Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.     

Selection of antimalarial drug resistance after intermittent preventive treatment of infants and children (IPTi/c) in Senegal

Senegal has since 2003 used sulphadoxine-pyrimethamine (SP) for Intermittent Preventive Treatment (IPT) of malaria in risk groups. However, the large-scale IPT strategy may result in increasing drug resistance. Our study investigated the possible impact of SP-IPT given to infants and children on the prevalence of SP-resistant haplotypes in the Plasmodium falciparum genes Pfdhfr and Pfdhps, comparing sites with and without IPTi/c. P. falciparum positives samples (n = 352) were collected from children under 5 years of age during two cross-sectional surveys in 2010 and 2011 in three health districts (two on IPTi/c and one without IPTi/c intervention) located in the southern part of Senegal. The prevalence of SP-resistance-related haplotypes in Pfdhfr and Pfdhps was determined by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)–ELISA. The prevalence of the Pfdhfr double mutant haplotypes (CNRN and CICN) was stable between years at < 10% in the control group (P = 0.69), while it rose significantly in the IPTi/c group from 2% in 2010 to 20% in 2011 (P = 0.008). The prevalence of the Pfdhfr triple mutant haplotype (CIRN) increased in both groups, but only significantly in the IPTi/c group from 41% to 65% in 2011 (P = 0.005). Conversely, the Pfdhps 437G mutation decreased in both groups from 44.6% to 28.6% (P = 0.07) and from 66.7% to 47.5% (P = 0.02) between 2010 and 2011 in the control and the IPTi/c groups, respectively. Combined with Pfdhfr, there was a weak trend for decreasing prevalence of quadruple mutants (triple Pfdhfr + Pfdhps 437G) in both groups (P = 0.15 and P = 0.34). During the two cross-sectional surveys, some significant changes were observed in the SP-resistance-related genes. However, since these changes were observed in the two groups, the IPTi/c strategy does only seem to have limited impact on resistance development and other factors as well. However, continuous monitoring will be needed, due to the up-scaling of the IPTi/c strategy in Senegal according to WHO recommendations.     

Durable Resistance to Crop Pathogens: An Epidemiological Framework to Predict Risk under Uncertainty

Increasing the durability of crop resistance to plant pathogens is one of the key goals of virulence management. Despite the recognition of the importance of demographic and environmental stochasticity on the dynamics of an epidemic, their effects on the evolution of the pathogen and durability of resistance has not received attention. We formulated a stochastic epidemiological model, based on the Kramer-Moyal expansion of the Master Equation, to investigate how random fluctuations affect the dynamics of an epidemic and how these effects feed through to the evolution of the pathogen and durability of resistance. We focused on two hypotheses: firstly, a previous deterministic model has suggested that the effect of cropping ratio (the proportion of land area occupied by the resistant crop) on the durability of crop resistance is negligible. Increasing the cropping ratio increases the area of uninfected host, but the resistance is more rapidly broken; these two effects counteract each other. We tested the hypothesis that similar counteracting effects would occur when we take account of demographic stochasticity, but found that the durability does depend on the cropping ratio. Secondly, we tested whether a superimposed external source of stochasticity (for example due to environmental variation or to intermittent fungicide application) interacts with the intrinsic demographic fluctuations and how such interaction affects the durability of resistance. We show that in the pathosystem considered here, in general large stochastic fluctuations in epidemics enhance extinction of the pathogen. This is more likely to occur at large cropping ratios and for particular frequencies of the periodic external perturbation (stochastic resonance). The results suggest possible disease control practises by exploiting the natural sources of stochasticity.     

Increased Respiratory Disease Mortality at a Microwave Popcorn Production Facility with Worker Risk of Bronchiolitis Obliterans

Background

Bronchiolitis obliterans, an irreversible lung disease, was first associated with inhalation of butter flavorings (diacetyl) in workers at a microwave popcorn company. Excess rates of lung-function abnormalities were related to cumulative diacetyl exposure. Because information on potential excess mortality would support development of permissible exposure limits for diacetyl, we investigated respiratory-associated mortality during 2000–2011 among current and former workers at this company who had exposure to flavorings and participated in cross-sectional surveys conducted between 2000–2003.

Methods

We ascertained workers'' vital status through a Social Security Administration search. Causes of death were abstracted from death certificates. Because bronchiolitis obliterans is not coded in the International Classification of Disease 10^th revision (ICD-10), we identified respiratory mortality decedents with ICD-10 codes J40–J44 which encompass bronchitis (J40), simple and mucopurulent chronic bronchitis (J41), unspecified chronic bronchitis (J42), emphysema (J43), and other chronic obstructive pulmonary disease (COPD) (J44). We calculated expected number of deaths and standardized mortality ratios (SMRs) with 95% confidence intervals (CI) to determine if workers exposed to diacetyl experienced greater respiratory mortality than expected.

Results

We identified 15 deaths among 511 workers. Based on U.S. population estimates, 17.39 deaths were expected among these workers (SMR = 0.86; CI:0.48-1.42). Causes of death were available for 14 decedents. Four deaths among production and flavor mixing workers were documented to have a multiple cause of ‘other COPD’ (J44), while 0.98 ‘other COPD’-associated deaths were expected (SMR = 4.10; CI:1.12–10.49). Three of the 4 ‘other COPD’-associated deaths occurred among former workers and workers employed before the company implemented interventions reducing diacetyl exposure in 2001.

Conclusion

Workers at the microwave popcorn company experienced normal rates of all-cause mortality but higher rates of COPD-associated mortality, especially workers employed before the company reduced diacetyl exposure. The demonstrated excess in COPD-associated mortality suggests continued efforts to lower flavoring exposure are prudent.     

Pseudoxanthoma Elasticum: Cardiac Findings in Patients and Abcc6-Deficient Mouse Model

Background

Pseudoxanthoma elasticum (PXE), caused by mutations in the ABCC6 gene, is a rare multiorgan disease characterized by the mineralization and fragmentation of elastic fibers in connective tissue. Cardiac complications reportedly associated with PXE are mainly based on case reports.

Methods

A cohort of 67 PXE patients was prospectively assessed. Patients underwent physical examination, electrocardiogram, transthoracic echocardiography, cardiac magnetic resonance imaging (CMR), treadmill testing, and perfusion myocardial scintigraphy (SPECT). Additionally, the hearts of a PXE mouse models (Abcc6^−/−) and wild-type controls (WT) were analyzed.

Results

Three patients had a history of proven coronary artery disease. In total, 40 patients underwent exercise treadmill tests, and 28 SPECT. The treadmill tests were all negative. SPECT showed mild perfusion abnormalities in two patients. Mean left ventricular (LV) dimension and function values were within the normal range. LV hypertrophy was found in 7 (10.4%) patients, though the hypertrophy etiology was unknown for 3 of those patients. Echocardiography revealed frequent but insignificant mitral and tricuspid valvulopathies. Mitral valve prolapse was present in 3 patients (4.5%). Two patients exhibited significant aortic stenosis (3.0%). While none of the functional and histological parameters diverged significantly between the Abcc6^−/− and WT mice groups at age of 6 and 12 months, the 24-month-old Abcc6^−/− mice developed cardiac hypertrophy without contractile dysfunction.

Conclusions

Despite sporadic cases, PXE does not appear to be associated with frequent cardiac complications. However, the development of cardiac hypertrophy in the 24-month-old Abcc6^−/− mice suggests that old PXE patients might be prone to developing late cardiopathy.     

Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest Streptococcus pneumoniae Clonal Complex in the MLST Database

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.     

Effects of Acute Exposure to Moderate Altitude on Vascular Function,Metabolism and Systemic Inflammation

Background

Travel to mountain areas is popular. However, the effects of acute exposure to moderate altitude on the cardiovascular system and metabolism are largely unknown.

Objectives

To investigate the effects of acute exposure to moderate altitude on vascular function, metabolism and systemic inflammation.

Methods

In 51 healthy male subjects with a mean (SD) age of 26.9 (9.3) years, oxygen saturation, blood pressure, heart rate, arterial stiffness, lipid profiles, low density lipoprotein (LDL) particle size, insulin resistance (HOMA-index), highly-sensitive C-reactive protein and pro-inflammatory cytokines were measured at 490 m (Zurich) and during two days at 2590 m, (Davos Jakobshorn, Switzerland) in randomized order. The largest differences in outcomes between the two altitudes are reported.

Results

Mean (SD) oxygen saturation was significantly lower at 2590 m, 91.0 (2.0)%, compared to 490 m, 96.0 (1.0)%, p<0.001. Mean blood pressure (mean difference +4.8 mmHg, p<0.001) and heart rate (mean difference +3.3 bpm, p<0.001) were significantly higher at 2590 m, compared to 490 m, but this was not associated with increased arterial stiffness. At 2590 m, lipid profiles improved (median difference triglycerides −0.14 mmol/l, p = 0.012, HDL +0.08 mmol/l, p<0.001, total cholesterol/HDL-ratio −0.25, p = 0.001), LDL particle size increased (median difference +0.45 nm, p = 0.048) and hsCRP decreased (median difference −0.18 mg/l, p = 0.024) compared to 490 m. No significant change in pro-inflammatory cytokines or insulin resistance was observed upon ascent to 2590 m.

Conclusions

Short-term stay at moderate altitude is associated with increased blood pressure and heart rate likely due to augmented sympathetic activity. Exposure to moderate altitude improves the lipid profile and systemic inflammation, but seems to have no significant effect on glucose metabolism.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01130948","term_id":"NCT01130948"}}NCT01130948     

A Systematic Review of Risk Factors Associated with Surgical Site Infections among Surgical Patients

Importance

Surgical site infection (SSI) complicates 2-5% of surgeries in the United States. Severity of SSI ranges from superficial skin infection to life-threatening conditions such as severe sepsis, and SSIs are responsible for increased morbidity, mortality, and economic burden associated with surgery. Staphylococcus aureus (S. aureus) is a commonly-isolated organism for SSI, and methicillin-resistant S. aureus SSI incidence is increasing globally.

Objective

The objective of this systematic review was to characterize risk factors for SSI within observational studies describing incidence of SSI in a real-world setting.

Evidence Review

An initial search identified 328 titles published in 2002-2012; 57 were identified as relevant for data extraction. Extracted information included study design and methodology, reported cumulative incidence and post-surgical time until onset of SSI, and odds ratios and associated variability for all factors considered in univariate and/or multivariable analyses.

Findings

Median SSI incidence was 3.7%, ranging from 0.1% to 50.4%. Incidence of overall SSI and S. aureus SSI were both highest in tumor-related and transplant surgeries. Median time until SSI onset was 17.0 days, with longer time-to-onset for orthopedic and transplant surgeries. Risk factors consistently identified as associated with SSI included co-morbidities, advanced age, risk indices, patient frailty, and surgery complexity. Thirteen studies considered diabetes as a risk factor in multivariable analysis; 85% found a significant association with SSI, with odds ratios ranging from 1.5-24.3. Longer surgeries were associated with increased SSI risk, with a median odds ratio of 2.3 across 11 studies reporting significant results.

Conclusions and Relevance

In a broad review of published literature, risk factors for SSI were characterized as describing reduced fitness, patient frailty, surgery duration, and complexity. Recognition of risk factors frequently associated with SSI allows for identification of such patients with the greatest need for optimal preventive measures to be identified and pre-treatment prior to surgery.     

HER2 Phosphorylates and Destabilizes Pro-Apoptotic PUMA,Leading to Antagonized Apoptosis in Cancer Cells

HER2 is overexpressed in 15–20% of breast cancers. HER2 overexpression is known to reduce apoptosis but the underlying mechanisms for this association remain unclear. To elucidate the mechanisms for HER2-mediated survival, we investigated the relationship between HER2 and p53 upregulated modulator of apoptosis (PUMA), a potent apoptosis inducer. Our results showed that HER2 interacts with PUMA, which was independent of HER2 activation. In addition, we observed that HER2 interacted with PUMA in both mitochondrial and non-mitochondrial compartments. We next examined whether HER2 phosphorylates PUMA. Notably, PUMA tyrosine phosphorylation has never been reported. Using an intracellular assay, we found PUMA to be phosphorylated in breast cancer cells with activated HER2. Via cell-free HER2 kinase assay, we observed that PUMA was directly phosphorylated by HER2. Activation of HER2 decreased PUMA protein half-life. To identify which of the three tyrosines within PUMA are targeted by HER2, we generated three PUMA non-phosphorylation mutants each with a single Tyr→Phe substitution. Results indicated that each PUMA single mutant had lost some, but not all phosphorylation by HER2 indicating that HER2 targets all three tyrosines. Consequently, we created an additional PUMA mutant with all three tyrosines mutated (TM-PUMA) that could not be phosphorylated by HER2. Importantly, TM-PUMA was found to have a longer half-life than PUMA. An inverse association was observed between HER2 and PUMA in 93 invasive breast carcinoma samples. We further found that TM-PUMA suppressed growth of breast cancer cells to a greater degree than PUMA. Also, TM-PUMA had a stronger propensity to induce apoptosis than PUMA. Together, our results demonstrate, for the first time, that PUMA can be tyrosine phosphorylated and that HER2-mediated phosphorylation destabilizes PUMA protein. The HER2-PUMA interplay represents a novel mechanism by which PUMA is regulated and a new molecular basis for HER2-mediated growth and survival of cancer cells.