BMI1 regulates human erythroid self-renewal through both gene repression and gene activation.

The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient numbers of in vitro-derived red blood cells (RBC) for clinical purposes. We and others have determined that BMI1, a member of the polycomb repressive complex 1 (PRC1), is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts (SREs). However, the mechanisms of BMI1 action remain poorly understood. BMI1 overexpression led to 10 billion-fold increase BMI1-induced (i)SRE self-renewal. Despite prolonged culture and BMI1 overexpression, human iSREs can terminally mature and agglutinate with typing reagent monoclonal antibodies against conventional RBC antigens. BMI1 and RING1B occupancy, along with repressive histone marks, were identified at known BMI1 target genes, including the INK-ARF locus, consistent with an altered cell cycle following BMI1 inhibition. We also identified upregulated BMI1 target genes with low repressive histone modifications, including key regulator of cholesterol homeostasis. Functional studies suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. These findings support the hypothesis that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand the pool of immature erythroid precursors for eventual clinical uses.

Competition for H2A.Z underlies the developmental impacts of repetitive element de-repression.

The histone variant H2A.Z is central to early embryonic development, determining transcriptional competency through chromatin regulation of gene promoters and enhancers. In addition to genic loci, we find that H2A.Z resides at a subset of evolutionarily young repetitive elements, including DNA transposons, long interspersed nuclear elements and long terminal repeats, during early zebrafish development. Moreover, increases in H2A.Z occur when repetitive elements become transcriptionally active. Acquisition of H2A.Z corresponds with a reduction in the levels of the repressive histone modification H3K9me3 and a moderate increase in chromatin accessibility. Notably, however, de-repression of repetitive elements also leads to a significant reduction in H2A.Z over non-repetitive genic loci. Genic loss of H2A.Z is accompanied by transcriptional silencing at adjacent coding sequences, but remarkably, these impacts are mitigated by augmentation of total H2A.Z protein via transgenic overexpression. Our study reveals that levels of H2A.Z protein determine embryonic sensitivity to de-repression of repetitive elements, that repetitive elements can function as a nuclear sink for epigenetic factors and that competition for H2A.Z greatly influences overall transcriptional output during development. These findings uncover general mechanisms in which counteractive biological processes underlie phenotypic outcomes.

HEXIM1 is an essential transcription regulator during human erythropoiesis.

ABSTRACT: Regulation of RNA polymerase II (RNAPII) activity is an essential process that governs gene expression; however, its contribution to the fundamental process of erythropoiesis remains unclear. hexamethylene bis-acetamide inducible 1 (HEXIM1) regulates RNAPII activity by controlling the location and activity of positive transcription factor ß. We identified a key role for HEXIM1 in controlling erythroid gene expression and function, with overexpression of HEXIM1 promoting erythroid proliferation and fetal globin expression. HEXIM1 regulated erythroid proliferation by enforcing RNAPII pausing at cell cycle check point genes and increasing RNAPII occupancy at genes that promote cycle progression. Genome-wide profiling of HEXIM1 revealed that it was increased at both repressed and activated genes. Surprisingly, there were also genome-wide changes in the distribution of GATA-binding factor 1 (GATA1) and RNAPII. The most dramatic changes occurred at the ß-globin loci, where there was loss of RNAPII and GATA1 at ß-globin and gain of these factors at γ-globin. This resulted in increased expression of fetal globin, and BGLT3, a long noncoding RNA in the ß-globin locus that regulates fetal globin expression. GATA1 was a key determinant of the ability of HEXIM1 to repress or activate gene expression. Genes that gained both HEXIM1 and GATA1 had increased RNAPII and increased gene expression, whereas genes that gained HEXIM1 but lost GATA1 had an increase in RNAPII pausing and decreased expression. Together, our findings reveal a central role for universal transcription machinery in regulating key aspects of erythropoiesis, including cell cycle progression and fetal gene expression, which could be exploited for therapeutic benefit.

Dietary Patterns of Children from the Amazon Region of Ecuador: A Descriptive, Qualitative Investigation.

Many young children in Ecuador suffer from high rates of malnutrition and stunting that affect their long-term growth and development. Little is known about the dietary patterns of children from the Amazon region who experience some of the highest rates of stunting (height-for-age) within Ecuador. Semi-structured interviews were conducted with 50 mothers of young children living in the Ecuadorian Amazon. In addition to descriptions of overall dietary patterns, three themes emerged from the interviews relating to strengths mothers have in feeding their children healthy diets: knowledge, autonomous and independent children, and supportive and responsive parenting. Five themes were found relating to barriers mothers have in feeding their children healthy diets. The first four themes concerned barriers (lack of knowledge of healthy foods, lack of access to healthy foods, not enough money, and child's health) related to multidimensional poverty. All these influenced the last theme found, namely, how difficult of an eater the child was. The implications of intervention efforts to reduce undernutrition and promote children's development by building on specific family and community strengths and identified barriers are also discussed in this paper.

A novel internal training program using Kern's 6-step approach to curriculum development for medical laboratory scientists training to be international quality assurance/quality control coordinators.

OBJECTIVE: Patient Safety Monitoring in International Laboratories (pSMILE) is a resource ensuring quality testing in clinical laboratories performing National Institutes of Health-funded HIV research requiring specific staff training. We demonstrate the development of an online asynchronous training model using Kern's 6-step approach to support pSMILE functions. METHODS: An existing curriculum was revamped to incorporate Kern's approach. Metrics for success were described in rubrics with feedback guiding improvements and updates. RESULTS: Curriculum updates took more than a year. Direct observations of skills informed curriculum changes. Module self-evaluations were reviewed to assess performance and the overall curriculum. The content, curriculum, and training documentation were deemed compliant with International Organization for Standardization (ISO) 9001:2015. CONCLUSION: Asynchronous training for highly skilled and self-directed staff is a novel way to deploy training while maintaining productivity of existing staff. Feedback and evaluation allowed for curriculum updates including previously underdeveloped topics. Kern's approach ensured that the needs of the sponsor, management, laboratories, and learners were met.

TXNIP loss expands Myc-dependent transcriptional programs by increasing Myc genomic binding.

The c-Myc protooncogene places a demand on glucose uptake to drive glucose-dependent biosynthetic pathways. To meet this demand, c-Myc protein (Myc henceforth) drives the expression of glucose transporters, glycolytic enzymes, and represses the expression of thioredoxin interacting protein (TXNIP), which is a potent negative regulator of glucose uptake. A Mychigh/TXNIPlow gene signature is clinically significant as it correlates with poor clinical prognosis in triple-negative breast cancer (TNBC) but not in other subtypes of breast cancer, suggesting a functional relationship between Myc and TXNIP. To better understand how TXNIP contributes to the aggressive behavior of TNBC, we generated TXNIP null MDA-MB-231 (231:TKO) cells for our study. We show that TXNIP loss drives a transcriptional program that resembles those driven by Myc and increases global Myc genome occupancy. TXNIP loss allows Myc to invade the promoters and enhancers of target genes that are potentially relevant to cell transformation. Together, these findings suggest that TXNIP is a broad repressor of Myc genomic binding. The increase in Myc genomic binding in the 231:TKO cells expands the Myc-dependent transcriptome we identified in parental MDA-MB-231 cells. This expansion of Myc-dependent transcription following TXNIP loss occurs without an apparent increase in Myc's intrinsic capacity to activate transcription and without increasing Myc levels. Together, our findings suggest that TXNIP loss mimics Myc overexpression, connecting Myc genomic binding and transcriptional programs to the nutrient and progrowth signals that control TXNIP expression.

Review of Medication Error Sources Associated With Inpatient Subcutaneous Insulin: Recommendations for Safe and Cost-Effective Dispensing Practices.

Background: The challenge of addressing variation in practice is underlined by variation in institutional operational and financial limitations, which ultimately directs institutional insulin dispensing strategy. Insulin therapy is multi-modal, and inpatient pharmacies may be responsible for simultaneously managing up to 8 formulations. While available as a tool for glycemic management in the inpatient setting, intravenous (IV) insulin and insulin pumps are out of scope for this review. Considerations when designing subcutaneous insulin dispensing processes include medication safety, infection control, pharmacy workflow, nursing workflow, drug cost, hardware cost, and hospital policy. Objective: This review provides an overview of common inpatient subcutaneous insulin dispensing practices, describes the regulations directing current practice, and discusses the benefits and risks of each dispensing practice. Finally, the review provides recommendations for subcutaneous insulin dispensing practices with respect to institutional limitations that minimize patient safety risk and consider the variable costs that practices incur. Methods: Common formulations will be presented along with medication safety considerations and potential medication administration errors to preface strategies for management. Conclusion: Nearing the centennial anniversary of the invention of insulin, the practice of dispensing subcutaneous insulin products has remained a core responsibility of the hospital pharmacy as a primary treatment option for Diabetes Mellitus. Despite the continued presence of this product as a universal component of the hospital formulary, subcutaneous insulin dispensing practices remain varied institution-to-institution.

Subtype-Independent ANP32E Reduction During Breast Cancer Progression in Accordance with Chromatin Relaxation.

BACKGROUND: Chromatin state provides a clear decipherable blueprint for maintenance of transcriptional patterns, exemplifying a mitotically stable form of cellular programming in dividing cells. In this regard, genomic studies of chromatin states within cancerous tissues have the potential to uncover novel aspects of tumor biology and unique mechanisms associated with disease phenotypes and outcomes. The degree to which chromatin state differences occur in accordance with breast cancer features has not been established. METHODS: We applied a series of unsupervised computational methods to identify chromatin and molecular differences associated with discrete physiologies across human breast cancer tumors. RESULTS: Chromatin patterns alone are capable of stratifying tumors in association with cancer subtype and disease progression. Major differences occur at DNA motifs for the transcription factor FOXA1, in hormone receptor-positive tumors, and motifs for SOX9 in Basal-like tumors. We find that one potential driver of this effect, the histone chaperone ANP32E, is inversely correlated with tumor progression and relaxation of chromatin at FOXA1 binding sites. Tumors with high levels of ANP32E exhibit an immune response and proliferative gene expression signature, whereas tumors with low ANP32E levels appear programmed for differentiation. CONCLUSIONS: Our results indicate that ANP32E may function through chromatin state regulation to control breast cancer differentiation and tumor plasticity. This study sets a precedent for future computational studies of chromatin changes in carcinogenesis.

Evaluating kidney function using a point-of-care creatinine test in Ugandan children with severe malaria: a prospective cohort study.

BACKGROUND: Acute kidney injury (AKI) disproportionately affects individuals in low-and middle-income countries (LMIC). However, LMIC-particularly countries in sub-Saharan Africa- are under-represented in global AKI research. A critical barrier in diagnosing AKI is access to reliable serum creatinine results. We evaluated the utility of a point-of-care test to measure creatinine and diagnose AKI in Ugandan children with malaria. METHODS: Paired admission creatinine was assessed in 539 Ugandan children 6 months to 4 years of age hospitalized with severe malaria based on blood smear or rapid diagnostic test. Creatinine levels were measured using isotope dilution mass spectrometry (IDMS)-traceable methods. The reference creatinine was measured using the modified Jaffe method by a certified laboratory and the point-of-care testing was conducted using an i-STAT blood analyzer (i-STAT1, with and without adjustment for the partial pressure of carbon dioxide). AKI was defined and staged using the Kidney Disease: Improving Global Outcomes criteria. RESULTS: The mean age of children was 2.1 years, and 21.6% of children were stunted. Mortality was 7.6% in-hospital. Over the entire range of measured creatinine values (<0.20mg/dL-8.4mg/dL), the correlation between the reference creatinine and adjusted and unadjusted point-of-care creatinine was high with R2 values of 0.95 and 0.93 respectively; however, the correlation was significantly lower in children with creatinine values <1mg/dL (R2 of 0.44 between the reference and adjusted and unadjusted i-STAT creatinine). The prevalence of AKI was 45.5% using the reference creatinine, and 27.1 and 32.3% using the unadjusted and adjusted point-of-care creatinine values, respectively. There was a step-wise increase in mortality across AKI stages, and all methods were strongly associated with mortality (p<0.0001 for all). AKI defined using the reference creatinine measure was the most sensitive to predict mortality with a sensitivity of 85.4% compared to 70.7 and 63.4% with the adjusted and unadjusted point-of-care creatinine values, respectively. CONCLUSIONS: Point-of-care assessment of creatinine in lean Ugandan children <4 years of age underestimated creatinine and AKI compared to the clinical reference. Additional studies are needed to evaluate other biomarkers of AKI in LMIC to ensure equitable access to AKI diagnostics globally.

Malaria-Associated Acute Kidney Injury in African Children: Prevalence, Pathophysiology, Impact, and Management Challenges.

Acute kidney injury (AKI) is emerging as a complication of increasing clinical importance associated with substantial morbidity and mortality in African children with severe malaria. Using the Kidney Disease: Improving Global Outcomes (KDIGO) criteria to define AKI, an estimated 24-59% of African children with severe malaria have AKI with most AKI community-acquired. AKI is a risk factor for mortality in pediatric severe malaria with a stepwise increase in mortality across AKI stages. AKI is also a risk factor for post-discharge mortality and is associated with increased long-term risk of neurocognitive impairment and behavioral problems in survivors. Following injury, the kidney undergoes a process of recovery and repair. AKI is an established risk factor for chronic kidney disease and hypertension in survivors and is associated with an increased risk of chronic kidney disease in severe malaria survivors. The magnitude of the risk and contribution of malaria-associated AKI to chronic kidney disease in malaria-endemic areas remains undetermined. Pathways associated with AKI pathogenesis in the context of pediatric severe malaria are not well understood, but there is emerging evidence that immune activation, endothelial dysfunction, and hemolysis-mediated oxidative stress all directly contribute to kidney injury. In this review, we outline the KDIGO bundle of care and highlight how this could be applied in the context of severe malaria to improve kidney perfusion, reduce AKI progression, and improve survival. With increased recognition that AKI in severe malaria is associated with substantial post-discharge morbidity and long-term risk of chronic kidney disease, there is a need to increase AKI recognition through enhanced access to creatinine-based and next-generation biomarker diagnostics. Long-term studies to assess severe malaria-associated AKI's impact on long-term health in malaria-endemic areas are urgently needed.

Regulation of RNA polymerase II activity is essential for terminal erythroid maturation.

The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone posttranslational modifications combined with chromatin immunoprecipitation coupled with high-throughput sequencing, Assay for Transposase Accessible Chromatin, and RNA sequencing. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA polymerase II "pausing" is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late-stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation and provide a novel paradigm for understanding normal and perturbed erythropoiesis.

Implementation of a pharmacy-focused morbidity, mortality, and improvement conference.

PURPOSE: Review lessons learned during the development and implementation of a pharmacy-focused Morbidity, Mortality, and Improvement conference at an academic medical center. SUMMARY: Since the early 1900s, Morbidity and Mortality conferences have provided a forum for clinicians to discuss medical errors and adverse outcomes. Many institutions have now added "improvement" to the conference title to emphasize the goal of approaching these conferences in a systems-oriented manner. To date, a gap remains in the literature evaluating the impact of a pharmacy-focused Morbidity, Mortality, and Improvement (MM&I) conference. The primary goal in establishing this pharmacy-focused conference was to foster and strengthen the culture of medication safety within our department. In establishing our program, we identified an opportunity to leverage pharmacy residents similar to a medical resident-facilitated conference. After gaining leadership buy-in, a core planning team was formed to identify events and create conference materials. Primary metrics to gauge the success of implementation included event reporting trends and medication-safety strategic initiative tracking. The first year of MM&I conferences provided forward momentum for our department's safety culture. Safety event reporting by pharmacy staff increased by 150% over the fiscal year, and more frontline staff expressed a personal interest in becoming involved in safety projects and initiatives outside of their normal shift responsibilities. CONCLUSION: We have learned several important lessons that may be helpful to others, the primary of which is that improving a culture of safety takes time.

Genome-wide chromatin accessibility is restricted by ANP32E.

Genome-wide chromatin state underlies gene expression potential and cellular function. Epigenetic features and nucleosome positioning contribute to the accessibility of DNA, but widespread regulators of chromatin state are largely unknown. Our study investigates how coordination of ANP32E and H2A.Z contributes to genome-wide chromatin state in mouse fibroblasts. We define H2A.Z as a universal chromatin accessibility factor, and demonstrate that ANP32E antagonizes H2A.Z accumulation to restrict chromatin accessibility genome-wide. In the absence of ANP32E, H2A.Z accumulates at promoters in a hierarchical manner. H2A.Z initially localizes downstream of the transcription start site, and if H2A.Z is already present downstream, additional H2A.Z accumulates upstream. This hierarchical H2A.Z accumulation coincides with improved nucleosome positioning, heightened transcription factor binding, and increased expression of neighboring genes. Thus, ANP32E dramatically influences genome-wide chromatin accessibility through subtle refinement of H2A.Z patterns, providing a means to reprogram chromatin state and to hone gene expression levels.

Malaria parasitemia among blood donors in Uganda.

BACKGROUND: Malaria remains a leading transfusion associated infectious risk in endemic areas. However, the prevalence of malaria parasitemia has not been well characterized in blood donor populations. This study sought to determine the prevalence of Plasmodium in red blood cell (RBC) and whole blood (WB) units after the rainy season in Uganda. METHODS AND MATERIALS: Between May and July 2018, blood was collected from the sample diversion pouch of 1000 WB donors in Kampala and Jinja, Uganda. The RBC pellet from ethylenediamine tetraacetic acid (EDTA) anticoagulated blood was stored at -80°C until testing. DNA was extracted and nested PCR was used to screen samples at the genus level for Plasmodium, with positive samples further tested for species identification. RESULTS: Malaria parasitemia among asymptomatic, eligible blood donors in two regions of Uganda was 15.4%; 87.7% (135/154) of infections were with P. falciparum, while P. malariae and P. ovale were also detected. There were 4.3% of blood donors who had mixed infection with multiple species. Older donors (>30 years vs. 17-19 years; aPR = 0.31 [95% CI = 0.17-0.58]), females (aPR = 0.60 [95% CI = 0.42-0.87]), repeat donors (aPR = 0.44 [95% CI = 0.27-0.72]) and those donating near the capital city of Kampala versus rural Jinja region (aPR = 0.49 [95% CI = 0.34-0.69]) had a lower prevalence of malaria parasitemia. CONCLUSIONS: A high proportion of asymptomatic blood donors residing in a malaria endemic region demonstrate evidence of parasitemia at time of donation. Further research is needed to quantify the risk and associated burden of transfusion-transmitted malaria (TTM) in order to inform strategies to prevent TTM.

Development of an electronic trigger tool at a children's hospital within an academic medical center.

PURPOSE: To evaluate the validity and reliability of select recommended triggers, defined as flags found on review of the medical record that prompt further investigation to determine the presence or absence of an adverse drug event (ADE), selected from a list initially constructed based on severity, frequency, and detectability of triggers within a pediatric population. METHODS: This was a single-center, retrospective cohort analysis of pediatric patients admitted to University of North Carolina (UNC) Children's Hospital who received trigger-associated medications between January 2015 and December 2016. Patient-care areas of the emergency department, operating rooms, and post-anesthesia care units were excluded. Trigger-detection encounters were evaluated by two reviewers using pre-established, consensus ADE criteria as determined by a panel of pediatric and medication safety specialists at UNC Medical Center. Events were categorized according to medication-related trigger and analyzed using descriptive statistics. RESULTS: A total of 3,836 positive triggers were included in this study. For the aggregate 12-part trigger tool package, 1,055 positive ADEs were identified, leading to a positive predictive value (PPV) of 27.5%. A 50% increase from baseline serum creatinine, resulting from co-administration of 2 or more nephrotoxic medications accounted for a total of 3,698/3,836 (96.4%). Incomplete documentation was the leading cause for event exclusion, 8/27 (30%). The triggers with the highest PPV included protamine 4/4 (100%), flumazenil 1/1 (100%), and vancomycin-related events 51/67 (76.1%), respectively. Phenytoin level >30 µg/mL or free level >2.5 µg/mL resulted in the lowest PPV, 1/12 (8.3%). CONCLUSION: This study lays the foundation for further studies to develop a robust pediatric trigger tool that may involve developing multi-element triggers, determining sensitivity and specificity of triggers, or mobilizing the trigger tool to an automated system. Trigger tools can be individualized to meet each institutions' needs and unique patient population.

The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28.

KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity.

Microfluidics: The future of microdissection TESE?

Non-obstructive azoospermia (NOA) is a severe form of infertility accounting for 10% of infertile men. Microdissection testicular sperm extraction (microTESE) includes a set of clinical protocols from which viable sperm are collected from patients (suffering from NOA), for intracytoplasmic sperm injection (ICSI). Clinical protocols associated with the processing of a microTESE sample are inefficient and significantly reduce the success of obtaining a viable sperm population. In this review we highlight the sources of these inefficiencies and how these sources can possibly be removed by microfluidic technology and single-cell Raman spectroscopy.

Optimization of microelectrophoresis to select highly negatively charged sperm.

PURPOSE: The sperm membrane undergoes extensive surface remodeling as it matures in the epididymis. During this process, the sperm is encapsulated in an extensive glycocalyx layer, which provides the membrane with its characteristic negative electrostatic charge. In this study, we develop a method of microelectrophoresis and standardize the protocol to isolate sperm with high negative membrane charge. METHODS: Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate prior to and following density gradient centrifugation (DGC), and evaluated for sperm DNA damage, and histone retention. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an ICSI needle and directly analyzed for DNA damage. RESULTS: When raw semen was analyzed using microelectrophoresis, 94 % were NCS. In contrast, DGC completely or partially stripped the negative membrane charge from sperm resulting PCS and neutrally charged sperm, while the charged sperm populations are increased with an increase in electrophoretic current. Following DGC, high sperm DNA damage and abnormal histone retention were inversely correlated with percentage NCS and directly correlated with percentage PCS. NCS exhibited significantly lower DNA damage when compared with control (P < 0.05) and PCS (P < 0.05). When the charged sperm population was corrected for neutrally charged sperm, sperm DNA damage was strongly associated with NCS at a lower electrophoretic current. CONCLUSION: The results suggest that selection of NCS at lower current may be an important biomarker to select healthy sperm for assisted reproductive treatment.

Micro-electrophoresis: a noninvasive method of sperm selection based on membrane charge.

OBJECTIVE: To develop a technique with the potential of isolating genetically fit sperm for assisted reproductive technology (ART) treatment without compromising its structural or functional competence. DESIGN: Observational study. SETTING: University hospital. PATIENT(S): Fifty patients undergoing infertility diagnosis and 88 couples undergoing ART treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate before and after density gradient centrifugation (DGC), and evaluated for sperm DNA damage, histone retention, and couples' ART outcomes. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an intracytoplasmic sperm injection needle and directly analyzed for DNA damage. RESULT(S): There was a reduction in the NCS population (95.10% ± 0.94% vs. 54.48% ± 2.39%) and an increase in the PCS population (4.28% ± 0.58% vs. 42.52% ± 2.36%) after DGC. The DNA damage was inversely proportional to %NCS (r(2) = -0.242) and directly proportional to the %PCS (r(2) = 0.206). When sperm were picked according to their charge and directly analyzed, sperm DNA damage was lower in the NCS population (3.9% ± 1.5%) compared with control (17.3% ± 3.2%) and %PCS populations (27.8% ± 6.0%). The %NCS was positively associated with fertilization rate (r(2) = 0.469) and blastocyst development (r(2) = 0.308) and inversely associated with embryo arrest (r(2) = -0.253). Implantation rate and clinical pregnancies were higher in patient groups with increased NCS. CONCLUSION(S): Selection of NCS through micro-electrophoresis has the potential to isolate sperm relatively free of DNA damage to be used in ART.

Training the public health workforce from Albany to Zambia: technology lessons learned along the way.

This article describes lessons learned by the University at Albany Center for Public Health Preparedness (UA-CPHP) in using three technologies to deliver preparedness training for public health professionals in New York State. These three technologies are: 1. Audience response system (ARS, or the "clicker" system)--Purchased to improve engagement of all participants in heterogeneous training audiences, it also markedly reduces staff time while improving training evaluation (cost: $4,500). 2. Satellite broadcast programs--UA-CPHP produced more than 50 broadcasts, which remain available as videostreams and/or podcasts. Viewership of archived programs sometimes surpasses that of the live event (cost estimate: $23,000 to $39,000). 3. Interactive online courses--Seventeen courses have registered more than 44,000 learners worldwide. The Pandemic Influenza course alone has reached more than 16,000 registrants from all 50 U.S. states and at least 56 other countries (cost estimate: $30,000 to $65,000). UA-CPHP's experience as a preparedness training center has confirmed that contemporary technology can be employed to improve and increase the reach of these training efforts. An additional finding was that, quite unintentionally, the intensive use of distance-based educational modalities designed to reach public health practitioners in New York State has afforded UA-CPHP a substantial national and international audience as well, and at no additional cost.