Bed Net Durability Assessments: Exploring a Composite Measure of Net Damage

Background

The durability of Long Lasting Insecticidal Nets (LLINs) in field conditions is of great importance for malaria prevention and control efforts; however, the physical integrity of the net fabric is not well understood making it challenging to determine overall effectiveness of nets as they age. The 2011 World Health Organization Pesticide Evaluation Scheme (WHOPES) guidelines provide a simple, standardized method using a proportional hole index (PHI) for assessing net damage with the intent to provide national malaria control programs with guidelines to assess the useful life of LLINS and estimate the rate of replacement.

Methods

We evaluated the utility of the PHI measure using 409 LLINs collected over three years in Nampula Province, Mozambique following a mass distribution campaign in 2008. For each LLIN the diameter and distance from the bottom of the net were recorded for every hole. Holes were classified into four size categories and a PHI was calculated following WHOPES guidelines. We investigate how the size, shape, and location of holes influence the PHI. The areas of the WHOPES defined categories were compared to circular and elliptical areas based on approximate shape and actual measured axes of each hole and the PHI was compared to cumulative damaged surface area of the LLIN.

Results

The damaged area of small, medium, large, and extra-large holes was overestimated using the WHOPES categories compared to elliptical areas using the actual measured axes. Similar results were found when comparing to circular areas except for extra-large holes which were underestimated. (Wilcoxon signed rank test of differences p< 0.0001 for all sizes). Approximating holes as circular overestimated hole surface area by 1.5 to 2 times or more. There was a significant difference in the mean number of holes < 0.5 cm by brand and there were more holes of all sizes on the bottom of nets than the top. For a range of hypothetical PHI thresholds used to designate a “failed LLIN”, roughly 75 to 80% of failed LLINs were detected by considering large and extra-large holes alone, but sensitivity varied by brand.

Conclusions

Future studies may refine the PHI to better approximate overall damaged surface area. Furthermore, research is needed to identify whether or not appropriate PHI thresholds can be used to deem a net no longer protective. Once a cutoff is selected, simpler methods of determining the effective lifespan of LLINs can help guide replacement strategies for malaria control programs.     

Posttranslational regulation of copper transporters

Copper is an essential but potentially harmful trace element involved in many enzymatic processes that require redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by posttranslational regulation of copper import and export through the copper import proteins hCTR1 and hCTR2 and the copper exporters ATP7A and ATP7B. Regulation of copper uptake and export is achieved by modulation of transporter expression, copper-dependent and copper-independent trafficking of the different transporters, posttranslational modifications, and interacting proteins. In this review we systematically discuss the contribution of these different mechanisms to the regulation of copper transport.     

Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion

Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O(2) that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H(2)O(2) generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.     

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 +/- 3.4% cell death. Hypothermia induction to 25 degrees C was most protective (14.3 +/- 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 +/- 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 +/- 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 microM) reversed these changes and abrogated hypothermia protection. In addition, the PKCepsilon inhibitor myr-PKCepsilon v1-2 (5 microM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cepsilon; nitric oxide synthase.     

Epsin: inducing membrane curvature

Epsin was originally discovered by virtue of its binding to another accessory protein, Eps15. Members of the epsin family play an important role as accessory proteins in clathrin-mediated endocytosis. Epsin isoforms have been described that differ in intracellular site of action and/or in tissue distribution, although all epsins essentially contribute to membrane deformation. Besides inducing membrane curvature, epsin also plays a key function as adaptor protein, coupling various components of the clathrin-assisted uptake and fulfils an important role in selecting and recognizing cargo. Furthermore, epsin possesses the ability to block vesicle formation during mitosis. To perform all these functions, epsin, apart from interacting with PtdIns(4,5)P2 via its ENTH domain, also engages in several protein interactions with different components of the clathrin-mediated endocytic system. Recently, RNA interference has successfully been exploited to generate a cell line constitutively silencing epsin expression, which can be used to study internalization of multiple ligands.     

Nitric oxide during ischemia attenuates oxidant stress and cell death during ischemia and reperfusion in cardiomyocytes

Nitric oxide (NO) has been implicated as a cardioprotective agent during ischemia/reperfusion (I/R), but the mechanism of protection is unknown. Oxidant stress contributes to cell death in I/R, so we tested whether NO protects by attenuating oxidant stress. Cardiomyocytes and murine embryonic fibroblasts were administered NO (10-1200 nM) during simulated ischemia, and cell death was assessed during reperfusion without NO. In each case, NO abrogated cell death during reperfusion. Cells overexpressing endothelial NO synthase (NOS) exhibited a similar protection, which was abolished by the NOS inhibitor N(omega)-nitro-l-arginine methyl ester. Protection was not mediated by guanylate cyclase or the mitochondrial K(ATP) channel, as inhibitors of these systems failed to abolish protection. NO did not prevent decreases in mitochondrial potential, but cells protected with NO demonstrated recovery of potential at reperfusion. Measurements using C11-BODIPY reveal that NO attenuates lipid peroxidation during ischemia and reperfusion. Measurements of oxidant stress using the ratiometric redox sensor HSP-FRET demonstrate that NO attenuates protein oxidation during ischemia. These findings reveal that physiological levels of NO during ischemia can attenuate oxidant stress both during ischemia and during reperfusion. This response is associated with a remarkable attenuation of cell death, suggesting that ischemic cell death may be a regulated event.