterol after 4 h in mice lacking SR-BI and/or CD36 and fed a high-fat/high-cholesterol diet is delayed to more distal regions of the small intestine. This effect probably arises because ATP binding cassette half transporters G5 and G8-mediated back flux of cholesterol from the BBM to the lumen of the small intestine limits absorption and causes the local cholesterol uptake facilitated by SR-BI and CD36 to become rate-limiting under this dietary condition.

A robust two-step PCR method of template DNA production for high-throughput cell-free protein synthesis

A two-step PCR method has been developed for the robust, high-throughput production of linear templates ready for cell-free protein synthesis. The construct made from the cDNA expresses a target protein region with N- and/or C-terminal tags. The procedure consists only of mixing, dilution, and PCR steps, and is free from cloning and purification steps. In the first step of the two-step PCR, a target region within the coding sequence is amplified using two gene-specific forward and reverse primers, which contain the linker sequences and the terminal sequences of the target region. The second PCR concatenates the first PCR product with the N- and C-terminal double-stranded fragments, which contain the linker sequences as well as the sequences for the tag(s) and the initiation and termination, respectively, for T7 transcription and ribosomal translation, and amplifies it with the universal primer. Proteins can be fused with a variety of tags, such as natural poly-histidine, glutathione-S-transferase, maltose-binding protein, and/or streptavidin-binding peptide. The two-step PCR method was successfully applied to 42 human target protein regions with various GC contents (38-77%). The robustness of the two-step PCR method against possible fluctuations of experimental conditions in practical use was explored. The second PCR product was obtained at 60-120 microg/ml, and was used without purification as a template at a concentration of 2-4 microg/ml in an Escherichia coli coupled transcription-translation system. This combination of two-step PCR with cell-free protein synthesis is suitable for the rapid production of proteins in milligram quantities for genome-scale studies.