Kristina Jackson Behan

Alternative splicing removes an Ets interaction domain from Lozenge during Drosophila eye development

Physical and functional characteristics of the RUNX family of transcription factors are conserved between vertebrates and the Drosophila protein Lozenge. The runt-homology domain responsible for DNA binding and also the C-terminus are both nearly identical between the two proteins. The mammalian and fly proteins heterodimerize with a non-DNA binding partner protein to form a core binding factor essential for gene regulation during cell differentiation. The mammalian protein RUNX1 (AML1/PEBP2αB) interacts with the transcription factor Ets-1 to increase DNA binding and transactivation potential. Alternative splicing of the mammalian RUNX1 removes a domain required for this cooperative transactivation. In this work we determine the structure of the lozenge transcription unit and map 21 mutations. We show that the lozenge transcript is alternatively spliced during eye development to remove an Ets interaction domain. Emphasis is placed on Pointed the Drosophila homolog of the vertebrate Ets-1 protein; both Lozenge and Pointed proteins are needed for the activation of prospero expression. We use site-directed mutagenesis and yeast two-hybrid analysis to show that conserved amino acids within the alternate Lozenge exon are important for interaction with Pointed. Furthermore, the ectopic expression of Lozenge is sufficient to rescue Prospero expression in the presence of the Pointed competitor, YanACT. We show that both lozenge isoforms are expressed during eye development and that the relative ratio of the transcripts for the two isoforms is sensitive to changes in Ras activity. We suggest that during eye development, Lozenge isoforms function in divergent roles, either interacting with Pointed on downstream targets or by functioning independently to establish distinct cell fates.

Reporting variant hemoglobins discovered during hemoglobin A1c analysis — Common practices in clinical laboratories

BACKGROUND Patients with variant hemoglobins may receive inaccurate results by some HbA(1c) methods. We examined reporting practices of clinical laboratories with respect to variant hemoglobins and limitations of methodology. METHODS A survey of reporting practices was published in LabMedicine, and circulated to directors of Clinical Laboratory Sciences programs. Websites of reference laboratories were reviewed. RESULTS One hundred thirty-five laboratories from 42 US states responded. 61.5% of those laboratories report only HbA(1c) value and reference interval; 5% of laboratories include methodology. 51% of laboratories use IE-HPLC, 47% use immunoassay and 2% use boronate affinity chromatography. Of laboratories using IE-HPLC, 39% routinely report the presence of hemoglobin variants, and 10% report variants only if they cause interference with the test. Of laboratories using immunoassay, only one appends the disclaimer that elevated HbF interferes with test results. All of the major reference laboratories report methodology on their websites; only 2 can detect hemoglobin variants. Six out of 7 reference laboratories state limitations of methodology on their websites. CONCLUSIONS There is no standardized reporting format for HbA(1c) that includes methodology, test limitations or notification of variant hemoglobins. An algorithm for detection and reporting of variant hemoglobins and test methodology is proposed based on best practices.

Mutations in lozenge and D-Pax2 invoke ectopic patterned cell death in the developing Drosophila eye using distinct mechanisms

Mutations in the lozenge gene of Drosophila melanogaster elicit a pleiotropic set of adult phenotypes, including severe compound eye perturbations resulting from the defective recruitment of photoreceptors R1/6 and R7, cone and pigment cells. In this study, we show that excessive patterned apoptosis is evident at the same developmental stage in these lozenge mutants. In lozenge null mutants, apoptosis occurs prior to lozenge-dependent cell fate specification. A second gene, D-Pax2, genetically interacts with lozenge. Interestingly, D-Pax2 mutants also exhibit increased cell death, but slightly later in development than that in lozenge mutants. Although expression of the caspase inhibitor p35 eliminates death in both lozenge and D-Pax2 mutants, the lozenge mutant eye phenotypes persist because other normal Lozenge functions are still lacking. D-Pax2 eye phenotypes, in contrast, are dramatically altered in a p35 background, because cells that normally differentiate as cone and primary pigment cells are subsequently transformed into secondary pigment cells. This study leads us to propose that Lozenge, aside from its known role in gene regulation of cell-specific transcription factors, is required to contribute to the repression of cell death mechanisms, creating a permissive environment for the survival of undifferentiated cells in early eye development. Lack of lozenge expression increases the likelihood that an undifferentiated cell will initiate its default death program and die prematurely. The ectopic cell death evident in D-Pax2 mutants appears to arise from the cell fate transformation of cone cells into secondary pigment cells, either autonomously or as a result of defective signalling.

The Relationship Between Waist Circumference and Biomarkers for Diabetes and CVD in Healthy Non-Obese Women. The Pensacola Study

Background: Waist circumference (WC) measurement has been shown to perform as well as or better than body mass index (BMI) in identifying women at risk for diabetes and cardiovascular disease (CVD). The goal of this study was to determine the relationship between WC and the classic biomarkers of risk in healthy women. Methods: Nondiabetic, non-obese, middle-aged women were categorized by WC quartiles. The correlation of WC to fasting plasma glucose (FPG), A1c, lipid profile, C-reactive protein (CRP) and white blood cell count (WBC) was examined. Results: Waist circumference correlated with triglycerides, CRP, cholesterol/HDL, non-HDL, LDL, and glucose, and inversely with HDL (r = 0.465, 0.414, 0.321, 0.299, 0.267, 0.279, 0.266, respectively; P = 0.000 for all), but not A1c or WBC. There was a trend of increasing glucose, LDL, and CRP with increasing WC. Quartile 4 showed the highest glucose, CRP, LDL, triglycerides, cholesterol/HDL ratio, and non-HDL. Conclusion: Increasing WC correlates with several biomarkers of risk for diabetes and CVD in healthy women.

Improving the Accuracy of Hemoglobin A 1c : Your Help Is Needed

Clinical laboratory personnel are important members of the diabetes treatment team, from the technical staff, to the supervisor, to the pathologist. The technicians and technologists measure glucose, hemoglobin A 1c (A1c), lipids, microalbumin, and liver function tests for diabetic patients to help those patients to decrease the risk of chronic complications. The laboratory staff and supervisors monitor quality control with a critical eye. The laboratory management brings in better instrumentation and methodology as soon as it is feasible. Questionable laboratory results are investigated and corrected. Quality assurance is not just a catchphrase, it is a way of life. So it is a cause for concern to all laboratory professionals that A1c results may be inaccurate, yet reported, in patients who have variant hemoglobins. The nature of the A1c inaccuracy, whether it is a spurious increase or decrease, is specific to the method used and to the variant hemoglobin (eg, HbS, HbC, HbE, HbF). An analysis of the capabilities of available methods is listed at a Web site maintained by the National Glycohemoglobin Standardization Program (NGSP). 1 The 2 major types of testing currently in use are high-performance liquid chromatography (HPLC) and immunoassay. High-performance liquid chromatography has an advantage in that the latest generation of analyzers can resolve and identify several forms of hemoglobin, including most variants, and it provides a chromatogram that shows peaks where the forms have been resolved. High-performance liquid chromatography analyzers also provide quantitation of those peaks. Immunoassay accuracy may be affected by some variant hemoglobins, but there is no visual interpretation of the results other than a number to aid the laboratory professional in evaluating accuracy. Immunoassay analyzers are simpler to use and, as a result, are used as point-of-care tests (POCT) in physicians’ offices. This can be problematic as physicians may be unaware that a particular diabetic patient has a hemoglobinopathy. Hemoglobin electrophoresis is not a recommended laboratory test for a new diabetic, according to the American Diabetes Association (ADA) Standards of Medical Care. 2 Moreover, diabetic individuals may not be aware that they have a hemoglobinopathy or that a known hemoglobin variant can affect their A1c results. Diabetic patients with hemoglobinopathies may receive inaccurate A1c results several times before the error and cause is discovered. This is a serious quality issue for all laboratory professionals. This report will discuss this system error in A1c testing and recommend a method to eliminate it. The recommendation will ask you, the reader, to make it happen. Collective data on laboratory practices provided by you will be used to determine testing and reporting practices and be used

ABO Discrepancy and Hemolytic Anemia Post Liver Transplant due to Passenger Lymphocyte Syndrome

Patient: 54-year-old male Chief Complaint: High fever, diarrhea, and skin rash History of Present Illness: The patient is blood type group AB, Rh(D) positive with nonalcoholic steatohepatitis (NASH). Due to the advanced nature of his disorder, he received a liver transplant; the donor was group B. The patient received 24 units of blood during and after surgery. Fourteen days post transplant he was discharged. Thirty one days post transplant the patient presented with the current symptoms. Hematology workup revealed pancytopenia with a critically low hemoglobin concentration. The blood bank workup revealed an ABO discrepancy and an apparent auto-anti-E antibody. (Table 1) Past Medical History: The patient has a history of diabetes mellitus. The patient had gastric bypass 25 years prior to the transplant, resulting in a weight loss of 115 pounds. One year ago the patient was diagnosed with NASH. 1. What are the patient’s most striking clinical and laboratory findings? 2. What is the nature of the ABO discrepancy? 3. What is the most likely cause of the intravascular hemolysis? 4. Is hemolytic anemia an unexpected complication of transplants? 5. What is the immunological basis for this condition? 6. What is the appropriate blood component therapy for this patient? 1. The most striking clinical findings of the patient were fever, diarrhea, and skin rash. These are indications of graft versus host disease (GVHD), a complication that is rarely seen after orthotopic liver transplantation.1 Acute GVHD occurs between 1 and 8 weeks after the transplant. Symptoms may include fever, diarrhea, and skin rash. Diarrhea is due to lymphocyte infiltration and destruction of the intestinal mucosa, leading to a loss of absorptive function.1 Pancytopenia is associated with GVHD in many cases. The neutropenia and thrombocytopenia may lead to a life-threatening infection.1 A skin biopsy of this patient was consistent with GVHD. The Hemoglobin …

Is the Correlation of HbA1c to Average Glucose Predictable in Individuals With Sickle Cell Trait

Hemoglobin A1c has a predictable correlation to blood glucose, and is used to estimate average glucose for the previous 2-3 months.1 Hemoglobin variants may invalidate HbA1c results in vitro by analytical interference or in vivo by altering RBC lifespan or glycation rate. Several researchers have examined analytical reproducibility in heterozygous Hemoglobin AS (HbAS) samples, i.e. from subjects with sickle cell trait (SCT), and determined which assays provide reproducible results.2-5 No study to date has compared HbA1c to 3 months of glucose measurements in persons with SCT and diabetes. We asked if the correlation between HbA1c and average glucose was altered by the presence of SCT.

Teaching Research Design and Practice One Bite at a Time in the MT/CLS Curriculum

The curriculum for clinical laboratory sciences (CLS) majors is tightly packed with basic and applied science, math, education, and management courses. In the past few years, clinical laboratory science program directors have been charged with adding a component on research to comply with the new #9B8 of the standards that programs must meet to pass accreditation by the National Accrediting Agency for Clinical Laboratory Sciences (now CLSI). The standard reads: “the CLS/MT will possess knowledge, skills and relevant experiences in research design/practice sufficient to evaluate published studies as an informed consumer.” 1 Graduate and professional schools spend years teaching their students how to evaluate research. How can CLS students pick it up as an add-on? This is akin to the riddle: “how do you eat an elephant? One bite at a time.” In the first bite, establish what you want the student be able to do with the new skills: the expected student learning outcomes (SLOs). For the second bite, design a series of research experiences that can be integrated into the established curriculum. In the third bite, measure student success at meeting the SLOs from both the instructor’s and student’s perspectives. For the fourth bite, assess the success of the project in its entirety and make plans to improve the process. These steps should sound familiar. They are plan, implement, assess, and improve. In a clinical laboratory, the implementation of a plan like this happens over a long period of time. In the CLS curriculum, it should also happen over time. What follows is a case study on how NAACLS standard #9B8 Research Design has been integrated into the clinical laboratory science curriculum at the University of West Florida in an active learning model embedded in core CLS courses, with an increasing level of student engagement and responsibility. The comprehensive plan also satisfies a component of the quality enhancement plan (QEP) of the university itself, which calls for a written plan for improvement in student skills of content, communication, project management, and critical thinking.

Normoglycemia May Encompass Two Subpopulations With Respect to Vascular Risk in Non-Obese Caucasian Women

Metabolic syndrome, or insulin resistance syndrome, refers tothe presence of a cluster of characteristics, and is defined by thepresence of 3 or more of the following: FPG >110 mg/dL, ab-dominal obesity, triglycerides >150 mg/dL, or HDL <40 mg/dLfor a man or <50 mg/dL for a woman, and hypertension.