Optimized quantification of intra-host viral diversity in SARS-CoV-2 and influenza virus sequence data.

High error rates of viral RNA-dependent RNA polymerases lead to diverse intra-host viral populations during infection. Errors made during replication that are not strongly deleterious to the virus can lead to the generation of minority variants. However, accurate detection of minority variants in viral sequence data is complicated by errors introduced during sample preparation and data analysis. We used synthetic RNA controls and simulated data to test seven variant-calling tools across a range of allele frequencies and simulated coverages. We show that choice of variant caller and use of replicate sequencing have the most significant impact on single-nucleotide variant (SNV) discovery and demonstrate how both allele frequency and coverage thresholds impact both false discovery and false-negative rates. When replicates are not available, using a combination of multiple callers with more stringent cutoffs is recommended. We use these parameters to find minority variants in sequencing data from SARS-CoV-2 clinical specimens and provide guidance for studies of intra-host viral diversity using either single replicate data or data from technical replicates. Our study provides a framework for rigorous assessment of technical factors that impact SNV identification in viral samples and establishes heuristics that will inform and improve future studies of intra-host variation, viral diversity, and viral evolution. IMPORTANCE When viruses replicate inside a host cell, the virus replication machinery makes mistakes. Over time, these mistakes create mutations that result in a diverse population of viruses inside the host. Mutations that are neither lethal to the virus nor strongly beneficial can lead to minority variants that are minor members of the virus population. However, preparing samples for sequencing can also introduce errors that resemble minority variants, resulting in the inclusion of false-positive data if not filtered correctly. In this study, we aimed to determine the best methods for identification and quantification of these minority variants by testing the performance of seven commonly used variant-calling tools. We used simulated and synthetic data to test their performance against a true set of variants and then used these studies to inform variant identification in data from SARS-CoV-2 clinical specimens. Together, analyses of our data provide extensive guidance for future studies of viral diversity and evolution.

T Cell-Intrinsic Interferon Regulatory Factor 1 Expression Suppresses Differentiation of CD4+ T Cell Populations That Support Chronic Gammaherpesvirus Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infection and are associated with B cell lymphomas. To establish chronic infection, these viruses usurp B cell differentiation and drive a robust germinal center response to expand the latent viral reservoir and gain access to memory B cells. Germinal center B cells, while important for the establishment of latent infection, are also thought to be the target of viral transformation. The host and viral factors that impact the gammaherpesvirus-driven germinal center response are not clearly defined. We show that the global expression of the antiviral and tumor suppressor interferon regulatory factor 1 (IRF-1) selectively attenuates the murine gammaherpesvirus 68 (MHV68)-driven germinal center response and restricts the expansion of the latent viral reservoir. In this study, we found that T cell-intrinsic IRF-1 expression recapitulates some aspects of the antiviral state imposed by IRF-1 during chronic MHV68 infection, including the attenuation of the germinal center response and viral latency in the spleen. We also discovered that global and T cell-intrinsic IRF-1 deficiency leads to an unhindered rise of interleukin-17A (IL-17A)-expressing and follicular helper T cell populations, two CD4+ T cell subsets that support chronic MHV68 infection. Thus, this study unveils a novel aspect of the antiviral activity of IRF-1 by demonstrating IRF-1-mediated suppression of specific CD4+ T cell subsets that support chronic gammaherpesvirus infection. IMPORTANCE Gammaherpesviruses infect over 95% of the adult population, last the lifetime of the host, and are associated with multiple cancers. These viruses usurp the germinal center response to establish lifelong infection in memory B cells. This manipulation of B cell differentiation by the virus is thought to contribute to lymphomagenesis, although exactly how the virus precipitates malignant transformation in vivo is unclear. IRF-1, a host transcription factor and a known tumor suppressor, restricts the MHV68-driven germinal center response in a B cell-extrinsic manner. We found that T cell-intrinsic IRF-1 expression attenuates the MHV68-driven germinal center response by restricting the CD4+ T follicular helper population. Furthermore, our study identified IRF-1 as a novel negative regulator of IL-17-driven immune responses, highlighting the multifaceted role of IRF-1 in gammaherpesvirus infection.

Gammaherpesvirus Usurps Host IL-17 Signaling To Support the Establishment of Chronic Infection.

Gammaherpesviruses establish lifelong infection and are associated with a variety of cancers, including B cell lymphomas. These viruses manipulate the B cell differentiation process to establish lifelong infection in memory B cells. Specifically, gammaherpesviruses infect naive B cells and promote entry of both infected and uninfected naive B cells into germinal centers, where the virus usurps rapid proliferation of germinal center B cells to exponentially increase its cellular latent reservoir. In addition to facilitating the establishment of latent infection, germinal center B cells are thought to be the target of viral transformation. In this study, we have uncovered a novel proviral role of host interleukin 17A (IL-17A), a well-established antibacterial and antifungal factor. Loss of IL-17A signaling attenuated the establishment of chronic gammaherpesvirus infection and gammaherpesvirus-driven germinal center response in a route of inoculation-dependent manner. Further, IL-17A treatment directly supported gammaherpesvirus reactivation and de novo lytic infection. This study is the first demonstration of a multifaceted proviral role of IL-17 signaling.IMPORTANCE Gammaherpesviruses establish lifelong infections in a majority of humans and are associated with B cell lymphomas. IL-17A is a host cytokine that plays a well-established role in the clearance of bacterial and fungal infections; however, the role of IL-17A in viral infections is poorly understood. In this study, we show that IL-17A signaling promoted the establishment of chronic gammaherpesvirus infection following the mucosal route of infection, viral lytic replication, and reactivation from latency. Thus, our study unveils a novel proviral role of IL-17A signaling in gammaherpesvirus infection.

Interferon Regulatory Factor 3 Supports the Establishment of Chronic Gammaherpesvirus Infection in a Route- and Dose-Dependent Manner.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections and are associated with several malignancies, including B cell lymphomas. Uniquely, these viruses manipulate B cell differentiation to establish long-term latency in memory B cells. This study focuses on the interaction between gammaherpesviruses and interferon regulatory factor 3 (IRF-3), a ubiquitously expressed transcription factor with multiple direct target genes, including beta interferon (IFN-ß), a type I IFN. IRF-3 attenuates acute replication of a plethora of viruses, including gammaherpesvirus. Furthermore, IRF-3-driven IFN-ß expression is antagonized by the conserved gammaherpesvirus protein kinase during lytic virus replication in vitro In this study, we have uncovered an unexpected proviral role of IRF-3 during chronic gammaherpesvirus infection. In contrast to the antiviral activity of IRF-3 during acute infection, IRF-3 facilitated establishment of latent gammaherpesvirus infection in B cells, particularly, germinal center and activated B cells, the cell types critical for both natural infection and viral lymphomagenesis. This proviral role of IRF-3 was further modified by the route of infection and viral dose. Furthermore, using a combination of viral and host genetics, we show that IRF-3 deficiency does not rescue attenuated chronic infection of a protein kinase null gammaherpesvirus mutant, highlighting the multifunctional nature of the conserved gammaherpesvirus protein kinases in vivo In summary, this study unveils an unexpected proviral nature of the classical innate immune factor, IRF-3, during chronic virus infection.IMPORTANCE Interferon regulatory factor 3 (IRF-3) is a critical component of the innate immune response, in part due to its transactivation of beta interferon (IFN-ß) expression. Similar to that observed in all acute virus infections examined to date, IRF-3 suppresses lytic viral replication during acute gammaherpesvirus infection. Because gammaherpesviruses establish lifelong infection, this study aimed to define the antiviral activity of IRF-3 during chronic infection. Surprisingly, we found that, in contrast to acute infection, IRF-3 supported the establishment of gammaherpesvirus latency in splenic B cells, revealing an unexpected proviral nature of this classical innate immune host factor.

Interferon Regulatory Factor 7 Attenuates Chronic Gammaherpesvirus Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections and are associated with a variety of malignancies, including lymphomas. Interferon regulatory factor 7 (IRF-7) is an innate immune transcription factor that restricts acute replication of diverse viruses, including murine gammaherpesvirus 68 (MHV68). Importantly, very little is known about the role of IRF-7 during chronic virus infections. In this study, we demonstrate that IRF-7 attenuates chronic infection by restricting establishment of gammaherpesvirus latency in the peritoneal cavity and, to a lesser extent, viral reactivation in the spleen. Despite the classical role of IRF-7 as a stimulator of type I interferon (IFN) transcription, there were no global effects on the expression of IFN-induced genes (ISGs) in the absence of IRF-7, with only a few ISGs showing attenuated expression in IRF-7-deficient peritoneal cells. Further, IRF-7 expression was dispensable for the induction of a virus-specific CD8 T cell response. In contrast, IRF-7 expression restricted latent gammaherpesvirus infection in the peritoneal cavity under conditions where the viral latent reservoir is predominantly hosted by peritoneal B cells. This report is the first demonstration of the antiviral role of IRF-7 during the chronic stage of gammaherpesvirus infection.IMPORTANCE The innate immune system of the host is critical for the restriction of acute viral infections. In contrast, the role of the innate immune network during chronic herpesvirus infection remains poorly defined. Interferon regulatory factor 7 (IRF-7) is a transcription factor with many target genes, including type I interferons (IFNs). In this study, we show that the antiviral role of IRF-7 continues into the chronic phase of gammaherpesvirus infection, wherein IRF-7 restricts the establishment of viral latency and viral reactivation. This study is, to our knowledge, the first to define the role of IRF-7 in chronic virus infection.

B Cell-Intrinsic Expression of Interferon Regulatory Factor 1 Supports Chronic Murine Gammaherpesvirus 68 Infection.

Gammaherpesviruses are ubiquitous pathogens that are associated with cancers, including B cell lymphomas. These viruses are unique in that they infect naive B cells and subsequently drive a robust polyclonal germinal center response in order to amplify the latent reservoir and to establish lifelong infection in memory B cells. The gammaherpesvirus-driven germinal center response in combination with robust infection of germinal center B cells is thought to precipitate lymphomagenesis. Importantly, host and viral factors that selectively affect the gammaherpesvirus-driven germinal center response remain poorly understood. Global deficiency of antiviral tumor-suppressive interferon regulatory factor 1 (IRF-1) selectively promotes the murine gammaherpesvirus 68 (MHV68)-driven germinal center response and expansion of the viral latent reservoir. To determine the extent to which antiviral effects of IRF-1 are B cell intrinsic, we generated mice with conditional IRF-1 deficiency. Surprisingly, B cell-specific IRF-1 deficiency attenuated the establishment of chronic infection and the germinal center response, indicating that MHV68 may, in a B cell-intrinsic manner, usurp IRF-1 to promote the germinal center response and expansion of the latent reservoir. Further, we found that B cell-specific IRF-1 deficiency led to reduced levels of active tyrosine phosphatase SHP1, which plays a B cell-intrinsic proviral function during MHV68 infection. Finally, results of this study indicate that the antiviral functions of IRF-1 unveiled in MHV68-infected mice with global IRF-1 deficiency are mediated via IRF-1 expression by non-B cell populations.IMPORTANCE Gammaherpesviruses establish lifelong infection in over 95% of all adults and are associated with B cell lymphomas. The virus's manipulation of the germinal center response and B cell differentiation to establish lifelong infection is thought to also precipitate malignant transformation, through a mechanism that remains poorly understood. The host transcription factor IRF-1, a well-established tumor suppressor, selectively attenuates MHV68-driven germinal center response, a phenotype that we originally hypothesized to occur in a B cell-intrinsic manner. In contrast, in testing, B cell-intrinsic IRF-1 expression promoted the MHV68-driven germinal center response and the establishment of chronic infection. Our report highlights the underappreciated multifaceted role of IRF-1 in MHV68 infection and pathogenesis.

B Cell-Intrinsic SHP1 Expression Promotes the Gammaherpesvirus-Driven Germinal Center Response and the Establishment of Chronic Infection.

Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections in the majority of adults worldwide. Chronic gammaherpesvirus infection has been implicated in both lymphomagenesis and, somewhat controversially, autoimmune disease development. Pathogenesis is largely associated with the unique ability of gammaherpesviruses to usurp B cell differentiation, specifically, the germinal center response, to establish long-term latency in memory B cells. The host tyrosine phosphatase SHP1 is known as a brake on immune cell activation and is downregulated in several gammaherpesvirus-driven malignancies. However, here we demonstrate that B cell- but not T cell-intrinsic SHP1 expression supports the gammaherpesvirus-driven germinal center response and the establishment of viral latency. Furthermore, B cell-intrinsic SHP1 deficiency cooperated with gammaherpesvirus infection to increase the levels of double-stranded DNA-reactive antibodies at the peak of viral latency. Thus, in spite of decreased SHP1 levels in gammaherpesvirus-driven B cell lymphomas, B cell-intrinsic SHP1 expression plays a proviral role during the establishment of chronic infection, suggesting that the gammaherpesvirus-SHP1 interaction is more nuanced and is modified by the stage of infection and pathogenesis.IMPORTANCE Gammaherpesviruses establish lifelong infection in a majority of adults worldwide and are associated with a number of malignancies, including B cell lymphomas. These viruses infect naive B cells and manipulate B cell differentiation to achieve a lifelong infection of memory B cells. The germinal center stage of B cell differentiation is important as both an amplifier of the viral latent reservoir and the target of malignant transformation. In this study, we demonstrate that expression of tyrosine phosphatase SHP1, a negative regulator that normally limits the activation and proliferation of hematopoietic cells, enhances the gammaherpesvirus-driven germinal center response and the establishment of chronic infection. The results of this study uncover an intriguing beneficial interaction between gammaherpesviruses that are presumed to profit from B cell activation and a cellular phosphatase that is traditionally perceived to be a negative regulator of the same processes.

Plasma lipidome reveals critical illness and recovery from human Ebola virus disease.

Ebola virus disease (EVD) often leads to severe and fatal outcomes in humans with early supportive care increasing the chances of survival. Profiling the human plasma lipidome provides insight into critical illness as well as diseased states, as lipids have essential roles as membrane structural components, signaling molecules, and energy sources. Here we show that the plasma lipidomes of EVD survivors and fatalities from Sierra Leone, infected during the 2014-2016 Ebola virus outbreak, were profoundly altered. Focusing on how lipids are associated in human plasma, while factoring in the state of critical illness, we found that lipidome changes were related to EVD outcome and could identify states of disease and recovery. Specific changes in the lipidome suggested contributions from extracellular vesicles, viremia, liver dysfunction, apoptosis, autophagy, and general critical illness, and we identified possible targets for therapies enhancing EVD survival.

LXR Alpha Restricts Gammaherpesvirus Reactivation from Latently Infected Peritoneal Cells.

Gammaherpesviruses are ubiquitous viruses that establish lifelong infections. Importantly, these viruses are associated with numerous cancers and lymphoproliferative diseases. While risk factors for developing gammaherpesvirus-driven cancers are poorly understood, it is clear that elevated viral reactivation from latency often precedes oncogenesis. Here, we demonstrate that the liver X receptor alpha isoform (LXRα) restricts gammaherpesvirus reactivation in an anatomic-site-specific manner. We have previously demonstrated that deficiency of both LXR isoforms (α and ß) leads to an increase in fatty acid and cholesterol synthesis in primary macrophage cultures, with a corresponding increase in gammaherpesvirus replication. Interestingly, expression of fatty acid synthesis genes was not derepressed in LXRα-deficient hosts, indicating that the antiviral effects of LXRα are independent of lipogenesis. Additionally, the critical host defenses against gammaherpesvirus reactivation, virus-specific CD8+ T cells and interferon (IFN) signaling, remained intact in the absence of LXRα. Remarkably, using a murine gammaherpesvirus 68 (MHV68) reporter virus, we discovered that LXRα expression dictates the cellular tropism of MHV68 in the peritoneal cavity. Specifically, LXRα-/- mice exhibit reduced latency within the peritoneal B cell compartment and elevated latency within F4/80+ cells. Thus, LXRα restricts gammaherpesvirus reactivation through a novel mechanism that is independent of the known CD8+ T cell-based antiviral responses or changes in lipid synthesis and likely involves changes in the tropism of MHV68 in the peritoneal cavity.IMPORTANCE Liver X receptors (LXRs) are nuclear receptors that mediate cholesterol and fatty acid homeostasis. Importantly, as ligand-activated transcription factors, LXRs represent potential targets for the treatment of hypercholesterolemia and atherosclerosis. Here, we demonstrate that LXRα, one of the two LXR isoforms, restricts reactivation of latent gammaherpesvirus from peritoneal cells. As gammaherpesviruses are ubiquitous oncogenic agents, LXRs may represent a targetable host factor for the treatment of poorly controlled gammaherpesvirus infection and associated lymphomagenesis.

Efficacy of Cell Wall-Deficient Spheroplasts Against Experimental Murine Listeriosis.

Various strategies adapted to develop an efficient vaccine against foodborne pathogen, Listeria monocytogenes, have met with little success. Spheroplasts (bacterial cell devoid of cell wall) are likely to undergo membrane-membrane fusion, leading to the delivery of their content to the cytosol of antigen-presenting cells, thus facilitating MHC class I antigen processing and presentation. In this study, we evaluated the prophylactic potential of Listeria spheroplast-based vaccine against experimental murine listeriosis in comparison with heat-killed Listeria (HKL) and archaeosome-entrapped Listeria whole-cell protein (LWCP). Compared with HKL, the spheroplast-based vaccine was found to evoke better Th1 response as exhibited by the presence of type 1 cytokines in the host (interferon-γ and IL-12) and a high IgG2a /IgG1 ratio. Robust lympho-proliferative efficacy was apparent in both spheroplast-immunized and archaeosome-entrapped LWCP-immunized groups. The upregulation of costimulatory and effector memory markers upon immunization with spheroplasts was found to be at par with that evoked by archaeosome-entrapped LWCP-immunized group. Central memory response in gated CD8(+) T cell was much higher in spheroplast-immunized animals when compared with archaeosome-entrapped LWCP group. The data presented here clearly demonstrate that spheroplasts evoked a robust immune response and offer better prophylactic potential against L. monocytogenes.

What big size you have! Using effect sizes to determine the impact of public health nursing interventions.

BACKGROUND: The Omaha System is a standardized interface terminology that is used extensively by public health nurses in community settings to document interventions and client outcomes. Researchers using Omaha System data to analyze the effectiveness of interventions have typically calculated p-values to determine whether significant client changes occurred between admission and discharge. However, p-values are highly dependent on sample size, making it difficult to distinguish statistically significant changes from clinically meaningful changes. Effect sizes can help identify practical differences but have not yet been applied to Omaha System data. METHODS: We compared p-values and effect sizes (Cohen's d) for mean differences between admission and discharge for 13 client problems documented in the electronic health records of 1,016 young low-income parents. Client problems were documented anywhere from 6 (Health Care Supervision) to 906 (Caretaking/parenting) times. RESULTS: On a scale from 1 to 5, the mean change needed to yield a large effect size (Cohen's d ≥ 0.80) was approximately 0.60 (range = 0.50 - 1.03) regardless of p-value or sample size (i.e., the number of times a client problem was documented in the electronic health record). CONCLUSIONS: Researchers using the Omaha System should report effect sizes to help readers determine which differences are practical and meaningful. Such disclosures will allow for increased recognition of effective interventions.

The effect of acute moderate-intensity exercise on the accuracy of air-displacement plethysmography in young adults.

BACKGROUND/OBJECTIVES: Air-displacement plethysmography (ADP) body composition systems utilize a precise pre-test protocol that must be followed for each trial. Previous research has shown that body temperature changes influence the results when using ADP as a measurement. The objective of this study is to determine the effect of post-exercise body temperature changes on body composition results using ADP. SUBJECTS/METHODS: Forty young adults (18-30 years) participated in the study (23 females, 17 males). Resting heart rate was measured to calculate exercise intensity. First, an ADP test was done according to the instructions outlined by the manufacturer. Upon completion of the ADP test, the subject exercised on a treadmill at moderate intensity (∼65% heart rate reserve) for 30 min to increase body temperature. Another ADP measurement followed the physical activity. Chamber temperature (CT) was measured during each of the trials using a thermistor to assess changes. Fat mass (FM), fat-free mass (FFM), percentage body fat (%BF) and CT were compared pre- and post exercise. RESULTS: Paired sample t-tests revealed significant differences (P<0.05) between pre-exercise %BF and post-exercise %BF (21.3 ± 9.8% vs 19.6 ± 10.2%), pre-exercise CT and post-exercise CT (22.7 ± 1.2 vs 23.0 ± 1.1 °C), and pre-exercise FM and post-exercise FM (14.9 ± 7.9 vs 13.8 ± 8.0 kg). FFM showed no significant difference. CONCLUSION: This investigation demonstrates the importance of following the manufacturer's recommended pre-test protocol as the accuracy of the ADP testing may be compromised, resulting in lower FM and %BF estimations.

PCR offers no advantage over culture for microbiologic diagnosis in cellulitis.

PURPOSE: Most cases of cellulitis are traditionally attributed to ß-hemolytic Streptococcus and Staphylococcus species, although in most cases, no organism is identified. Development of PCR using the conserved bacterial 16 S rRNA DNA permits identification of bacteria independent of conventional culture approaches and prior use of antibiotics. METHODS: We used PCR-based techniques to identify cellulitis etiology using aspirate samples from affected skin. Saline was infiltrated and aspirated at the site of greatest erythema or at the cellulitic border. Samples were tested for 16 S rRNA DNA, and organism-specific probes used to identify bacteria commonly seen in skin infections. RESULTS: Aspirates from 32 patients were studied, and 16 S rRNA DNA was detected in nine of these patient samples (28.1%). Bacterial species were identified by PCR methods in six of these nine samples (66.6%), with S. aureus and methicillin-resistant S. aureus (MRSA) identified in four and two, respectively, of these samples. Of the patients with positive aspirate bacterial cultures (3/9, 33.3%), S. aureus and coagulase-negative Staphylococcus (CoNS) were present on cultures of two of the three (both 66.6%) positive samples. Only in one of the three positive bacterial cultures did the PCR method detect the same organism as was detected by culture. Among patients with positive provider-collected clinical cultures, MRSA was the predominant organism (11/18, 61.1%) and when present, it was found as the sole organism. Where S. aureus or Streptococcus species were detected by molecular methods, clinical cultures yielded a positive result as well. CONCLUSIONS: PCR-based techniques do not appear to be more sensitive than aspirate cultures for the detection of pathogens in cellulitis.

Human hematopoietic progenitor cells grow faster under rotational laminar flows.

We report significant and reproducible growth acceleration of human progenitor cells when exposed to rotational flow when compared with stationary conditions. Nonenriched CD34+ umbilical cord derived human hematopoietic progenitor cells were cultured in Petri dishes located at different radial distances with respect to the central axis of a rotating platform. Growth dynamics under 3 or 5 rpm agitation was compared against that observed under typical stationary conditions. Cells cultured at 3 or 5 rpm exhibited (a) the absence of a latency phase, (b) an increase in final cell concentrations by 54-58.5%, and (c) reduced doubling time in their exponential phase by 12-16% in comparison with stationary culture. Cells grown under rotational agitation were confirmed to remain CD34+ by PCR. These results document a significant positive effect of exposure to laminar flow fields on the growth of human hematopoietic progenitor cells.

Endurance and strength training outcomes on cognitively impaired and cognitively intact older adults: a meta-analysis.

BACKGROUND: Dementia is a common syndrome in the geriatric population. Subsequent impairment of cognitive functioning impacts the patient's mobility, ADLs, and IADLs. It is suggested that older persons with lower levels of cognition are less likely to achieve independence in ADLs and ambulation (1-2). Frequently, nursing home residents are viewed as too frail or cognitively impaired to benefit from exercise rehabilitation. Often, persons with Mini Mental State Score (MMSE) score below 25 are excluded from physical rehabilitation programs. However, Diamond (3) and Goldstein (4) concluded that geriatric patients with mild to moderate cognitive impairment were just as likely as cognitively intact patients to improve in functional abilities as a result of participation in exercise rehabilitation programs. PURPOSE: The objective of this study is to compare, through a meta-analysis endurance and strength outcomes of Cognitively Impaired (MMSE < 23) and Cognitively Intact (MMSE superior 24) older adults who participate in similar exercise programs. METHODS: Published articles were identified by using electronic and manual searches. Key search words included exercise, training, strength, endurance, rehabilitation, cognitive impairment, cognition, MMSE, older adult, aged, and geriatrics. Articles were included if the were from RCTs or well-designed control studies. RESULTS: A total of 41 manuscripts met the inclusion criteria. We examined 21 exercise trials with cognitively impaired individuals (CI=1411) and 20 exercise trials with cognitively intact individuals (IN=1510). Degree of cognitive impairment is based on the reported MMSE score. Moderate to large effect sizes (ES = dwi, Hedges gi) were found for strength and endurance outcomes for the CI groups (dwi = .51, 95% CI= .42- .60), and for the IN groups (dwi = .49, 95% CI= .40- .58). No statistically significant difference in ES was found between the CI and IN studies on strength (t=1.675, DF= 8, P= .132), endurance (t=1.904, DF= 14, P=.078), and combined strength and endurance effects (t=1.434, DF= 56, P= .263). CONCLUSIONS: These results suggest that cognitively impaired older adults who participate in exercise rehabilitation programs have similar strength and endurance training outcomes as age and gender matched cognitively intact older participants and therefore impaired individuals should not be excluded from exercise rehabilitation programs.

The migration of physicians from sub-Saharan Africa to the United States of America: measures of the African brain drain

Background: The objective of this paper is to describe the numbers; characteristics; and trends in the migration to the United States of physicians trained in sub-Saharan Africa. Methods: We used the American Medical Association 2002 Masterfile to identify and describe physicians who received their medical training in sub-Saharan Africa and are currently practicing in the USA. Results :More than 23of America's 771 491 physicians received their medical training outside the USA; the majority (64) in low-income or lower middle-income countries. A total of 5334 physicians from sub-Saharan Africa are in that group; a number that represents more than 6of the physicians practicing in sub-Saharan Africa now. Nearly 86of these Africans practicing in the USA originate from only three countries: Nigeria; South Africa and Ghana. Furthermore; 79were trained at only 10 medical schools. Conclusions: Physician migration from poor countries to rich ones contributes to worldwide health workforce imbalances that may be detrimental to the health systems of source countries. The migration of over 5000 doctors from sub-Saharan Africa to the USA has had a significantly negative effect on the doctor-to-population ratio of Africa. The finding that the bulk of migration occurs from only a few countries and medical schools suggests policy interventions in only a few locations could be effective in stemming the brain drain

The North-West Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort.

AIMS: To determine the incidence of, and clinically relevant risk factors for, new foot ulceration in a large cohort of diabetic patients in the community healthcare setting. METHODS: Diabetic patients (n = 9710) underwent foot screening in six districts of North-west England in various healthcare settings. All were assessed at baseline for demographic information, medical and social history, neuropathy symptom score, neuropathy disability score, cutaneous pressure perception (insensitivity to the 10 g monofilament), foot deformities, and peripheral pulses. Two years later, patients were followed up via postal questionnaire to determine the incidence of new foot ulcers. Cox's proportional hazards regression analysis was used to determine the independent, relative risk of baseline variables for new foot ulceration. RESULTS: New foot ulcers occurred in 291/6613 patients who completed and returned their 2-year follow-up questionnaire (2.2% average annual incidence). The following factors were independently related to new foot ulcer risk: ulcer present at baseline (relative risk (95% confidence interval)) 5.32 (3.71-7.64), past history of ulcer 3.05 (2.16-4.31), abnormal neuropathy disability score (> or = 6/10) 2.32 (1.61-3.35), any previous podiatry attendance 2.19 (1.50-3.20), insensitivity to the 10 g monofilament 1.80 (1.36-2.39), reduced pulses 1.80 (1.40-2.32), foot deformities 1.57 (1.22-2.02), abnormal ankle reflexes 1.55 (1.01-2.36) and age 0.99 (0.98-1.00). CONCLUSIONS: More than 2% of community-based diabetic patients develop new foot ulcers each year. The neuropathy disability score, 10 g monofilament and palpation of foot pulses are recommended as screening tools in general practice.

Comparison of pregnancy outcome in mares among methods used to evaluate and select spermatozoa for insemination.

An artificial insemination dose for mares consisting of 500 million progressively motile spermatozoa is considered "standard" by most clinicians. However, little information is available directly comparing pregnancy outcome among methods of evaluating and selecting spermatozoa for insemination. The objective of this study was to determine if the method of spermatozoal evaluation and selection influences fertility as measured by pregnancy outcome. Mares were inseminated with 100 or 500 million spermatozoa that were selected for progressive motility, normal morphology, hypoosmotic swelling or absolute number regardless for evaluation method or quality. Thirty-two breeding cycles were tested for each treatment group and at each spermatozoal dose. Pregnancy outcomes were 44 and 41%, 55 and 41%, 39 and 31%, and 45 and 41%, for the 100 and 500 million progressively motile, morphologically normal, hypoosmotic swelling positive and absolute number treatment groups, respectively. Pregnancy outcome did not differ among methods of spermatozoal evaluation and selection for artificial insemination in the 100 (P=0.52) or 500 (P=0.78) million spermatozoa groups. Also the total number of spermatozoa and the absolute number of progressively motile, morphologically normal or hypoosmotic swelling positive spermatozoa inseminated, were not closely associated with pregnancy outcome in the 100 (P=0.24, 0.29, 0.33 and 0.38, respectively) or 500 (P=0.20, 0.84, 0.50 and 0.74, respectively) million spermatozoa groups. In this study, we found that the method of spermatozoal evaluation did not offer an advantage for pregnancy when used to select spermatozoa for insemination at the doses tested. These results were surprising, as we expected there would be differences among the evaluation methods. Instead, we found that evaluating spermatozoa offered no advantage for pregnancy over simply inseminating with a specified number of spermatozoa not selected for any particular characteristic under the conditions of our experiment.

Using immunohistochemistry to study plant metabolism: the examples of its use in the localization of amino acids in plant tissues, and of phosphoenolpyruvate carboxykinase and its possible role in pH regulation.

To understand many aspects of the metabolism of complex plant structures such as leaves, fruit and roots it is important to understand how metabolic processes are compartmentalized between tissues. The aim of this article is to show how immunohistochemistry, in conjunction with biochemical and physiological studies, is useful in understanding both the function of an enzyme in a tissue and metabolic processes occurring in plant tissues. This is illustrated by two examples. Firstly, the use of immunohistochemisty in the localization of amino acids in plant tissues is described. Secondly, the use of immunohistochemistry in understanding the function of an enzyme in a tissue and the metabolic processes occurring within the tissue is described. To illustrate this the example of phosophoenolpyruvate carboxykinase (PEPCK), an enzyme which is present in many plant tissues in which its function is unknown, is used. Evidence is provided that PEPCK may play a role in pH regulation in tissues active in the metabolism of nitrogen.