Characterisation of the cold-shock response in Mycobacterium smegmatis

The response of Mycobacterium smegmatis to a cold shock was investigated in order to gain insight into the stress responses of members of the genus Mycobacterium. Mycobacterium smegmatis cultures were shocked from 37°C to 30°C, 25°C, 15°C, and 10°C and the effects on both growth (ATP concentration, culture turbidity, colony-forming units) and metabolism (incorporation of ¹⁴C-leucine and ³H-uracil) were investigated. The magnitude of the cold-shock response was found to be dependent upon the degree of the cold shock. A cold shock to 10°C had the greatest effect and resulted in a "lag period" of 24 hours in both the growth and metabolism of the culture. The synthesis of proteins was reduced 20-fold during this period, indicating at block in translation. The cold-shock response in Mycobacterium smegmatis was an adaptive response with growth eventually being resumed at the colder temperature, but at a reduced rate. Using the techniques of one-dimensional sodium-dodecyl-sulphate polyacrylamide gel electrophoresis and two-dimensional protein gel electrophoresis, ³⁵S-methiononine-labelled proteins that were synthesised during the cold shock were analysed. At least fourteen radio-labelled proteins were induced during the first 24-hour period and these demonstrated two distinct patterns of cold-shock induced expression: transient and continuous. Depending upon the pattern of expression and size, the cold-shock proteins were classified as "cold-induced proteins", "cold-shock proteins" or "cold-acclimation proteins". CipM, a 27kDa protein, was identified as the major cold-shock protein through one-dimensional protein electrophoresis. From N-terminal sequence data generated from a protein (CipM.1) within this band, a corresponding degenerate DNA probe was used to isolate cipM.1. This gene was cold-inducible, with mRNA levels transiently increasing 5-7 fold after a 37°C to 10°c cold-shock. Homologues of this cold-shock gene are found in the genomes of Mycobacterium tuberculosis and Mycobacterium leprae. The corresponding mycobacterial proteins showed homology at the N-terminus to the HU~ subunit of HU of Escherichia coli and possessed similar C-terminal praline, lysine and alanine degenerate repeats to the mycobacterial heparin-binding hemagglutinin. The response of several mycobacterial cold-shock gene homologues to a cold shock was also investigated, by northern-hybridisation and S1 nuclease analysis. The cspA homologue of Mycobacterium smegmatis demonstrated a 16-24 fold transient induction in mRNA levels following a 37°C to 10°C temperature-shift, while gyrA mRNA levels were maintained at a constant level throughout the cold shock. Although some similarities were demonstrated between the cold-shock response of Escherichia coli and Mycobacterium smegmatis, definite differences occur in the proteins that are involved in the adaptive stages of the response

Citrulline metabolism in cultured fibroblasts : citrullinemia analysis and nitric oxide production

A citrullinemic fibroblast cell line was used in this study to investigate two biochemical pathways involving citrulline. In the first section, the genetic mutation responsible for the argininosuccinate synthetase (-ASS) deficiency (1-5% activity) in this cell line was investigated. PCR analysis of the ASS cDNA revealed that the mRNA coding region (1236bp) was intact, showing no signs of major rearrangements. The ASS cDNA (1307bp) was cloned and sequenced and showed the presence of a single base mutation at position 1045bp, which represented a G->A transition. This mutation resulted in a glycine -> serine amino acid substitution at position 324 in the ASS subunit protein sequence. Although this glycine residue was not found to occur in any potential substrate binding sites, it was shown to be highly conserved among species, indicating a possible role of this amino acid in ASS catalytic activity. In the second section, the presence of the nitric oxide pathway in fibroblasts was investigated. Inducible nitric oxide synthase activity was assayed by measuring the production of ¹⁴C-citrulline from ¹⁴C-arginine after cytokine stimulation. By using the citrullinemic cell line (ASS deficient) any citrulline that may be produced by this pathway would accumulate, allowing detection. Under the assay conditions that were tested, no detectable ¹⁴C-citrulline was formed. Evidence suggests that human fibroblasts have the potential to synthesise nitric oxide, although a more sensitive assay system may need to be employed (longer cytokine activation, nitrite/nitrate detection)

Efficiency of Buccal DNA sampling device in the mortuary

Identification of forensic DNA samples by short tandem repeat (STR) profiling is currently an essential component of criminal investigations and can aid in linking perpetrators to crimes as well as identifying missing individuals or unidentified remains. In South Africa, recent amendments to legislation have allowed for the mandatory acquisition of reference DNA samples from certain offenders in order to populate the new National Forensic DNA Database. A novel method for the collection of buccal samples, the EasiCollect device, has been proposed to facilitate the collection of these DNA samples, replacing blood collecting devices as the standard method of DNA collection. Subsequently, this device has been introduced into South African state mortuaries to assist in the identification of deceased individuals. In order to ascertain if this device is suitable for use in the postmortem setting, an investigation was performed to compare the main methodology currently utilised within South African mortuaries, namely femoral blood transferred to ‘Fast Technology for Analysis of nucleic acids’ (FTA) cards, and buccal cells obtained using the EasiCollect device. DNA yields and STR genotyping results were compared between the two collection methods in thirty deceased individuals. Buccal samples provided a significantly greater DNA yield than blood samples, while no significant difference was observed between the qualities of the sample types as measured by the 260/280 nm ratio. Full STR profiles were obtained from all blood and buccal samples, with amplification efficiency showing limited DNA degradation and PCR inhibition in these samples, and only 3% of samples giving potentially disputable results. Numerous issues surrounding the collection of blood samples, however, indicated that this method is not optimal for use in the mortuary, with the EasiCollect device providing a more practical and robust method for the collection of DNA samples in the mortuar

Cancer/Testis Antigen Expression Panel Incorporating MAGEC1 and BAGE2 Predicts Multiple Myeloma Disease Stage and Severity

To provide a single molecular assay that could be used to easily stage Multiple Myeloma patients at diagnosis, we investigated the association between the simultaneous expression of 7 Multiple Myeloma-associated Cancer/Testis Antigens and biochemical parameters that are currently used for disease staging. We analysed the mRNA expression of MAGEC1, MAGEA3/A6, BAGE2, PRAME, NYESO1, SSX2 and PAGE by qualitative reverse transcription PCR using RNA extracted from diagnostic bone marrow samples from 39 patients covering the Multiple Myeloma disease continuum and compared this to levels of key biochemical parameters at diagnosis. We found that the Cancer/Testis Antigen panel was expressed in a specific order that was specifically associated with the severity of disease. This allowed the Cancer/Testis Antigens expression profile to successfully place the patient clearly into either stage I or stage III of the disease, with further sub-stratification in the stage III grouping. In addition, we putatively identified MAGEC1 expression as a confirmatory diagnostic marker for symptomatic Multiple Myeloma and clearly associated BAGE2 expression exclusively with stage III disease. We also demonstrated the novel finding of PAGE expression in Multiple Myeloma, with an association with more advanced disease. We suggest that this particular molecular Cancer/Testis Antigen panel can be used at diagnosis as a single test to clearly stage patient

Online Patient Portals: If You Build It, Who Will Come?

Research Objective: Many primary care practices have purchased electronic health records (EHRs) and accompanying patient portals. The role online portals may play in quality and outcome improvement will depend not only on who is using such technology, but how it is used. We evaluate the characteristics of patients using an online portal in comparison to those not using, and examine the portal features and functionalities accessed by users.Study Design: Observational, cohort study for which data were obtained from EHR and health system administrative data. Patient-level data (including demographic information, service use, and portal activation and use) were joined with information characterizing clinics in which patients received care (e.g., medical teaching on site, size, and urban/suburban location). The primary study outcome, portal use, was defined by the initiation of at least one online session. Among users, user-initiated clicks were used to determine specific features accessed. Logistic regression models with random effects were fit using the PROC GLIMMIX procedure (SAS software, Version 9.4) to test the role of clinic- and patient-level variables on patient portal activation. Subjects were blocked by physician, nested within clinic, and the Laplace method was used for likelihood approximation.Population Studied: Study eligible patients were aged 18 years and older with an office visit between 4/1/2013 and 3/31/2014 to a primary care physician practicing in one of the 26 primary care clinics of an integrated delivery system serving Detroit, Michigan and the surrounding suburban areas (N=20,282 patients).Principal Findings: As implemented in December 2012, the online portal enabled users to securely schedule appointments, receive appointment reminders, pay bills online, view lab and other test results, manage information about their health, and communicate with care teams via a secure messaging system. Cohort patients were on average 68.7 years of age (SD=14.7), predominately white (65%) or black (30%) race, and 60% female. Within 18 months of implementation, 33% had an activated account, with African Americans (OR=0.50, 95% CI 0.46-0.56), Hispanics (OR=0.63, 95% CI 0.47-0.84), those over aged 70 years (OR=0.48, 95% CI 0.44-0.52), and those preferring a language other than English (OR=0.43, 95% CI 0.31-0.59) less likely to be a portal user. Patients who were married (OR=0.55, 95% CI 1.44-1.67) and more connected with the clinic, as measured by visit frequency and health maintenance visit use, were more likely to be portal users (OR=1.08, 95% CI 1.05-1.10 and OR=1.39, 95% CI 1.27-1.52, respectively). Among users, the medical record access and management feature (95.9%) was most commonly accessed, most often to obtain laboratory testing results (91.7%). The majority of users also accessed appointment management (76.6%) and messaging (59.1%) functionalitiesConclusions: While the diversity of functions accessed by those with a portal account bodes well for the ability of portals to engage patients, without purposeful intervention enhancements to care delivery afforded by portals may be inaccessible to many, including racial/ethnic minorities and those less connected to traditional care services.Implications for Policy or Practice: Online portals have the potential to extend care beyond the confines of traditional office visits, but inattention to who uses portals may exacerbate known disparities in health care access and outcomes

Examiners\u27 decision‐making processes in observation-based clinical examinations

Background: Objective structured clinical examinations (OSCEs) are commonly used to assess the clinical skills of health professional students. Examiner judgement is one acknowledged source of variation in candidate marks. This paper reports an exploration of examiner decision making to better characterise the cognitive processes and workload associated with making judgements of clinical performance in exit‐level OSCEs. Methods: Fifty‐five examiners for exit‐level OSCEs at five Australian medical schools completed a NASA Task Load Index (TLX) measure of cognitive load and participated in focus group interviews immediately after the OSCE session. Discussions focused on how decisions were made for borderline and clear pass candidates. Interviews were transcribed, coded and thematically analysed. NASA TLX results were quantitatively analysed. Results: Examiners self‐reported higher cognitive workload levels when assessing a borderline candidate in comparison with a clear pass candidate. Further analysis revealed five major themes considered by examiners when marking candidate performance in an OSCE: (a) use of marking criteria as a source of reassurance; (b) difficulty adhering to the marking sheet under certain conditions; (c) demeanour of candidates; (d) patient safety, and (e) calibration using a mental construct of the \u27mythical [prototypical] intern\u27. Examiners demonstrated particularly higher mental demand when assessing borderline compared to clear pass candidates. Conclusions: Examiners demonstrate that judging candidate performance is a complex, cognitively difficult task, particularly when performance is of borderline or lower standard. At programme exit level, examiners intuitively want to rate candidates against a construct of a prototypical graduate when marking criteria appear not to describe both what and how a passing candidate should demonstrate when completing clinical tasks. This construct should be shared, agreed upon and aligned with marking criteria to best guide examiner training and calibration. Achieving this integration may improve the accuracy and consistency of examiner judgements and reduce cognitive workload

The Use of MAGE C1 and Flow Cytometry to Determine the Malignant Cell Type in Multiple Myelom

The malignant cell phenotype of Multiple Myeloma (MM) remains unclear with studies proposing it to be either clonotypic B or proliferating plasma cells. Cancer/testis antigen MAGE C1 is being extensively studied in MM and it has been suggested that it is involved in the pathogenesis of the cancer. Therefore, we report on the use of MAGE C1 to determine the malignant cell phenotype in MM using flow cytometry. Bone marrow aspirate (BM) and peripheral blood (PB) was collected from twelve MM patients at diagnosis, as well as three MM disease-free controls. Mononuclear cells were isolated using density-gradient centrifugation, and stabilized in 80% ethanol, before analysis via flow cytometry using relevant antibodies against B cell development cell-surface markers and nuclear MAGE C1. MAGE C1 expression was observed consistently in the early stem cells (CD34+) and early pro-B to pre-B cells (CD34+/−/CD19+), as well as the proliferating plasma cells in both the MM PB and BM, while no expression was observed in the corresponding control samples. Monoclonality indicated a common origin of these cell types suggesting that the CD34+/MAGE C1+ are the primary malignant cell phenotype that sustains the downstream B cell maturation processes. Furthermore, this malignant cell phenotype was not restricted to the BM but also found in the circulating PB cells. Introductio

Development of a flow cytometric method to detect the presence of mutated nucleophosmin 1 in acute myeloid leukemia

Objective/Background: Nucleophosmin 1 (NPM1) plays multiple roles in cell growth and proliferation. Deletion/insertion mutations in exon 12 of NPM1 (NPM1-DIM), commonly found in patients with acute myeloid leukemia (AML), alter the C-terminal amino acids and disrupt the normal nucleocytoplasmic shuttling function of the protein, which in turn leads to disease pathogenesis. However, this altered function as a result of NPM1-DIM positivity is actually associated with a significantly better response to therapy and overall survival, and thus it is of clinical relevance to investigate the mutation status at diagnosis. Our objective was to design a reliable flow cytometry assay to detect mutated NPM1 in peripheral blood (PB) samples from AML patients, using a polyclonal mutation-specific antibody. Methods: A commercially available NPM1 mutation-specific polyclonal antibody in combination with a secondary goat antirabbit antibody was used to detect the C-terminal-mutated NPM1 by flow cytometry. OCI/AML3 (+) cell line and clinical PB controls were used to optimize the assay and determine sensitivity, reliability, and reproducibility parameters. The assay was then tested on a small cohort of 12 AML patients at diagnosis and compared with NPM1-DIM testing on a standard polymerase chain reaction (PCR) platform. Results: Flow cytometry using the polyclonal antibody was able to reliably detect mutated NPM1 populations of at least 10%. Using an objective analysis of the mean fluorescent intensity, clear positive and negative mutated cell populations could be distinguished using the clinical AML samples. From the analysis of 12 patients, 2 were found to be positive using this assay, which corresponded with conventional PCR methodology. Conclusions: Flow cytometry may be used to detect NPM1 C-terminal mutations in AML patients using a polyclonal anti-NPM1 antibody, allowing rapid mutation status determination at diagnosis. Keywords: Acute myeloid leukemia, Flow cytometry, Nucleophosmin

Schematic representation of the co-expression of B cell maturation markers and MAGE C1 in the relevant PB B-lymphocyte sub-populations of MM patients and donors.

<p>MAGE C1 expression was observed in the very early stages of B cell maturation involving the CD34⁺ and CD19⁺ as well as the proliferating plasma cells in the PB. Additionally no MAGE C1 expression was observed in the late B cells (CD20⁺) in the PB. The percentage indicated is the number of cells in a specific cell population expressing MAGE C1 (as defined by a specific cell-surface antigen). Kappa/Lambda light chain expression as well as CD117, CD10 and CD27 antibodies were not investigated in the PB. MM 1 to 12 as well as D1 to 3 indicates the different MM patient and donor profiles, respectively.</p