Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry

Metals often determine the chemical reactivity of the proteins to which they are bound. Each cell in the body tightly maintains a unique metalloproteomic profile, mostly dependent on function. This paper describes an analytical online flow injection quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method, which was applied to profiling the metal-binding proteins found in primary cultures of neurons and astrocytes. This method can be conducted using similar amounts of sample to those used for Western blotting (20-150 μg protein), and has a turnaround time of <15 minutes. Metalloprotein standards for Fe (as ferritin), Cu and Zn (as superoxide dismutase-1) were used to construct multi-point calibration curves for online quantification of metalloproteins by SEC-ICP-MS. Homogenates of primary neuron and astrocyte cultures were analysed by SEC-ICP-MS. Online quantification by external calibration with metalloprotein standards determined the mass of metal eluting from the column relative to time (as pg s-1). Total on-column Fe, Cu and Zn detection limits ranged from 0.825 ± 0.005 ng to 13.6 ± 0.7 pg. Neurons and astrocytes exhibited distinct metalloprotein profiles, featuring both ubiquitous and unique metalloprotein species. Separation and detection by SEC-ICP-MS allows appraisal of these metalloproteins in their native state, and online quantification was achieved using this relatively simple external calibration process. © 2013 The Royal Society of Chemistry

Constitutively Enhanced Lymphatic Pumping in the Upper Limbs of Women Who Later Develop Breast Cancer-Related Lymphedema.

BACKGROUND: It has previously been shown that the lymph drainage rate in both upper limbs is greater in women destined to develop breast cancer-related lymphedema (BCRL) than in those who do not develop BCRL, indicating a constitutive predisposition. We explored constitutive differences further by measuring the maximum lymphatic pump pressure (Ppump) and the rate of (99m)Tc-Nanocoll transport generated by the contractile upper limb lymphatics before and after breast cancer surgery in a group of women who were followed for 2 years to determine their eventual BCRL or non-BCRL status. METHODS AND RESULTS: Ppump and tracer transport rate were measured by lymphatic congestion lymphoscintigraphy in the ipsilateral upper limb in 26 women pre- and post-breast cancer surgery. BCRL occurred in 10/26 (38.5%) cases. Ppump in the women who later developed BCRL (40.0 ± 8.2 mmHg) was 1.7-fold higher than in those who did not develop BCRL (23.1 ± 10.8 mmHg, p = 0.001). Moreover, the rate of lymph tracer transport into the forearm was 2.2-fold greater in the women who later developed BCRL (p = 0.052). Surgery did not significantly reduce Ppump measured 21 weeks postsurgery, but impaired forearm tracer transport in pre-BCRL women by 58% (p = 0.047), although not in those who did not develop BCRL. CONCLUSIONS: Women destined to develop BCRL have higher pumping pressures and lymph transport, indicating harder-working lymphatics before cancer treatment. Axillary lymphatic damage from surgery appears to compromise lymph drainage in those women constitutively predisposed to higher lymphatic pressures and lymph transport

Test–retest stability of patient experience items derived from the national GP patient survey

PURPOSE: The validity and reliability of various items on the GP Patient Survey (GPPS) survey have been reported, however stability of patient responses over time has not been tested. The purpose of this study was to determine the test-retest reliability of the core items from the GPPS. METHODS: Patients who had recently consulted participating GPs in five general practices across the South West England were sent a postal questionnaire comprising of 54 items concerning their experience of their consultation and the care they received from the GP practice. Patients returning the questionnaire within 3 weeks of mail-out were sent a second identical (retest) questionnaire. Stability of responses was assessed by raw agreement rates and Cohen's kappa (for categorical response items) and intraclass correlation coefficients and means (for ordinal response items). RESULTS: 348 of 597 Patients returned a retest questionnaire (58.3 % response rate). In comparison to the test phase, patients responding to the retest phase were older and more likely to have white British ethnicity. Raw agreement rates for the 33 categorical items ranged from 66 to 100 % (mean 88 %) while the kappa coefficients ranged from 0.00 to 1.00 (mean 0.53). Intraclass correlation coefficients for the 21 ordinal items averaged 0.67 (range 0.44-0.77). CONCLUSIONS: Formal testing of items from the national GP patient survey examining patient experience in primary care highlighted their acceptable temporal stability several weeks following a GP consultation.Funding was provided by Health Services and Delivery Research Programme (Grant No. RP-PG-0608-10050)

Changing Epidemiology of Serious Bacterial Infections in Febrile Infants without Localizing Signs

Objective: Historically, management of infants with fever without localizing signs (FWLS) has generated much controversy, with attempts to risk stratify based on several criteria. Advances in medical practice may have altered the epidemiology of serious bacterial infections (SBIs) in this population. We conducted this study to test the hypothesis that the rate of SBIs in this patient population has changed over time. Patients and Methods: We performed a retrospective review of all infants meeting FWLS criteria at our institution from 1997–2006. We examined all clinical and outcome data and performed statistical analysis of SBI rates and ampicillin resistance rates. Results: 668 infants met criteria for FWLS. The overall rate of SBIs was 10.8%, with a significant increase from 2002–2006 (52/ 361, 14.4%) compared to 1997–2001 (20/307, 6.5%) (p = 0.001). This increase was driven by an increase in E. coli urinary tract infections (UTI), particularly in older infants (31–90 days). Conclusions: We observed a significant increase in E. coli UTI among FWLS infants with high rates of ampicillin resistance. The reasons are likely to be multifactorial, but the results themselves emphasize the need to examine urine in all febrile infants,90days and consider local resistance patterns when choosing empiric antibiotics

Prime movers : mechanochemistry of mitotic kinesins

Mitotic spindles are self-organizing protein machines that harness teams of multiple force generators to drive chromosome segregation. Kinesins are key members of these force-generating teams. Different kinesins walk directionally along dynamic microtubules, anchor, crosslink, align and sort microtubules into polarized bundles, and influence microtubule dynamics by interacting with microtubule tips. The mechanochemical mechanisms of these kinesins are specialized to enable each type to make a specific contribution to spindle self-organization and chromosome segregation

How robust are the natural history parameters used in chlamydia transmission dynamic models? A systematic review

Transmission dynamic models linked to economic analyses often form part of the decision making process when introducing new chlamydia screening interventions. Outputs from these transmission dynamic models can vary depending on the values of the parameters used to describe the infection. Therefore these values can have an important influence on policy and resource allocation. The risk of progression from infection to pelvic inflammatory disease has been extensively studied but the parameters which govern the transmission dynamics are frequently neglected. We conducted a systematic review of transmission dynamic models linked to economic analyses of chlamydia screening interventions to critically assess the source and variability of the proportion of infections that are asymptomatic, the duration of infection and the transmission probability. We identified nine relevant studies in Pubmed, Embase and the Cochrane database. We found that there is a wide variation in their natural history parameters, including an absolute difference in the proportion of asymptomatic infections of 25% in women and 75% in men, a six-fold difference in the duration of asymptomatic infection and a four-fold difference in the per act transmission probability. We consider that much of this variation can be explained by a lack of consensus in the literature. We found that a significant proportion of parameter values were referenced back to the early chlamydia literature, before the introduction of nucleic acid modes of diagnosis and the widespread testing of asymptomatic individuals. In conclusion, authors should use high quality contemporary evidence to inform their parameter values, clearly document their assumptions and make appropriate use of sensitivity analysis. This will help to make models more transparent and increase their utility to policy makers

Autistic behavior in boys with fragile X syndrome: social approach and HPA-axis dysfunction

The primary goal of this study was to examine environmental and neuroendocrine factors that convey increased risk for elevated autistic behavior in boys with Fragile X syndrome (FXS). This study involves three related analyses: (1) examination of multiple dimensions of social approach behaviors and how they vary over time, (2) investigation of mean levels and modulation of salivary cortisol levels in response to social interaction, and (3) examination of the relationship of social approach and autistic behaviors to salivary cortisol. Poor social approach and elevated baseline and regulation cortisol are discernible traits that distinguish boys with FXS and ASD from boys with FXS only and from typically developing boys. In addition, blunted cortisol change is associated with increased severity of autistic behaviors only within the FXS and ASD group. Boys with FXS and ASD have distinct behavioral and neuroendocrine profiles that differentiate them from those with FXS alone and typically developing boys

Preoperative cardiac risk assessment in geriatric patients with hip fractures: an orthopedic surgeons’ perspective

Hip fracture is one of the most common orthopedic conditions and is associated with significant morbidity and mortality. With a progressively aging population, the annual incidence of hip fracture is expected to increase substantially. Emerging evidence suggests that early surgery (<24 h) minimizes complications secondary to immobilization, including orthostatic pneumonia and venous thromboembolism. Delayed surgical repair (>48 h) has been consistently demonstrated to be associated with an increased risk of 30-day and 1-year mortality. Nonetheless, early surgery necessitates a shorter time for preoperative medical preparation, in particular cardiac assessment. Patients who undergo emergent orthopedic surgery are therefore at greater risk of perioperative cardiac events than those who undergo elective surgery. In addition, the prompt triage system for preoperative cardiac assessment not only identifies patients at high risk of perioperative cardiac complications but also reduces unnecessary cardiac consultations for low-risk patients. We review the current recommendations for preoperative cardiac assessment adapted for patients with hip fracture and describe our current triage system for preoperative cardiac consultation

Reproductive abnormalities in mice expressing omega-3 fatty acid desaturase in their mammary glands

The Caenorhabditis elegans n-3 fatty acid desaturase (Fat-1) acts on a range of 18- and 20-carbon n-6 fatty acid substrates. Transgenic female mice expressing the Fat-1 gene under transcriptional control of the goat β-casein promoter produce milk phospholipids having elevated levels of n-3 polyunsaturated fatty acids (PUFA). However, females from this line were also observed to have impaired reproductive performance characterized by a smaller litter size (2.7 ± 0.6 vs. 7.2 ± 0.7; P < 0.05) than wildtype controls. While there is a close association between PUFA metabolism, prostaglandin biosynthesis, and fertility; reproductive problems in these mice were unanticipated given that the Fat-1 transgene is primarily expressed in the lactating mammary gland. Using multiple approaches it was found that Fat-1 mice have normal ovulation and fertilization rates; however fewer embryos were present in the uterus prior to implantation. Small litter size was also found to be partly attributable to a high incidence of post-implantation fetal resorptions. Embryo transfer experiments revealed that embryos developing from oocytes derived from transgenic ovaries had an increased rate of post-implantation resorption, regardless of the uterine genotype. Ovary transplantation between Fat-1 and C57BL/6 wildtype females revealed that non-ovarian factors also contributed to the smaller litter size phenotype. Finally, surgical removal of the mammary glands from juvenile Fat-1 mice increased the subsequent number of implantation sites per female, but did not lessen the high rate of post-implantation resorptions. In conclusion, we herein report on a system where an exogenous transgene expressed predominately in the mammary gland detrimentally affects female reproduction, suggesting that in certain circumstances the mammary gland may function as an endocrine regulator of reproductive performance