Poor circulation stability and inadequate cell membrane penetration are significant impediments in the implementation of nanocarriers as delivery systems for therapeutic agents with low bioavailability. This research discusses the fabrication of a biocompatible poly(lactide-co-glycolide) (PLGA) based nanocarrier with cationic and hydrophilic surface properties provided by natural polymer chitosan and coating polymer polyethylene glycol (PEG) for the entrapment of the hydrophobic drug disulfiram. The traditional emulsification solvent evaporation method was compared to a microfluidics-based method of fabrication, with the optimisation of the parameters for each method, and the PEGylation densities on the experimental nanoparticle formulations were varied. The size and surface properties of the intermediates and products were characterised and compared by dynamic light scattering, scanning electron microscopy and X-ray diffraction, while the thermal properties were investigated using thermogravimetric analysis and differential scanning calorimetry. Results showed optimal particle properties with an intermediate PEG density and a positive surface charge for greater biocompatibility, with nanoparticle surface characteristics shielding physical interaction of the entrapped drug with the exterior. The formulations prepared using the microfluidic method displayed superior surface charge, entrapment and drug release properties. The final system shows potential as a component of a biocompatible nanocarrier for poorly soluble drugs. Nanostructured materials have demonstrated efficiency at improving bioavailability and reducing toxicity of bio-actives when implemented in the design of drug delivery systems . SynthetNaturally occurring polymers such as chitosan can mimic the extracellular matrix of biological systems ,8,9. ChiIn the design of advanced nanocarriers there are generally two main regions of the nanoparticle structure that influences the biocompatibility, i.e., the nanoparticle core and the corona . The corStudies have shown that this can be circumvented by coating such nanoparticles with chitosan ,15. Low PEG chains also introduce a degree of steric repulsion which further protects the nano-system from the RES . Hereby,In order to fully exploit the individual properties of PLGA, chitosan and PEG for the design of an optimised nano-enabled drug delivery system, the particle size, dispersity, surface charge and structural organisation need to be precisely controlled. These characteristics are influenced in part by the preparation methods used, and it has been shown that the fabrication techniques can determine the overall functionality of these nano-systems , with thIn this study, we compared two methods for the synthesis of PLGA-chitosan-PEG nanoparticles to encapsulate the model hydrophobic drug disulfiram (DSF) which is used clinically for the treatment of alcohol abuse  and, mord,l-lactide-co-glycolide) (PLGA) , polyvinyl alcohol (PVA) , disulfiram (model drug), dialysis tubing (MWCO 14 KDa), PBS buffer tablets (pH = 7.2), tween80 and mannitol were purchased from Sigma-Aldrich . Polyethylene glycol (PEG2000), glacial acetic acid, acetone, chloroform, dichloromethane, and acetonitrile were purchased from Merck (Pty) Ltd., . All reagents were of analytical grade.Polymers, namely poly(Various nanoparticle formulations were prepared using a single-emulsion oil in water (O/W) solvent evaporation approach . Four inv/v) glacial acetic acid to produce a 0.25% (w/v) concentration as the aqueous phase. The PEG-based formulations were prepared using 2% PEG and the PVA-based formulations were prepared using either 0.5%, 1% or 2% PVA. These constituted the aqueous phase and were prepared prior to the addition of the organic phase. The organic phase in each case was prepared by dissolving 100 mg of PLGA in 1 mL of either acetone, chloroform or dichloromethane and added dropwise under stirring to the aqueous phase. For the DSF-loaded formulations, 10 mg of DSF was added to the organic phase before addition to the aqueous phase. These concentrations and ratios were optimal for PLGA and DSF as previously reported [For each CHT-based formulation, 25 mg of CHT was agitated in 1% (reported . The emuw/v) mannitol to observe the effect of cryopreservation.For the sonicated formulations, the emulsion was sonicated for 2 min using a probe sonicator at 130 W with 40% amplitude.  before the evaporation step. Thereafter the formulation was centrifuged  for 30 min at 5000 rpm at 4 °C and washed thrice with distilled water to remove any unentrapped drug. The resultant pellets were redispersed in 4 mL of distilled water and then sonicated for 10 min to produce free-flowing nanoparticles that were frozen at −80 °C and then lyophilized  for 24 h. A portion of sample of each formulation was mixed with 5% . The defining characteristics of the nanoparticles produced by this approach were compared to those of the emulsification solvent evaporation method. The Nanoassemblr utilizes microchannels in a cartridge that allows for the mixing of organic and aqueous phase in precisely controlled speeds and ratios under laminar flow. The organic phase comprised 10 mg/mL PLGA and 1 mg/mL disulfiram dissolved in acetonitrile using the favourable ratio of polymer to drug (10:1) described earlier, and a concentration of organic phase polymer which was shown to produce relatively low particle size using this instrument .w/v), PEG (2% w/v) and PVA (2% w/v) in the aqueous phase. The particle size and stability were compared using flow rate ratios of 1:1, 1:3 and 1:5 and flow rates of 8, 12 and 15 mL min−1. Thereafter, DSF-loaded and native nanoparticles were prepared using CHT (0.25% w/v) and PEG (2% w/v) and loaded nanoparticles were prepared with 0.25% (w/v) chitosan only, and 0.25% (w/v) chitosan with 4%, 6% and 8% (w/v) PEG, using a flow rate of 8 mL min−1. The downstream processing protocol of centrifugation, washing, and lyophilization was followed as per the emulsification solvent evaporation method.Formulations were prepared using CHT (0.25% The nanoparticles were dissolved (1 mg of each formulation in 1 mL of Millipore water) and analysed using dynamic light scattering and photon correlation spectroscopy at a fixed angle of 90° on a ZetaSizer NanoZS  particle size analyzer. Samples were diluted and sonicated before measurement, with the sample temperature maintained at 25 °C throughout the analyses. The particle size, polydispersity index and zeta potential values were used to screen formulations and select the most favourable preparation parameters such as the organic solvent, the concentration of stabilizer and sonication for the emulsification solvent evaporation approach. The flow rate ratio and total flow rate was used to screen formulations prepared by the microfluidics-based approach using the NanoAssemblr .−1 with a constant pressure of 110 psi. The chemical structures of each nanoparticle formulation prepared by either the emulsification solvent evaporation and microfluidic methods were compared.The native polymers and different nanoparticle formulations were subjected to Fourier Transform Infra-Red (FTIR) analysis to assess the chemical integrity and stability of nanoparticles produced from the two approaches. Characteristic peaks were compared to confirm the molecular vibrations of the polymers in the formulations. The FTIR spectra were recorded using a PerkinElmer  spectrometer equipped with a single reflection diamond MIRTGS detector. Samples were processed by a universal attenuated total reflectance (ATR) polarization accessory at a resolution of 4 cm−1. The samples were maintained in an inert environment by constant nitrogen purging throughout the experiment. Thermograms were plotted as percentage mass against temperature. The phase transitions of the fabricated loaded and blank nanoparticles and native polymers were investigated using a differential scanning calorimeter (DSC) . These analyses provide insight about the thermally induced phase changes that the samples undergo, and hence characterizes the variation in thermal and physical properties with change in the constituent materials of the samples. The nanoparticles and drug samples of ~5 mg were weighed into aluminium crucibles which were sealed and then heated over a temperature range of 0 to 300 °C, at a heating rate of 10 °C min−1. The samples were maintained in an inert N2 gas atmosphere. Calorimetry spectra were recorded as a function of heat flow against temperature.In order to characterize the thermal properties of the nanoparticles, thermogravimetric analysis (TGA) was undertaken using a TG analyser , whereby the temperature range at which the formulation samples degraded were determined. Native DSF and DSF-loaded nanoparticle samples were exposed to 30 °C and then heated at a rate of 10 to 800 °C min−1. Powder X-ray diffractometry (XRD) analyses indicate the degree of crystallinity and amorphous nature of the polymers and nanoparticles and hence provides insight on their surface properties and behaviour.Lyophilized DSF-loaded samples and the native polymers were finely milled. PVA crystals were dissolved in distilled water and dried to form a film. Each sample was finely packed and smoothed into a sample holder for analysis using a benchtop MiniFlex 600  powder X-ray diffractometer. CuKα radiation at 40 kV and 15 mA was employed and data was recorded using a 2θ scan range of 10–60° at a scan rate of 10° minLyophilized samples were re-dispersed in distilled water and a drop of each sample was placed on aluminium stubs and dried for 48 h. Solid samples were mounted on double sided carbon tape. In order to induce electrical conduction, samples were coated in a vacuum with a fine layer of gold using a sputter coater. Each sample was then analysed on a SIGMA 03-39 field emission scanning electron microscope  at 5–15 kV acceleration voltage under an argon atmosphere.Due to the hydrophobic nature of disulfiram a UV spectrophotometric method of analysis was adapted from . Brieflyv/v) Tween 80 and agitated for 72 h at 37 °C. At predetermined time intervals , 1 mL of the external release medium was withdrawn and replaced with an equal volume of drug-free medium to maintain sink conditions. Parameters were kept constant for all formulations assessed and samples were filtered prior to DSF analysis at 216 nm.In vitro release of DSF from the nanoparticle formulations were determined using a dialysis tubing method . The tesData for preparation and characterization of nanoparticles and drug release studies were processed and analyzed by Origin software . All results were represented by the mean ± standard error of three experiments.Even though the abnormal tumour vasculature allows the penetration of particles <400 nm in size , particlw/v) PVA, sonication time of 2 min, and dichloromethane as the organic phase, as shown in Figure 4. For a full list of results, refer to Solvent evaporation using acetone in the presence of no stabilizing agent were the parameters initially used for the preparation of the nanoparticles. While this method produced nanoparticles in a desired size range for the unloaded and disulfiram loaded PLGA nanoparticles, as previously reported , the sizw/v) PVA, 2% (w/v) PEG and 0.25% (w/v) chitosan, and an organic phase of 10 mg/mL PLGA, with a PLGA:DSF ratio of 10:1 in acetonitrile. The best formulation (with a size of 218 nm and PDI of 0.165) was obtained using a flow rate ratio of 3:1 aqueous:organic phase and a total flow rate of 8 mL per minute, as shown by The parameters were optimized using an aqueous phase of 2% (w/v) PVA and sonication time of 2 min. The microfluidic formulations were prepared using a flow rate of 3:1 and a total flow rate of 8 mL min−1. In this method, the mixing of the two phases were automated and controlled by the machine and hence the results obtained were highly reproducible. The uniformity of the dispersions was much better compared to the solvent emulsions method across all formulations as shown by the relatively low PDI values (0.191–0.258) and size disparity (218–330 nm) of the microfluidic preparations in The emulsification solvent evaporation formulations were prepared using dichloromethane as a solvent, 1% (w/v) PEG in the aqueous phase, as shown by w/v) PEG displayed similar characteristics as that of the 6% (w/v) PEG. In formulations prepared by both methods, the uncoated PLGA nanoparticles have negative zeta potentials. The emulsification solvent evaporation uncoated nanoparticles display a larger negative surface charge, which is due to the larger relative amount of PLGA used in this formulation. When coated with chitosan, there is an increase in surface charge of both formulations, while there is an initial decrease when PEG (2% (w/v)) is added, then an increase until the PEG concentration reaches 6% (w/v), and a decrease once again when the PEG concentration is increased to 8% (w/v). The entrapment efficiencies of both formulations are high with PEG concentrations of 4 and 6% (w/v) and peak when the concentration of PEG is 6% (w/v). The entrapment efficiency of the PEG 6% formulations prepared by the microfluidics method (78.7%) was 8.4% higher than those of the solvent evaporation method (72.6%).Both emulsification solvent evaporation and microfluidic prepared formulations showed optimal properties when prepared with 6%  PEG resulted in particles with the highest entrapment efficiencies, suggesting a stabilizing effect on the amphiphilic structure, leading to a larger amount of hydrophobic drug-polymer interactions within the PLGA core.In theory, the larger the surface area, the more amino groups are present and therefore the more positive the zeta potential of the nanoparticle. The high surface charge of the nanoparticles also prevents particle aggregation in aqueous solution due to electrostatic repulsion . The incThe optimal entrapment efficiencies were similar for the formulations prepared by both methods, and this could be due to the optimal PLGA:disulfiram ratio of 10:1 ,38 that Reports describe low PEG surface density in a mushroom conformation and as PEG density increases, the PEG chains reconfigure into a brush conformation ,41. WithWith higher concentrations of PEG, the conformations change from mushroom to brush, with steric repulsions between PEG chains causing reduced lateral interactions between PEG chains and chitosan surface, allowing for end chain PEG interactions only, as shown by −1, stretching of carbonyl groups at 1775 cm−1, as well as stretching of single carbon-hydrogen (C–H), methylene (CH2), and methyl (CH3) bonds between 2885 cm−1 and 3000 cm−1. A methylene stretching peak due to ester bonds is also observed at 2955 cm−1 [−1, a peak corresponding to methyl groups at 2800 cm−1, a carbonyl stretching peak at 1775 cm−1, and broad peaks due to C–H bending from 600 cm−1 to 1400 cm−1. The disulfiram spectrum shows a peak due to methyl stretching at 2975 cm−1, a peak at 1500 cm−1 due to C–H stretching, a peak at 1250 cm−1 due to C=S stretching and 1150–1200 cm−1 due to C–C stretching. Peaks at 950 cm−1 and 800 cm−1 were due to C–N and C–S stretching respectively, while peaks at 500–625 cm−1 were due to S–S bending [−1 due to hydroxyl and amine stretching, and a characteristic amide peak at 1550 cm−1 [−1, a strong peak at 2800 cm−1 due to C–H stretching, and a characteristic peak at 1125 cm−1 can be observed due to C–O–C stretching vibrations [955 cm−1 . In the  bending . In the 550 cm−1 . In the brations .The FTIR spectra show no new peaks on nanoparticles as coatings are added, and the spectra are superimpositions of the starting materials, with no peak shifts or new peaks, indicating no formation of new covalent bonds. This confirms that the coatings interact by physical adsorption only . The nanThe degradation of disulfiram occurred at 225 °C and degraded completely by 250 °C. The thermal degradation profiles of the formulations prepared by both methods were very similar. The blank nanoparticles prepared by both methods began degrading at 275 °C, with a slow degradation step until 410 °C, at which temperature 30% of the blank and 40% of the loaded nanoparticles had degraded, and a second step from 410 °C until complete degradation at 440 °C. There was still 8% of residue of the loaded nanoparticles by the end of the experiment. The loaded nanoparticles followed the degradation profile of the blank nanoparticles closely, as shown in The thermogram for disulfiram show characteristic endotherms at 74 °C and 190–250 °C corresponding to melting and degradation events, respectively . The theThe thermal and physical state of the nanoparticles influence its drug loading and release characteristics. The thermogravimetric analysis and differential scanning calorimetry data show that all the thermal events due to disulfiram were not displayed by any of the nanoparticle formulations, indicating that the loaded nanoparticles have adopted the thermal characteristics of the blank nanoparticles and that disulfiram is found physically dispersed in the nanoparticle system. The similarity of the formulations prepared by both methods indicate that they have similar physical states, which could be another reason for the similar entrapment efficiency observed in As shown by XRD provides information of surface characteristics of nanoparticles, which is important for interactions within the circulatory system . As illuThe use of mannitol to cryopreserve the samples had no effect except dilution and the samples that were evaporated on stubs were unstable and prone to cracking under the microscope. Hence all images were captured using solid lyophilized samples with no cryoprotectant. Both methods of preparation produced particles with spherical morphologies. The chitosan coating provides a positive charge which favours increased interaction with negative cell membranes. As previously reported , when thThe drug release profile of the emulsification solvent evaporation and microfluidics prepared nanoparticles compared to native disulfiram is shown in By 4 h, disulfiram had released 10% of its concentration into the buffer, with negligible release thereafter. Both nanoparticle formulations demonstrated biphasic release patterns, with initial burst release followed by sustained release. The nanoparticles prepared by the emulsification solvent evaporation and microfluidic methods released maxima of 40% by 8 h, and 70% by 12 h, of their disulfiram content, respectively.Release behaviour is biphasic, with initial burst release caused by drug that is poorly bound by polymeric matrix or adsorbed on nanoparticle surface, while subsequent sustained release from PLGA core is further controlled by chitosan and PEG surface layers. PLGA degrades into its constituent co-polymers, lactic acid and glycolic acid, by hydrolysis of its ester linkages, while chitosan swells, providing a hydrogel-like layer which controls drug diffusion from the surface . The proWe have designed and prepared polymeric nano-systems with a hydrophobic core, a cationic component and surface density of PEG chains that have resulted in favourable size, dispersity, surface charge and entrapment efficiencies. The hydrophobic PLGA core encouraged a high degree of carrier-drug interactions due to favourable drug: polymer ratio, while the chitosan and PEG coatings provided cationic and hydrophilic properties which are important for enhanced biocompatibility. The formulations prepared by solvent evaporation and microfluidics technique were physically, thermally and chemically similar. However, the difference in their physical surface properties was due in part to the difference in polymer concentrations which was an outcome of the method parameters. The formulations prepared by both methods entrapped the drug in an amorphous state, and did not retain any of its thermal or chemical properties, suggesting that the entrapped drug was efficiently shielded. The formulations also displayed cationic properties while maintaining the PEG groups on the nanoparticle surface. However, the microfluidics-based formulations displayed more desirable characteristics in terms of more positive surface charge which is associated with higher cancer cell uptake and toxicity, high entrapment efficiency with favourable drug binding and release properties, and higher cumulative and sustained release. These properties would make it highly effective for the delivery of drugs with low bioavailability, and could have applications for enhanced membrane permeation while retaining the greater benefits of antifouling effects in a passively targeted nanocarrier. Future directions of study with these formulations could include comparison of the hydrophilic properties that the different density of PEG shields convey to the delivery system, investigation of protein adsorption to the surface of the nanoparticles, and cytotoxicity and cellular uptake assays to investigate biocompatibility and therapeutic activity.


Post cardiac arrest syndrome is associated with high morbidity and mortality, which is related not only to a poor neurological outcome but also to respiratory and cardiovascular dysfunctions. The control of gas exchange, and in particular oxygenation and carbon dioxide levels, is fundamental in mechanically ventilated patients after resuscitation, as arterial blood gases derangement might have important effects on the cerebral blood flow and systemic physiology.2) levels is strongly underestimated, as its alterations quickly affect also the changes of intracellular pH, and consequently influence metabolic energy and oxygen demand. Hypo/hypercapnia, as well as mechanical ventilation during and after resuscitation, can affect CO2 levels and trigger a dangerous pathophysiological vicious circle related to the relationship between pH, cellular demand, and catecholamine levels. The developing hypocapnia can nullify the beneficial effects of the hypothermia. The aim of this review was to describe the pathophysiology and clinical consequences of arterial blood gases and pH after cardiac arrest.In particular, the pathophysiological role of carbon dioxide (COAccording to our findings, the optimal ventilator strategies in post cardiac arrest patients are not fully understood, and oxygen and carbon dioxide targets should take in consideration a complex pattern of pathophysiological factors. Further studies are warranted to define the optimal settings of mechanical ventilation in patients after cardiac arrest. However, the pathophysiology of cardiac arrest and its systemic effects, as well as the relationship between arterial blood gases, hemodynamic management, and ventilation during and after resuscitation is complex. Evidence regarding the optimal oxygen or carbon dioxide (CO2) targets is limited, and the relationship between PaO2 and PaCO2 targets in the context of catecholamine administration and pH changes is not fully understood. Also, the pathophysiological effects of CO2 are underestimated in the literature; variations of carbon dioxide levels can quickly change the extracellular-intracellular concentration of H+, and modifications in pH and acidosis have extraordinary importance on systemic homeostasis [The aim of cardiopulmonary resuscitation is to promptly restore spontaneous circulation in order to avoid hypoxic ischemic brain injury. In this context, ventilation has the goal to maintain an appropriate level of arterial oxygen  [Patients after cardiac arrest may often present with a combination of metabolic and respiratory disturbances, where lactic acidosis is the main cause of metabolic acidosis (defined as pH ≤ 7.35 and lactatemia > 2.0 mmol lHypoperfusion and acidotic disturbances during return of spontaneous circulation (ROSC) result in a misbalance between lactate production and removal; the dissociation of lactic acid produces hydrogen ions which are used for the oxidative phosphorylation and adenosine Tri-Phosphate (ATP) production , 5. In aHowever, acid-base disturbances imply a larger spectrum of clinical consequences. Acute lactic acidosis depresses left ventricular contractility, triggers the release of endogenous catecholamine, and decreases the responsiveness of the left ventricle to the actions of catecholamine . TherefoThe pathophysiological effects of acidosis on cardiomyocytes are well described ; extrace2 and PaO2 levels in post cardiac arrest patients are predictors for hospital mortality.After successful resuscitation and ROSC, achieving appropriate levels of oxygen and carbon dioxide in arterial blood is necessary to avoid further hypoxia and secondary damage  Figs. 11, 2, 3. 2 levels are associated with low pH and almost immediate sodium-potassium (Na+/K+) ATPase dysfunction, with consequent ion imbalance. Thus, hypoxemia induces mitochondrial damage, which determinates systemic inflammation with energy failure and cell apoptosis [Re-oxygenation after the no flow phase is a major stimulant for myocardial catecholamine release . Decreaspoptosis . On the poptosis .2) [2 > 200 mmHg (26.7 kPa). Elevated PaO2 may increase oxidative stress, releases reactive oxygen species (ROS), and predisposes to mitochondrial dysfunction that finally results in molecular injury including deoxyribonucleic acid (DNA) damage and worsened organ function.Hyperoxia is usually observed after cardiac arrest as consequence of intubation and artificial ventilation with high fraction of oxygen (FiO2) . Mild hyElevated ribonucleic acid (RNA) expression of nitric oxide synthase (iNOS) was observed during hyperoxia when compared to normoxia in animal models of ischemia/reperfusion injury. Furthermore, pro apoptotic factors such as cyclooxygenase-2 (COX-2), toll like receptor 4 (TLR-4), and caspase-3 protein expression were increased, with a strong impact on apoptosis and inflammation mechanisms leading to myocardial injury .2 supplementation compared with high O2 supplementation. Recent data show an increased accumulation of hypoxic-induced factor (HIF) during exposure to oxygen FiO2 > 30% in the brain tissue, hepatocytes, and myocardium [Animal studies demonstrated improved outcome and lower neuronal damage using low Oocardium –23.Translated in the clinical practice, hyperoxia toxicity, with overproduction of ROS and oxidative cellular injury, can determinate elevated levels of superoxide, inhibits hypoxic pulmonary vasoconstriction, affects the adsorption of atelectasis, and is cause of apoptosis, necrosis, and direct ROS toxicity (Lorrain Smith effect) on alveolar capillary barrier which predisposes to hemorrhagic pulmonary edema; also, hyperoxia leads to vasoconstriction of arteries to distant organ thus causing coronary spasm and ischemic events .2 may be a marker of post ROSC cerebral ischemia in nontraumatic cardiac arrest as PaCO2 levels directly regulate cerebral blood flow and cerebrovascular diameter, with every 1 mmHg decrease of PaCO2 determining a decrease of approximately 3% in cerebral blood flow. Hypocarbia may cause cerebral vasoconstriction that worsens ischemic injury and should be avoided to preserve cerebral blood flow [2 50–55 mmHg) may improve cerebral oxygenation when compared with normocarbia (PaCO2 35–45 mmHg), and Beitler et al. [2 can have detrimental effects on patients’ outcome, especially if related to hypercapnic acidosis [Youn-Jung Kim et al.  suggesteood flow , 27. On ood flow , 29 suggr et al.  in a recacidosis , 32.Catecholamines are principally used in cardiac arrest to achieve hemodynamic stabilization and to improve systemic blood pressure and cardiac output in hemodynamically unstable patients. However, recent studies showed that epinephrine administration is associated with post-resuscitation myocardial dysfunction and worse outcome . SynthetCatecholamines can also affect peripheral vascular resistances through myosin light-chain kinase, which regulate smooth muscle cells myosin activity. The alpha 1 receptors activation induce vasoconstriction with increased risk of coronary spasms and can determinate reduction of splanchnic perfusion, lower mucosal pH, and higher hepatic vein lactate , 40. VasFinally, catecholamine release, with consequent important alteration in microcirculatory system, impairs cardiac fatty-acid metabolism, and mitochondrial metabolism, and can activate immunology response. Neuronal and immune responses are rapidly activated during cardiac arrest and trigger a pathway of systemic neuroendocrine and immune responses with uncontrolled immune reaction because of inadequate hormonal suppression and disturbance between anti-inflammatory and proinflammatory responses. Catecholamines and their metabolites influence every aspect of innate and adaptive immune response, because immune cells express both alpha and beta adrenoreceptors . In part2 alterations during resuscitation can quickly cause abrupt intracellular and extracellular pH changes; hypocapnia, as consequence of hyperventilation, increases the demand of metabolic energy and O2, while ATP production is often reduced, thus determining a sympathetic-like activation which further increases the energy demand  [The pH derangements strongly influence the function of many enzymes as well as the active and passive cytoplasmic transport of ions, especially of calcium (Ca2+) , 45. The2+) . On the 2+) .A vicious circles can therefore occur, triggered by hypocapnia, which determinates a lack of energy substrate together with cardiac and pulmonary failure  Fig. 4)4).The increased production of catecholamines is a compensatory phenomenon in acidosis, and there is a positive feedback between catecholamine production and H+ concentration: the catecholamine-sensitivity is very low in acidosis, though their production is high. However, when compensatory mechanisms and treatment improve acidosis and an alkalotic pH develops, the catecholamine levels remain still high; the synergism of the high levels of catecholamines and pH can poison systemic and immunological functions through the previously described mechanisms , 49.2, and lactate. In particular, the production of lactate depends on three mechanisms: hypoxia/anoxia, with activation of anaerobic pathway (lactate/pyruvate); ATP demand and rate of metabolism , which is pH dependent; and intracellular pH [Thus, the beneficial or harmful effect of catecholamines depends on different factors including pH, levels of PaCOlular pH . Also, w2 levels, as acidosis can slow down the enzymatic intracellular and membrane processes by reducing energy demand-we should avoid raising PaCO2 above normal level because this will lead to metabolic disorders in the long term [Although from these pathophysiological concepts, it is clear that the best way to terminate this vicious circle is to elevate PaCOong term .Post cardiac arrest syndrome results in different mechanisms of injury, including ischemia/reperfusion injury, oxidative stress, microcirculatory disturbances with coagulopathy, and inflammation which may increase the risk for multiple organ failure Table .Table 1During resuscitation, the relationship between tissue oxygenation, energy demand, and cellular function is complex and strictly related to intracellular pH.Evidences regarding the levels and changes in oxygen and carbon dioxide during and after cardiac arrest as well as of optimal carbon dioxide and oxygenation targets are scarce, but the above discussed pathophysiological changes during cardiac arrest may help to guide clinical practice Fig. .Fig. 5SDuring cardiac arrest, oxygen and carbon dioxide levels depend on the cause of pre arrest ; two recent studies showed that during cardiopulmonary resuscitation, patients presenting with hypoxia had lower survival rates than those with normoxia or hyperoxia; in these studies, hypercapnia and respiratory acidosis were common, but no association between the severity of acidosis or hypercapnia with outcome was found , 52. TheOther authors showed that hyperventilation and hypocapnia during cardiopulmonary resuscitation can worsen outcome by reducing cardiac output and pulmonary blood flow , 44.2 100% and a tidal volume of 500 ml, and respiratory rate of 12, minimizing acute changes on PaCO2 [Therefore, current recommendations suggest to avoid hypoxia during cardiac arrest patients by using high FiOon PaCO2  Fig. 5)2 100% an2 administration, which should be titrated in order to avoid both hypoxia and hyperoxia. The use of high FiO2 = 100% can cause extreme hyperoxia and can result in higher release of neuron specific enolase, a biomarker of brain injury, when compared to lower FiO2 [In the post resuscitation phase, attention should be paid on FiOwer FiO2 , 47. Penwer FiO2  in an ex2 values after 48 or 72 h from ROSC.Ebner et al.  showed t2 of 4.5–6.0 kPa [Hypocapnia should be avoided in order to optimize cerebral perfusion and mitigate ischemia-reperfusion injury due to post cardiac arrest oxidative stress, but the correction of hypocapnia should take in consideration the pH, catecholamine administration, and intracellular pH. In this phase, also the correction of hypercapnia should be performed with caution; some authors even suggest a correlation between good outcome and moderate hypercapnia by a potential effect of attenuation in the above mentioned excitotoxicity processes . However–6.0 kPa , 51, but the frequency of ARDS in this population is not well characterized. Two hypotheses have been proposed to explain the development of ARDS in these patients: the first theory includes the indirect activation and proliferation of neutrophils with progressive inflammatory alveolar infiltrates by activation of the immune system after cardiac arrest. Another hypothesis focuses on direct epithelial barrier damage related to ischemia reperfusion injury and endothelial cells activation and dysfunction . The conGas exchange impairment after cardiac arrest can be also related to myocardial dysfunction and consequent pulmonary edema, as well as impairment of the immune system with increased predisposal to infections, with frequency up to 70% . FinallyAltogether, attention should be paid in the optimization of mechanical ventilation, in particular in the difference phases of cardiac arrest, during and in the post resuscitation period Fig. , aiming The pathophysiological effects of cardiac arrest on systemic homeostasis are complex. The appropriate ventilator strategies are not fully understood, and oxygen and carbon dioxide targets should take in consideration several pathophysiological factors including the intracellular and extracellular pH as well as the use of catecholamines and hypothermia. In general, hypoxia (< 60 mmHg) and hyperoxia (> 300 mmHg) should be avoided as well as hypocapnia (< 30 mmHg) and hypercapnia (> 50 mmHg). Large prospective randomized controlled studies are warranted to further explore the pathophysiological basis of this theory and define the optimal setting of mechanical ventilation in patients after cardiac arrest.


Aedes aegypti populations on a global scale, which has significantly compromised control actions. Insecticide resistance, and its underlying mechanisms, has been investigated in several countries, mostly in South American and Asian countries. In Africa, however, studies reporting insecticide resistance are rare and data on resistance mechanisms, notably knockdown resistance (kdr) mutations, is scarce. In this study, the recently described V410L kdr mutation is reported for the first time in old world Ae. aegypti populations, namely from Angola and Madeira island. Two additional kdr mutations, V1016I and F1534C, are also reported for the first time in populations from Angola and Cape Verde. Significant associations with the resistance phenotype were found for both V410L and V1016I individually as well as for tri-locus genotypes in the Angolan population. However, no association was found in Madeira island, probably due to the presence of a complex pattern of multiple insecticide resistance mechanisms in the local Ae. aegypti population. These results suggest that populations carrying the same kdr mutations may respond differently to the same insecticide, stressing the need for complementary studies when assessing the impact of kdr resistance mechanisms in the outcome of insecticide-based control strategies.The extensive use of insecticides for vector control has led to the development of insecticide resistance in  Aedes-transmitted arboviral infections has been vector control, which is primarily based on the use of chemical insecticides. However, extensive use of insecticides has led to the development of insecticide resistance, undermining the sustainability of control programs. Mutations in the voltage-gated sodium channel gene have been associated with knockdown resistance in many insect species including Aedes aegypti. In Africa, in spite of the use of insecticides for vector control in many countries, data on insecticide resistance and its underlying mechanisms remain scarce. In this study, we report for the first time the occurrence of a recently described kdr mutation, V410L, in Old World Ae. aegypti from Angola and Madeira island. Two other kdr mutations, V1016I and F1534C, were also identified in populations from Angola and Cape Verde, extending our knowledge about the distribution of these mutations in Africa. We found significant associations between kdr genotypes and the resistance phenotype but only in the Angolan population. These results suggest that populations carrying the same kdr mutations may respond differently to the same insecticide, stressing the need for complementary studies when assessing the impact of kdr resistance in the outcome of insecticide-based vector control.One of the pillars for the prevention of  Aedes aegypti, and for mitigating the impact of these infections on human populations. During an outbreak, insecticide-based vector control interventions are usually intensified to reduce mosquito abundance and interrupt human-vector contact. However, increased use of insecticides may result in the selection of mosquitoes carrying genetic traits associated with insecticide resistance. The emergence of insecticide resistance in natural vector populations can ultimately affect the efficacy of insecticide-based vector control.Vector control has been a mainstay for preventing diseases caused by arboviruses transmitted by kdr) [Ae. aegypti, a total of 10 kdr mutations have been reported [Ae. aegypti populations from Asia, South America and, to a lesser extent, Africa [Target site insensitivity is a major mechanism of insecticide resistance that results from point mutations in genes encoding proteins at the specific site where an insecticide binds, typically in the nervous system . These mkdr) . In Ae. , Africa .kdr mutation, V410L, located in domain I of segment 6 of the VGSC, was described for the first time in a pyrethroid-resistant Ae. aegypti laboratory strain originating from Rio de Janeiro, Brazil [Xenopus oocytes revealed that V410L significantly reduced sensitivity to both type I and type II pyrethroids. Genotyping of natural populations from Northeast Brazil did not reveal the presence of this mutation [Ae. aegypti from Colombia, with frequencies ranging from 0.06 to 0.36 [In 2017, a novel , Brazil . Functiomutation . However to 0.36 .Aedes-borne arboviral infections. Such was the case for a dengue outbreak that occurred in Cape Verde, in 2009, which affected eight of the 10 islands of this archipelago with more than 21,000 cases reported and four deaths [https://www.ncbi.nlm.nih.gov/pubmed/23784016). In addition to the above mentioned outbreaks, Angola and Cape Verde were also affected by Zika outbreaks in 2015 and 2016, and cases of congenital Zika syndrome causing microcephaly were reported in both countries [In recent years, Africa has been experiencing major outbreaks of r deaths . In 2015r deaths . The couca. 680 km east off the coast of Morocco, it belongs administratively to Portugal and the European Union, raising concern about the risk of the introduction of both vectors and viraemic cases to mainland Europe.On Madeira Island, a Dengue outbreak involving 2,168 notified cases occurred in 2012–2013 . AlthougAe. aegypti from the city of Praia, the capital of Cape Verde, revealed resistance to DDT but susceptibility to all other insecticides tested [kdr alleles were detected [Ae. aegypti population of Madeira Island was found to be resistant to all major insecticide classes currently used in public health practices. This population displays multiple mechanisms of resistance, including the presence of V1016I and F1534C kdr mutations at a high frequency [As a response to these outbreaks, vector control programs tend to intensify insecticide-based interventions as a means of rapidly halting arbovirus transmission. This may lead to emergence of insecticide resistance in the local vector populations. Insecticide susceptibility tests carried out in 2009 on s tested . Subsequdetected . The locrequency .Ae. aegypti populations remains scarce [kdr mutation. Considering that this mutation has shown potential to significantly reduce sodium channel sensitivity to both type I and II pyrethroid and to increase resistance in combination with F1534C [Ae. aegypti populations, as its presence may greatly undermine the utility of a wide variety of pyrethroid insecticides, which are currently the major insecticide class used in vector control.In Africa, information on mechanisms of insecticide resistance in s scarce . The pres scarce  and in Bs scarce , althougs scarce , Cape Ves scarce . None ofkdr loci of Ae. aegypti populations from Angola, Cape Verde and Madeira Island, collected either during or after the onset of arboviral outbreaks. The objectives were: i) to determine the presence and frequency of the V410L, V1016I and F1534C mutations and ii) to investigate the association of these mutations with the pyrethroid and DDT resistance phenotype.In this study, we genotyped the In Cape Verde, mosquito collections were carried out from September 2017 to March 2018 in two islands: Santiago and Maio. In Santiago, mosquitoes were sampled in Praia (N 14º 55’ 15” W 23º 30’ 30”), the capital of the country, and São Lourenço dos Órgãos (SLO) (N 15º 6’ W 23º 60’). In Maio, sampling was performed in the main city, Maio Village (N 15º 13’ W 23º 10’). In Angola, collections took place in October-December 2016, in the cities of Huambo (S 12º 46’ E 15º 44’) and the capital, Luanda (S 8º 50’ 18” E 13º 14’ 4”). On Madeira island, mosquitoes were collected in Funchal (N 32º 39’ W 16º 55’) between September and November 2013 [Aedes aegypti surveillance program and their geo-localization is available at http://iasaude.sras.gov-madeira.pt/naomosquito/. In Cape Verde, collections were performed with 40 ovitraps distributed to cover most of the area of each locality surveyed. Collections in Luanda were carried out using 49 ovitraps covering an area of ca. 70 km2 in the Great Luanda region. Larval collections with dips and pipettes were performed in two areas of the city of Huambo, the city centre and one semi-rural area.In all but one of the localities surveyed, mosquito eggs were collected using ovitraps, adapted from the model of Faye & Perry . OvitrapCollected eggs (or larvae) were transported to local laboratory facilities and reared to adults using standard protocols for mosquito rearing .Mosquito samples from Cape Verde (Maio and Santiago islands), Madeira island and from Luanda-Angola were phenotyped for their susceptibility to deltamethrin 0.05% and permethrin 0.75% using WHO test kits and protocols ,22. In Mvgsc gene amplified using the primers Ae410F1 and Ae410R1 described in [2, 0.4 Mm dNTP mix, 100 nM of each primer, 1 unit GoTaq G2 Flexi DNA Polymerase  and 10 ng template DNA. The cycling conditions consisted of denaturation at 94 °C for 2 min, followed by 35 cycles of denaturation at 94 °C for 40 s, primer annealing at 58 °C for 45 s, and extension at 72 °C for 1 min, with a final extension of 10 min at 72 °C. The PCR products were purified using Antarctic phosphatase (NEB) and Exonuclease I (NEB) to remove unincorporated dNTPs and primers. The purified PCR products were diluted 1:30 and subjected to direct sequencing in an ABI Prism 3500XL system at the DNA sequencing platform of the Aggeu Magalhães Institute/FIOCRUZ (Brazil) or in an ABI 3730 XL instrument at the STAB VIDA DNA Sequencing Service . Sequencing was performed in both directions using the primers Ae410F2 and Ae410R1 [2, 0.4 Mm dNTP mix, 1 unit GoTaq G2 Flexi DNA Polymerase  in a total volume of 25 μL. Each PCR assay included positive controls for each homozygous and heterozygous genotypes that had been previously confirmed with direct sequencing and a negative control (no DNA template). PCR products were visualized in a agarose gel 4% stained with Greensafe Premium  by transillumination under UV . Pairwise estimates of linkage disequilibrium coefficients and associated chi-squared tests between loci were calculated using LINKDOS [To assess the association between  LINKDOS , followi LINKDOS ,27.Ae. aegypti were genotyped, 91 of which were from Madeira Island, 168 from Cape Verde  and 49 from the island of Maio) and 95 from Angola (67 from Luanda and 28 from Huambo). Of the total sample, mutation V410L was screened by direct sequencing in 91 individuals from Madeira, 67 from Cape Verde and 95 from Angola. Sequences obtained from Ae. aegypti mosquitoes from Madeira are available in GenBank with accession numbers MK656133-MK656223. Apart from the V410L, no other genetic polymorphism was detected in the 104 bp fragment analysed for Madeira population. The genotype and allele frequencies of each kdr mutation are shown in In total, 314 Pairwise linkage disequilibrium analysis revealed significant genotypic associations between positions 1016 and 410 in all populations tested . For poset al. [Mortality rates for the insecticides tested varied between regions, being highest in the populations of Cape Verde islands (89.9%-100.0%) and lowest in Luanda-Angola (2.6%-7.4%). These results are detailed in et al. .kdr mutations and the resistant phenotype. In Luanda, there were significant associations between resistance phenotypes and genotypic frequencies for mutations V1016I and V410L, but not for F1534C  so thatkdr mutations were also found in Ae. aegypti from two outer West African archipelagos, Madeira and Cape Verde. In Cape Verde, mutation V410L was not detected whereas mutations V1016I and F1534C were found at low frequency (≤ 3.0%) in Santiago, the main island of the archipelago. Previous analysis of samples collected in the same island in 2007, 2010 and 2012 did not reveal the presence of any of these mutations [kdr mutations in this island. Whether these mutations represent introductions or independent mutation events is unknown. A recent phylogeographic study suggests that Ae. aegypti from Cape Verde originated from populations of Senegal [kdr mutations from neighbouring regions of mainland West Africa, specifically of mutation F1534C found in the city of Praia, where an international airport and port are located. On the other hand, the detection of the V1016I mutation in the remote inland locality of São Lourenço dos Órgãos argues in favour of an independent mutation event. The recent emergence of kdr mutations in Cape Verde may also explain the low frequency of resistant-associated alleles despite increased insecticide pressures imposed by vector control since the Dengue epidemic of 2009 [In addition to mainland Africa, utations ,29. This of 2009 .kdr mutations analysed were present in Madeira Island. One of these mutations, F1534C, was fixed while the others displayed moderate frequencies. These kdr frequencies do not seem to be fully explained by insecticide pressures, given that vector control in Madeira has been predominately based on the elimination of mosquito breeding sites. A more plausible explanation is that kdr frequencies reflect those of the source populations that colonized Madeira Island [Ae. aegypti was recently introduced into Madeira Island from South-American source populations, notably from Venezuela [Ae. aegypti populations displaying high frequency of the F1534C and varying levels of the V1016I mutation [In contrast to Cape Verde, the three a Island . Phylogeenezuela . This remutation .kdr alleles and the resistant phenotype to deltamethrin. This was evident for tri-locus genotypes and individually for mutations V410L and V1016I. The lack of association of mutation F1534C with resistance is consistent with previous observations suggesting that this mutation alone confers low levels of resistance but co-evolved with V1016I yielding higher levels of resistance [kdr survey in Ae. aegypti from Angola, so that no historical data are available for comparison. In Angola, insecticides have been widely used for vector control since the DDT-based malaria eradication campaigns implemented in the 1950s [In Luanda, Angola, significant associations were found between resistant-associated sistance ,33. Linkhe 1950s ,36.Rij) found in Luanda (0.07–0.33) were generally much lower than those obtained in Mexico , which kdr alleles and pyrethroid resistance in Madeira island. This result was unexpected but it probably reflects the complex pattern of multiple insecticide resistance mechanisms present in the local Ae. aegypti population [kdr, a recessive trait, microarray-based gene expression analysis provided evidence for metabolic and cuticular resistance mechanisms, which may disrupt the statistical association between phenotypes and kdr genotypes. Also noteworthy is that the highest linkage coefficient between V410L and V1016I was found in Madeira island, a result that agrees with the hypothesis of these mutations being introduced in the island from a few colonizing individuals already carrying multiple resistance associated alleles [In contrast with the observations in Luanda and Mexico, there were no associations between resistance associated pulation . In addi alleles .kdr mutation in populations of Ae. aegypti from the old world for the first time. As also observed in South America, this mutation appears to coevolve with V1016I providing substantial higher levels of resistance to pyrethroid in the population of Angola. However, this was not the case of Madeira island, where association between kdr and pyrethroid resistance is probably disrupted by the coexistence of multiple resistance mechanisms. These findings suggest that populations carrying kdr mutations may respond differently to pyrethroid. Further studies will be required to assess the real impact of kdr mechanisms in the outcome of insecticide-based control of Ae. aegypti.In summary, we report the occurrence of the V410L S1 Table(DOCX)Click here for additional data file.


Takabuti, was a female who lived in ancient Egypt during the 25th Dynasty, c.660 BCE. Her mummified remains were brought to Belfast, Northern Ireland, in 1834 and are currently displayed in the Ulster Museum. To gain insight into Takabuti’s ancestry, we used deep sampling of vertebral bone, under X-ray control, to obtain non-contaminated bone tissue from which we extracted ancient DNA (aDNA) using established protocols. We targeted the maternally inherited mitochondrial DNA (mtDNA), known to be highly informative for human ancestry, and identified 38 single nucleotide variants using next generation sequencing. The specific combination of these SNVs suggests that Takabuti belonged to mitochondrial haplogroup H4a1. Neither H4 nor H4a1 have been reported in ancient Egyptian samples, prior to this study. The modern distribution of H4a1 is rare and sporadic and has been identified in areas including the Canary Islands, southern Iberia and the Lebanon. H4a1 has also been reported in ancient samples from Bell Beaker and Unetice contexts in Germany, as well as Bronze Age Bulgaria. We believe that this is an important finding because first, it adds to the depth of knowledge about the distribution of the H4a1 haplogroup in existing mtDNA, thus creating a baseline for future occurrences of this haplogroup in ancient Egyptian remains. Second, it is of great importance for archaeological sciences, since a predominantly European haplogroup has been identified in an Egyptian individual in Southern Egypt, prior to the Roman and Greek influx (332BCE). Translation of the inscriptions on her coffin identify her as the daughter of Nespare, a priest of Amun at Thebes, and his wife Taseniret.Takabuti lived in Thebes, Egypt, during the 25th Dynasty (c. 660 BCE). Her mummified remains and the coffin containing them were brought to Belfast, Northern Ireland, in 1834 and are currently displayed in the Ulster Museum  with exceptionally well-preserved dentition from which an age at death of 25–30 years was estimated. The decayed bandages, which had likely been dipped in resin, had covered the entire body including the head, arms, legs and feet .Four days after examination, the body was rewrapped in Irish linen, leaving the head, one arm and one foot exposed. More comprehensive studies of the mummy, carried out by X-ray imaging in 1987 and computerized tomography (CT) scanning in 2006, confirmed the age of 25–30 years at time of death and revealed a healthy individual with no evidence of serious childhood illness.1, DNA degradation in the Egyptian climate once limited the usefulness of this approach on mummified individuals2. Next generation sequencing, preceded by targeted hybridization capture can be used to enrich for particular components of the human genome3. Such techniques, have changed how DNA data can be obtained, providing rich analytical data from which sex4 , kinship5 and population affinity6 can be inferred. Using established hybridization capture methods and next generation sequencing we identified the mitochondrial haplotype of Takabuti and compare her haplogroup with the mitochondrial haplogroups previously identified in ancient Egyptian mummies.Although mitochondrial DNA (mtDNA) sequencing can be used to study population affinities of archaeological human remainsThe three bone biopsies were combined in one tube, and generated bone powder of 50 mg in total. Sample was then subjected to DNA extraction and library preparation followed by two rounds of in-solution hybridization capture directed at the mtDNA NC_012920.1) and sequenced using Illumina technology. The library prepared after the first round of capture gave 18,903,330 sequence reads, of which 81,384 were initially mapped to the mtDNA . This work was performed on bone tissue removed by minimally invasive techniques necessitating of only milligram quantities of bone and which minimised contamination. We therefore have confidence that the haplogroup we report for Takabuti is correct and not affected by modern contamination.14. The H4a1 variant possessed by Takabuti is relatively rare with a modern distribution including ~ 2% of a southern Iberian population15 , ~ 1% in a Lebanese population12 and ~ 1.5% of multiple Canary Island populations13.When interpreting our findings within the broader genetic record, the H super-haplogroup is the most common mtDNA lineage in Europe and is found also in parts of present-day Africa and western Asia10 , and one individual from early Bronze Age Bulgaria15 illustrating both the rare occurrence and sporadic distribution of this haplogroup.Until our work neither H4 nor H4a1 has been reported in ancient Egyptian samples. However, in the archaeological record H4a1 has been reported in sixth–fourteenth century CE remains sourced from the Canary Islands, and three additional ancient DNA samples, two from Bell Beaker and Unetice contexts (2500–1575 BCE) at Quedlinburg and Eulau, both in Saxony-Anhalt, Germany17. Superimposed on this are individuals like Takabuti with rare haplogroups, which have not been previously identified in the background.The overall perspective from an examination of 97 samples from ancient Egypt with a mitochondrial haplogroup Fig.  is of a Perhaps the most intriguing aspect of our findings, which is of great archaeological interest and importance, is the observation of a predominantly European haplogroup in an Egyptian individual located in Southern Egypt. What is fascinating is that the individual pre-dates the Roman and Greek influx (332BCE). At face value, the current genetic evidence suggests a high degree of isolation from migration into Southern Egypt. However, this finding challenges that assertion, suggesting that further investigative work could be carried out to gain a better understanding of the genetic makeup of ancient Southern Egypt.5, pushing back the earliest observation of this haplogroup in Egyptian mummies by 500 years. Similarly, a study by Loreille et al., 20184 pushed back the recorded chronology of the U haplogroup in ancient Egypt by almost 1000 years. Therefore, single-case studies add to existing knowledge in this field, challenging and updating our current understanding.The simplified representation of mitochondrial haplogroups in ancient Egypt in Fig. 6 and the importance of those findings to better understand and interpret ancient Egyptian populations.Our results add to the growing body of reports demonstrating the utility of hybridization capture as a means of obtaining authentic ancient DNA sequences from Egyptian mummiesSampling was performed at the Ulster Museum in the gallery where the mummy is on display, using strict anti-contamination controls. Following isolation of the display area, the mummy was placed on a medium-density fibreboard supported by trestles that were constructed in situ for the purpose of sampling Fig. . Personn−2 for 2 × 5 min, with 180° rotation between exposure) before use. Personal protective equipment included Tyvek forensic suits, face masks, hair nets, goggles, boot covers and two pairs of sterile gloves. DNA extraction was carried out in a Class II biological safety cabinet, and sequencing libraries and polymerase chain reaction (PCR) mixes were prepared in a laminar flow cabinet. DNA extraction was accompanied by a DNA-free negative control  followed by a DNA-free PCR negative control and a library preparation negative control, the latter controls set up with water rather than DNA extract5. To test for potential contamination during sampling and DNA processing, mouth swabs were taken from all individuals present during the tissue sampling and from individuals working in the Manchester ancient DNA labs, and these samples were anonymized and the mtDNA for each sample was typed. Samples were obtained by informed consent and all steps in this process were performed in accordance with the University of Manchester ethics regulations. Institutional governance check confirms that the work was conducted in accordance with the University of Manchester policy.Samples were transferred to the Manchester Institute of Biotechnology where DNA extraction and sequencing library preparation were performed in a set of physically isolated, restricted access laboratories, each equipped with an ultrafiltered air supply system maintaining positive displacement pressure. The laboratories were periodically sterilized by UV irradiation when not in use. All surfaces were cleaned with 5% sodium hypochlorite solution and 70% ethanol, and all utensils and equipment such as pipettes were treated with DNA-Away before and after use. Consumables such as tubes were UV irradiated  N-lauroylsarcosine to the extraction buffer and 5 M NaOAc and 5 M NaCl to the PB buffer20.DNA was extracted from 50 mg of bone powder  following the procedure of Dabney21 directed at the mtDNA hypervariable region I (HVRI) loci  to assess DNA preservation and endogenous DNA content, as well as a multiplex PCR using previously established primers22 targeting the amelogenin locus (NC_000023.11:g.11314994_11315100 (chrX) and NC_000024.10:g.6738028_6738138 (chrY)) to confirm the sex of the mummy  and sequenced from both ends by the Sanger method . Sequences were mapped to the rCRS using Geneious v8.1.823. The amelogenin test failed probably due to poor preservation, whereas preliminary PCRs showed no variations.Preliminary analyses involved two overlapping PCRs25 including one library and one extraction negative control. Only the sample library was enriched twice by in-solution hybridization capture (Arbor Biosciences) according to the manufacturer's instructions for degraded samples, using a baitset covering the entire mitochondrial genome. A total of 12 and 15 cycles of post-capture PCR was performed with the enriched products using the IS5 (5′-AATGATACGGCGACCACCGA-3′) and IS6 (5′-CAAGCAGAAGACGGCATACGA-3′) primers. Libraries (one enriched and two non-enriched) were quantified by qPCR (Roche LightCycler 480) and fluorimetry (Qubit 2.0), and their length distribution assessed using a bioanalyzer (Agilent). The concentration of the extraction and library controls were undetectable and therefore were not enriched or sequenced. The remaining library was then purified using the MinElute Purification kit (Qiagen) and both enrichment rounds sequenced using the MiSeq Reagent kit (v3) 2×75. Sequence data are curated at the European Nucleotide Archive under project accession number PRJEB38492.Three dual-indexed NGS libraries were prepared26 specifying the following parameters: trimns, minlength 25, trimqualities, minquality 30, collapse. Reads with overlaps of at least 11 bp were collapsed into single sequences, whereas reads that did not overlap were processed separately using BEDTools v2.26.027 and only confirmed forward and reversed pairs retained. Mapping was performed with BWA v.0.7.5a-r40528 using default aln, samse and sampe commands with the exception of the l1024 parameter which is suggested for ancient DNA data. The dataset was mapped against the rCRS reference genome29. Subsequently, the sam files were cleaned and sorted by coordinate system and converted to bam format using default CleanSam and Sortsam commands from Picard Tools v2.18.27 (https://broadinstitute.github.io/picard). Mapped reads were extracted with Samtools v1.230 using the parameters b, q30, and F4. Removal of duplicate sequences was performed with the script aweSAM collapser (https://gist.github.com/jakeenk/). Single nucleotide variations (SNVs) were called using HaplotypeCaller from GATK v.431 and validated using Geneious v.R823. The depth of SNV coverage was set at DP > 4 and only those SNVs with a 100% read coverage were retained. Mitochondrial DNA haplogroup assignment was performed using Haplogrep32 sand data authenticity was assessed with MapDamage v2.033. BLAST34 searches were carried out online (https://blast.ncbi.nlm.nih.gov/Blast.cgi).Raw data were demultiplexed using BBmap v38.07 (sourceforge.net/projects/bbmap/) and adapter sequences removed using AdapterRemoval v2.1


Anterior segment optical coherence tomography (AS OCT) is a helpful tool used to diagnose and manage many corneal conditions, but its use has not been reported in case of peripheral ulcerative keratitis (PUK). The aim of this study is to describe AS OCT findings in cases of PUK.Retrospective observational case series of six eyes presenting with a PUK and proven systemic vasculitis. Clinical course, slit lamp photographs, and AS OCT findings were the main outcomes.P = 0.03).The AS OCT findings were found to correlate with the ocular disease’s level of activity. In the acute stage, an absence of corneal epithelium, a scrambled appearance of the anterior stroma and a heterogeneous stromal reflectivity were observed. During the reduction of disease level activity, an irregular hyporeflective epithelium, a smoother anterior stroma, and a homogenous hyperreflective stroma were seen. At the healed stage, a filling of the corneal defect by a hyporeflective thick epithelium, the persistence of the hyperreflective underlying stroma, and a demarcation line were observed. The mean total corneal thickness at last follow-up was significantly thicker (509 ± 147 μm) compared with the mean corneal thickness at onset (408 ± 131 μm; AS OCT provides an assessment of structural changes occurring in PUK, useful for its diagnosis and monitoring. Peripheral ulcerative keratitis (PUK) is a rare form of corneal inflammation leading to corneal ulceration and stromal destruction. Its incidence is reported as three cases per million per year . The mosAnterior segment optical coherence tomography (AS OCT) is a relatively new imaging modality providing a transformative shift in the imaging field towards a better evaluation, diagnosis and management of many anterior segment diseases , 9. The This retrospective descriptive monocentric case series included patients who presented with a PUK between December 2011 and October 2018 to the tertiary Ophthalmology Department at Cochin Hospital in Paris, France. At baseline, the diagnosis was made at slit lamp examination by two cornea specialists  according to the commonly applied definition of PUK, i.e. the presence of a crescent-shaped destructive inflammatory process of the corneal stroma within the peri-limbic area, associated with an epithelial defect, the presence of stromal inflammatory cells and stromal keratolysis . AssociaData collected at baseline, 1 week, 1 month and 3 months were demographics, best corrected distance visual acuity (CDVA) with Snellen chart, slit lamp examination with fluorescein staining, anterior segment photography, and AS OCT. The clinical evaluation of corneal healing as a main result of treatment efficacy was based on non-progression of the corneal thinning, filling of the ulcer crater with negative fluorescein staining and a decrease of pain and ocular discharge. Recurrence or unresponsiveness to treatment were defined as persistent pain and ocular discharge, progression of the corneal thinning, and persistent fluorescein staining. Using AS OCT, the disease activity was monitored by the evaluation of the corneal thickness at the thinnest point and structural changes of the corneal ulcer. Specific anatomic features observed on AS OCT at each stage of the disease were outlined. In parallel, detailed medical history including previous systemic disease, and treatment were recorded. Physical examination along with complete serologic evaluation were performed at baseline to detect an underlying infectious or auto-immune condition.ImageJ software 1.52 version . The measurements were performed at the thinnest point at onset. The caliper was placed perpendicular to the corneal endothelium. At last follow-up, the measurements were made at the same location, following the same protocol. Visual verification was performed on the infrared image provided by the AS OCT device by the cornea specialist (CB) who performed the measurement, to ensure that the same location was used at last follow-up.Slit lamp photographs of the cornea were performed using the Canon 20D camera with a Haag-Streit flash unit for anterior segment photography (acquisition software: Eye Cap v7) and AS OCT was performed with the anterior segment module of Spectralis OCT . The wavelength was 870 nm, the A-line rate was 24 kHz with an axial resolution of 7 μm. At each time-point, 5 to 10 horizontal scans of 12-mm were acquired at a rate of 40 frames per scan . The scaVisual acuities were converted to LogMAR for statistical analysis. Wilcoxon paired test was used for continuous data comparison. XLSTAT Addinsoft, version 2018.55292  statistical and data analysis was used to perform the analysis.2) pulses were added until healing of PUK. Topical therapy consisted of steroids drops  three times a day tapered for 3 months, antibiotic drops (azithromycin dihydrate 15 mg/g) two times a day for 3 months and artificial tears  as needed. All patients were treated medically, no significant change in CDVA (P = 0.18) was noted and no cases of perforation or death were observed.A total of six eyes of six patients including four women (66.7%) and 2 men (33.3%) with a mean age at diagnosis of 50.3 ± 14.7 years (range 33 to 74) and a mean follow-up of 4.4 ± 3.8 months were analyzed. The Table Figure P = 0.03) (Table P = 0.62). The epithelial thickness was often not measurable at onset given the epithelium was absent due to an epithelial defect when measured at the thinnest point.In all cases, thinning of the cornea was visible with achievable objective measurements of the remaining posterior stroma Fig. . The mea3) Table . There wAt the initial evaluation, AS OCT findings of active disease were: 1) The epithelial layer was not visible, 2) The surface of the anterior stroma was irregular with a scrambled appearance, 3) The reflectivity of the underlying stroma was variable - iso, hyper or hyporeflective, disorganized and heterogenous. After an initial intense systemic therapy, between day 7 and 21, AS OCT findings showed healing patterns: 1) The epithelial layer was irregular and hyporeflective, 2) The anterior stroma was smoother with a resolution of the scrambled appearance, 3) The reflectivity of the underlying stroma was more homogenous and hyperreflective. After 1 month, the AS OCT findings showed healed patterns: 1) The epithelial layer was hyporeflective and thicker, filling the area of corneal thinning, 2) The underlying stroma was hyperreflective and regular, 3) A demarcation line between the healed and unaffected stroma was visible (Fig. In three cases, the same AS OCT findings than the one seen during the initial active stage were retrieved during the follow-up, and used to diagnose a recurrence of the PUK: an epithelial defect, an irregular scrambled anterior stromal surface, a heterogenous reflectivity of the remaining stroma and a progressive corneal thinning. After treatment intensification, the same AS OCT findings than the one seen at healed stage were retrieved: a thick intact epithelium, hyperreflectivity of the remaining stroma and a demarcation line Fig. , confirmThe gold standard to diagnose PUK is currently with slit lamp biomicroscopy . Our stuOther peripheral corneal lesions, infectious or inflammatory, can mimic a PUK, and AS OCT has been shown to be helpful in allowing an accurate diagnosis. For example, its usefulness in the setting of infectious keratitis has been described, with specific AS OCT features such as retrocorneal plaques and stromal necrosis , 14. ThiIn our series the diagnosis of PUK was certain as laboratory work-up found a systemic vasculitis in all cases, whereas classically 50% of PUK cases have an associated collagen vascular disease . Our finAS OCT has become an important tool in the evaluation and management of many corneal and anterior segment diseases, allowing a detailed evaluation in a non-contact and safe way . In caseUsing these imaging techniques, a better understanding of the pathophysiology of PUK can be achieved. The mechanisms of keratolysis are complex and currently poorly understood. The peripheral cornea and limbus reside close to conjunctival blood vessels and lymphatic channels, and with more Langherans cells and C1 components than the central cornea, are an ideal area for immune complex deposition as signs of active collagen vascular disease . These dConclusions that can be drawn are limited due to the retrospective data and small sample size. Thickness measurements were made manually and the reproducibility of the measurements between examinations were visually assessed. In the future, the use of AS OCT devices implemented with eye-tracking and automated epithelial and corneal thickness mapping softwares could help define its place and identify severity criteria and risk factors of recurrences. AS OCT allowed visualization of morphologic changes, but the sensitivity and specificity of AS OCT in the diagnosis and management of PUK remain to be established.We have shown AS OCT signs of PUK. The use of AS OCT was helpful in monitoring the disease activity through corneal thickness evolution and recognizing specific patterns. It allowed for an early diagnosis of recurrences with subsequent successful modification of treatment. This highlight the utility of AS OCT as an adjunctive modality that can provide non-invasive imaging guidelines for the diagnosis and management of PUK.


Oryza sativa L.) is the major staple food. Therefore, increasing concentrations of these micronutrients in rice grain represents a sustainable solution to hidden hunger. This study aimed at enhancing concentration of Zn, I, Fe and Se in rice grains by agronomic biofortification. We evaluated effects of foliar application of Zn, I, Fe and Se on grain yield and grain concentration of these micronutrients in rice grown at 21 field sites during 2015 to 2017 in Brazil, China, India, Pakistan and Thailand. Experimental treatments were: (i) local control (LC); (ii) foliar Zn; (iii) foliar I; and (iv) foliar micronutrient cocktail . Foliar-applied Zn, I, Fe or Se did not affect rice grain yield. However, brown rice Zn increased with foliar Zn and micronutrient cocktail treatments at all except three field sites. On average, brown rice Zn increased from 21.4 mg kg–1 to 28.1 mg kg–1 with the application of Zn alone and to 26.8 mg kg–1 with the micronutrient cocktail solution. Brown rice I showed particular enhancements and increased from 11 μg kg–1 to 204 μg kg–1 with the application of I alone and to 181 μg kg–1 with the cocktail. Grain Se also responded very positively to foliar spray of micronutrients and increased from 95 to 380 μg kg–1. By contrast, grain Fe was increased by the same cocktail spray at only two sites. There was no relationship between soil extractable concentrations of these micronutrients with their grain concentrations. The results demonstrate that irrespective of the rice cultivars used and the diverse soil conditions existing in five major rice-producing countries, the foliar application of the micronutrient cocktail solution was highly effective in increasing grain Zn, I and Se. Adoption of this agronomic practice in the target countries would contribute significantly to the daily micronutrient intake and alleviation of micronutrient malnutrition in human populations.Widespread malnutrition of zinc (Zn), iodine (I), iron (Fe) and selenium (Se), known as hidden hunger, represents a predominant cause of several health complications in human populations where rice ( Globally, an estimated 2 billion people suffer from micronutrient malnutrition , especially in the developing world. Particular micronutrient deficiencies include Fe, Zn, Se and I which result in severe health complications  as well Iron deficiency has been suggested to be the most common micronutrient disorder in human populations, and is often associated with anemia . The mosDespite the use of iodized salt since decades, human I deficiency has been reported to be still prevalent in a number of countries. An estimated 2 billion people are at risk of I deficiency . Even, sLow availability of soil micronutrients to crop plants and consequent inadequate dietary intake of micronutrients have been discussed as the root cause for the high incidence of micronutrient deficiencies in humans . AccordiAmong the approaches for enrichment  of micronutrient in staple foods, genetic  and agronomic  approaches are used widely. After long-term successful efforts, the International HarvestPlus program has developed and released new Zn- or Fe-biofortified wheat and rice varieties containing additional Zn or Fe, at about 10 mg per kg grain . Also, mThe contribution of root absorption and root-to-grain transportation of Zn and Fe during the grain filling stage is minimal under field conditions compared to the greenhouse conditions with always high amount of available micronutrients and optimal water . This inIn the present study, we aimed to investigate the foliar applications of Zn, I, Fe and Se alone or together in a cocktail solution on the grain concentrations of these micronutrients in rice grown under different soil and crop management conditions of five major rice growing countries over two years by using seven different rice cultivars. Indeed, there are many publications about the effect of soil and/or foliar applications of micronutrients, usually alone or 2 combined elements, on grain concentration of micronutrients in food crops . To the A field experiment on rice was conducted over two cropping seasons in 2015 to 2017 at a total of 21 field sites of five countries, i.e., Brazil, China, India, Pakistan and Thailand. Except for Brazil, where rice was cultivated under upland conditions, in all other locations rice was grown under flooded conditions with 10 to 20 cm standing water above the soil surface for most of the growing season. The rice cultivars grown at various field sites were commonly cultivated in the respective countries . Initial4⋅7H2O in spray solution); iii) local control + foliar application of I (0.05% KIO3 in spray solution); and iv) local control + simultaneous foliar spray of micronutrients  in a cocktail solution containing 0.5% ZnSO4⋅7H2O + 0.05% KIO3 + 0.02% Fe-EDTA + 0.001% Na2SeO4. The field experiment was laid out in a randomized complete block design with four replications. The foliar solution of each treatment, varying between 500 to 800 L ha–1, was applied twice, i.e., at the panicle initiation (about one week prior to heading) and the early grain milk stages. The foliar treatments were applied at late afternoon time until the solution started to run-off from the leaves as described by 2 central area of each plot.The experiment comprised four treatments: i) local control ; ii) local control + foliar application of Zn  for the determination of Zn, Fe and Se. For I analysis, brown rice samples were extracted in TMAH at 90 °C using a closed-vessel microwave reaction system  as described earlier by Subsamples of brown rice  from each plot were secured for analysis of Zn, I, Fe and Se. Brown rice samples were rinsed and washed with distilled de-ionized (DDI) water and dried first with tissue papers and then at about 45 °C in a forced-draft oven to constant weight. Dried brown rice grains were digested in HNOThe significance of the effects of different foliar treatments on the dependent variables was determined using one-factor ANOVA at 0.05 probability level of least significant difference (LSD) test using SAS software . For overall effectiveness, the data sets across locations and years were compared by the paired t test using SPSS 13.0 for Windows. The linear models were used to evaluate correlations among various parameters using MS Excel.–1 at Lambari location of Brazil in 2017 to 11.46 Mg ha–1 at Jiangsu location of China in 2015  yields varied drastically across 21 field sites across 14 locations in five countries. For example, in case of the local control treatment , rice grain yield varied from 1.94 Mg ha in 2015 . Foliar reatment .–1 at the Ludhiana field site of India in 2017 to 26.9 mg kg–1 at CMU field site of Thailand in 2015 (P < 0.05), except for at Ludhiana field site of India in both years and at Gurdaspur field site of India in 2016 ; the overall magnitude of increase in grain Se concentration was 4-fold .The relationships between soil extractable micronutrient concentrations and their grain concentrations are presented in –1 and 14 to 176 mg kg–1, respectively . This result clearly indicates that when applied together with three other micronutrients, the leaf absorption and transportation of Zn in the grain are not seriously affected by the other micronutrients present in the same spray solution. Foliar application of KIO3 alone had no effect on rice grain Zn and even tended to increase grain Zn  and was observed with the application of I alone or in combination with other micronutrients in the cocktail. There were no clear antagonistic effects of other micronutrients on grain I accumulation when sprayed together . On average, the increase in grain Se with foliar cocktail treatment was 4-fold  and two foliar sprays (@ 600 L cocktail solution ha–1) the amount sprayed was about 5 g Se ha–1 which resulted in marked increases in grain Se. As rice is a predominant staple cereal in many countries of the world, especially in developing countries, Se-enriched rice through the agronomic biofortification could be a sustainable Se source for the human populations living in Se-deficient regions.The foliar application of the micronutrient cocktail solution was also effective in increasing grain Se concentration at all field sites in five countries . By applying the same micronutrient cocktail solution used in the present study, In contrast to Zn, I and Se, foliar spray of the micronutrient cocktail solution did not affect grain Fe concentration compared with the local control treatment, except for one field site in Brazil, i.e., at Lambari in 2016 (White rice (polished rice) represents the common form for human consumption; however, its nutritional value is lower than brown rice due to loss of significant amount of minerals during polishing, especially micronutrients such as Fe . TherefoFoliar application of the cocktail micronutrient solution resulted in significant enrichments of Zn, I and Se in brown rice grains under a wide range of environmental and soil conditions of five major rice producing countries. Based on the data published by Statista GmbH the studied five countries produce almost 300 million metric tons of milled rice which corresponds to about 60% of world production of milled rice per year . Thus, aThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose: To determine the effect of different types of Rigid Gas Permeable (RGP) contact lenses on ocular aberrations in patients with keratoconus.Methods: Eighteen eyes of young patients with mild to moderate keratoconus were selected. General ocular examinations such as refraction, visual acuity, and ocular aberrations were performed. Three types of RGP contact lenses, i.e., Boston, Senso Select and Wohlk, were fitted using the cross over method. Repeated measures analysis of variance and Mauchly’s test of sphericity were used to compare the average of residual high order aberrations and visual acuity after fitting each type of lens.Results: Vertical coma was -0.271 ± 0.37 µm before fitting and decreased to 0.081 ± 0.08 µm with Boston, 0.098 ± 0.08 µm with Senso Select and 0.124 ± 0.08 µm with Wohlk contact lens . The mean RMS (root mean square) for high order aberrations decreased from 0.526 ± 0.43 µm before fitting to 0.256 ± 0.09 µm with Boston, 0.263 ± 0.12 µm with Senso Select, and 0.304 ± 0.10 µm with Wohlk contact lens . The mean RMS for low order aberrations decreased from 1.480 ± 0.78 µm before fitting to 0.703 ± 0.43 µm with Boston, 0.802 ± 0.39 µm with Senso Select, and 0.760 ± 0.45µm with Wohlk . Conclusion: Despite achieving optimal fit and good visual acuity with these different RGP lenses, in keratoconus patients, their performance is different in reducing ocular aberrations. The estimated prevalence of keratoconus in the Caucasian population is about 1/ 2000 [2]. In a study conducted on the Iranian population selected from Tehran citizens, the prevalence of keratoconus has been estimated at 0.8% [3]. Several studies have reported that in eyes with keratoconus, irregular astigmatism, corneal and ocular aberrations individually are larger than in normal eyes . Due to the localization of conical protrusion of thinned cornea, out of the high order aberrations, third order vertical coma is in negative sign and it is significantly more than in normal eyes [1]. Since the quality of retinal images depends on the ocular aberrations, reduction of visual performance in affected patients is clearly described by large magnitudes of high order aberrations. Therefore, reducing ocular aberrations can improve the visual quality in these patients [6]. Keratoconus is a kind of inflammatory and progressive corneal dystrophy that is usually bilateral and asymmetric. This condition usually leads to high amount of myopic astigmatism [7]. Contact lens fitting remains the basic option for the correction of high amounts of induced irregular astigmatism by keratoconus and could be an appropriate alternative to surgical intervention in moderate cases . There are different methods of keratoconus management according to the intensity of disease progression. Generally, spectacle correction is recommended in early stages, contact lenses are used in mild to moderate stages, and surgery is indicated in severe stages . RGP contact lenses are currently the most appropriate option for the correction of high order aberrations like spherical aberration, coma, curvature of field, distortion, trefoil, tetra foil and secondary astigmatism induced by keratoconus . The most successful lenses are still the corneal rigid gas permeable (RGP) contact lenses . Different types of RGP contact lenses, manufacturing and fitting methodologies are the most important factors to choose the suitable contact lenses. Corneal specifications in central and paracentral zones, contact lens design, material, and interactions between the contact lens and the anterior corneal surface are some of the important parameters that influence an optimal fitting. Because of poor centration of the contact lens, corneal scar and staining, and reduced visual acuity are some complications of suboptimal fitting of corneal warpage . Currently, different types of RGP contact lens design, containing multicurve and aspheric with unique and variable asphericity in surfaces impose some difficulties regarding the achievement of appropriate fit between practitioners . The purpose of this study was to determine the impact of three types of RGP contact lenses commonly used by Iranian practitioners, i.e., the Boston, Senso Select, and Wohlk, on visual acuity, low and high order aberration, vertical coma, and secondary corneal astigmatism.It has been suggested that contact lens design is the first and the most effective factor in achieving optimal fitting [10]. The reason for choosing mild to moderate keratoconus is that in advanced keratoconus with corneal scars and deformation, corneal contact lenses do not provide acceptable visual acuity and may be intolerable for some patients; therefore, in these cases, more specific designs of contact lenses, like scleral lenses, are recommended [16]. The inclusion criteria were a best corrected visual acuity of 20/ 40 or better, no history of ocular diseases including cataract, corneal scarring, corneal grafts, corneal collagen cross linking, and other inflammatory or infectious ophthalmic disorders except for keratoconus. In this cross-over before-after study, 18 eyes of 11 patients aged 18 to 35 years, with mild to moderate keratoconus, were investigated. The patients were examined by a corneal specialist in the Eye Hospital and were referred to the contact lens clinic with a diagnosis of keratoconus after clinical and paraclinical examinations. The diagnoses of keratoconus were confirmed through imaging modalities. Keratoconus diagnosing was performed according to keratoconus severity score described by CLEK study . Maximum centration, suitable movement and adequate tear film interchange were evaluated by the fluorescein pattern on slit lamp examinations with cobalt blue light.The maximum time to stabilization for each RGP contact lens was approximately 10-15 min. Then, subjective over-refraction and aberrometry measurements were performed [17]. For the corneal curvature to resume its normal shape, 5-10 min was spared after each RGP lens removal [6].Optimal fitting characteristics were verified by the three-point touch fitting method including mild touch on the apex of the cone and two other touch areas on the corneal mid periphery . Although RGP lenses are still the best choice to improve visual performance in patients with moderate keratoconus, different available designs of RGP contact lenses and their proficiency of centration in proportion with corneal topography is challenging [18]. Our goal in this study was to show that each RGP contact lens reduced aberrations by different mechanisms. In this investigation, RGP lenses were used in a group of young patients with mild to moderate keratoconus because previous studies reported that the disease progresses in young adults. Therefore, proper prescription is essential in these patients to achieve an appropriate visual acuity and to improve their quality of life. Likewise, in early stages of keratoconus, the manifest refraction of these patients shows an increase in both myopia and irregular astigmatism, commonly ranging between 2.00 and 8.00 D, with absence of parallelism of keratometry mires with a mean corneal power under 52.00 D  and design of posterior surface of RGP lenses have been mentioned in providing good centration [20].In this study, the three RGP contact lenses changed coma aberrations, especially vertical coma, in such a way that the direction of vertical coma changed from negative to positive. These directional changes could have influenced the reduction in total HOA RMS, especially in patients with negative vertical coma. These findings are similar to those were reported previously by Choi et al. . The differences found between three RGP contact lenses can be due to some factors such as corneal centration, the ratio of the base curve radius in relation to the diameter, and tear film volume. Therefore, a suitable lens design also provides optimum fit, reduces residual aberrations, and improves visual acuity [Table 3, the subsequent visual loss of keratoconus was corrected with different mechanisms by RGP contact lenses: The Senso Select contact lens showed the best performance in reducing secondary corneal astigmatism in comparison with two other lenses, whereas, it was not successful in reducing low order aberrations. The Wohlk contact lens reduced all parameters to a moderate amount, and the Boston contact lens reduced low and high order aberrations and vertical coma maximally, but it did not reduce secondary corneal astigmatism compared to the Senso Select. Unlike whatever mentioned about aberration changes, the differences in aberrations reduction were not significant between these three types of RGP contact lenses. As presented in Table 2).The result of this study showed that the three RGP lenses, i.e., the Boston, Senso Select, and Wohlk, improved visual acuity after optimum fit. The differences in visual acuity improvement were not significant. The mean base curves were different for optimal fit in these RGP lenses that reflect the effect of the design of these contact lenses (One of the limitations of this study was that in spite of extensive correspondence and search, the details of these contact lenses were not available, because the details of the lens design are privileged information. In this regard, this study focused on clinical outcomes of each RGP contact lens. A strength of this study was that it compared different clinical parameters pre and post fitting with three RGP contact lenses. Furthermore, each keratoconus patient was fitted with three RGP lenses and compared to him/ herself. Table 3, the performance of these three types of RGP contact lenses in improving the visual acuity was the same and they had the same function in terms of correcting aberrations. In summary, our results indicated that although the design of these three types of RGP contact lenses are different, all of them improved visual performance by reducing ocular aberrations and there is no preference in selecting the best lens for keratoconus patients.In conclusion, this investigation showed that after an optimal fit with the Boston, Senso Select, and Wohlk RGP lenses, they all could equally improve visual acuity in keratoconus patients, while their impacts on ocular aberration were different depending on the initial aberrations of their cornea and RGP contact lens design. According to Conflict of InterestNone of the authors has any ﬁnancial interest in the rigid gas permeable contact lenses mentioned.Financial SupportNone.