Atrial premature beats are frequently diagnosed during pregnancy (PR); supraventricular tachycardia (SVT)  is less frequently diagnosed. For acute therapy, electrical cardioversion with 50–100 J is indicated in all unstable patients (pts). In stable SVT, the initial therapy includes vagal maneuvers to terminate tachycardias. For short-term management, when vagal maneuvers fail, intravenous adenosine is the first choice drug and may safely terminate the arrhythmia. Ventricular premature beats are also frequently present during PR and benign in most of the pts; however, malignant ventricular tachyarrhythmias  may occur. Electrical cardioversion is necessary in all pts who are in hemodynamically unstable situation with life-threatening ventricular tachyarrhythmias. In hemodynamically stable pts, initial therapy with ajmaline, procainamide or lidocaine is indicated. In pts with syncopal VT, VF, VFlut or aborted sudden death, an implantable cardioverter-defibrillator is indicated. In pts with symptomatic bradycardia, a pacemaker can be implanted using echocardiography at any stage of PR. The treatment of the pregnant patient with cardiac arrhythmias requires important modifications of the standard practice of arrhythmia management. The goal of therapy is to protect the patient and fetus through delivery, after which chronic or definitive therapy can be administered. Both supraventricular and ventricular tachyarrhythmias are not uncommon during pregnancy.2 However3911et al.[et al.[Supraventricular or ventricular tachyarrhythmias can become more frequent during pregnancy or they may develop for the first time. An incre1et al. reportedl.[et al. studied l.[et al. In womenl.[et al. Similar l.[et al.21et al.[P = ns) and sinus tachycardia (heart rate >100/min)  were relatively rare, whereas there was a high frequency of sinus arrhythmias in both groups . Isolated atrial premature beats (APB) were seen in 56% in G I and 58% in G II (P = ns), complex APBs  or SVT  were observed rarely. Isolated ventricular premature beats (PVC) were recorded in 49% G I and 40% G II patients (P = ns), whereas the incidence of multifocal PVCs was higher in G I (12%) than in G II (2%) (P < 0.05). Ventricular tachycardia (VT) or ventricular fibrillation (VF) were not recorded in any of the patients.[Shotan et al. assessedpatients.Before initiating therapy, it is important to correctly diagnose the type and mechanism of the underlying arrhythmia so that the proper therapeutic modalities can be implemented. The pregnant patient with arrhythmias usually seeks medical attention because of palpitations, light-headedness, shortness of breath or anxiety. Clues for correct diagnosis and treatment come from findings during physical examination and correct analysis of the electrocardiogram (ECG). Knowing the ECG features of the different types of narrow (QRS width <0.12 s) or wide (QRS width >0.12 s) tachycardias are of extreme importance to obtain ECG documentation of the arrhythmia so that the pregnant woman can receive the correct treatment.Intrauterine, all types of arrhythmias can occur. They are frequently intermittent and may disappear until delivery or the neonatal period.23 Fetal 2428et al. documented intrauterine arrhythmias with the use of fetal electrocardiography in 1968. Currently, fetal echocardiography is the best method and remains the cornerstone for in utero diagnosis of arrhythmias.[The description of intrauterine AFlut by Carr and McLure in 1931 is probably the first published report. Blumenthal hythmias. For diffsyst < 100 mmHg). Clinical studies of verapamil in pregnant women have not demonstrated adverse effects on either the patient or the fetus. However, intravenous administration of verapamil carries a risk of precipitating maternal hypotension and secondary hypoperfusion. In addition, verapamil is capable of causing fetal bradycardia, high-degree AV block and hypotension. Pregnancy is also related to an increased frequency of arrhythmias in previously asymptomatic patients with Wolff- Parkinson-White syndrome.[Narrow-QRS tachycardia is a cardiac rhythm with a rate faster than 100 beats/min and a QRS duration of <0.12 s. The pati33syndrome. Thereforsyndrome. There arsyndrome. In a versyndrome.Any arrhythmia can occur in the pregnant woman, and the frequency and symptomatic severity of arrhythmias may be increased during pregnancy. Although atrial fibrillation (AF) and atrial flutter (AFlut) are very frequent arrhythmias in adult non- pregnant patients, AF and AFlut are unusual in the absence of structural heart disease. ObviouslAtrial premature beats (APB) in pregnant woman with structurally normal hearts are benign. Atrial pOne of the most important problems in intensive care, emergency medicine and cardiac rhythmology are to take care of pregnant patients with recurrent VT, ventricular flutter or VF. Management of cardiac arrest due to life-threatening ventricular tachyarrhythmias is essential to prevent sudden cardiac death in the mother and the fetus. However, treatment of the underlying arrhythmia requires a correct diagnosis. This is possible in the majority of patients using a 12-lead surface electrocardiogram.Since a drug given for the treatment of SVT may be deleterious to a patient with VT, the differential diagnosis of a broad QRS tachycardia is critical. Wide-QRS complex tachycardias (QRS duration >0.12 s) often pose a difficult diagnostic and therapeutic problem. Errors aAlthough sustained (duration >30 s) VT is rare in pregnant women, there are some reports that VT (when occurring) originates in the patient with a normal heart mainly from the right ventricular outflow tract. Idiopath45Life-threatening ventricular fibrillation (VF) or ventricular flutter (VFlut) can occur in any stage of pregnancy and is associated with a high risk of sudden cardiac death. In patients with VF or VFlut, DC defibrillation is the method of choice (100-360 J). Prompt cardiopulmonary resuscitation and early defibrillation by either DC-countershock or an automated external defibrillator (AED) significantly improve the likelihood of successful resuscitation from VF. For longVentricular premature beats (VPB) in pregnant woman with structurally normal hearts are benign and therapy is usually not necessary. Patient P = ns). Intrauterine death was 8.0% in fetal AFlut and 8.9% in fetal SVT (P = ns).Management of fetal arrhythmias is very difficult and requires cooperation of different consultants . The problem with fetal tachyarrhythmias is the risk of hydrops fetalis and subsequent death. Supravenet al. reported a 25-year-old pregnant woman with persistent fetal tachycardia (rate 267 beats/min) and subsequent hydrops fetalis.[et al. described 3 cases with hydrops fetalis due to supraventricular tachyarrhythmias, successfully treated with amiodarone and digoxin or the combination of digoxin, procainamide and propranolol.[The treatment of fetal arrhythmias is possible by treating the mother or by treating the fetus directly. Antiarrhythmic agents that have been used to treat fetal arrhythmias include digoxin, β-blocking agents, verapamil, procainamide and quinidine. In addition, in cases of fetal ventricular tachyarrhythmias class I and III antiarrhythmic agents have been advocated.13 Recent fetalis. The womapranolol.et al. described 60 cases with fetal arrhythmias, of which 26 cases (43%) were with hydrops fetalis and 34 cases (57%) without. When tachyarrhythmias were refractory to transplacental treatment, fetal therapy was performed with direct umbilical drug administration.[maternal therapy fails to suppress or sufficiently decreases the rate of fetal tachyarrhythmias, direct drug administration to the fetus is mandatory. Direct fetal treatment regimes have been used that consists of intraperitoneal and/or umbilical intravenous administration of different drugs. In addition, umbilical drug administration allows not only direct treatment but also drug monitoring. Hansmann stration. Of thosestration.56 DespitDuring pregnancy, an increased incidence of maternal cardiac arrhythmias is observed, ranging from clinically irrelevant isolated atrial or ventricular premature beats to debilitating SVT and VT or VF. In all pregnant patients with tachyarrhythmias, evaluation of the underlying etiology and the degree of left ventricular function (dysfunction) is essential. Correct treatment of arrhythmias in the intensive care patient should be based on understanding the causal mechanism. In pregnant women with maternal and/or fetal arrhythmias, therapeutic strategies should be based on interdisciplinary cooperation . In general, acute therapy of arrhythmias during pregnancy is similar to that in the nonpregnant patient. However, special consideration should be given to potential teratogenic and hemodynamic adverse effects on the fetus. With this in mind, a successful pregnancy, for both mother and the fetus, can usually be the result.


The occurrence of atrial tachycardias (AT) is a direct function of the volume of atrial tissue ablated in the patients with atrial fibrillation (AF). Thus, the incidence of AT is highest in persistent AF patients undergoing stepwise ablation using the strategic combination of pulmonary vein isolation, electrogram based ablation and left atrial linear ablation. Using deductive mapping strategy, AT can be divided into three clinical categories viz. the macroreentry, the focal and the newly described localized reentry all of which are amenable to catheter ablation with success rate of 95%. Perimitral, roof dependent and cavotricuspid isthmus dependent AT involve large reentrant circuits which can be successfully ablated at the left mitral isthmus, left atrial roof and tricuspid isthmus respectively. Complete bidirectional block across the sites of linear ablation is a necessary endpoint. Focal and localized reentrant AT commonly originate from but are not limited to the septum, posteroinferior left atrium, venous ostia, base of the left atrial appendage and left mitral isthmus and they respond quickly to focal ablation. AT not only represents ablation-induced proarrhythmia but also forms a bridge between AF and sinus rhythm in longstanding AF patients treated successfully with catheter ablation. Atrial fibrillation (AF) is no longer a formidable rhythm since ablationists challenged this notorious arrhythmia more than a decade ago in their unprecedented quest for sinus rhythm (SR) . AblatioBased on our observation and also that of others, the likelihood of occurrence of ATp is probably a function of the volume of tissue ablated for AF. Importantly, recent expansion of AF ablation has caused a surge in the incidence of ATp. Incidence of ATp after PV isolation has been variably reported from 2.9% to 10% . NotablyThe Working Group Report classified atrial flutter/tachycardia into macroreentrant and focal, the clinically relevant categories . It is oATp can occur intraprocedurally and/or postprocedurally (early or delayed). There is a mechanistic difference between the ATs occurring intraprocedurally and postprocedurally. It has been observed that the mechanism of ATp depends upon the clinical type of AF and the ablation strategy, to a great extent.Using a stepwise approach to ablation of longstanding AF, PV isolation is followed by electrogram-based atrial defragmentation. During this stage of ablation, usually, there is an increase in the AF cycle length (AFCL) compared to baseline AFCL. Often, at this very stage, AF converts into slower atrial tachycardia or flutter (CL ≥ 200 ms approximately). Thus, AT is observed as a transitional rhythm from AF to SR before the third and final step of linear ablation is undertaken . Such inAfter performing AF ablation, usually, variable degree of inflammatory state exists in the atria. It takes about 6–12 weeks to form a fibrous scar at the site of ablative lesion. Post-AF ablation, this is usually observed as a blanking period marked by transient but recurrent nonclinical atrial arrhythmias. As inflammation heals, inhomogeneous areas of nonuniform scar interspersed with healthy and/or partially damaged atrial tissue emerge . Thus, aIn contradistinction to intraprocedural ATp which occur before linear ablation, most of the post procedural ATp develop after linear ablation of longstanding AF.It has been observed that the volume of tissue ablation probably dictates the incidence of ATp. The volume of tissue ablated is directly related to the clinical type of AF. In paroxysmal AF, PV isolation is enough to cure AF in majority of the patients. Since the lesions are limited around PV ostium in the antrum, the PV ostia are the likely sites of focal ATp. Moreover, circumferential lesions involve higher volume of tissue than segmental lesions explaining a substantial contribution of the former to the rising incidence of ATp.For longer lasting forms of AF, wherein circumferential PV isolation is combined with linear ablation in the roof, posterior left atrium or posterolateral mitral isthmus, macroreentry is established most likely around the mitral annulus or ipsilateral pulmonary venous antrum. Mitral annular and roof dependent flutters are the consequential ATp in the same order of frequency. Cavotricuspid isthmus (CTI) dependent ATp is another likely macroreentrant tachycardia although it is the least common variety.Localized circuits get established in small areas most commonly at the venous ostia , left septum, mouth of left atrial appendage (LAA), posterior left atrium and posterolateral mitral isthmus . Such localized small circuits give rise to focal ATp from these sites. Left atrium (LA) adjoining anterior mitral annulus is an uncommon site of focal ATp , 22. UnlClinically, the diagnostic and localizing value of surface ECG is debatable in atrial tachycardias arising after ablation. Since, the magnitude and direction of vector of atrial activation are tremendously influenced by the differential conduction velocity and low voltage areas of extensively ablated atria, surface P waves do not provide consistent information on the mechanism of ATp and the location of focal ATp. Therefore, regular ECG clues cannot be applied effectively to patients subjected to extensive ablation of the atria.In the electrophysiology laboratory, it is important to diagnose the mechanism underlying ATp to achieve successful termination. As a preliminary step, it is important to acquire the information on previously ablated sites and determine if clear end point  had been achieved. If patient is in SR during the repeat procedure, the evaluation for completeness of previously performed PV isolation should be the first step. If there is any evidence of PV reconnection, PV isolation should be completed before targeting other areas. If linear ablation involving CTI was performed previously, bidirectional block should be reconfirmed. Repeat ablation may be attempted to ascertain block.During ongoing tachycardia, we adopt and recommend a three-stepped approach  to diagnUsing the electrograms recorded from LAA and coronary sinus (CS) for one minute, the mean cycle length and the range of variation in cycle length over 1 minute is assessed. If the variation is >15% of the mean cycle length, focal mechanism is the most likely diagnosis. On the other hand, variation <15% does not rule out focal mechanism. In the latter situation, next step should be used to diagnose or rule out macroreentrant ATp.perimitral ATp is the most likely possibility  and the anterior and posterior walls activate low to high, counterclockwise (or clockwise) sibility . roof dependent ATp rotating counterclockwise (or clockwise) around the antrum of right PVs is the most likely possibility. If the left atrial activation pattern is lateral to septal both anteriorly and posteriorly and the anterior and posterior walls activate low to high and high to low (or vice versa), respectively, roof dependent ATp rotating counterclockwise (or clockwise) around the antrum of left PVs is the most likely possibility. From therapeutic standpoint, we do not need to categorize roof dependent circuit as right or left , respectively,  or left . CTI dependent flutter would be septal to lateral and low to high both on the anterior and posterior walls. In contrast, clockwise CTI dependent flutter would demonstrate high to low activation on the anterior and posterior walls of the left atrium. The left atrial activation pattern in typical counterclockwise Entrainment of ATp is a confirmatory step in the diagnosis of macroreentry. Importantly, if the ATp is entrained from three (at least two segments to avoid inadvertent termination or conversion to another tachycardia due to rapid pacing) distinct atrial segments  with postpacing interval (PPI) < 30 ms at each of the sites, macroreentry is confirmed. If this is not the case, macroreentry is ruled out and focal tachycardia is the only possible diagnosis.Considering that macroreentrant ATp would usually represent one of the following three: (1) mitral isthmus dependent flutter , (2) roof dependent flutter, and (3) cavotricuspid isthmus dependent flutter, let us deductively infer each of these possibilities based on left atrial and CS activation patterns.The atrial activation pattern reflects the direction of centrifugal propagation of ATp from the focal source. In a single tachycardia cycle, the activation pattern of CS  and the left atrial activation pattern  which do not conform to macroreentrant mechanism provide a useful guide for mapping the focal source. Recording fragmented electrogram spanning over 50–75% of tachycardia cycle length is suggestive of site of localized small circuit . The entrainment response for focal ATp is characteristically distinct from macroreentry. Of note, in contrast to macroreentrant circuit which is spread over more than one atrial segment, small circuit can be localized to one atrial segment only. On entrainment, PPI continues to get shorter as pacing site gets closer to the focus. This phenomenon provides an important guide to segmental localization of focal ATp . PPI < 3In contrast to typical focal ATs arising in normal hearts, focal ATp demonstrating automatic mechanism of initiation and maintenance of tachycardia is less commonly encountered in post-AF ablation patients. High variability in AT cycle length is characteristically described for automatic tachycardias with spontaneous onset and offset with gradual warm up and decline, respectively. The centrifugal atrial activation pattern can be helpful in mapping the source of the focus. Instead of continuous low voltage fragmented local activity on electrograms, a sharp and high voltage presystolic potential with high negative dV/dT monophasic (QS shaped) unipolar electrogram will be obtained at the site of origin of AT. Entrainment of automatic ATs is not feasible. Overdrive pacing of automatic tachycardias will have a variable response from the same site of pacing. Postoverdrive pacing recovery time of automatic focus is highly variable. As against it, fixed postpacing interval can be obtained after entrainment of focal AT due to localized reentry.Three dimensional electroanatomical mapping systems are not indispensable for diagnosis and ablation of ATp. For those who are conversant with the fluoroscopic anatomy of atria and their venous inputs and perform electrogram guided ablation of ATp expeditiously, 3D systems do not offer any specific advantage in majority of the cases. Very often, more than one ATp sequentially occurs before restoration of SR. Conversion of target tachycardia to another tachycardia will entail reconstruction of activation maps every time it happens. Besides, after building the maps, entrainment maneuvers cannot be dispensed with for establishing and/or confirming the diagnosis. Moreover, critical areas of low amplitude signals may be ignored as noise. This can cause disparity between clinical tachycardia and reconstructed electroanatomical map especially for localized reentrant ATp. Though it is a matter of operator preference, 3D systems are not essential for ATp ablation. Instantaneous multielectrode epicardial mapping systems have the advantage of building atrial activation maps within 8–10 minutes noninvasively. Sustenance of tachycardia is not mandatory. Preprocedural diagnosis regarding the mechanism and source of ATp may be obtained noninvasively. However, clinical utility in this area will require validation.Usually, AF ablation is undertaken in patients who fail to respond to drug therapy. The response to drug therapy may improve postablation in patients with recurrent AF. However, ventricular rate gets faster during ATp than AF. Often, ATp is very stable and sustained. Patient tolerance is worse for ATp than AF for such reasons. Though there are no randomized trials involving drug therapy in ATp, it is observed that ATp do not effectively respond to the drugs. On the other hand, ablation offers cure for the arrhythmia. Consequentially, catheter ablation is preferred over medical management for ATp.The ablation of focal ATp is accomplished more easily than the diagnosis. The reverse holds true for macroreentrant ATp. Successful termination of target tachycardia is the common endpoint for both the types of ATp. Conversion of target tachycardia to another tachycardia with same/different cycle length and different atrial activation pattern is frequently encountered before eventual restoration of SR. Therefore, close monitoring of electrograms during tachycardia ablation is very important. Finer changes in activation pattern and cycle length may be the only subtle clues towards requirement of repeating diagnostic maneuvers including the entrainment response.Focal ATp are ablated at the site of origin. Using saline irrigated tip catheter and a target temperature upto 42°C, radiofrequency energy with power of upto 30 W can be delivered for 30–60 seconds at each point with continuous monitoring of electrograms. Transient acceleration of tachycardia following the commencement of energy-delivery may occur when ablation is localized at the right spot. If tachycardia fails to terminate or convert to another tachycardia after at least 60 s of energy delivery, further mapping is performed to localize a better site for ablation. Macroreentrant ATp are targeted by performing linear ablation. CTI ablation is performed conventionally for CTI dependent flutters. Linear ablation of the left atrial roof is performed by joining the ostia of two superior pulmonary veins at the most cranial aspect of LA. It terminates both the right and the left sided roof dependent flutter. Perimitral flutter is targeted by performing linear ablation at the posterolateral mitral isthmus. Ablation of mitral isthmus is performed by withdrawing the catheter from the ventricular side of the mitral isthmus  to the left inferior PV. Ablation within the CS on the epicardial side of the mitral isthmus is performed when bidirectional conduction block cannot be achieved across the isthmus after at least 20 minutes of endocardial energy application. Power of up to 35 W endocardially and upto 25 W epicardially is required to achieve transmural lesion in most resistant cases. The endpoint is creation of line of complete block. Confirmation of bidirectional block is undertaken following restoration of SR.Block across the mitral isthmus linear ablation is demonstrated by differential pacing from distal and proximal CS dipoles sequentially and recording the time of activation in LAA. If LAA activation is earlier with proximal CS pacing than more distal CS pacing, it is suggestive of unidirectional block. If subsequent pacing from LAA shows that the activation in CS proceeds from proximal dipole distally, it is conclusive of bidirectional block . EstimatUsing the algorithm mentioned above, 97% ATp were diagnosed in less than 11 minutes of mapping duration in a recently published report from our center. Incoherent mapping due to widespread areas of altered electrogram characteristics/scars and noninducibility after inadvertent termination of tachycardia caused difficulty in diagnosis. All the diagnosed cases were treated successfully. Complete line of block at mitral isthmus, left atrial roof and CTI was achieved in 95%, 97%, and 100% cases, respectively. At follow-up of 21 ± 10 months, recurrence of sustained ATp was observed in 5%. After repeat ablation procedure, SR was maintained in 95% patients .It is proven beyond doubt that ATp is partly an iatrogenic proarrhythmia. Since the volume of ablation drives the probability of occurrence of ATp, limiting the ablation within the atrium can help reduce the incidence of certain forms of ATp. Judicious use of ablative energy can help minimize the incidence of reentrant and focal ATp. Macroreentrant ATp develop frequently in patients with incompletely blocked previous linear ablation. Ensuring that bidirectional block is achieved and documented after linear ablation can prevent recurrent macroreentry. An important lesson learnt from AF ablation procedures is that application of energy unscrupulously and indiscriminately is not helping cure AF. Therefore, limiting ablation to achieve isolation of PVs in paroxysmal AF and PV isolation plus necessary defragmentation and linear ablation in persistent and long lasting forms of AF can retard the epidemically growing pace of ATp.In the era of AF ablation, there is an increase in the incidence of both macroreentrant and focal ATs. Extensive substrate modification generates focal areas of slow conduction and low voltage capable of sustaining localized reentry, a novel mechanism of reentrant atrial arrhythmias post-AF ablation. ATp is also the final common pathway towards restoration of SR in catheter ablation of long lasting AF. Ascertaining bidirectional block after linear ablation and minimizing the volume of tissue ablation can help reduce the incidence of ATp. Using conventional tools and electrogram guidance for catheter ablation, deductive algorithm presented here can meticulously diagnose and cure ATp.


Plasmodium chabaudi has proven of great value in the analysis of fundamental aspects of host-parasite-vector interactions implicated in disease pathology and parasite evolutionary ecology. However, the lack of gene modification technologies for this model has precluded more direct functional studies.The rodent malaria parasite in vitro culture methods to yield P. chabaudi schizonts for transfection and conditions for genetic modification of this rodent malaria model are reported.The development of P. chabaudi gene-integrant lines that constitutively express high levels of green fluorescent protein throughout their life cycle have been generated.Independent P. chabaudi is now possible. The production of genetically distinct reference lines offers substantial advances to our understanding of malaria parasite biology, especially interactions with the immune system during chronic infection.Genetic modification of  Plasmodium have led to substantial advances in malaria research [Plasmodium gallinaceum, followed by Plasmodium falciparum [Plasmodium berghei [Plasmodium yoelii [P. berghei has been the focus for the development of technical advances in transfection [The development of transfection technologies in research -3. The flciparum ,5. Howev berghei -9, and h berghei -13. Stabm yoelii  but P. bsfection .P. berghei causes a rapid and virulent infection leading to widespread tissue pathology and early death without effective host immune control making the investigation of host adaptive immune responses and chronic malaria infections challenging [P. berghei clones limits its use as a model for investigations of the evolution and ecology of host-parasite interactions [P. berghei through the mosquito vector occurs at lower temperatures (18–21°C) and is longer (21 days) than transmission of human or other rodent malaria parasites (> 24°C for 10–14 days) and P. falciparum. This may influence studies of parasite biology and development in the vector as parasite-vector interactions are sensitive to temperature .P. chabaudi could provide a more relevant model for investigating anti-malarial host immune responses because infections are usually controlled by host immunity .  chronic -26. Alsodynamics -30. In aa models .P. chabaudi will, therefore, provide opportunities for experimental and analytical advances in fields as diverse as immunology and evolutionary ecology. This paper presents the first report of the generation of fluorescent P. chabaudi lines that constitutively express high levels of Green Fluorescent Protein (GFP) throughout their life-cycle.The development of gene transformation technologies for i.p. with 1 × 107 P. chabaudi parasites from clone AJ4916. 500 μl of blood containing ring and early trophozoite-stage parasites were collected by cardiac puncture at 3 days post infection (5–10% parasitaemia). Parasites were cultured for 17–18 hours at a 1.5% dilution in complete culture medium  containing 25% heat inactivated foetal calf serum (Gibco), at pH 7.25) in the presence of 10% O2, 5% CO2, 85% N2, at 32°C in upright 200 ml flasks (Ikawa) in a horizontal shaking incubator at 30 rpm. Parasitized blood forming a layer at the bottom of the flask was gently removed and centrifuged at 1500 rpm for 30 sec. 5 μl of pelleted cells (1 × 106 schizonts) were used per transfection.To obtain parasites for transfection, male MF1 mice (10 weeks) were infected Gfp was introduced into the genome of P. chabaudi parasites, using the PbGFPCON plasmid previously described [2O was added to 100 μl of Amaxa nucleofectortm test solution 88A6 (Basic Parasite Nucleofectortm solution 2) in the manufacturers' cuvette (Amaxa Biosystems). 5 μl of schizont mix was added and electroporation was carried out in an Amaxa nucleofectortm using program U33. After electroporation, 50 μl of pre-warmed complete culture media was added and the transfection mix was immediately injected i.v. into an MF1 mouse. Recipient mice received 35 μg/ml pyrimethamine in their drinking water (pH 3.5–5) for seven days post infection; the minimum pyrimethamine dose required to clear P. chabaudi infections of clone AJ in 24 hours . Giemsa-stained smears were scanned every one to two days and parasites that produced patent infections (days 14–20) were immediately passaged to further mice for the production of stabilate stocks.escribed . 5 μg ofssu-rrna locus at the 5' region, amplification was carried out with primer Pc5'F (TTGTAAGAACGTGCTTGGTG) that is specific for P. chabaudi ssu-rrna sequence on P. chabaudi genome contig827, in the target region, and primer Pl5'R (TTCCCAGTCACGACGTTG) that anneals to P. berghei d-ssu rrna sequence in the PbGFPCON plasmid. To demonstrate integration into the contig827 ssu-rrna locus at the 3' region, amplification was carried out with primer Pc3'R (AGAGCCCAGCGATGAC) that is specific for P. chabaudi contig827 ssu-rrna sequence in the integration site, and primer Pl3'F (CAATGATTCATAAATAGTTGGAC) that anneals to P. berghei d-ssu rrna sequence in the PbGFPCON plasmid. To demonstrate the presence of tg-dhfr sequence, amplification was carried out with primers L190 (CGGGATCCATGCATAAACCGGTGTGTC) + L191; CGGGATCCAAGCTTCTGTATTTCCG. To amplify circular PbGFPCON plasmid, amplification was carried out with primers PlF2 (AATCATGACTTCTGTCACTGC) and Pl5'R. Primers Pc5'R and Pc3'R anneal specifically to sequences within P. chabaudi contig827 ssu-rrna and not to sequences within the PbCON vector. Probe template for the detection of tgdhfr by Southern blots was amplified using primers L190/L191. DNA was digested with HindIII and NheI, transferred to Hybond N+ membrane (Amersham) and hybridized according to the manufacturers methods. Wet preparations of live parasites expressing GFP were visualized using Openlab digital imaging (Improvision). The development and progression of parasitaemia in transformed parasites was compared to the wild type ancestor by following six mice infected with 106 parasitized red blood cells for each line. Infections were monitored daily to collect red blood cell density and parasitaemia data until day 14 post infection, when the acute phase parasites had been cleared. The infection dynamics of the two lines were analysed using linear mixed-effects models, which account for repeated measures across infections. One mouse from each line was euthanized (day 10 and 11) so these infections did not contribute data for the whole time course.For genetic analysis of the integration locus, DNA was isolated as previously described  and SoutPlasmodium is thought to be the free merozoites, released at schizogony, that are not surrounded by red blood cell cytoplasm and membranes.P. berghei may, therefore, be relatively amenable to genetic transformation because schizonts developing in reticulocytes do not rupture in in vitro culture conditions, so high numbers can be purified. These rupture, releasing merozoites, during electroporation. In contrast, P. chabaudi schizonts, developing in mature red blood cells, do not arrest in culture and cannot be purified in such large numbers. For this reason, the transformation efficiency of P. chabaudi is likely to be considerably lower that that of P. berghei. The major improvements in rodent malaria parasite transformation efficiency obtained with the Amaxa Nucleofectortm technology [P. chabaudi schizonts suggested to us that transformation of P. chabaudi may now be possible.The optimal stage for DNA uptake by chnology , howeverP. chabaudi schizonts, the in vitro culture protocol described by Mackinnon et al [P. chabaudi ring-stage parasites/young trophozoites develop over 17–18 hours into a population of parasites that contain > 10% mature schizonts  for selection of transgenic parasites  between the P. berghei d-ssu-rrna target region and P. chabaudi ssu-rrna sequences on genome contig827, indicating that these are orthologous loci. Linearized PbGFPCON DNA was introduced into the parasite genome by electroporation and recombinant parasites that express tghfr were selected by treatment with pyrimethamine.To obtain appropriate numbers of on et al  was adaps Figure . Schizonette pyrR for seleCON cassette into the P. chabaudi genome was confirmed by PCR analysis in four independent lines and line '2.3' was selected for further genetic and phenotypic analysis. In P. chabaudi line 2.3, PbGFPCON integrated into the genome of contig 827 rRNA subunit, orthologous to the P. berghei c or d-ssu-rrna that have previously been shown to be non-essential genes in rodent malaria parasites [Integration of the PbGFPin vivo asexual dynamics of line 2.3 was not significantly different to wild type P. chabaudi AJ4916 ancestral parasites that had undergone comparable numbers of passages = 0.02; P = 0.901). The patterns observed throughout infections were similar, though parasitaemia of line AJ4916 was significantly lower on days 8 and 9 post infection  = 4.96; P < 0.0001). The lines did not differ in the patterns or the average levels of anaemia they caused  = 0.001; P = 0.975). Although reversion to the wild-type genotype was observed at a low rate after multiple blood-stage passages, all Pc-GFPCON (line 2.3) parasites observed at day 8 of infection were GFP-positive.The Plasmodium chabaudi is reproducibly accessible for genetic transformation at an efficiency that is sufficient for genomic integration of introduced genes. The development of technologies that allow disruption or modification of gene expression in P. chabaudi, thus, opens the way for direct functional analysis of parasite proteins throughout both acute and chronic stages of an in vivo malaria infection, including those that have been implicated in modulation of the host immune response [P. chabaudi parasite lines also offers the opportunity for imaging of direct interactions between the parasite and host cells within a variety of host tissues.response . The genGFP: green fluorescent protein; Tgdhfr: Toxoplasma gondii Dihydrofolate reductase.The authors declare that they have no competing interests.Both authors conceived and designed the project and prepared the manuscript. SR prepared and characterized parasites and JT undertook the transfection and molecular analyses. All authors read and approved the final manuscript.


The homeostatic control of the cellular proteome steady-state is dependent either on the 26S proteasome activity or on the lysosome function. The sex hormone 17β-estradiol (E2) controls a plethora of biological functions by binding to the estrogen receptor α (ERα), which is both a nuclear ligand-activated transcription factor and also an extrinsic plasma membrane receptor. Regulation of E2-induced physiological functions  requires the synergistic activation of both transcription of estrogen responsive element (ERE)-containing genes and rapid extra-nuclear phosphorylation of many different signalling kinases . Although E2 controls ERα intracellular content and activity via the 26S proteasome-mediated degradation, biochemical and microscopy-based evidence suggests a possible cross-talk among lysosomes and ERα activities. Here, we studied the putative localization of endogenous ERα to lysosomes and the role played by lysosomal function in ERα signalling. By using confocal microscopy and biochemical assays, we report that ERα localizes to lysosomes and to endosomes in an E2-dependent manner. Moreover, the inhibition of lysosomal function obtained by chloroquine demonstrates that, in addition to 26S proteasome-mediated receptor elimination, lysosome-based degradation also contributes to the E2-dependent ERα breakdown. Remarkably, the lysosome function is further involved in those ERα activities required for E2-dependent cell proliferation while it is dispensable for ERα-mediated ERE-containing gene transcription. Our discoveries reveal a novel lysosome-dependent degradation pathway for ERα and show a novel biological mechanism by which E2 regulates ERα cellular content and, as a consequence, cellular functions. While protein synthesis always requires gene transcription and mRNA translation, cells have evolved different physiological mechanisms to regulate proteolysis and thus protein turnover. Indeed, degradation of intracellular proteins occurs via targeted  26S proteasome activation and extra-cellular proteins are eliminated through a vesicular system that ultimately addresses them to the lysosomes. Remarkably, in recent years, this notion has been refined by the recognition that also intracellular soluble proteins can be shuttled to lysosomes for degradation via a non-vescicular system. Thus, beside the homeostatic control of protein turnover, the regulatory mechanisms of proteostasis networks could represent also master organizers of signal transduction circuits The functions of the cellular proteome are controlled by a homeostatic steady-state, which is granted by the balance between protein synthesis and degradation   e.g., ERK/MAPK; PI3K/AKT)  and the realization of the E2-induced cellular effects both in cell lines  e.g., cell migration) The estrogen receptor α (ERα) is a ligand-activated transcription factor that belongs to the nuclear hormone receptor super-family. ERα, together with the other receptor subtype (ERβ) mediates the pleiotropic effects of the sex hormone 17β-estradiol (E2) that include many physiological processes such as growth, development, and differentiation. In particular, the E2:ERα complex molecular actions are a function of ERα intracellular localization: in the nucleus, the activated ERα drives transcription not only of those genes that contain the estrogen-response element (ERE) within their promoters but also of non-ERE-containing genes through the stimulation of the activity of specific transcription factors  also controls E2-induced ERα degradation The existing paradigm defines that the E2-dependent control of ERα intracellular concentration contributes to the regulation of the pleiotropic effects elicited by E2 in several target tissues. Regulation of ERα stability depends on the activation of the 26S proteasome and is intrinsically connected with the ability of the E2-activated receptor to regulate gene transcription i.e., MCF-7 and T47D-1 mammary adenocarcinoma cells). Our results indicate that ERα degradation requires lysosomal function in addition to the 26S proteasome activity and that lysosomes are implicated in the regulation of the E2-depedent signalling to cell proliferation.In addition to 26S proteasome, some relationships among lysosomes and ERα have been reported in different cell lines In order to understand a potential interplay among lysosomes and ERα, we started by investigating the 26S proteasome-dependent ERα degradation. To this purpose adenocarcinoma (MCF-7) cells were treated for 2 hrs with E2 in the presence or in the absence of the pre-treatment with different doses of Mg-132, an inhibitor of 26S proteasome activity . As expeThese data confirm that ERα breakdown is regulated by the 26S proteasome but additionally suggest the presence of other degradation mechanisms in the control of ERα intracellular levels.i.e., Sp-1 ERα) Because we observed that the 26S proteasome activity is only partially required for E2-induced ERα degradation, we next studied the role of lysosomes in the control of ERα intracellular level. To fulfil this task, we evaluated if ERα could localize to lysosomes by employing an ERα antibody, which highlights cytoplasmic ERα in breast cancer cells , which recognizes an epitope located within the ERα N-terminus. Confocal microscopy analysis demonstrated that the anti-ERα Sp-1 antibody stains MCF-7 cells both in the nucleus and in the cytoplasm while anti-ERα D12 antibody stains only the nucleus of MCF-7 cells . ERα barely co-localizes with LAMP-2 and lysotraker in MCF-7 cells under basal conditions while E2 treatment determines a time-dependent increase in cytoplasmic co-localization of ERα with either lysosomal markers that reached a maximum after 2 hrs of hormone treatment  with increased ERα cytoplasmic localization  that barely affect MCF-7 cell viability , a drug that inhibits lysosomal enzymes by changing endosomes and lysosomes internal pH iability  induced iability  and timeiability  dependeniability ’ and 3E.iability .The change in endosomal pH caused by chloroquine has also the consequence to impede the fusion of endosomes to lysosomes Altogether, these data strongly indicate that cytoplasmic ERα is addressed to the lysosomal compartment in an E2-dependent manner and that lysosomal function is implicated in the control of ERα cellular content.Because ERα degradation contributes to E2-induced ERα gene transcription in vitro and in vivoIt is now accepted that the extra-nuclear plasma membrane localized ERα directs the activation of the rapid E2 signalling e.g., cyclin D1) and in parallel up-regulate the level of the anti-apoptotic and pro-survival protein Bcl-2 Many groups including our own have clarified that the E2-induced ERα extra-nuclear activity is required for E2-induced cell proliferation. In particular, the E2-dependent activation of ERK/MAPK and PI3K/AKT pathways control the transcription of specific cell cycle regulated genes  do not affect breast cancer cell viability and are the minimum sufficient amount that determines the time- and dose-dependent (Also in ERα-containing cells the exposure to 17β-estradiol (E2) results in a ligand-dependent reduction of the total receptor content. In this way, E2 determines the amount of ERα intracellular levels by controlling receptor turnover and synchronizes ERα activities with the cellular response. The mechanism underlying ERα elimination requires the activation of the 26S proteasome. Indeed, both apoERα and E2-activated receptor undergo proteasomal degradation ependent  accumulaependent , a membrependent . In turni.e., 10 µM)  was observed in lysososmes of HepG2 cells by electron microscopy Accordingly, we report here that lysosomes contribute to the E2-dependent control of ERα intracellular content . Indeed,, 10 µM) , which a, 10 µM)  and [28], 10 µM)  and time, 10 µM)  E2-inducThe accumulation of ERα observed in the presence of the lysosome-distrupting function drug chloroquine rapidly occurs after E2 administration (30 min) and remains significant up to 8 hrs of hormone treatment , possibli.e., anti-ERα Sp-1 antibody) {i.e., H2_NES - Nessi) that is abundantly and artificially located in the cytoplasm {i.e., up to 2 hrs) also induces a progressive reduction in the co-localization of the ERα with the EEA1  that originate from the plasma membrane feed in the endosomes e.g., lysosomes; nucleus). In this respect, published evidence suggests both an active mechanism for E2 internalization into cells and an endocytic shuttling for the membrane-localized ERα Another finding presented here is the fact that the cytoplasmic ERα localizes at the lysosomes . This stthe EEA1 . In parathe EEA1 , occurs.the EEA1  further In conclusion, the findings reported here reveal a novel role for lysosomes in E2-induced ERα degradation as well as in those ERα activities required for E2-dependent breast cancer cell proliferation. Remarkably, our data, together with the recognition that the activity and the cellular concentration of the receptor for glucocorticoids are at least in part under the control of lysosomes Human breast adenocarcinoma cells (MCF-7 and T47D-1) −8 M), Mg-132 (1 µM), chloroquine (Clo) (10 µM) or EGF (1 µg/ml). Cell number counts, protein extraction, biochemical assays were performed as previously described Cells were grown in 1% charcoal-stripped fetal calf serum medium for 24 h and then stimulated with E2 at the indicated time points; where indicated, inhibitors  were added 30 min before E2 administration. Unless otherwise indicated, cell were treated with E2 (10The pcDNA 3.1 flag-ERα was previously described 5′-CATCGACGTCCCTCCAGAAGAG-3′ (forward) and 5′-CTCTGGGACTAATCACCGTGCTG-3′ (reverse), for human cyclin D1 5′- AACTACCTGGACCGCTTCCT-3′ (forward) and 5′- CCACTTGAGCTTGTTCACCA-3′ (reverse), for human cathepsin D 5′-GTACATGATCCCCTGTGAGAAGGT-3′ (forward) and 5′-GGGACAGCTTGTAGCCTTTGC-3′ (reverse), for human progesterone receptor (PR) 5′-AAATCATTGCCAGGTTTTCG-3′ (forward) and 5′-TGCCACATGGTAAGGCATAA-3′ (reverse), for human GAPDH 5′-CGAGATCCCTCCAAAATCAA-3′ (forward) and 5′-TGTGGTCATGAGTCCTTCCA-3′ (reverse). Total RNA was extracted from cells using TRIzol Reagent  according to the manufacturer’s instructions. To determine gene expression levels, cDNA synthesis and qPCR were performed using the GoTaq 2-step RT-qPCR system  in a ABI Prism 7900 HT Sequence Detection System  according to the manufacturer’s instructions. Each sample was tested in triplicate, the experiment repeated twice and the gene expression normalized for GAPDH mRNA levels.The sequences for gene-specific forward and reverse primers were designed using the OligoPerfect Designer software program . The following primers were used: for human pS2 MCF-7, T47D-1 and ERα-transfected HeLa cells were plated and stained as previously described i.e., vinculin or tubulin) intensity. In all analyses, p values <0.01 were considered significant, but for densitometric analyses where p was <0.05. Data are means of at least three independent experiments +/− SD.A statistical analysis was performed using the ANOVA test with the InStat version 3 software system . Densitometric analyses were performed using the freeware software Image J by quantifying the band intensity of the protein of interest respect to the relative loading control band  or lysotracker (B) both in the presence and in the absence of E2 (10 nM–2 hrs). Figures show one unique confocal plane. All co-staining procedures were described in details in the Material and Methods section. (C) Time course analysis of T47D-1 cells treated with E2 (10 nM) at the indicated time points both in the presence and in the absence of chloroquine (Clo–10 µM). Loading control was done by evaluating vinculin expression in the same filter. Figure shows representative blots of three independent experiments. (D) Western blot analysis of cyclin D1 (Cyc D1) and Bcl-2 expression levels in T47D-1 cells treated with E2 (10 nM–24 hours) both in the presence and in the absence of chloroquine (Clo–10 µM). Loading control was done by evaluating tubulin expression in the same filter. Figure shows representative blots of three independent experiments.(TIF)Click here for additional data file.Figure S2H2_NES ERα characterization. (A) Schematic of the point mutations introduced in the hinge region of the ERα (TIF)Click here for additional data file.


Visceral leishmaniasis is a lethal parasitic disease transmitted by phlebotomine sand flies. The largest focus of VL in Ethiopia is located in the lowland region bordering Sudan, where the epidemiology is complicated by the presence of thousands of seasonal agricultural workers who live under precarious conditions.We conducted two parallel case-control studies to identify factors associated with VL risk in residents and migrants. The studies were conducted from 2009 to 2011 and included 151 resident cases and 157 migrant cases, with 2 matched controls per case. In multivariable conditional regression models, sleeping under an acacia tree at night (odds ratios (OR) 5.2 [95% confidence interval 1.7–16.4] for residents and 4.7 [1.9–12.0] for migrants), indicators of poverty and lower educational status were associated with increased risk in both populations. Strong protective effects were observed for bed net use . For residents, living in a house with thatch walls conferred 5-fold and sleeping on the ground 3-fold increased risk. Among migrants, the risk associated with HIV status was borderline significant and sleeping near dogs was associated with 7-fold increased risk.Preventive strategies should focus on ways to ensure net usage, especially among migrant workers without fixed shelters. More research is needed to understand migration patterns of seasonal labourers and vector bionomics. Visceral leishmaniasis is a lethal parasitic disease transmitted by sand flies. The largest focus of VL in Ethiopia is located in the lowland region bordering Sudan, where hundreds of thousands of agricultural workers migrate for work every year during the planting and harvest seasons. We conducted two parallel studies in residents and migrants to determine the living conditions and behaviors that put people at higher risk of VL risk. We found that sleeping under an acacia tree at night, indicators of poverty and lower educational status were associated with increased risk in both populations. Sleeping under a bed net was protective. For residents, living in a house with thatch walls and sleeping on the ground increased risk of VL. Among migrants, the risk associated with HIV status was borderline significant and sleeping near dogs was associated with increased risk. Preventive strategies should focus on ways to ensure net usage, especially among migrant workers without fixed shelters. More research is needed to understand migration patterns of seasonal labourers and vector behavior. Leishmania donovani has also been found in dogs and wild mammals in Sudan and Ethiopia Visceral leishmaniasis (VL) is a lethal parasitic disease transmitted by phlebotomine sand flies. The typical clinical picture is a chronic systemic illness with fever, weight loss, splenomegaly, hepatomegaly, and bone marrow suppression with pancytopenia. East Africa is second only to the Indian subcontinent in annual VL incidence Phlebotomus orientalis, found in association with cracked black cotton-clay soils and acacia-balanites forest In 2005, a new VL epidemic was reported in Libo Kemkem in the highlands of north-western Ethiopia, where only a handful of cases had been reported in the past The studies were conducted in Kafta-Humera district, Tigray regional state, bordered on the west by Sudan, and on the north by the Tekezé River which separates Ethiopia from Eritrea . Kafta-HThe study protocols were approved by the ethical review committees of the Ethiopian Public Health Association (resident protocol) and the Tigray Regional State Health Bureau (migrant protocol). Each adult participant provided written informed consent. A parent or guardian provided consent on behalf of child participants. All research activities adhered to the principles expressed in the Declaration of Helsinki. Approval to record existing hospital data on HIV status was given only for the migrant protocol; these data are therefore missing for the resident protocol.Because the resident and migrant populations differed substantially in terms of practical options for valid control selection and some aspects of exposure history, we performed the two investigations independently. An effort was made to maintain key variables in both investigations, but the difference in living circumstances required some questions to be worded differently.Médecins Sans Frontières (MSF)-Holland until to June 2009. After June 2009, responsibility for VL treatment was assumed by regular hospital staff. This hospital is the main VL treatment facility for the area and treated 348 VL cases in 2009, 505 in 2010 and 405 in 2011. For both studies, VL case definitions followed the hospital diagnostic algorithm. A past VL case was a patient with at least 2 weeks of fever plus weight loss and/or splenomegaly, diagnosed by KAH based on positive DAT  or rK39 dipstick and treated with antileishmanial drug with clinical resolution of symptoms; or a patient with Leishmania amastigotes demonstrated in splenic aspirate. A current VL case was a patient diagnosed with illness characterized by at least 2 weeks of fever plus weight loss and/or splenomegaly, plus Leishmania amastigotes in the relevant aspirate and/or positive DAT or rK39 dipstick result. Both studies included 2 controls per case matched by sex and age group . A meta-analysis estimated rK39 and DAT sensitivities of 79% and 93% in East African studies, with specificities of 85% and 96% respectively Cases were ascertained for both studies at the Kashay Aberra Hospital (KAH) which contained a VL treatment center operated by We used a community-based strategy to recruit controls for the resident study. Because a community-based approach was impossible for the migrant study due to the transience of the migrants, we recruited controls for this study in the hospital on services other than the VL treatment service. The two studies were therefore conducted independently, although an effort was made to include key variables in both questionnaires. The target sample size was determined in PS  . Based the assumption of 30% exposure among controls, we estimated that 150 cases and 300 controls were required for each study to achieve 90% power, with 95% confidence, and 5% precision to detect a variable with an odds ratio of 1.9. Details specific to each study are included below.kebeles . Controls were selected from the two nearest neighboring houses to the case. Candidate control households were excluded and the next neighboring house selected if there was no household member meeting the age and sex matching criteria, or if there had been a case of VL in the household in the last 5 years. Candidate control individuals were screened with a focused medical history to rule out past or current VL. Those with past VL by history were excluded as controls. Persons with suspected untreated VL found in the course of fieldwork were referred to the hospital for diagnosis and treatment, and were eligible to be included as cases if confirmed.Resident case patients were recruited from May to June 2009. Resident cases were identified both retrospectively from hospital records and prospectively for patients diagnosed during the recruitment period. A line listing of VL patients treated over the previous 12 months was compiled with the collaboration of MSF-Holland and the study team sought patients in the five most affected Cases and controls for the migrant study were recruited prospectively in KAH from October 2009 to February 2011, with recruitment concentrated in the months with higher migrant influx. Migrants were defined as persons living in the area temporarily for economic purposes. Patients who met the VL case definition above and reported themselves to be migrants were eligible to participate. Controls were recruited among migrants presenting to KAH with any condition other than VL or malaria, and were recruited within a week of the corresponding case. Two age-group- and sex-matched controls were recruited for each case. Negative results by rK39 rapid test were required for inclusion. The most common diagnoses among potential controls were malaria, pneumonia, trauma and HIV-related conditions. Potential controls diagnosed with malaria  were excluded.Structured questionnaires were used to collect the data. For the resident study, trained field workers administered the questionnaire under the supervision of study team members. For the migrant study, trained hospital nurses familiar with VL diagnosis and treatment procedures administered the questionnaires. One hospital supervisor coordinated data collection in collaboration with the study team. Data were single-entered in databases designed using Epidata® software and managed by the study data manager. All questionnaires were reviewed in the field and the database and questionnaire compared by two independent observers. The data were analysed using univariable and backwards stepwise multivariable conditional regression with Wald 95% confidence limits, to account for the matched design in SAS 9.2 . The Kruskal-Wallis test was used for comparison of continuous variables. Variables with p<0.10 in the univariable analyses was tested in multivariable model. Interaction between variables tested using interaction terms.Each study recruited slightly more than 150 VL cases  with two matched controls per case . Among rIn univariate analyses. factors associated with elevated risk in both studies included sleeping outside, on the ground or under an acacia tree , 3 and 4Some significant risk factors were found only in one of the studies. In the resident study, having house walls constructed from grass thatch was associated with an odds ratio of 4.5 compared to walls made from earth or other materials . Data onIn multivariable conditional regression models, both datasets demonstrated elevated risk associated with sleeping under an acacia tree at night (odds ratio (OR) 5.22 [95% confidence interval 1.66–16.42] and 4.74 [1.88–11.99] for residents and migrants respectively), indicators of poverty  and lower educational status . Strong Although the northern lowland border with Sudan has long been known as the most important VL-endemic zone in Ethiopia, our data represent the first systematic evaluations of risk factors for disease in the area. Several overarching themes emerge from both the resident and migrant data. Poverty and low educational status are strong underlying factors in both studies. Environmental associations also emerged, including sleeping under acacia trees and in more exposed settings . A protective effect of bed net use was seen in both studies, and supports the policy of net use as a control strategy. The massive scale-up of insecticide-treated nets for malaria control launched by the Ministry of Health in 2005 may therefore have collateral benefits for VL control Humera is located near the Eritrean and Sudanese borders, acting as a node for cross-border trade and traffic. An estimated 250,000 migrant workers come to the area each farming season . Many migrants come from the highlands, where until recently there was no reported VL transmission. Return of newly infected migrant workers was hypothesized to have triggered the VL epidemic in Libo Kem Kem and Fogera in the mid-2000s P. orientalis has generally been considered exophilic and exophagic P. orientalis, with high sand fly densities found in association with acacia-balanites forest Risk associated with nocturnal outdoor exposure was also seen in our earlier study in Libo Kem Kem We found no association with animal ownership in our resident analysis, in contrast to findings in Libo where dog ownership was linked to increased risk and Sudan where both dogs and cattle appeared as risk factors for VL The two studies we performed used different recruitment methods for logistical reasons, and this difference may have affected characteristics of the control groups, in particular. The use of village controls, as in the resident study, is generally considered optimal in terms of comparability of case and control groups. We chose resident controls with no history of kala-azar in the household in the previous 5 years, because of the strong household clustering seen in VL, the fact that many of the factors evaluated are household-level variables, and the high likelihood that household members of kala-azar cases might have undetected subclinical VL infection Finally, in our studies as in many others, VL risk was strongly associated with poverty and factors linked to poverty Checklist S1STROBE checklist.(DOCX)Click here for additional data file.


The endocannabinoid system is comprised of cannabinoid receptors (CB1 and CB2), a group of endogenous neuromodulatory lipids (endocannabinoids), and the machinery for their biosynthesis, metabolism, and transit that are involved in a variety of physiological processes including pain, appetite, memory, inflammatory and immune responses. In the brain, endocannabinoids are primarily involved in retrograde signaling, being synthesized and released from postsynaptic neurons to stimulate CB1 receptors located on the presynaptic neurons. Here, we provide an overview of the current research on the transport of endocannabinoids across plasma membranes and their intracellular trafficking with an emphasis on various potential targets for developing therapeutic drugs.The two most extensively studied endocannabinoids are anandamide (AEA or N-arachidonylethanolamine) and 2-arachidonylglycerol (2-AG). They are believed to be synthesized “on-demand” in response to various physiological stimuli to modulate intracellular secondary messengers upon receptor activation. Imbalances in the endocannabinoid system, either in the central nervous system (CNS) or peripheral tissues, is associated with many types of pathologies. In particular, changes in tissue concentrations of anandamide and 2–arachidonoylglycerol have been observed in pain, inflammation, obesity, neurological and immunological disorders.During neurotransmission, stringent regulation of the receptor mediated signaling is required along with rapid removal of the endocannabinoids from the synaptic cleft. A growing body of evidence has shown that endocannabinoids can rapidly cross the plasma membrane followed by carrier-mediated transport to their intracellular sites of sequestration or hydrolysis in different subcellular locations ,2. SeverFor many years it was understood that transport of lipid molecules such as long-chain fatty acids through the cell membrane occurred by passive diffusion. However, a fundamental shift in understanding has occurred and now it is generally accepted that fatty-acids cross the cell membrane by a protein-mediated mechanism involving either specific transporter(s) and/or carrier protein(s). Pharmacological studies  indicateThe structural similarity of endocannabinoids to fatty acids suggests that they may share similar transport mechanisms. Contemporary models based on the characterization of “fatty-acid transporters” are comprised of, but not limited to, plasma membrane-associated fatty-acid binding proteins (FABPs), the cytosolic and circulating FABPs as well as a family of fatty-acid transport proteins FATP 1–6). These intracellular carrier proteins can provide potential targets for the development of therapeutics that modulate lipid-signaling pathways. Currently, the functions of individual components of the fatty-acid transporter model are being elucidated, and intracellular carrier proteins that shuttle the endocannabinoid anandamide from the plasma membrane to its intracellular targets have been identified. These include fatty acid binding proteins, albumin, heat shock protein 70, and the fatty acid amide hydrolase-like anandamide transporter protein (FLAT) –6. It ha. These iA number of compounds that inhibit the cellular uptake of anandamide have been developed; however, in most cases the mechanism by which they inhibit cellular uptake has yet to be fully determined. Even so, it has been demonstrated that AM404, AM1172, VDM11 and UCM707 as well as OMDM1, OMDM2 and LY2183240, can significantly reduce the cellular uptake of anandamide. Moreover, one or more of these compounds have been found to decrease carcinoma cell proliferation and tumor growth, and are effective in animal models against indicators of inflammation, pain, multiple sclerosis, colitis and anxiety, diarrhea. In addition, both AM404 and VDM11 were found to inhibit the reinstatement of nicotine-seeking behavior in rats, and that AM404 also prevents the development of nicotine dependence in rats. Evidence has also been obtained, from murine models, that AM404 may be effective against obsessive-compulsive disorder. Though AM404 has off-target interactions that inhibit COX-1, COX-2 and prostaglandin synthesis, this compound has been found to reduce signs of visceral pain in mice in a manner that appears to be CB1 receptor-mediated and is a molecule with overall interesting therapeutic effects that make it a candidate for further studies. It has also been reported that SB-Fl-26, an inhibitor of the intracellular fatty acid binding proteins, displays antinociceptive activity in a mouse model of inflammatory pain. Continuing effort in the design and development of pharmacological agents that inhibit anandamide uptake can serve to control lipid-signaling pathways, inflammatory responses and metabolic dysfunction, offering a new class of multi-indication pharmacophore with high potential for future therapeutic applications in the prevention and treatment of metabolic associated disease.


N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.The oligosaccharyltransferase is the signature enzyme for  N-glycosylation refers to the covalent attachment of glycans to target proteins at asparagine residues located within a conserved sequon . The oligosaccharyltransferase (OST) is the signature enzyme of the N-glycosylation pathways in all three domains of life [ of life –3. In bo of life  and AglB of life , respect of life . Both Pg of life ,4.N-glycosylation pathway in Archaea has been best studied, in terms of both glycan structure and genetic analysis of the biosynthesis and assembly of the glycan, in three distinctive model organisms, namely the methanogen Methanococcus maripaludis, the extreme halophile Haloferax volcanii and the thermoacidophile Sulfolobus acidocaldarius [N-linked glycans in archaea are S-layer proteins [N-glycosylation pathway involves the sequential addition of sugar monomers onto a dolichol-type lipid carrier embedded in the cytoplasmic membrane, followed by a flipping of the lipid-linked glycan across the membrane. Finally, on the external face side of the cytoplasmic membrane, AglB transfers the glycan from the lipid carrier onto the acceptor protein en bloc [N-glycan has been shown to occur in a very limited number of archaeal species [The aldarius ,7,8. Glyaldarius ,9,11. Thproteins –14, archproteins –19 and pproteins –22. The  en bloc . Further species ,25.N-acetyl-glucosamine, N-acetyl-galactosamine or a simple hexose [Mc. maripaludis and Hfx. volcanii, aglB deletion mutants have been isolated [S. acidocaldarius [Among the three domains, it appears that glycan structures as well as the nature of the linking sugar and the lipid carrier are most variable in Archaea . In diffe hexose . The lipe hexose ,24,26–30isolated ,12,31 whaldarius .Archeoglobus fulgidus and Pyrococcus furiosus. In in vitro experiments, neither enzyme could process the lipid-linked glycan of the other organism [Haloarcula marismortui, Halobacterium salinarum and Haloferax mediterranei could all functionally replace the oligosaccharyltransferase activity in a Hfx. volcanii ΔaglB strain. While all of these species are extreme halophiles, their respective AglBs, though structurally similar, all transfer lipid-linked glycans in their native cells that are distinct from that found in Hfx. volcanii. In addition, in at least some cases, the heterologous AglB had to accommodate a different linking sugar or dolichol length to be effective, demonstrating a relaxed specificity of the enzymes.The specificity of AglB for different sugar-donor substrates was first reported for enzymes from organism , indicatorganism . SpecifiaglB [aglB within a given organism indicated that, at least in some cases, distinct versions of the enzyme are present that possess, for example, variations in the catalytic motif WWDXG. Other studies have indicated that not all versions of AglB are constitutively expressed in organisms that have multiple copies [Hfx. volcanii, two distinct N-linked glycans have been reported, depending on the salt concentration of the medium [aglB mutant even though only a single aglB gene is detected in the sequenced genome [aglB with such low sequence similarity to known aglBs that it escaped detection or a novel mechanism/enzyme for the transfer of the low salinity glycan. The presence of multiple AglBs in a single organism, some not constitutively expressed, some with variations in conserved motifs, suggest that they may be involved in different transfer reactions with different substrates. Such is the case in Trypanosoma brucei, where there are three single subunit OSTases, all with distinct donor and acceptor specificities [The overwhelming majority of archaea (166/168 sequenced genomes examined) contain an identifiable aglB , includie copies ,24. In te medium . The trad genome  suggestificities .Mc. maripaludis, N-glycosylation of archaellins  also required for archaella assembly [fla operon, EarA [In rchaella ,39) is crchaella ,40,41. Irchaella . In mutarchaella . In vitrhe donor . While che donor . The latassembly . This ocon, EarA .Mc. maripaludis strain deleted for aglB. This information contributes to the substrate specificity/variability of AglB and may aid in studies designed to use Archaea for glyco-engineering purposes [In this contribution, we examine the ability of several heterologous AglBs to functionally compensate for the oligosaccharyltransferase activity in a purposes ,45.Methanococcus maripaludis S2 Δhpt (Mm900) [Methanococcus voltae PS, Methanothermococcus thermolithotrophicus DSM2095 were all grown in Balch Medium III [2/H2 (20:80). Methanocaldococcus jannaschii JAL-1 was grown in the minimal medium described by Ferrante et al. [Mc. maripaludis and Mc. voltae were incubated at 35°C, Mtc. thermolithotrophicus at 60°C and Mcc. jannaschii at 80°C. For complementation studies, Mc. maripaludis ΔaglB harbouring the various complementation vectors were grown in nitrogen-free medium containing puromycin (2.5μg/ml) for plasmid selection and supplemented with either L-alanine (10mM) or NH4Cl (10mM) as sole nitrogen source [Escherichia coli Top10 cells , used for various cloning steps, were grown at 37°C in Luria-Bertani (LB) broth or agar with ampicillin added (100μg/ml) for plasmid selection when required. (Mm900) , Methanodium III  under a e et al. . Mc. marn source . EscheriaglB in Mc. maripaludis was re-created using pKJ574 and methodology previously reported [aglB-14-9.An in-frame deletion of reported . FollowiaglB, from Mc. voltae (GenBank accession ABD17750), was amplified by PCR  using primers listed in Mc. voltae cells as template. The forward and reverse primers had either NsiI or MluI restriction sites added, respectively. For Mtc. thermolithotrophicus, the aglB sequence has not been previously reported. A BLAST search using the AglB protein sequence from Mc. maripaludis as bait, retrieved WP_018154595 in GenPept as the only hit  in Mtc. thermolithotrophicus DSM 2095. The gene (GenBank accession NZ_AQXV01000054.1) was amplified by PCR  using the primers listed in M. thermolithotrophicus as template. The aglB gene from Mcc. jannaschii (MJ_RS08150) was amplified by PCR  using the primers listed in Mcc. jannaschii as template. The forward primer in this case included a single nucleotide change to remove an internal NsiI restriction site while leaving the amino acid sequence unchanged. The aglB gene from Hfx. volcanii (HVO_RS12050) was amplified by PCR  with the primers listed in Hfx. volcanii aglB and Sulfolobus acidocaldarius aglB (NC_007181.1) were also synthesized with a C-terminal FLAG-tag using Mc. maripaludis codon preferences while avoiding NsiI and MluI sites . Each of the aglB genes was digested with NsiI and MluI and cloned into pHW40 previously digested with the same restriction enzymes to generate the complementation plasmids used in this study  or alanine (nif promoter induced). At least three transfers in nitrogen-free medium supplemented with alanine were done prior to analysis.n method . The comΔaglB mutant cells carrying either pKJ1240  or pKJ1251  was extracted using a High Pure RNA Isolation Kit (Roche Life Science), followed with an additional DNase digest using an Ambion™ TURBO DNA-free Kit (Invitrogen). The presence of the aglBsa or aglBhv transcript in the RNA extract was detected using a OneStep RT-PCR Kit (Qiagen) with 10 ng of the total RNA extract as template and the corresponding gene-specific primers listed in ΔaglB mutant cells carrying either pKJ1240 or pKJ1251 as template and the corresponding gene-specific primer pair, but omitting the reverse transcription step were conducted to rule out the possibility of DNA contamination of the RNA template.Total RNA from ΔaglB mutant cells carrying plasmid-borne aglBsa (pKJ1240) as template and the aglBhv primer pair and RNA extract from ΔaglB mutant cells carrying plasmid-borne algBhv (pKJ1251) as template and the aglBsa primer pair to further exclude the possibility of non-specific amplification of the two primer pairs.PCR experiments using pKJ1240 or pKJ1251 as template and the corresponding gene-specific primer pairs were performed to confirm the amplicon size and primer specificity in the RT-PCR. In addition, RT-PCR experiments were also conducted using RNA extract from Mc. maripaludis Mm900, Mc. maripaludis ΔaglB-14-9 and the various Mc. maripaludis ΔaglB-14-9 strains carrying complementation plasmids were separated by SDS-PAGE (12.5% acrylamide gels) and transferred to Immobilon-P membrane  and examined by western blotting using anti-FlaB2 specific antibodies, as previously described [4Cl or alanine prior to the western blotting experiments. Detection of FLAG-tagged versions of Hfx. volcanii and S. acidocaldarius AglB in whole cell lysates of the appropriate complementation strains was attempted using anti-FLAG antibodies .Whole cell lysates of escribed . ComplemMc. maripaludis strains after staining with 2% (w/v) phosphotungstic acid, pH 7.0, as previously described [Transmission electron microscopy was performed on the various escribed .http://www.enzim.hu/hmmtop/) [The presence of transmembrane domains in AglB proteins selected for this study was predicted using HMMTOP (hmmtop/) .N-glycosylation pathway in Archaea, namely the transfer of a glycan from its lipid carrier to the target protein [aglB deletion strain in Mc. maripaludis, attempts were made to complement that strain with a wildtype copy of aglB expressed in trans. However, it was not possible to confirm that the expressed wildtype copy of aglB could rescue the deletion strain since the aglB mutant quickly stopped synthesis of FlaB2 upon repeated serial transfers [AglB is the oligosaccharyltransferase that performs the most conserved and critical terminal step in the  protein . Followiransfers . This turansfers .aglB deletion and immediately store aliquots of the mutant, designated ΔaglB-14-9, at -80°C while it was still synthesizing FlaB2 that could be detected in western blots. Using pKJ677 expressing wildtype Mc. maripaludis aglB from a nif promoter, it was possible to show complementation of the deleted aglB in the ΔaglB-14-9 mutant. This was initially demonstrated by western blotting where the faster migrating version of the reporter glycoprotein FlaB2 in ΔaglB-14-9 was fully restored to wildtype size following growth of the complemented strain in nitrogen-free medium supplemented with alanine, where transcription from the nif promoter is induced  and from other more distantly related archaeal species in which the N-glycosylation system has been best studied, namely S. acidocaldarius and Hfx. volcanii. Successful complementation would also indicate, in cases where it was not yet known (Mtc. thermolithotrophicus and Mcc. jannaschii), that the putative aglB gene did encode an active oligosaccharyltransferase. The topology of AglB proteins include a typical 13 transmembrane helices in the N-termini with extracellular loops between transmembrane helixes, and a soluble C-terminal domain located in the extracellular side of the cytoplasmic membrane where the catalytic site is located [Mc. maripaludis AglB to the other AglBs used in this study is presented in Mc. voltae, the thermophilic Mtc. thermolithotrophicus  and the hyperthermophilic Mcc. jannaschii . These methanogens, as well as Mc. maripaludis, are all stringent anaerobes that grow optimally at neutral pH with 1–4% NaCl. Hfx. volcanii is an extreme halophile growing optimally in 1.5–2.5M NaCl, at pH 7 and 45°C. S. acidocaldarius, on the other hand, is a thermoacidophile, growing optimally at 70–75°C and pH 2–3. Since the catalytic site in AglB is predicted to be orientated extracellularly [Hfx. volcanii and S. acidocaldarius may have specific adaptations to function in high salt or low pH that would not be required for the methanogen AglBs.To study the promiscuous nature of archaeal AglBs, the  located –56. A pallularly , the enzMtc. thermolithotrophicus was most similar to AglB of Mc. maripaludis, being almost 60% identical (and 77.5% similar) and almost identical in length . AglBs from Mc. voltae and Mcc. jannaschii were larger by 65 and 81 amino acids, respectively and slightly less than 50% identical to the Mc. maripaludis enzyme. Most of the extra length in the Mcc. jannaschii enzyme is found in one large insertion of about 70 amino acids (between amino acids 227 and 299), when compared to the Mc. maripaludis AglB. This insertion is predicted to contain 2 additional transmembrane helices, resulting in a total of 15, compared to 13 for AglBs of Mc. maripaludis, Mc. voltae and Mtc. thermothithotrophicus. The AglBs from the two non-methanogens tested, Hfx. volcanii and S. acidocaldarius, were much less similar to the Mc. maripaludis AglB, being only 17.9 and 19.9% identical, respectively. In addition, these two oligosaccharyltransferases were much different in length compared to the Mc. maripaludis AglB. The Hfx. volcanii AglB was almost 200 amino acids larger than the Mc. maripaludis enzyme while that of S. acidocaldarius was almost 100 amino acids smaller.AglB of Hfx. volcanii it is GND and in S. acidocaldarius it is GFD. In all cases, this motif is found in the first extracellular loop in the N-terminus of the protein. The second motif, WWDXG, is necessary for catalysis and found in the extracellular C-terminal domain. In the four methanogens, this motif is identical, WWDNG, while in both Hfx. volcanii and S. acidocaldarius the sequence of this motif is WWDYG. Most variation is found in the third motif, the DK (DXXK or the relaxed version DXXMXXX(M/I)) or MI (MXXIXXX(I/V/W) motif [N-linked glycosylation sequon (N-X-S/T) and is found adjacent to the WWDXG motif in the tertiary structure of the enzymes. The DK motif is found in most AglB proteins (as well as in eukaryotic Stt3) although variations in the motif have been reported in terms of an insertion of 4–14 amino acids that interrupts the DK motif in some archaea (DXXMX(4–14)K), including in certain extreme halophiles. Hfx. volcanii AglB has a 5 amino acid insertion here. S. acidocaldarius AglB has the DK motif (DIAK) which is typical of Crenarchaeota. In lieu of the DK motif, some archaea  have an MI motif at the same position which is thought to perform the same function as the DK motif. This is the case for the four methanogens in this study. The three key amino acids are conserved in all four cases (MXXIXXXW) although there is some variation in the X positions  motif . This moositions . Since tMc. maripaludis ΔaglB mutant, the initial screen to indicate successful complementation was by western blotting after growth of the complemented ΔaglB-14-9 strain in nitrogen-free medium supplemented with alanine or NH4Cl. Only a limited number of archaeal N-linked glycan structures are known [Mc. voltae has an N-linked glycan structure been reported. It is a trisaccharide with significant similarity to the tetrasaccharide of Mc. maripaludis [N-acetylated. In the case of Mc. voltae, this position is held by an N-acetyl-glucosamine while in Mc. maripaludis the linking sugar in the glycan is N-acetyl-galactosamine. Transformation of the ΔaglB-14-9 strain with pKJ1229 containing Mc. voltae aglB, followed by growth in alanine supplemented nitrogen-free medium indicated that the Mc. voltae enzyme was capable of restoring the lost oligosaccharyltransferase activity leading to the production of wildtype sized FlaB2s  from a whole genome shotgun sequence (GenBank accession No. NZ_AQXV01000019) with high sequence identity to aglB from Mc. maripaludis. Despite being a thermophile with optimal growth near 60°C, this gene from Mtc. thermolithotrophicus was able to very effectively restore wildtype size FlaB2  found in the Mcc. jannaschii AglB lacking in the remaining three methanogen AglBs. The lack of complementation may be due simply to the activity profile of the Mcc. jannaschii AglB in regards to temperature. Complementations of the aglB mutant of Mc. maripaludis were done at 35°C and 40°C while Mcc. jannaschii grows optimally above 80°C. Growth of Mc. maripaludis can occur to 45°C but above 42°C the reporter protein we use, FlaB2 archaellin, is not made [aglBs were cloned into the same site of the same vector and transcribed from the same nif promoter, we cannot rule out that the lack of activity demonstrated was due to degradation/instability of the expressed Mcc. jannaschii AglB. Furthermore, the lipid composition of the cytoplasmic membrane of Mcc. jannaschii is known to vary considerably depending on the growth temperature. At the low end of its growth range (45°C), the lipids are mostly typical diethers, but as the growth temperature is raised to 75°C the proportion of tetraether lipids and a novel macrocyclic diether lipid dramatically increase [Mcc. jannaschii aglB to complement in Mc. maripaludis may thus be due to faulty insertion of the AglB into the strictly diether lipids in the cytoplasmic membrane of Mc. maripaludis [Mc. voltae or Mtc. thermolithotrophicus since both these organisms have membrane lipids that are also diethers [Mtc. thermolithotrophicus, no N-linked glycan structures have been reported for Mcc. jannaschii and, likewise, there are no reports on the structures of dolichol phosphate carriers so it is possible that AglB in Mcc. jannaschii would naturally recognize a unique glycan structure possibly with a different linking sugar or a different lipid carrier and was not able to utilize the Mc. maripaludis versions.All attempts to show complementation of the Δccessful . The fainot made . Howeverincrease . The inaipaludis . This wodiethers . As withaglBs from S. acidocaldarius and Hfx. volcanii , RT-PCR demonstrated that the mRNA for each aglB was present in the respective complementations .The other two failed complementations used the volcanii . These tand AglB . For thentations . Expressreported , Taguchin E.coli . Express degrees . The aglHfx. volcanii, the N-linked glycosylation system is very complicated. Completely different glycans are found depending on the salt content of the growth medium. At high salt (3.4M NaCl), a pentasaccharide  is made [aglB gene is reported for Hfx. volcanii but this AglB is only required for the transfer of the pentasaccharide but not the low salt tetrasaccharide [Hfx. volcanii AglB to that of Mc. maripaludis is much lower (17.9/ 31.0%) than that shown by the Mc. voltae and Mtc. thermolithotrophicus enzymes and the halophile enzyme is much longer. In addition, the DK/MI motif is completely different in halophiles and methanogens. The DK motif has been shown to be catalytically important both in yeast Stt3 [P. furiosus and A. fulgidus [Hfx. volcanii, the sequence is the modified DM version of the motif, DWQMASTDAK. Methanococci have the MI motif at the corresponding position in AglB believed to perform the same role as the DK/DM motif and in Mc. maripaludis the MI motif is MTSIASVW. In PglB of Campylobacter jejuni, the MI motif (MSLIFSTV) has been shown by mutational analysis to be important for catalytic activity with only chemically similar amino acids tolerated at the conserved positions [Hfx. volcanii and not properly folded in the 2% NaCl-containing medium used for Mc. maripaludis. It is also possible that the halophile AglB was misfolded and either degraded or did not insert properly into the cytoplasmic membrane, although the membrane lipids of Hfx. volcanii are diether types like that in Mc. maripaludis [Hfx. volcanii AglB complementation can also be envisioned. One is the difference in the linking sugar in the glycans of Hfx. volcanii and Mc. maripaludis. However, it is known that the Hfx. volcanii aglB can be replaced with aglB from different extreme halophiles that can transfer glycans with either a hexose or N-acetylhexuronic acid as a linking sugar so one would expect that the AglB from Hfx. volcanii would accommodate a GalNAc linking sugar. Another unlikely reason for the failure of the Hfx. volcanii AglB to complement could lie in the nature of the dolichol carrier, but the Hfx. volcanii carrier is C55-C60 dolichol phosphate with saturated isoprenes at the α and ω positions [Mc. maripaludis it is a C55 dolichol phosphate that has two sites of saturation, presumed to be the α and ω positions too [In  is made . The fir is made ,25. Here is made . Intriguccharide . The oliast Stt3  as well fulgidus ,56. In Hositions . Given tositions , it is pipaludis . Other lositions  and in MS. acidocaldarius AglB. In S. acidocaldarius, the N-linked glycan is a complex tri-branched hexasaccharide containing the unusual sulfated sugar, sulfoquinovose and with a linking sugar of N-acetylglucosamine [Mc. maripaludis. Even with an AglB from the much more closely related hyperthermophile Mcc. jannaschii, we were unable to show complementation. Furthermore, in its native environment AglB of S. acidocaldarius would be inserted into a cytoplasmic membrane comprised of tetraether lipids, including ones that could contain up to 4 cyclopentane rings [Hfx. volcanii AglB, the one from S. acidocaldarius shows low sequence identity and similarity to that of Mc. maripaludis (19.9%/34.1%). The S. acidocaldarius AglB has the DK motif (DIAK) rather than the MI motif of Mc. maripaludis AglB. Furthermore, there are potentially significant differences in the substrates encountered. The linking sugar in the glycan of S. acidocaldarius is GlcNAc and not GalNAc as in Mc. maripaludis. In addition, the dolichol carrier normally recognized by AglB in S. acidocaldarius was recently shown to be an unusual, short (C45) dolichol pyrophosphate [S. acidocaldarius AglB towards the Mc. maripaludis dolichol carrier, identified as C55 dolichol phosphate [The other failed complementation occurred with the cosamine . Many pone rings ,68. Cytone rings ,70. As whosphate  and not hosphate  and thishosphate .in vivo experiments with extreme halophiles [in vitro with the hyperthermophiles A. fulgidus and P. furiosus [Mc. voltae AglB to transfer glycans of similar but different composition, including glycans with either GlcNAC or GalNAc as the linking sugar. We have also identified for the first time the AglB from the thermophilic methanogen Mtc. thermolithotrophicus and demonstrate that it can functionally replace the oligosaccharyltransferase activity in the Mc. maripaludis ΔaglB mutant. As there are currently no published data on the nature of N-linked glycans in Mtc. thermolithotrophicus, it is not possible to state how different the Mc. maripaludis glycan is from that naturally transferred by the Mtc. thermolithotrophicus AglB. We have begun studies to determine the N-glycan attached to archaellins in this thermophilic methanogen to address this issue. Attempts to complement using AglBs from more distantly related and more extremophilic archaea for which data on N-linked glycosylation systems is well known were unsuccessful, although the possible reasons for this are many. The accumulation of data on the relaxed nature of substrates accepted by various AglBs will aid in any efforts to develop archaea as platforms for glycoengineering [This study extends to methanogens the examination of AglB promiscuity previously reported in lophiles  and in vfuriosus . We haveineering .S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.


Dengue fever is the most important arboviral infection that affects humans, particularly in tropical and subtropical regions. Here, we provide the first comprehensive overview of the severity of dengue epidemics in French Guiana.3 were associated with dengue severity.We monitored hospitalized cases between 2008 and 2013. Detailed clinical features and biological parameters were collected on a daily basis from all cases. Among the 1,356 cases, 216 (16%) were classified according to the WHO 2009 classification as dengue without warning signs (WS), 926 (68%) were classified as dengue with WS and 214 (16%) were classified as severe dengue. The severity rates were similar between the three major epidemics that occurred during the study period, whereas the hospitalization rate was highest in 2013. Fluid accumulation, aspartate aminotransferase (ASAT) counts>193 IU/L and platelet counts<75,000 cells/mmOur findings provide a recent epidemiological description of the severity of dengue epidemics in French Guiana. These results highlight the potential impacts and consequences of implementing the WHO 2009 classification on hospital activity. Future studies should include virological and immunological investigations of well-documented serum samples. Aedes mosquitoes. Recent estimates indicate that dengue fever is the most important arthropod-borne human disease, with infections primarily occurring in tropical and subtropical regions; there are 390 million dengue infections per year worldwide, of which 96 million are clinically apparent , [32–62] and [>62]. A quantitative variable “delta-hemat” was calculated using the equation Between October 2008 and December 2013, 1,356 hospitalized cases were included in the study, of which 243 were hospitalized during the 2009 epidemic, 115 in 2010 and 694 in 2013 . As showAs shown in A comparison of the clinical category distributions revealed an increase in the hospitalization rate in 2013 (4.3% vs. 1.8% in 2009 and 1.3% in 2010) . By cont−3). The 2013 epidemic had the highest impact on hospital admissions during the study period, with 694 hospitalizations. However, the duration of hospitalization did not show any significant variation between epidemics , whereas the 2010 epidemic had a lower severity rate (0.3%) . Overallpidemics .3, transaminases level higher than 62 UI/L or a variation in hematocrit levels . The 2009 epidemic was caused predominantly by DENV-1 (67.6%) and DENV-4 (27.0%). DENV-2 was isolated throughout 2010, although the predominance of DENV-1 and DENV-4 were 48.8% and 43.9%, respectively. In 2013, a clear predominance of DENV-2 was observed, representing 96.7% of hospitalized cases. The distributions of the four serotypes were significantly different betweenclinical categories (P = 0.049). The DENV-2 serotype was more strongly associated with dengue severity  than was DENV-1 . DENV-1 was therefore over-represented among dengue cases without WS, whereas DENV-2 was under-represented in this category.Transaminase (ASAT and ALAT) levels progressively increased between days 1 and 5 days of the illness , with daThis study was the first of its kind conducted in French Guiana describing the clinical and biological features associated with hospitalized dengue cases. The five-year system of monitoring hospitalized cases allowed us to describe the dynamics, magnitude and severity of the three epidemics that occurred since 2008. The impacts of the outbreaks on hospital activity were considerable in 2013, moderate in 2009 and minimal in 2010. The hospitalization rate was the highest during the 2013 outbreak (4.2%), which was noteworthy in terms of duration and number of estimated cases.The retrospective implementation of the WHO 2009 classification for both epidemics among hospitalized cases enabled us to compare the severity of the epidemics. Although the number of admissions substantially increased in 2013, the severity rates were similar in 2009 and 2013. These results may be linked to increased awareness due to the WHO 2009 classification recommending that patients with any WS should be admitted for close observation ,24, and Health care policies recommend immediate admission for vulnerable patients  with dengue-like syndrome to avoid severe illness. Professional practices following these recommendations could have explained the small number of infants with severe forms of the disease (7%). In addition, many studies have noted that secondary infection with another serotype could be a risk hazard for SD –30. ThisAccording to the univariate analysis, we found that the presence of petechia, epistaxis, clinical fluid accumulation, hepatomegaly, low pulse rate and abdominal pains were associated with SD. At day 5 of the illness, low albuminuria, natremia and serum total protein levels as well as low platelet counts were associated with SD. Patients with SD had higher ASAT and ALAT levels and higher hematocrit variations at day 5 than patients classified with dengue without WS or with WS. We found that low platelet count and transaminases levels were significantly associated with SD. Recent studies regarding the 2009 WHO classification confirmed these results ,32. In aOne limitation of this study was that inclusion was restricted to hospitalized cases, which does not allow us to generalize our findings to all dengue cases. Indeed, our results regarding the factors associated with SD could be different if ambulatory cases were included in the study.One of the strengths of our study was that all hospitalized cases between November 2008 and December 2013 admitted at any of the three hospitals for the whole territory were enrolled ina large study population , giving our results good power and representation. Additionally, a single clinician classified all of the cases, leading to high quality data and avoiding biases related to variations in medical practices. In addition, the daily data collection from the time of admission until discharge allowed us to describe the clinical evolution and to study the clinical and biological features throughout the hospitalization period. We cannot rule out the possibility that clinical signs and symptoms used in the WHO 2009 system may not have been stringently collected prior to its implementation. Nevertheless, clinical features collected during the study period appeared consistent with the proper implementation of WHO 2009 classification.The pathophysiology of SD appears to be multifactorial, entailing complex interactions among viral factors, host genetics and the immunologic background of the host, the most important being prior to exposure to dengue virus. Hyperendemicity with different serotypes is believed to be one of the most significant factors influencing dengue severity . Future We presented a comprehensive epidemiological description of the severity of dengue epidemics in French Guiana. We also identified potential new clinical and biological predictive factors of dengue severity, although further work in identifying other virological and immunological markers is needed to improve our knowledge of the risk factors in French Guiana.