Recent evidence suggests that endogenous arginase activity potentiates airway responsiveness to methacholine by attenuation of agonist-induced nitric oxide (NO) production, presumably by competition with epithelial constitutive NO synthase for the common substrate, L-arginine. Using guinea pig tracheal open-ring preparations, we now investigated the involvement of arginase in the modulation of neuronal nitric oxide synthase (nNOS)-mediated relaxation induced by inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation.Electrical field stimulation -induced relaxation was measured in tracheal preparations precontracted to 30% with histamine, in the presence of 1 μM atropine and 3 μM indomethacin. The contribution of NO to the EFS-induced relaxation was assessed by the nonselective NOS inhibitor L-NNA (0.1 mM), while the involvement of arginase activity in the regulation of EFS-induced NO production and relaxation was investigated by the effect of the specific arginase inhibitor nor-NOHA (10 μM). Furthermore, the role of substrate availability to nNOS in EFS-induced relaxation was measured in the presence of various concentrations of exogenous L-arginine.EFS induced a frequency-dependent relaxation, ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz, which was inhibited with the NOS inhibitor L-NNA by 78.0 ± 10.5% at 0.5 Hz to 26.7 ± 7.7% at 8 Hz . In contrast, the arginase inhibitor nor-NOHA increased EFS-induced relaxation by 3.3 ± 1.2-fold at 0.5 Hz to 1.2 ± 0.1-fold at 4 Hz , which was reversed by L-NNA to the level of control airways in the presence of L-NNA . Similar to nor-NOHA, exogenous L-arginine increased EFS-induced airway relaxation .The results indicate that endogenous arginase activity attenuates iNANC nerve-mediated airway relaxation by inhibition of NO generation, presumably by limiting L-arginine availability to nNOS. The inhibitory nonadrenergic noncholinergic (iNANC) nervous system is the most effective bronchodilating neural pathway of the airways. Inhibition of nitric oxide synthase (NOS) markedly reduces the iNANC relaxation of both guinea pigs -3 and huNO is generated by a family of NOS isoforms that utilize the semi-essential amino acid L-arginine, oxygen and NADPH as substrates to produce NO and L-citrulline . Three iAnother L-arginine metabolizing enzyme is arginase, which hydrolyzes L-arginine to L-ornithine and urea. Arginase is classically considered to be an enzyme of the urea cycle in the liver, but also occurs in extrahepatic tissues, including the lung ,13. Two A role for arginase in the iNANC system has been found in internal anal sphincter  and peniThe role of endogenous arginase in the regulation of iNANC-derived NO generation in the airways has not yet been investigated. In the present study, we demonstrated that endogenous arginase activity and L-arginine availability are importantly involved in the modulation of iNANC nerve-mediated NO-production and relaxation of guinea pig tracheal smooth muscle.ad libitum, while a 12-h on/12-h off light cycle was maintained.Male specific pathogen free HsdPoc:Dunkin Hartley guinea pigs , weighing 500 – 800 g, were used in this study. The animals were group-housed in individual cages in climate-controlled animal quarters and given water and food All protocols described in this study were approved by the University of Groningen Committee for Animal Experimentation.2 and 5% CO2. The composition of the KH-solution in mM was: NaCl 117.50; KCl 5.60; MgSO4 1.18; CaCl2 2.50; NaH2PO4 1.28; NaHCO3 25.0 and D-glucose 5.50; pH 7.4. The trachea was prepared free of serosal connective tissue. Twelve single proximal tracheal open-ring preparations were mounted for isotonic recording (0.3 g preload) between two parallel platinum point-electrodes in water-jacketed (37°C) organ baths containing 20.0 ml of gassed KH-solution and indomethacin (3 μM), which remained present during the whole experiment to eliminate any influence of prostanoids.The guinea pigs were sacrificed by a sharp blow on the head. After exsanguination, the trachea was removed from the larynx to the bronchi and rapidly placed in a Krebs-Henseleit (KH) buffer solution of 37°C, gassed with 95% Oω-nitro-L-arginine , the specific arginase inhibitor Nω-hydroxy-nor-L-arginine , a combination of both inhibitors, or L-arginine  were applied 30 min prior to histamine-addition. In line with previous observations [After a 30 min equilibration period, tracheal preparations were relaxed with isoprenaline (0.1 μM) to establish basal tone. After a washout period of 30 min with three washes with fresh KH solution, maximal contraction of the tracheal preparations to histamine was determined with cumulative additions of the agonist . After washout (30 min), the tracheal preparations were precontracted with histamine to 30% of the maximal histamine-induced tone in the presence of atropine (1 μM) to prevent EFS-induced cholinergic airway contraction. On the plateau, biphasic EFS  was applied and frequency response curves (0.5 – 16 Hz in doubling steps) were recorded. Per preparation, one frequency response curve was performed. When used, the nonselective NOS inhibitor Nrvations , neitherAll individual relaxations elicited by EFS were estimated as peak height of the EFS-induced response, and were expressed as a percentage of maximal relaxation as established in the presence of isoprenaline. The contribution of NO to the EFS-induced relaxation was determined by the effect of the NOS inhibitor L-NNA. Similarly, the role of arginase activity in the modulation of EFS-induced airway relaxation was determined by the effect of the arginase inhibitor nor-NOHA. The role of substrate availability in EFS-induced airway relaxation was assessed by measuring the responses in the presence of various concentrations of exogenous L-arginine.P < 0.05.All data are expressed as means ± s.e.m. Statistical significance of differences was evaluated using a paired or unpaired two-tailed Student's t-test as appropriate, and significance was accepted when ω-nitro-L-arginine, (-)-isoprenaline hydrochloride and L-arginine hydrochloride were obtained from Sigma Chemical Co. . Nω-hydroxy-nor-L-arginine was kindly provided by Dr J.-L. Boucher (Université Paris V).Histamine dihydrochloride, indomethacin, atropine sulphate, NP < 0.01 all; Fig. In guinea pig tracheal open-ring preparations, EFS induced a frequency-dependent relaxation of histamine-induced tone ranging from 6.6 ± 0.8% at 0.5 Hz to 74.6 ± 1.2% at 16 Hz. Incubation with the NOS inhibitor L-NNA caused a significant inhibition of the EFS-induced relaxation at 0.5 to 8 Hz, particularly at the lower frequencies. The effect of L-NNA ranged from 78.0 ± 10.5% inhibition at 0.5 Hz to 26.7 ± 7.7% inhibition at 8 Hz , to the level of control preparations in the presence of L-NNA alone . In the presence of 5.0 mM L-arginine, a significant increase in EFS-induced relaxation was observed at all frequencies compared to untreated preparations (Using perfused tracheal preparations, we have previously demonstrated that endogenous arginase activity is involved in the regulation of agonist-induced airway constriction by inhibition of NO production, presumably by competition with cNOS for L-arginine . In the In line with previous observations , it was The observation that exogenous L-arginine significantly enhanced the EFS-induced airway smooth muscle relaxation confirms that L-arginine is indeed a limiting factor in EFS-induced, NO-mediated airway smooth muscle relaxation under basal conditions. Remarkably, the effect of nor-NOHA was similar to that observed in the presence of the maximally effective L-arginine concentration, indicating that endogenous arginase activity is a major factor in regulating the neural NO-mediated airway smooth muscle relaxation.Aspergillus fumigatus [Recently, we discovered that increased arginase activity is importantly involved in the pathophysiology of asthma by contributing to the allergen-induced NO-deficiency and subsequent airway hyperresponsiveness to methacholine after the early asthmatic reaction, by limiting the availability of L-arginine for cNOS to produce bronchodilating NO . Arginasumigatus . Moreoveumigatus , while iumigatus . In guinumigatus . Thus, iA role for arginase by restricting the L-arginine availability for nNOS in iNANC nerves has also been proposed in the pathophysiology of erectile dysfunction . In suppThis is the first demonstration that endogenous arginase activity is functionally involved in iNANC nerve activity in the airways, by attenuating the generation of nNOS-derived NO. Disturbance of this novel regulation mechanism of airway responsiveness might be involved in the pathophysiology of allergic asthma.ω-nitro-L-arginine; NADPH, nicotinamide adenine dinucleotide phosphate; nNOS, neuronal nitric oxide synthase; nor-NOHA, Nω-hydroxy-nor-L-arginine; VIP, vasoactive intestinal polypeptidecNOS, constitutive nitric oxide synthase; EFS, electrical field stimulation; eNOS, endothelial nitric oxide synthase; iNANC, inhibitory nonadrenergic noncholinergic; iNOS, inducible nitric oxide synthase; KH, Krebs-Henseleit; L-NNA, NThe authors declare that they have no competing interests.HMa designed and coordinated the study, performed a major part of the experiments, performed the statistical analysis and drafted the manuscript. MAT assisted substantially in performing the experiments. JZ participated in the design of the study, interpretation of results and final revision of the manuscript. HMe conceived of the study, participated in its design and direction, as well as in preparing the manuscript. All authors read and approved the final manuscript.


Obstetric palsy is the injury of the brachial plexus during delivery. Although many infants with plexopathy recover with minor or no residual functional deficits, some children don't regain sufficient limb function because of functional limitations, bony deformities and joint contractures. Shoulder is the most frequently affected joint with internal rotation contracture causing limitation of abduction, external rotation. The treatment comprises muscle release procedures such as posterior subscapularis sliding or anterior subscapularis tendon lengtening and muscle transfers to restore the missing external rotation and abduction function.We evaluated whether the preoperative abduction degree affects functional outcome. Between 1998 and 2002, 46 children were operated on to restore shoulder abduction and external rotation. The average age at surgery was 7.6 years and average follow up was 40.8 months. We compared the postoperative results of the patients who had preoperative abduction less than 90° (Group I: n = 37) with the patients who had preoperative abduction greater than 90° (Group II: n = 9), in terms of abduction and external rotation function with angle measurements and Mallet classification. We inquired whether patients in Group I needed another muscle transfer along with latissimus dorsi and teres major transfers.In Group I the average abduction improved from 62.5° to 131.4° (a 68.9° ± 22.9°gain) and the average external rotation improved from 21.4° to 82.6° (a 61.1° ± 23°gain). In Group II the average abduction improved from 99.4°to 140°(a40.5° ± 16°gain) and the average external rotation improved from 33.2°to 82.7° (a 49.5° ± 23.9° gain). Although there was a significant difference between Group I and II for preoperative abduction (p = 0.000) and abduction gain in degrees (p = 0.001), the difference between postoperative values of both groups was not significant (p = 0.268). There was also no significant difference between the two groups in the preoperative external rotation, the external rotation gain and the postoperative external rotation .Obstetric palsy patients with shoulder sequela who had a preoperative abduction less than 90°hadas good functional results using latissimus dorsi, teres major muscle transfer and subscapularis muscle release as the patients who hada preoperative abduction greater than 90°. Although the extent and severity of the deformity in obstetrical palsy may vary from patient to patient, shoulder is the most frequently affected joint. Many clinical and radiological classification systems had been described to address the problem and tailor the solution at the shoulder -5. AmongUsually release and transfer procedures are seperated depending on the age of the patient and condition of the shoulder but in older age group (>4 years), it is preferred to combine the shoulder release and muscle transfer simultaneously -8.Some authors perform other muscle transfers like levator scapulae and trapezius along with latissimus dorsi and teres major only for total flail shoulders , others In this study, we compared the results of combined release and tendon transfer operations performed at the same stage because of the late presentation of the cases, for shoulder abduction and external rotation in two groups of patients who did not have any surgical treatment before. In one group preoperative abduction degree was less than 90°, while in the other group it was equal or more than 90°. We compared the gains of abduction and external rotation in two groups and evaluated whether the preoperative abduction degree affects the functional outcome and in patients with preoperative abduction degree <90° and also another muscle transfer is needed along with latissimus dorsi and teres major transfers.Between 1998 and 2002, 46 children with obstetrical brachial plexus palsy who had no surgical treatment before  were operated to restore shoulder abduction and external rotation in our clinic. The average age at surgery was 7.6 years (3–16). The patients had an average follow up of 40.8 months (range 24 to 60 months).Involvement of right side was seen in 30 patients and the left side in 16 patients. No patient had bilateral involvement. All of the patients were vaginally delivered with vertex presentations. Obstetrical history revealed that most mothers were multiparous and 8 of the patients were delivered with the help of forceps or vacuum. The mean birth weight of the patients was 4.5 kg (3–6.6 kg).The pool of the patients consisted 12 of patients with C5–C6 spinal root involvement, while 11 of the patients had additional C7 involvement. Finally 23 of the patients had total brachial plexus roots involvement. Accompanying birth complications were fracture of clavicle in one case, injury of sternocleidomastoideus muscle in one case and Horner's Syndrome in 2 cases.Table 1 see  summarizp < 0.05.We compared the postoperative results of the patients who had preoperative abduction less than 90°  with the patients who had preoperative abduction equal or more than 90° , in terms of abduction and external rotation function with angle measurements and Mallet classification. The statistical analysis was performed with analysis of variance (ANOVA) and Student's t-test. Statistical significance was presumed at Preoperative and postoperative active and passive range of motion degrees of abduction and external rotation were measured, videos were recorded during shoulder abduction and external rotation and Mallet scores Figure  were notRadiography of the shoulder in adduction and 90° abduction and axial magnetic resonance imaging of the shoulder was performed. Shoulder deformity was classified according to Waters-Peljovich grading system, Table Most of the times, preoperative physical examination revealed weakness of the deltoid muscle and external rotators as well as co-contraction of pectoralis major, latissimus dorsi and teres major muscles at the anterior and posterior margin of the axillary fossa during shoulder elevation specially in Group I patients.® Plus (Johnson & Johnson) which had size 2 green ethibond polyster sutures on it, was applied at the insertion point of infraspinatus muscle on the rotator cuff  to 131.4° (90°–165°) and the average external rotation degree improved from 21.4° (0°–80°) to 82.6° (30°–95°) in Group I. We obtained 68.9° ± 22.9° (109%) gain for abduction and 61.1° ± 23° (285%) gain for external rotation. The difference between preoperative and postoperative values of abduction and external rotation was significant .The average abduction degree improved from 99.4° (90°–110°) to 140° (110°–170°) and the average external rotation degree improved from 33.2° (0°–65°) to 82.7° (45°–90°) in Group II. We obtained 40.5° ± 16° (40%) gain for abduction and 49.5° ± 23.9° (149%) gain for external rotation. The difference between preoperative and postoperative values of abduction and external rotation was significant . The results were summarized in Table Although difference between preoperative abduction and abduction gain values in terms of degrees of Group I and Group II were significant , the difference between postoperative values of both groups was insignificant .There was also no significant difference between the preoperative external rotation, the external rotation gain and the postoperative external rotation for both groups .The results of both groups were shown in graphics in Figure Mean Mallet scores increased from 2.8 to 3.9 for global abduction, from 2.5 to 3.9 for global external rotation, from 2.1 to 3.6 for hand to head and from 2.5 to 3.5 for hand to mouth for Group I. Hand to back Mallet score decreased from 2.5 to 2.2. All the differences between preoperative and postoperative values were found significant according to the Student's t-test .Mean Mallet scores increased from 3.5 to 4 for global abduction, from 2.8 to 4 for global external rotation, from 2.7 to 4 for hand to head and from 3.2 to 3.5 for hand to mouth for Group II. Hand to back Mallet score decreased from 2.8 to 2.1. All the differences, apart from hand to mouth score, between preoperative and postoperative values were found significant according to the Student's t-test . The difference between preoperative and postoperative hand to mouth scores was found insignificant .While the difference between preoperative Mallet scores of Group I and Group II concerning abduction, hand to head and hand to mouth was significant , the difference was insignificant for external rotation and hand to back scores . The difference between postoperative values of both groups was insignificant .The results were summarized in Table Example cases from each group can be seen in Figure Internal rotation contracture is the most frequent and important secondary deformity of the shoulder in birth palsy. The problem is sometimes addressed by muscle release procedures such as the posterior subscapular slide or an anterior subscapularis tendon lenghtening operations. Once passive external rotation is improved, the child is later assessed for muscle transfers to reconstruct active external rotation if necessary .According to Chang et al  there arHowever, there are still many controversies concerning donor muscle choice for transfer, timing and operative tecniques of palliative surgical theraphy for the shoulder deformity.The infraspinatus muscle works to center the humeral head in the glenoid throughout elevation. External rotation of the shoulder allows greater arm elevation by clearing the greater tuberosity from impingement by the coracoacromial arch. External rotation of the humerus also positions the long head biceps centrally to aid in its function as humeral stabilizer and loosens the inferior glenohumeral ligaments, thereby allowing greater arm elevation. Hence the infraspinatus muscle plays a key role in shoulder elevation as a humeral head stabilizer, an active abductor, and an external rotator of the shoulder .The importance of transferring the teres major and latissimus dorsi as one conjoined tendon and anchoring into the posterior aspect of the greater tuberosity at the insertion of the infraspinatus similar to Hoffer method is augmentation of the weakened infraspinatus. Transfer with this technique instead of rerouting around humeral neck enables a stronger external rotator power because of the increased mechanical advantage at its insertion in the humeral head as opposed to the humeral shaft. The reason for the dramatic improvement of shoulder abduction after latissimus muscle transfer is probably because the transfer enhances the stabilizing effect of the rotator cuff which enables the deltoid to act more effectively, this phenomenon was called "force couple" effect by Phipps and Hoffer .In many centers, muscle release procedures are performed before the age of two years, however for older children tendon transfer to restore abduction and external rotation is added . It is aGilbert  suggesteMuhlig et al  describeAs all of our patients were older than 2 years of age, we performed latissimus dorsi and teres major transfer at the same session with subscapularis and pectoralis major release.In total flail shoulders, despite a certain degree of innervation, the functional results of shoulder corresponds to zero with the absence of a strong latissimus dorsi. In that condition, the levator scapulae muscle is utilised as an intrinsic stabilizer of glenohumeral joint and trapezius muscle is used as a prime mover for shoulder abduction with or without latissimus dorsi and teres major transfers .Gilbert in his series of 44 patients with transfer of latissimus dorsi, the improvement of abduction was satisfactory in the shoulders which preoperatively coded as Grade III  or more, but not in those coded as Grade II  or less. Hence he thought it may be necessary to add a concomitant transfer of the trapezius to the patients whose abduction of the shoulder was weak or absent .Chen et al  assertedAl-Qattan  performeIt is our opinion that in our Group I patients, the co-contraction between shoulder abductors  and adductors  and also subscapularis muscle tightness cause limitation of shoulder elevation. If antagonistic muscles (teres major and latissimus dorsi) are transferred for the paretic muscles (infraspinatus) and the pectoralis major and subscapularis muscles are released with preserving their shoulder stability function, these children can have as succesfull postoperative shoulder abduction and external rotation as the children in Group II, who has less cross innervation hence better preoperative abduction value. Group I and II patient had almost similar postoperative mean abduction  and external rotation values .Extensive dissection of latissimus dorsi and teres major muscles from the surrounding structures gave us the opportunity to utilize both muscles for transfer, without any difficulty during the passage of the conjoined tendon through the tunnel which was prepared between long head of triceps and deltoid muscle, and also during reinsertion to the humerus.Several authors reported recurrences of the deformity in terms of reduction of external rotation and abduction gain. Two of the 12 children in Al-Qattan series  and threWe believe that in cases who has congruent glenohumeral joint (Type I-III Waters-Peljovich grading system), and deltoid muscle strength of M3–M4  but weak or absent external rotation, if the latissimus dorsi and teres major muscles have sufficient strength (M3 or more), the ideal procedure is transfer of latissimus dorsi and /or teres major onto the posterior aspect of the greater tuberosity of humerus, at the insertion of the infraspinatus. So we are not totally convinced about adding trapezius muscle transfer concomitantly with the latissimus dorsi + teres major transfer session. We rather preserve this muscle for the patients that could not achieve enough shoulder abduction after the first operation.Since our study was not randomised to treatment, the groups did not comprise equal number of patients, the assessments were not performed by independent observers but by a physiotherapy group of our team at postoperative 24 – 60 months (not at the same time for everyone), we would like to interpret our conclusions cautiously.Almost near normal shoulder function can still be reached in children who could not receive primary early neural reconstruction, by combined muscle release and muscle transfer operations which are performed before the severe glenohumeral deformities occur.We found out that the patients with obstetric palsy shoulder sequela who had a preoperative abduction value less than 90° could have good functional results as the patients who had preoperative abduction values equal or more than 90°, with latissimus dorsi, teres major muscle transfer and subscapularis muscle release.None declared.All authors participated in the design of the study, operations and drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Preoperative and postoperative range motion degrees and Mallet scores of the patients. Patients with bold numbers are in Group II with preoperative abduction values ≥ 90° and the others are in Group I with preoperative abduction values < 90° . Passive range of motion degrees are in parenthesis.Click here for file


Hyblaea puera) is a pest moth of teak woodlands in India and other tropical regions (e.g. Thailand) and is of major economic significance. This pest is of major concern as it is involved in complete defoliation of trees during the early part of the growing season. Defoliation does not kill teak trees, but it results in huge amount of timber loss. Teak defoliator outbreaks are a regular annual feature in most teak plantations in India and it is extremely difficult to predict the exact time and place of occurrence of these outbreaks. Evidence from the study of the population dynamics of H. puera indicated habitual, short range movements of emerging moth populations, suggesting that these populations have spread to larger areas, generation after generation, affecting the entire teak plantations. We were therefore interested in investigating the temporal and spatial relationship among various population groups in Nilambur, Kerala (India) and address the cause of outbreak at the landscape level.The Teak defoliator . We have used this method extensively to evaluate the species specificity, reproducibility and to discriminate among the three different characterised populations of teak defoliator.H. puera do not play a major role in contributing to large-scale infestations. With respect to the hypotheses put forward regarding the origin of outbreaks of the moth, this study confirms the role of migration in outbreak causation, while negating the belief that endemic populations aggregate to cause an epidemic.This method also allowed us to comment with some certainty that the endemic teak defoliator,  Tectona grandis L.) is a very valuable timber species, and is a member of the moist deciduous and dry deciduous forest types. Teak plantations are threatened by two major pests: the Teak Defoliator (Hyblaea puera Cramer) Lepidoptera: Hyblaeidae and the Teak Skeletonizer  syn.). H. puera is less widely distributed in the tropics: in Oriental and Australian regions ; in the West Indies; and in South and parts of East Africa  and RAPDs, it was possible to discriminate the micro- and macro geographical heterogeneity [S. avenae on wheat and cocksfoot grass, Dactylis glomerata (L.) were observed during the early months of infestation, which declined as the season progressed, largely as a result of genetic drift and local movement between adjacent host species [Bemisia tabaci (Gennadius) populations studied throughout the world [Solenopsis invicta (Buren) using different classes of markers, it was confirmed that both mitochondrial and nuclear markers display the same hierarchical structure [Age groups, sexes, life history variants, etc. and the processes including birth, death, immigration and emigration as different phenotypic classes have been very well defined . While sogeneity . Highly  species . Monophyhe world . While etructure . Distinctructure .The present study using RAGEP-PCR provides a tool for a logical continuation of the earlier work to trace the relationship of endemic, epicenter and epidemic populations of the teak defoliator. The dendrogram produced from nuclear RAGEP clearly indicates that the endemic insects are not involved in causing the epidemic; however, they are apparently involved in the localized spread by building up small epicenter populations. Similarly, while evaluating the observation based on mitochondrial RAGEP's, it is further apparent that endemic populations were not involved in causing the epidemic. This suggests that all the epidemic insects, which are spatially distinct, but temporally co-occurring, share the same gene pool.Aubeonymus mariafranciscae (Roudier), which had diverged recently [Listronotus bonariensis (Kuschel) in New Zealand, was traced to the eastern coast of South America [Oryzaephilus surinamensis (L.) [Lymantria dispar (L.) [S. avenae [Simulium spp. [Drosophila subobscura (Collin) [Drosophila athabasca Sturtevant and Dobzhansky, and Drosophila azteca Sturtevant and Dobzhansky has been extensively studied by allozymes, mitochondrial DNA (mtDNA) and RAPD markers.Randomness of genome amplification methods have been efficiently used in constructing the phylogenetic history in the weevil, recently , whilst  America . Use of sis (L.) , charactpar (L.) , host ba. avenae , and gen. avenae , has beeium spp. , whilst (Collin)  in the wH. puera coexist during the outbreak season. The degree of variability observed for RAGEPs also argues that this technique could be useful for a variety of questions, including individual identification, strain identification and phylogenetic analysis.With the Teak defoliator, earlier studies based on temporal and spatial distribution of the larvae indicated that the epicenters were not constant over the years and did not represent highly favourable local environments . The preH. puera epicenter populations would help prevent large-scale outbreaks of the teak defoliator in teak plantations. Therefore, appropriate strategies should be adopted to control the epicenter populations, which occurs in a smaller area. This appears to be a more practical and economical approach for teak defoliator management when compared with management of the pest in the total plantation area covering thousands of hectares. Thus the molecular markers detected using RAGEP-PCR can enhance the understanding of insect population dynamics and aid in tracing the spread and cause of epidemics.The present results appear to validate the hypothesis, that control of H. puera. Larval samples were collected from the infestation sites. Whenever fifth instar larvae were available, ten larvae were preserved in 70 % alcohol and stored deep frozen at -20°C. If only lower stages were available, i.e., third or fourth instar, they were reared up to 5th instar in the laboratory. Ten 5th instar larvae were preserved for DNA isolation from each sample site, whilst the remaining larvae were reared into the next generation. Each sample was assigned a code number containing the details of Year / Month / Date / Block / Grid / Generation for further reference. Using the duration of each instar , the temporal data on outbreaks were examined to see whether each subsequent epidemic could be explained on the basis of a previous outbreak. The details of location of pest incidence and extent of infestation were later transferred to the field map in order to understand the spatial pattern of infestation , 0.25 mM of dNTPs (Amersham) and 12 pM of primer per reaction. Primers were initially screened for polymorphism and repeatability. Amplifications were performed in similar cycling conditions in a Thermocycler (Biorad) programmed as follows: initial denaturation at 95°C for 5 min., followed by 45 cycles of cycle denaturation at 94°C for 1 min., annealing at 36°C for 1 min., extension at 72°C for 2 min. and final extension at 72°C for 5 mins. The amplification products were separated using 1.2% agarose gel in 0.5 × TBE buffer with ethidium bromide staining to visualize the product separation using a Bio-Rad's Fluor S imager. The molecular weight of each band was estimated by comparing with a co-migrating 100-bp ladder (Amersham). RAGEP fingerprints of each sample from different regions were then interpreted using Fingerprint type module of Bionumerics software .Both nuclear and mitochondrial DNA RAGEP amplifications were performed in a total volume of 30 μl. Each reaction consisted of 1x Taq buffer with 1.5mM MgClH. puera with those from the Teak skeletonizer, E. machaeralis , Leaf roller, Sylepta derogata (F.), Leaf folder, Cnaphalocrocis medinalis (Guenée), and the Silkworm, Bombyx mori (L.).A preliminary screening with 50 nuclear RAGEPs and 37 mitochondrial RAGEP primers were evaluated for polymorphism and repeatability. Only 11 nuclear Table- from eacThe polymorphic content for nuclear and mitochondrial primers were analyzed using Bionumerics software . Band seCN and MB performed the molecular studies and are responsible for the interpretation of molecular data whilst TV and VVS performed the field data collection and are responsible for spatial and temporal data interpretation. All authors read and approved the final manuscript. MB and VVS conceived and designed the study.


RNA-mediated gene silencing has emerged in recent years as an important mechanism for regulating gene expression. Some of the key discoveries have been made in plants Although the word ‘revolution’ should not be used lightly in science, there is no other way to describe the recent explosion in our awareness and understanding of RNA-mediated gene silencing pathways. The central player in RNA-mediated gene silencing is a double-stranded RNA (dsRNA) that is chopped into tiny RNAs by the enzyme Dicer. The tiny RNAs associate with various silencing effector complexes and attach to homologous target sequences (RNA or DNA) by basepairing. Depending on the protein composition of the effector complex and the nature of the target sequence, the outcome can be either mRNA degradation, translational repression, or genome modification, all of which silence gene expression . PresentCaenorhabditis elegans . Unexpectedly, the first transgene complex, which was stably active on its own, often became silenced in the presence of the second . The proNeurospora crassa , sequence-specific DNA methylation that triggered TGS, and RNA-directed DNA methylation. They had also proposed models for PTGS involving dsRNA , small RNeurospora identified common components in the respective silencing pathways (RNAi was discovered in experiments designed to compare the silencing activity of single-stranded RNAs (ssRNAs) (antisense or sense) with their dsRNA hybrid. While only marginal silencing of a target gene was achieved after injecting worms with the individual strands, injection of a sense–antisense mixture resulted in potent and specific silencing . This unpathways . This inpathways . PTGS isDrosophila systems demonstrated that 21–23 nucleotide ‘short interfering'RNAs (siRNAs), derived from cutting longer dsRNA, can guide mRNA cleavage  RNAs corresponding to silenced target genes in transgenic plants . They prcleavage ; identifcleavage ; and idecleavage   moves from cell to cell and through the vascular system to induce silencing at distant sites, was initially detected in plants in 1997  and late in 1998 . One of  in 1998 . A recen in 1998 .Arabidopsis has four Dicer-like (DCL) proteins, in contrast to mammals and worms, whose genomes encode only one Dicer protein (Arabidopsis to include at least three active members. An important goal has been to determine the functions of individual family members. Previous studies in Arabidopsis have shown that DCL1 is needed for processing miRNA precursors important for plant development (PLoS Biology delineates distinct functions for DCL2, DCL3, and RDR2. Nuclear-localized DCL3 acts with RDR2 to generate short RNAs that elicit DNA and histone modifications; DCL2 produces short RNAs active in antiviral defense in the cytoplasm of cells. This study illustrates nicely how RNA silencing components have diversified in plants to carry out specialized functions.Although RNA-mediated gene silencing pathways are evolutionarily conserved, there are various elaborations in different organisms. For example, the plant  protein . The RDRelopment , but notelopment . The papBy identifying small RNAs as agents of gene silencing that act at multiple levels throughout the cell, molecular biologists have created a new paradigm for eukaryotic gene regulation. Plant scientists have figured prominently in RNA-mediated silencing research. Instrumental to their success was the early ability to produce large numbers of transgenic plants, which displayed a rich variety of gene silencing phenomena that were amenable to analysis. The agricultural biotechnology industry provided incentives to find ways to stabilize transgene expression and use transgenic approaches to modulate plant gene expression and to genetically engineer virus resistance. As exemplified by the petunia cosuppression experiments, nonessential plant pigments provide conspicuous visual markers that vividly reveal gene silencing. The history of gene silencing research shows once again that plants offer outstanding experimental systems for elucidating general biological principles.


Why three non-governmental organizations are launching an alternative to the World Health Report Mobilising civil society around an alternative World Health Report At the World Health Assembly in May 2003, three civil society groups—the People's Health Movement, the Global Equity Gauge Alliance, and Medact—discussed the need for civil society to produce its own alternative to the World Health Organisation's World Health Report. We felt strongly that we needed to produce a global health report that had equity and the right to health at its heart. We also needed a way to monitor the performance of global health institutions themselves. The idea of an alternative to the World Health Report has developed into an initiative called the Global Health Watch, which we are launching next year.http://www.medact.org) is a United Kingdom–based global health charity, undertaking education, research, and advocacy on conflict, poverty, and the environment.Medact (http://www.gega.org.za) was created to participate in and support an active approach to monitoring health inequalities and promoting equity within and between societies. The Alliance currently includes 11 member-teams, called Equity Gauges, located in ten countries in the Americas, Africa, and Asia.The Global Equity Gauge Alliance (http://www.phmovement.org) is a global network of activists, organisations, and social movements. Its goal is to re-establish health and equitable development as top priorities in local, national, and international policy-making, with comprehensive primary health care as the strategy to achieve these priorities.The People's Health Movement Monitoring of international promises on aid and debt reliefSection F: Summary and Strategies for ActionThis is how the Watch will be alternative: it will present options for health policy-makers that question the dominant reform agenda that emphasises market-driven and diseased-based approaches to health care. A policy bias against government action and a lack of creative thinking about how governments can shape health care markets to work in favour of equity and social inclusion are unfortunate features of global health debates. More recently, the emphasis has been placed once again on campaigns against specific diseases such as HIV/AIDS and tuberculosis, despite the universally acknowledged importance of building and maintaining health systems that can respond to the broader needs of patients.We hope the Watch will present some alternative and imaginative thinking about how health services can respond creatively to the many challenges they face, with a strong focus on basic principles of equity and universality and avoiding top-down disease-focussed programmes that neglect the broader determinants of health. We have invited some of the most interesting and innovative thinkers in health policy—from both developing and developed countries and from academia and civil society—to help us achieve these objectives.The Watch will also be ‘alternative’ in another sense—it will act as a regular monitor of the policies, governance, and funding of the institutions affecting global health, including the World Health Organisation and World Bank, something no other health report undertakes. We hope to offer proposals for reform, as well as to stimulate further action by civil society to make these institutions more accountable and responsive to the needs of the poor and vulnerable.It is important to say that the three networks and organisations that have convened the Watch are really just its initiators. In the end we hope the Watch will be backed by as many individuals, organisations, and social movements as possible, strengthening the links between civil society organisations across countries and across health-related sectors, and increasing the power and influence of the report itself. Already, many have expressed their interest in the project, and their willingness to contribute: through writing chapters, contributing case studies, and launching the Watch and promoting it in their country when it is finished. Groups from India and Brazil are planning parallel national Watches.We plan to launch the Watch at the second People's Health Assembly, which will be held in Ecuador in July 2005. We don't want this report to be addressed just to health activists or health policy-makers or academics. If we are going to create change we need to capture the imagination of the broader health professional community and the public at large. That is why we encourage readers to get involved and tell others about the Watch and to use it to throw down a challenge to those who call the shots at national and international levels.


Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant  and susceptible  mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of αPseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.These results show that  Xenopus laevis oocytes [+ transport from the alveoli to the interstitium is the main force that drives water out of the alveoli [+ transport is also important in adults for lung liquid clearance [The epithelial sodium channel (ENaC) is expressed in epithelial cells of several tissues involved in salt and water reabsorption. The channel is composed of three related subunits  that are able to reconstitute a functional channel when expressed in  oocytes ,2. ENaC  oocytes ,3-5, dis oocytes ,3,5, lun oocytes -8, ear e oocytes ,10, papi oocytes -13, eyes oocytes , chondro oocytes  and diff oocytes . ENaC sy oocytes ,18. In t alveoli ,20. This alveoli . Its imp alveoli . Na+ tralearance ,23.in vitro, following stimulation with steroids, β-agonists, catecholamines, and agents that increase cAMP concentration [+ transport compared to normal cells [Increased ENaC expression has been detected in the lung and in alveolar epithelial cells ntration -27. αENantration ,6,27. Itntration ,29 and dntration ,31. Seventration -34. Althal cells ,36. Thisal cells ,38.Pseudomonas aeruginosa is a bacterium occuring naturally in a wide range of environments such as in soil, fresh and seawater, plants and decomposing organic matter [Pseudomonas can promote nosocomial lung infection after artificial ventilation [Pseudomonas aeruginosa is the main source of chronic lung infection in CF patients [c matter . Althougc matter . Pseudomtilation  and is atilation . Chronictilation  where Pspatients .Pseudomonas aeruginosa in the lung of anaesthetised rabbits has been reported to promote acute pneumonia, resulting in alveolar epithelial injury, loss of epithelial barrier integrity, lung edema, pleural empyema and pleural effusions within 8 h of infection [P. aeruginosa entrapped in agar beads. In this model, the lungs of susceptible mouse strains develop severe lung infection with a strong inflammatory response and some lung edema [Pseudomonas by itself has been shown to inhibit active sodium absorption in cultured airway epithelial cells [+-K+-ATPase.Instillation of nfection . A more ng edema ,45. Pseual cells . Here, wPseudomonas entrapped in agar beads was instilled in the lung of resistant  and susceptible  mouse strains, and the expression of α, β, γENaC and α1 Na+-K+-ATPase mRNA was studied by Northern blotting in lungs infected between 3 hours and 14 days.P. aeruginosa  was entrapped in agar beads, and 50-μl suspensions containing 2 × 105 to 1 × 106 CFU/ml were instilled intratracheally in male mice of resistant (BALB/c) or susceptible  strains as described previously [Clinical strain 508 of eviously ,45.2 inhalation at different time points after P. aeruginosa instillation in the lungs. The trachea was cannulated, and the lungs were washed seven times with 1 ml PBS. Total cell counts were conducted in a hemacytometer. Differential cell counts were made by Diff-Quick staining (American Scientific Products) of Cytospin preparations. Number of animals: day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3.BAL were performed as described elsewhere with a few modifications . The inf1Na+-K+-ATPase, glyceraldehyde-3-phosphate dehydrogenase (GADPH) or 18S rRNA). To detect αENaC mRNA, the blots were hybridized with 764-bp mouse αENaC cDNA (His-445 to stop codon) [1Na+-K+-ATPase probe was a gift from Dr. J. Orlowski  and consisted of a NarI-StuI 332-bp fragment coding from nucleotide 89 to 421 (from the 5'UTR to Arg-61) of the rat kidney and brain α isoform [The lungs from infected mice were harvested between 3 h to 14 days after infection, homogenized in 5 ml of 4 M guanidine isothiocyanate, and centrifuged on a cesium chloride gradient . Fifteenp codon) . The α1N isoform . For qua isoform . The bloBetween each round of hybridization, the membranes were stripped by treatment with 0.1 X SSC, 1% SDS and 2.5 mM EDTA at 95°C. The blots were allowed to cool gradually with agitation for 30 min at room temperature. The membranes were then rinsed with 5 X SSC and rehybridized. Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied.+, K+-ATPase mRNA expresion, the comparisons between groups were analyzed by Wilcoxon signed rank non-parametric test using Statsview software . Probability p values < 0.05 were considered to be significant.For the BAL cell count, the data are presented as means ± SE (standard error). For ENaC and NaPseudomonas instillation in the lung of C57BL/6 mice was monitored by studying the number of total cells in BAL at different times after infection. As shown in Figure The inflammation process evoked by Pseudomonas embedded in agar beads was administered intratracheally in resistant (BALB/c) and susceptible strains of mice  as described previously [Pseudomonas infection [1 Na+-K+-ATPase mRNA coding for the catalytic domain of the sodium pump, but could not find any significant change during infection  and -susceptible  mouse strains. Modulation of ENaC expression is therefore most likely not a genetic marker linked to the susceptibility of mouse strains to establishment of a chronic infection with Pseudomonas.The instillation of ammation ,45 and lammation  secondar strains ,45. The g injury , and becg injury , we testithelium ,23. The Pseudomonas infection affected ENaC mRNA with a pattern consisting of increased expression at 24 h, followed by a marked decrease on day 3 post-infection. The change in ENaC mRNA was related to bacterial infection, since agarose beads alone failed to evoke any modulation of these RNA. The three ENaC subunits were modulated with a similar profile, with some noticeable differences, however. Although αENaC mRNA expression tends to increase by day 1, the most noticeable feature brought by Pseudomonas infection to αENaC mRNA was the significant decreases after 3 days, 7 days and 14 days post-infection. To the best of our knowledge, this is the first report demonstrating that bacterial infection in vivo can lead to modulation of ENaC mRNA expression. Recently, αENaC mRNA expression was found to be downregulated in the mouse lung after 7 and 14 days of adenoviral infection [nfection . Furthernfection  reportednfection . All thePseudomonas infection. There was a significant increase in the mRNA expression on day 1 post-infection, followed, as for αENaC, by a decreased expression on day 3 and day 7 post-infection. Different stoichiometries have been proposed for ENaC. One model suggests a 2α, β, γ ratio [Xenopus laevis oocytes increases amiloride-sensitive Na+ current by 100% compared to αENaC alone [+ transport across the airway epithelium and a reduced height of the airway surface liquid [Pseudomonas infection, could certainly influence amiloride-sensitive current and lung liquid clearance as in αENaC KO mice rescued by transgenic expression of αENaC that has a lower expression of ENaC in the lung [The β ENaC subunit was also modulated by  γ ratio ,54 where γ ratio  or nonam γ ratio ,56. AlthaC alone ,58. GeneaC alone . Lung liaC alone , but is aC alone . Increase liquid . For allthe lung ,63. In sthe lung .Pseudomonas infection. Acute bacterial pneumonia in rats has been shown to increase alveolar epithelial fluid clearance [Pseudomonas infection could be of potential clinical significance to understand the slow improvement in some ARDS patients.The general biphasic modulation of ENaC mRNA expression with an increase at 24 h followed by a decrease thereafter is an interesting finding that could explain some contradictory reports concerning ENaC expression in lung following learance ,66 when 1 Na+-K+-ATPase mRNA was unaffected in the course of lung infection. This is similar to what has been reported during adenovirus lung infection [1 Na+-K+-ATPase. In ischemia reperfusion injury, there was also no modulation of α1 Na+-K+-ATPase expression despite significant ENaC downregulation [+-K+-ATPase is an important element in lung liquid clearance, however, by being one of the key generator of membrane potential, the enzyme also affects other channels and ion transport process. It is possible that by modulating ENaC expression and not α1 Na+-K+-ATPase, Pseudomonas infection alters the Na+ transport system but does not change other important cell functions meditated by Na+-K+-ATPase. Furthermore, despite a similar mRNA expression level, there could be a fall in protein content or activity of the sodium pump. Additional experiments are necessary to answer this question.In contrast to α and β ENaC, αnfection  where αEgulation . These rSeveral studies report that in lung epithelial cells, viral infection ,68, mycoPseudomonas infection modulates ENaC mRNA expression. This modulation is independent of mouse strain susceptibility to establishment of chronic infection with Pseudomonas. Although there is an elevation of ENaC expression after 24 h, the most important feature is probably the long-lasting decrease of αENaC transcripts on days 3 and 7 post-infection. The lung inflammation induced by Pseudomonas infection therefore seems to favour a reduction in the expression of an essential element involved in lung liquid clearance as well as the regulation of airway surface liquid volume.We have shown in this report that Pseudomonas instillation, RNA extraction and Northern blotting of RNA sample. The BAL recovery as well as PMN and macrophage counting was performed by CG. YB and DR designed and co-ordinated the study. All authors read and approved the final manuscript.AD performed the hybridization, the statistical analysis of the blots and wrote the manuscript. DG performed the


We tested the hypothesis that the tube cuff is inadequately inflated when manometers are not used.Cuff pressure in endotracheal (ET) tubes should be in the range of 20–30 cm H2O.With IRB approval, we studied 93 patients under general anesthesia with an ET tube in place in one teaching and two private hospitals. Anesthetists were blinded to study purpose. Cuff pressure in tube sizes 7.0 to 8.5 mm was evaluated 60 min after induction of general anesthesia using a manometer connected to the cuff pilot balloon. Nitrous oxide was disallowed. After deflating the cuff, we reinflated it in 0.5-ml increments until pressure was 20 cmH2O). Only 27% of pressures were within 20–30 cmH2O; 27% exceeded 40 cmH2O. Although it varied considerably, the amount of air required to achieve a cuff pressure of 20 cmH2O was similar with each tube size.Neither patient morphometrics, institution, experience of anesthesia provider, nor tube size influenced measured cuff pressure (35.3 ± 21.6 cmHWe recommend that ET cuff pressure be set and monitored with a manometer. Consecutive available patients were enrolled until we had recruited at least 10 patients for each endotracheal tube size at each participating hospital. All tubes had high-volume, low-pressure cuffs.At the University of Louisville Hospital, at least 10 patients were evaluated with each endotracheal tube size  that was connected to the pilot balloon of the endotracheal tube cuff via a three-way stopcock. This type of aneroid manometer is nearly as accurate as a mercury manometer, but easier to use . Pressur2O was achieved. The entire process required about a minute.A syringe attached to the third limb of the stopcock was then used to completely deflate the cuff, and the volume of air removed was recorded. The cuff was considered empty when no more air could be removed on aspiration with a syringe. The cuff was then progressively inflated by injecting air in 0.5-ml increments until a cuff pressure of 20 cmH2O as a function of tube size. Outcomes were compared by tube size, provider, and hospital with either an ANOVA  or the Kruskal-Wallis statistic . Compliance of the cuff system was evaluated by linear regression of measured cuff pressure vs. measured cuff volume. Data are presented as means (SD) or medians [interquartile ranges] unless otherwise noted; P < 0.05 was considered statistically significant.Our primary outcomes were 1) measured endotracheal tube cuff pressures as a function of tube size, provider, and hospital; and 2) the volume of air required to produce a cuff pressure of 20 cmH2O; only 27% of the patients had measured pressures within the recommended range of 20–30 cmH2O. Fifty percent of the values exceeded 30 cmH2O, and 27% of the measured pressures exceeded 40 cmH2O. Thus, 23% of the measured cuff pressures were less than 20 mmHg. Measured cuff volume averaged 4.4 ± 1.8 ml. Neither measured cuff pressure nor measured cuff volume differed among the hospitals .CRNAs (n = 72), anesthesia residents (n = 15), and anesthesia faculty (n = 6) performed the intubations. There were no statistically significant differences in measured cuff pressures among these three practitioner groups (2O) and the volume of air (ml) retrieved at complete deflation of the cuff; this showed a linear pressure-volume relationship: Pressure= 7.5. Volume+2.7, r2 = 0.39  than outside the range. It was nonetheless encouraging that we observed relatively few extremely high values, at least many fewer than reported in previous studies [Previous studies suggest that the cuff pressure is usually under-estimated by manual palpation. For example, Braz et al.  observedz et al.  observed studies . This reMeasured cuff inflation pressures were virtually identical at the three study sites: one academic center and two private hospitals. These data suggest that management of cuff pressure was similar in these two disparate settings. Interestingly, there was also no significant or important difference as a function of provider – measured cuff pressures were virtually identical whether filled by CRNAs, residents, or attending anesthesiologists. Our results thus fail to support the theory that increased training improves cuff management.We evaluated three different types of anesthesia provider in three different practice settings. Although we were unable to identify any statistically significant or clinically important differences among the sites or providers, our results apply only to the specific sites and providers we evaluated. While it is likely that these results are fairly representative, it is obvious that results would not be identical elsewhere because of regional practice differences.et al. [2O did not differ significantly as a function of endotracheal tube size. These data suggest that tube size is not an important determinant of appropriate cuff inflation volume. A caveat, though, is that tube sizes were chosen by clinicians in our study and presumably matched patient size; results may well have differed if tube size had been randomly assigned. We intentionally avoided this approach since our purpose was to evaluate cuff pressures and associated volumes in three routine clinical settings.Fernandez et al.  found thA limitation of this study is that cuff pressure was evaluated just once 60 minutes after induction of anesthesia. Because nitrous oxide was not used, it is unlikely that the cuff pressures varied much during the first hour of the study cases. We recognize that people other than the anesthesia provider who actually conducted the case often inflated the cuffs. However, a full hour was plenty of time for the provider to have checked and adjusted cuff pressure to a suitable level.2O independent of tube size for the same type of tube. However, there was considerable patient-to-patient variability in the required air volume. Measuring actual cuff pressure thus appears preferable to injecting a given volume of air.We observed a linear relationship between the measured cuff pressure and the volume of air retrieved from the cuff. The regression equation indicated that injected volumes between 2 and 4 ml usually produce cuff pressures between 20 and 30 cmHCuff pressure should be measured with a manometer and, if necessary, corrected.The author(s) declare that they have no competing interests.SP oversaw day-to-day study mechanics, collected data on many of the patients, and wrote an initial draft of manuscript.DIS contributed to study design, data analysis, and manuscript preparation.PM, SW, and AV recruited patients and performed many of the measurements.JD conceived of the study and participated in its design.AW contributed to protocol development, patient recruitment, and manuscript preparation.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To reach and grasp an object in space on the basis of its image cast on the retina requires different coordinate transformations that take into account gaze and limb positioning. Eye position in the orbit influences the image's conversion from retinotopic (eye-centered) coordinates to an egocentric frame necessary for guiding action. Neuroimaging studies have revealed eye position-dependent activity in extrastriate visual, parietal and frontal areas that is along the visuo-motor pathway. At the earliest vision stage, the role of the primary visual area (V1) in this process remains unclear. We used an experimental design based on pattern-onset visual evoked potentials (VEP) recordings to study the effect of eye position on V1 activity in humans.We showed that the amplitude of the initial C1 component of VEP, acknowledged to originate in V1, was modulated by the eye position. We also established that putative spontaneous small saccades related to eccentric fixation, as well as retinal disparity cannot explain the effects of changing C1 amplitude of VEP in the present study.The present modulation of the early component of VEP suggests an eye position-dependent activity of the human primary visual area. Our findings also evidence that cortical processes combine information about the position of the stimulus on the retinae with information about the location of the eyes in their orbit as early as the stage of primary visual area. In humans, goal-directed movements to an object in space are improved by foveal vision, namely by gaze anchoring on the object . Since mvs. lower visual field stimulation [for review see [The C1 component reverses classically in polarity for upper view see ]. ConseqOnce the C1 component was characterized for both upper and lower quadrant visual fields, the effect of eye position on C1 amplitude was measured only for both left and right lower quadrants visual field that is for the most salient C1 component which we observed.Mean peak latencies of C1 was calculated in each subject in response to both left and right lower visual quadrants and for both IN3 and IN4 recording sites. They ranged between 94.8 ms and 98.0 ms that are consistent with the C1 latency range previously observed in numerous studies ,15. For We observed that checkerboard presented in the lower visual field elicited VEPs with modulated amplitudes of the C1 component in respect of eye position. Grand averaged VEPs over the 20 subjects in response to flashed stimuli in the right lower quadrant of the visual field for three different eye positions (0° and 20° both left- and rightward) are shown in Figure In other words, the eye position effect was observed at the occipital recording site contralateral to the direction of the eye deviation. Equivalent VEPs but reverse eye-position effects were obtained in response to left field stimuli. A one-way Friedman repeated measures analysis of variance was conducted for each recording site and for each visual stimulation revealing a significant main effect of eye position on the amplitude of the C1 component. Complete data obtained at both recording sites, for each lower visual field are shown in Figure The overall result of the present study revealed that the amplitude of first major component of VEP elicited by checkerboard (C1) is modulated by the eye position. The previous data linking the C1 component to a striate cortex generator, namely V1 activity (see introduction), led us to suggest that eye position influences the earliest cortical stage of visual processing.One may first suggest that the present results may be explained by the difficulty of maintaining eccentric fixation, which may have altered the pattern of fixational eye movements, such as microsaccades . RecentThe question also arises if the effects of changing C1 amplitude may be due to oculomotor signals and/or retinal disparity that is to the difference in the position of the visual stimulus on each retina related to relative monitor distance. The absence of any parametric effect for the C1 amplitude between 10° and 20° of eye eccentricity may indicate that eye position effects are not due to the difference in the horizontal retinal disparity, but one may argue that a putative relationship between the horizontal disparity and the C1 amplitude is not linear. We evaluated the difference of both horizontal and vertical disparity between the different eye's deviations in our study  from extrastriate and/or oculomotor structures while the initial C1 sensory response (50–90 ms) was not modulated by attention -15,19-22Finally, Trotter et al. (1999) have shown that an eye position signal  is involved in the neural modulation process dealing with the eye position-dependent visual response observed in area V1 of behaving monkeys. Oculomotor signals coming from extraocular muscle afferents and/or corollary discharges are considered as the substrate of such an eye position signal and have been previously described in V1 ,24.The present results and previous works obtained from neural recordings in monkeys indicate that changes in eye position can modify response properties in V1 that is at the earliest cortical stage of visual processing. Among the visuo-motor processing allowing accurate reaching and grasping movements in space on the basis of the image seen by the retina, the primary visual area may be therefore one of the first cortical relay to convert the image in eye-centered coordinates into a target location by taking into account the position of the eyes in the orbit. It endorses some recent arguments pointing out that V1 could no longer be considered only in relation to the pattern of light falling on the retina but appears to be a cortical area in which contextual influences take place too .Twenty right-handed healthy volunteers with normal visual acuity  participated in VEP recordings after they provided their written informed consent. The study was approved by the Basse-Normandie ethics committee .-1 of spatial frequency) flashed against a black background (ISI = 500–1000 ms) and delivered by a visual stimulator . The subjects were instructed to fixate continuously the fixation dot and to keep their attention on it, for each quadrant of the visual field so that the projection of the stimulus on the retina was equivalent whatever was the deviated eye position. The edges of the monitor and the space up to 1 meter around the monitor were masked with an opaque black sheet preventing any cue perception in the room except the flashing checkerboard.The subjects were seated on a swivel armchair with their head stabilized with a headrest. An experimenter slowly rotated the armchair and locked it in one of the five different angles: 0°, 10° and 20° both left- and rightward from the center of the monitor leading to five different eye positions Figure . The verWith respect to the purpose of the present study, we recorded VEP from the scalp using the two occipital intermediate sites (IN3 and IN4) of the modified 10–20-system montage  with botWe evaluated the difference of disparity between the different eye's deviations Figure . The difUsing the dot product, for the left eye, α can be expressed as following:a1e1.a1e2 = |a1e1| |a1e2| cos(α)     (1)x,y,z) centred on the cyclopean axis (c):Expressing of the coordinates of the vectors a1e1 and a1e2 in the canonical reference (x a1e2x + a1e1y a1e2y + a1e1z a1e2z     (2)a1e1.a1e2 = a1e1and combining both (1) and (2) led to:x+a/2 cos(θ)) + (d +a/2sin(θ))2 / |a1e1| |a1e2|     (3)cos(α) = (a/2 cos(θ))) + (d -a/2sin(θ))2 / |a1e1| |a1e2|     (4)cos (β) = (-a/2cos(θ)), and combining both (3) and (4) allows to estimate the magnitude of the disparity (η = β-α) in function of the distance from the fixation point for each given eye's deviation. Therefore, we plotted the magnitude of disparity with each distance (e) and for each eye's deviation  normalized by the disparity calculated for the central eye position (θ = 0°, Figure C1 : early component of visual evoked potentialfMRI : functional magnetic resonance imagingIN3 and IN4 : occipital intermediate recording sitesISI : inter-stimulus intervalV1 : primary visual areaVEP : visual evoked potentialAll authors designed the study. FA and OE carried out the experiments and generated the method for data analysis. OE performed the statistical analysis and generated the method for estimation of the magnitude of disparity. FA and LP wrote the first versions of the manuscript. All authors read, discussed and approved the final version of the manuscript.


Zierath and Hawley discuss how different fiber types affect muscle metabolism and what the signals are that regulate muscle phenotype Skeletal muscle demonstrates a remarkable plasticity, adapting to a variety of external stimuli , includiIn most mammals, skeletal muscle comprises about 55% of individual body mass and plays vital roles in locomotion, heat production during periods of cold stress, and overall metabolism . Thus, ka, and the more anaerobic (glycolytic) fiber is termed FTb. Under aerobic conditions (sufficient oxygen supply to the working muscles), energy is produced without the production of lactate. Under anaerobic conditions (insufficient oxygen supply to the working muscles), energy is produced via the glycolytic pathway, which results in lactate accumulation and in turn limits anaerobic exercise. Thus, muscle fibers can be classified in terms of contractile and metabolic properties , is typically around 55%, with FTa fibers being twice as common as FTb fibers  of 14 elite male long distance runners, 18 good (but not world-class) male long distance runners, and 19 untrained men. The elite group included Olympic medal winners (p < 0.05). The values found for several of the elite runners were the highest observed in human muscle (> 92% ST). Moreover, the ST fibers from the elite runners were 29% larger than FT fibers (p < 0.05), and both ST and FT fibers were larger in the good runners than in the untrained men. Because of the marked hypertrophy (bulk increase) of the ST fibers in the elite runners, the cross-sectional area composed of these fibers was greater than either the good runners or the untrained subjects . When the data from the elite and good runners was combined, a positive correlation between the proportion of ST fibers and the best 6-mile performance time was noted .In this regard, the results of  winners  and Amer2 uptake . Indeed, while an athlete's muscle fiber type is an important morphological component and is related to several contractile and metabolic properties  are more likely to determine the upper limits of endurance capacity  had 50% versus 98% ST muscle fibers. Subsequent work  revealedties see , other pcapacity .The close coupling between muscle fiber type and associated morphological, metabolic, and functional properties is not confined to athletic ability. Insulin sensitivity also correlates with the proportion of ST oxidative fibers . Specifi2+/calmodulin-activated phosphatase implicated in nerve activity-dependent fiber-type specification in skeletal muscle, directly controls the phosphorylation state of the transcription factor NFAT, allowing for its translocation to the nucleus and leading to the activation of slow-type muscle proteins in cooperation with myocyte enhancer factor 2 (MEF2) proteins and other regulatory proteins (2+/calmodulin kinase activity is also upregulated by slow motor neuron activity, possibly because it amplifies the slow-type calcineurin-generated responses by promoting MEF2 transactivator functions and enhancing oxidative capacity through stimulation of mitochondrial biogenesis (Skeletal muscle fiber-type phenotype is regulated by several independent signaling pathways . These iproteins . Calciumogenesis .PLoS Biology (PGC1-α, a transcriptional coactivator of nuclear receptors important to the regulation of a number of mitochondrial genes involved in oxidative metabolism, directly interacts with MEF2 to synergistically activate selective ST muscle genes and also serves as a target for calcineurin signaling . New dat Biology  reveals PLoS Biology (The transition from aerobic to anaerobic metabolism during intense work requires that several systems are rapidly activated to ensure a constant supply of ATP for the working muscles. These include a switch from fat-based to carbohydrate-based fuels, a redistribution of blood flow from nonworking to exercising muscles, and the removal of several of the byproducts of anaerobic metabolism, such as carbon dioxide and lactic acid. Some of these responses are governed by transcriptional control of the FT glycolytic phenotype. For example, skeletal muscle reprogramming from a ST glycolytic phenotype to a FT glycolytic phenotype involves the Six1/Eya1 complex, composed of members of the Six protein family . Moreove Biology , a key rWith the 2004 Olympics still fresh on our minds, many will ask: Who has the right stuff to go the distance? Athletes like Olympic champion Frank Shorter are clearly exceptional and represent an extreme in human skeletal muscle phenotype. Realistically, few of us can ever hope to run a marathon in world-class time. However, there may be cause for some optimism for the average mortal, since endurance exercise training in healthy humans leads to fiber-type specific increases in the abundance of PGC-1 and PPAR-α protein in skeletal muscle . Moreove


Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses.Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity.The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting. Acute respiratory tract infections are major causes of morbidity and mortality. In 2000, lower respiratory tract infections were globally the number one infectious cause of disability adjusted life-years . The comAs part of service development it was necessary to provide an alternative to virus culture for testing immunofluorescence negative respiratory specimens. Historically and indeed currently immunofluorescence  and viruIncreasingly however, the sensitivity of nucleic acid amplification techniques for diagnosis has become recognised -10. HoweOne such challenge is the different optimal annealing temperatures of the primer sets for each prospective virus target. The ABI PRISM 7000 real-time facility from Applied Biosystems addresses this by using bundled software to design primer/probe combinations that use a common amplification protocol. However this approach is compromised by the inability of software to allow for target heterogeneity. In addition it does not allow users to adopt clinically validated primer sets from the literature.To address these problems we adopted an alternative approach through the development of a generic touchdown amplification protocol. Touchdown protocols involve a pre-designed stepped reduction in the annealing temperature used for primer-to-template binding, which introduces a competitive advantage for specific base-pair priming over non-specific priming . A detai3 copies per ml for human rhinovirus type 1b to 4.2 × 103 copies/ml for RSV-A. Specificity was ascribed through reproducibility, i.e. specimens which were repeatedly positive, following our standard clinical reporting algorithm [Before deciding on the layout of the molecular strip, as described in the methods, we undertook a wide range of preliminary validation steps for each primer set. The complexity of the strip makes it impossible to fully evaluate using the classical approach of applying an individual gold standard to each virus type. Classically this approach works well where a single target is under investigation . Howeverlgorithm , were relgorithm . In addiTo test the feasibility of its routine use we needed to clinically validate its performance in a routine setting on specimens tested in parallel with our standard immunofluorescence protocol for the diagnosis of acute virus respiratory infections. Although the routine immunofluoresence panel lacked capacity for the detection of rhinoviruses, human metapneumovirus and CoV-229E, these were included on the strip for clinical reasons during the period of the study. These findings and their implications are reported.A total of 99 viruses were detected in 84/222 specimens from a total of 79/183 patients and 4/5 National External Quality Assurance Scheme (NEQAS) controls; immunofluorescence did not detect the parainfluenza virus type 2 virus in one of the NEQAS specimens. Viruses were detected in all of the specimen types processed. The molecular strip detected virus in: 16/36 (44.4%) broncho-alveolar lavages, 62/120 (51.6%) nasopharyngeal secretions, 11/35 (31.4%) sputa and 10/31 (32.2%) combined throat and nasal swabs. Immunofluorescence detected virus in: 6/36 (16.6%) broncho-alveolar lavages, 23/120 (19.1%) nasopharyngeal secretions, 1/35 (2.8%) sputa and 1/31 (3.2%) combined throat and nasal swabs.The median age of male and female patients where virus was detected was 3 y (range 2 weeks – 79 years) and 4 y (5 weeks – 81 years) respectively. Sixteen viruses were detected in 14/27 (51.8%) specimens, confirming a respiratory virus in 12 out of 24 (50%) patients investigated in general practice. Seventy-nine viruses were detected in 70/191 (36.6%) specimens, confirming a respiratory virus in 67 out of 159 (42.1%) patients investigated in hospital. Of the 16 viruses detected in specimens from the community, PCR detected all 16 in contrast to a single identification, influenza A (H3), by immunofluorescence.PCR identified one or more viruses in specimens from 84 of the 183 patients and the 4 NEQAS positive specimens, detecting a total of 99 viruses as shown in Table Dual infections were detected in 11/79 (13.9%) patients. The dual infections were: influenza A (H3) and adenovirus (4); influenza A (H3) virus and rhinovirus (2); influenza A (H1) and adenovirus (1); adenovirus and rhinovirus (3); respiratory syncytial virus type B and rhinovirus (1).Nine patients had more than one specimen taken on the same day in which a virus was detected in at least one specimen by PCR. For 5 of the patients the same virus was detected in each of the 2 specimens. The viruses identified were rhinovirus (3), adenovirus (1) and parainfluenza type 3 (1); the latter was also immunofluorescence positive. In 2 cases a rhinovirus was detected in only one of the specimens. As part of a separate rhinovirus validation protocol one of these specimens was subjected to retesting coupled with limited sequencing of the 5' non-coding region amplicon which confirmed the presence of a rhinovirus sequence. Additionally, premature twin boys admitted to intensive care on the same day with severe bronchiolitis, both had evidence of acute rhinovirus infection by PCR. Limited sequencing of the 5' non-coding region of these viruses as part of the rhinovirus validation protocol confirmed the presence of an identical sequence of rhinovirus in both specimens.Immunofluorescence identified a virus in specimens from 28 of the 183 patients and 3/4 NEQAS positive specimens (16.4%), detecting a total of 31 viruses as shown in Table Although touchdown PCR has been used successfully to help overcome some of the uncertainties associated with the thermal amplification of microbial nucleic acid targets -22, its In this study the large number of targets is the main problem encountered. Using an empirical approach a series of amplification steps linked to a stepped reduction in annealing temperature from 55°C to 46°C was constructed. This proved extremely resilient when used with a wide range of primer sets and included the apparent anomaly of putting adenovirus through an initial reverse transcription step to stream line all of the targets on to a single strip; we have previously reported this approach for testing group F adenovirus alongside norovirus, astrovirus and rotavirus . The touWhere multiple viral targets are sought in clinical practice, we believe that it is only feasible to correlate the performance of the new assay in a head-to-head comparison with that already in routine use. Unfortunately for many clinical laboratories there is an elusion of testing for a wider range of viruses than is the case, by the inoculation of cell lines with a theoretical ability to grow the respective viruses. The annual reports of most clinical laboratories of one of the commonest human respiratory viruses, human rhinovirus, is an example of this; using the touchdown protocol we now report approximately 450 HRV infections per annum. The under reporting of adenovirus by standard methods  and the 3 and 2 × 103 copies per ml) with a generic matrix set (copy number sensitivity of 1 × 103 copies per ml).The ability to simultaneously validate the performance of multiple molecular primer sets in a routine clinical setting is a major accomplishment of the current methodological approach. The results demonstrated that a range of primers from both the medical literature and from in-house development could be amplified with a single generic touchdown protocol. It therefore confirmed the feasibility of directly incorporating primer sets into a standard operating procedure without the necessity for the individual optimisation of cycling parameters. As such the study results should facilitate primer selection and formal critical evaluation as here described. As an example of this enhanced flexibility we have recently replaced the primer sets for influenza A H1 and H3  cross-contamination during manual extraction and (b) contamination of second round adjacent wells with product from a first round positive amplification. The use of the QIAGEN BioRobot for the extraction of all specimens reduced the former while the nature of the strip prevents the latter, since all the wells have separate mixes Table . With boThe results obtained were very encouraging. Although the strip was constructed to detect a wider range of viruses than immunofluorescence, over the period of validation it almost doubled (59 versus 31) the number of viruses that could have been detected by immunofluorescence, including a positive NEQAS specimen which was missed by immunofluorescence. Of this group of viruses the detection of adenovirus showed the most dramatic increase, an observation we have also previously made in a separate study  and whicAs mentioned in the introduction a factor which often impacts negatively on a laboratory's decision to use molecular diagnostics is one of cost. It is worth considering that no matter which assay is chosen for use, it will attract the same overheads needed to provide the infrastructure of a laboratory set-up i.e. building, utilities, staff and equipment. In this regard there are no cheap tests and to use reagent costs as the sole factor in determining which assay to use is somewhat perverse. While the reagent costs of the strip are higher that commercial immunofluorescence reagents by a factor of 3, including extraction, this would undoubtedly narrow if immunofluorescence were capable of closing the pathogen gaps that currently exist e.g. HRV, hMPV. Currently using this approach, we have been able to replace both immunofluorescence and viral culture and this ability makes molecular diagnostics a more cost effective method for diagnosing viral infections. Taking into account the superior range, sensitivity, ability to quantify and speed of molecular techniques it is incredible how little they are used in routine laboratories. With the advent of SARS and the threat of avian influenza, this deficit is now beginning to disturb health care planners at the highest level.Because specimen sampling was not contiguous seasonal peaks were not detected, accounting for the small numbers of respiratory syncytial virus detected and the lack of detection of human metapneumoviruses, parainfluenza virus type 1 and coronavirus 229E; subsequent (unpublished) data from the routine use of the molecular strip support an important role for human metapneumovirus in acute respiratory infections and the sporadic nature of infections caused by parainfluenza type 1 and coronavirus 229E.Several interesting observations need highlighting. First, for immunofluorescence to perform reliably it was essential that a good nasopharyngeal specimen was available. The use of throat and/or nasal swabs with immunofluorescence alone is inappropriate. Second, immunofluorescence was very poor at detecting viruses from patients in the community, again almost certainly because of the universal use of swabs in that setting. Third, the rapid results of immunofluorescence were complemented by the touchdown protocol which can report definitive results within 24 hours, considerably faster than culture. Fourth, the molecular strip was better at detecting multiple infections. Even allowing for the inability of immunofluorescence to detect rhinoviruses, it should have detected the mixed adenovirus and influenza virus infections. Although immunofluorescence is capable of diagnosing dual infections, its routine use along with culture probably grossly underestimates their prevalence. The most plausible explanation is that the molecular technique detects infections where one of the viruses is below the detection threshold of immunofluorescence. These low level viruses are either just starting or more likely reaching the end of an infectious episode (latency is less likely) and this raises the previously unaddressed question of their role in viral respiratory pathogenesis. Fifth, the extent of rhinovirus infections was very significant. Their clinical significance ranged from acting as a definitive respiratory pathogen to a less certain role when acting as the most frequently detected co-pathogen in mixed infections.In conclusion the use of the touchdown protocol with pre-dispensed and quality checked primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses for immunofluorescence negative specimens. Immunofluorescence results were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol is a convenient and reliable method of testing for multiple viruses in a routine setting. Its generic nature makes it especially useful for introducing test repertoire modifications e.g. incorporating primers for the newly identified coronaviruses SARS-CoV and HCoV-NL63.A total of 222 specimens were included in the validation between January 2002 and June 2003, including 14 from an influenza surveillance scheme. The specimens were collected from 183 patients including: 103 male, median age 3.5 y (7 m – 84 y); 80 female patients, median age 9 y (7 m – 84 y); both male and female ages were skewed towards the lower age ranges, and 5 national external quality assurance scheme (NEQAS) specimens . One hundred and fifty-nine patients were in hospital and 24 were in the community at the time of sampling. Specimens tested consisted of a wide range of specimens including: broncho-alveolar lavage (36), nasopharyngeal secretions (120), sputum (35) and combined throat and nasal swabs (31).Nasopharyngeal secretions, broncho-alveolar lavage and sputum specimens were received in dry sterile containers at ambient temperature. Upon receipt they were re-suspended in 2 ml of virus transport medium (VTM) consisting of phosphate buffered saline pH 7.1, bovine serum albumin 7.5 μg/ml, penicillin G sodium 1000 units/ml, streptomycin sulphate 1000 μg/ml and amphotericin B 2.5 μg/ml. Throat and nasal swabs were received in 2 ml of VTM and vortexed on arrival to release cells attached to the fibres of the swab. An aliquot of 410 μl was taken off for extraction after which the specimens were centrifuged at 2600 g for 5 min and the resulting cell deposits air-dried on glass multi-well slides and fixed in acetone prior to testing. Immunofluorescence was set up on the respiratory specimens using commercial reagents according to the manufacturer's instructions, and was able to detect: influenza A, influenza B, respiratory syncytial virus, adenovirus, parainfluenza type 1, parainfluenza type 2 and parainfluenza type 3 .A volume of 200 μl of the respiratory specimen suspension was extracted on a QIAGEN BioRobot 9604 using the Blood and Body Fluid Vacuum Protocol of the QIAamp DNA Blood Kit . This protocol allows the co-extraction of both RNA and DNA simultaneously.Simultaneous amplification of all targets was facilitated by using a standard 8 well multi-well PCR strip to which all mixes were pre-dispensed and stored frozen; this format is referred to in the paper as the "respiratory strip" because of the respiratory nature of the targets. The respiratory strip targeted the following 12 common respiratory viruses: influenza A H3), influenza A (H1), influenza B, respiratory syncytial virus type A, respiratory syncytial virus type B, adenovirus, coronavirus 229E, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, human rhinovirus and human metapneumovirus. The final configuration of the single and multiplex primer mixes in the 8 well strip are shown in Table , influenEach primer master-mix was made-up and titrated against a known positive control before being aliquoted and dispensed into its respective well of the 8-well microtube strip. The strips were stored frozen at -20°C until used. A positive control was also aliquoted and stored separately at -20°C until used. For the duration of the study the positive control was the cloned target of parainfluenza virus type 1; a negative control was not deemed necessary.4, 1 unit AMV reverse transcriptase, 1 unit Tfl DNA polymerase, 0.2 mM each deoxynucleoside triphosphate  and 1 μM outer primers.First round volumes were made-up in Access RT-PCR buffer  and in the final 10 μl volume contained the following reagent amounts: 1.5 mM MgSO2, 50 mM KCl, 0.1% Triton X-100, 0.2 mM of each deoxynucleoside triphosphate , 0.25 units of Taq DNA polymerase (Promega) and 0.2 μM inner primers.Second round volumes were made-up in Taq Buffer B (Promega) and in the final 10 μl volume contained the following final amounts: 10 mM Tris-HCl (pH 9.0), 3.5 mM MgClFirst round amplification was performed on 2 μl of extract added to 8 μl of first round primer master-mix per well. Second round amplification was performed on 0.2 μl of the first round reaction added to 9.8 μl of second round primer master-mix per well; a multi-channel pipette facilitated the transfer of the 8 volumes in one step. The positive control was run on the eighth well of each strip. The second round products were run on ethidium bromide stained 2% agarose gels and photographed. Specimens were reported positive when respectively the correct size bands and the positive control bands were present.Amplification was carried out on a range of thermal cyclers including the Applied Biosystems GeneAmp 2400 and 9700 series and a DNA Engine Tetrad PTC 225 . The first and second round amplification protocols consisted of 36 identical cycles with the exception that (a) a reverse transcription step of 48°C-10 min preceded the first round and (b) a hot-start preceded the second round by transferring the strip directly from ice to a thermal cycler held at 94°C. The touchdown protocol consisted of 6 steps as follows: (a) initial denaturation (94°C-2 min); (b) 3 cycles of denaturation (94°C-30 s), annealing (55°C-30 s) and extension (72°C-30 s); (c) 3 cycles of denaturation (94°C-30 s), annealing (52°C-30 s) and extension (72°C-30 s); (d) 20 cycles of denaturation (94°C-30 s), annealing (49°C-30 s) and extension (72°C-30 s); (e) 10 cycles of denaturation (94°C-30 s), annealing (46°C-30 s) and extension (72°C-30 s); (f) 72°C for 5 mins.PVC: Touchdown and molecular strip design and manuscript preparation.GMO: Early application of touchdown cycling to respiratory samples.HJO'N: Study protocol and manuscript preparation.CMcC: Study protocol and manuscript preparation.DDEO: Routine application of touchdown protocol during study.FM: Routine application of touchdown protocol during study.SJM: Routine application of touchdown protocol during study.SAF: Primer-mastermix manufacture and quality control.DEW: Early application of touchdown cycling to respiratory samples and manuscript preparation.MF: Routine application of immunofluorescence protocol during study.JS: Design and validation of primers for human metapneumonavirus detection.


When James Watson and Francis Crick reported the structure of DNA in 1953, the mechanism of inheritance was instantly apparent. The complementary pairing of the DNA bases in the double helix, the pair famously wrote, “immediately suggests a possible copying mechanism for the genetic material.” The structure helped explain one of the central problems of modern biology: how does genetic material get faithfully replicated and then passed on from generation to generation? It was long thought that DNA is the only unit of inheritance.Since then, it's become clear that molecules of DNA are packaged into highly organized superstructures that themselves are inherited. These structures play a significant role in the regulation of genes by preventing or facilitating protein–DNA interactions. In the eukaryotic cell (a cell with a nucleus), DNA exists as long threadlike molecules—a typical human cell contains some 6.5 feet (2 meters) of DNA—that associate with a variety of proteins to form a network called chromatin. Genomic DNA wraps around specialized DNA-packing proteins called histones to form nucleosomes, which condense chromatin into chromosomes and thereby influence chromosome behavior. Chromosomes are in turn packaged in increasingly higher levels of organization, with some parts being dispersed and others condensed. The most condensed region is called heterochromatin, or silent chromatin. Gene expression is largely silent in these regions, since the proteins required for transcription can't access DNA to transcribe genes when chromosomes are so tightly packed. Other regions of chromosomes exist in an extended state, called euchromatin. This is the most genetically active state; with genes exposed, transcription can easily occur.PLoS Biology, Jasper Rine, Hiten Madhani, and colleagues identify and characterize the function of a protein complex that helps deposit the variant H2A.Z onto euchromatin in yeast.As chromatin shifts between these states, it influences gene expression, largely through the interactions of histones and large protein complexes that together assemble, remodel, and modify chromatin. Since proper cell function depends largely on activating the right gene at the right time, mechanisms have evolved that protect active genes from the intrusions of silencing structures like heterochromatin. Both euchromatin and heterochromatin respond to mechanisms that resist encroachments of the opposite state. One mechanism involves replacing “canonical”  histones with a histone variant. Previous work on yeast from Hiten Madhani and colleagues had shown that one histone variant, called H2A.Z, is found specifically in euchromatin and prevents silent chromatin from spreading into adjacent euchromatic regions. While researchers have characterized some of the mechanisms that deposit canonical histones onto euchromatin, they knew little about the mechanisms that deposit variant histones. In this issue of To investigate which proteins help direct H2A.Z to specific chromosomal locations, the authors isolated H2A.Z, along with whatever proteins were associated with it, from yeast cell extracts. They determined that 15 proteins were true binding partners of H2A.Z and that 13 of them form a complex called SWR1-Com. The largest subunit of this complex, called Swr1p, belongs to a well-known family of adenosine triphosphate (ATP)-dependent chromatin remodeling enzymes (they use the energy of ATP to power remodeling) that provide access to DNA in chromatin. Rine, Madhani, and colleagues show that protein subunits of SWR1-Com associate specifically with the histone variant H2A.Z. By comparing the gene expression profiles of yeast mutants lacking the H2A.Z-encoding gene with mutants lacking the Swr1p-encoding gene, the authors show that H2A.Z depends on the SWR1-Com protein complex to function. Most importantly, they show that SWR1-Com is required in living cells to deposit H2A.Z onto euchromatin. Interestingly, the authors note, SWR1-Com shares subunits with a histone-acetylating enzyme involved in the regulation of transcription  and with another chromatin remodeler, which suggests that biochemical modifications of the subunits on histone “tails” may play a role in replacing H2A with H2A.Z.This histone–protein complex, the authors conclude, represents a chromatin remodeling machine with a novel function, revealing a new role for Swr1p-type enzymes and a novel mechanism of genome regulation. By preventing the spread of silent chromatin into transcriptionally active chromosomal regions—the result of the interaction described here—this mechanism allows the cell's gene expression program to operate with precision and on schedule. Since chromosomes can be inherited by daughter cells in this active state, such mechanisms ensure that gene expression programs essential for ongoing fundamental processes like embryogenesis and cellular differentiation proceed without interference.