DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA.Using a cocktail of different carbohydrases, a method was developed that enables a complete digestion of the plant cell walls and subsequent DNA release. Optimized conditions for the digestion reaction minimize DNA shearing and digestion, and maximize DNA release from the plant cell. The method gave good results in 125 of the 156 tested species.In combination with conventional DNA isolation techniques, the new enzymatic method allows to obtain high-yield, high-molecular weight DNA, which can be used for many applications, including genome characterization by AFLP, RAPD and SSR. Automation of the protocol (from leaf disks to DNA) is possible with existing workstations. DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall surrounding the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA. The field of plant molecular biology is therefore at a disadvantage, especially when an automated high-throughput system for the isolation of PCR-ready genomic DNA is required in population genetics, species identification, biodiversity investigation, selection screening, food control and plant biotechnology. QIAGEN GmbH has developed a 96-well grinding method (MagAttract 96 Plant kit), but it requires a special mixer mill, a centrifugation step and consequently is not fully automatable.Large scale automatable DNA mini-prep facilities were recently offered by several companies for animal tissues . However, because of their cell wall, the automation of the isolation of DNA from plants needs improvements. Whereas animal tissues need only a lysis buffer containing detergents and proteinase K to release their DNA, plant tissues need in addition a mixture of carbohydrase enzymes able to digest the cell wall. Enzymatic digestion of the cell wall of leaf tissues is routinely used for the production of protoplasts but this approach was never adapted for routine isolation of DNA from plant tissue.Trichoderma longibrachiatum, which digests the cell walls in order to liquefy the tissue without the need of grinding. The enzymatically released DNA is then isolated with commercially available magnetic beads.We describe here a new method for the lysis of plant tissues using a powerful cocktail of enzymes isolated from ® DNA DIRECT™ Universal kit . Fig. Humulus lupulus, Vitis vinifera, Narcissus pseudonarcissus, Tilia sp., Lilium henryi and Helleborus dumetorum) the amount of loaded DNA was equal to or higher than 120 ng. As only 25% of the isolated DNA were loaded into the agarose gel, it can be estimated that the method permits the isolation of approximately 50 to 500 ng of genomic DNA from a leaf disk, depending on the species.Leaf disks from 24 different species were digested by 5 μl of the enzymatic cocktail in 50 μl of digestion buffer. Thirty μl of liquid containing cell debris were drawn up and released DNA was isolated using Dynabeads® Magnetic 96 Plant System kit (Promega) to isolate the released DNA . Fig. Arabidopsis, Begonia, Brassica, Beta, Alium, Nicotiana, Triticum, Piper ...etc. However, incubation times need to be increased for Fragaria, Ribes, Oryza, Soya, Zea ...etc. Thus, although 1 to 3 hours are generally enough, the incubation time for a given species is not foreseeable and the appropriate length of digestion has to be determined experimentally before undertaking large-scale DNA isolations.In experiments described above, the cell wall digestion was done overnight for convenience. In order to follow the release of DNA at different times of enzymatic digestion, three 5 mm leaf disks from dry leaves of Aster amellus and Ilex aquifolium and from seeds of Allium porum , using the Wizard® Magnetic 96 Plant System kit (Promega). Fig. Ilex aquifolium by a CTAB-based extraction method  and by the enzymatic method described here (lines 6–10) using the Wizard® Magnetic 96 Plant System. In both cases the amount of loaded DNA is one fifth of the DNA corresponding to a leaf disk of approximately 3 mg (dry weight). The amount of isolated DNA is similar for both methods.The method is highly reproducible. Fig. Ilex at 50°C. This indicates that the digestion mix does not contain active DNase in the condition used for digestion, and that in the case of Ilex endogenous plant DNase are inactivated by the digestion mix.As indicated by Fig. Ilex aquifolium, Aster amellus and Solanum tuberosum. The primers have been designed for the genus Ilex. Thus the few negative PCR in other species probably result from primer mismatch and not from polymerase inhibition. RAPD amplifications are also shown  a digestion mix alone, or (2) a digestion mix "contaminated" with a Ilex aquifolium leaf disk removed after 10 min and further incubated overnight at 50°C, or (3) a mix digesting a Ilex aquifolium leaf disk overnight at 50°C  or bacterial template could be detected. Instead, Ilex aquifolium templates are detected. Indeed, the ITS PCR product found in the digestion mix "contaminated" with a Ilex aquifolium leaf disk removed after 10 min and further incubated overnight at 50°C (line 10) has the same size as ITS of Ilex (larger than ITS of Trichoderma). Further sequencing demonstrated that this PCR product was an ITS sequence of Ilex and not of Trichoderma (data not shown). Similarly, the prokaryotic 16S rDNA sequence obtained from the mix digesting an Ilex aquifolium leaf disk overnight at 50°C (line 19) represented the plastid (prokaryotic) 16S rDNA of Ilex (data not shown), and not a bacterial sequence.The enzymatic cocktail is produced from 0°C Fig. . No fung® gene card). In conclusion, the advantage of our enzymatic non-grinding method of DNA extraction compared with the above-described methods is that a large amount of high-quality DNA is isolated and that it is fully automatable.To the best of our knowledge, only a few non-grinding methods for isolation of DNA from plant tissue have been proposed, but only low amounts of DNA are generally obtained. Jhingan  followedIn a paper on the comparative analysis of different DNA extraction protocols from plant tissues, Csaikl et al.  wrote thQuercus represents such a case and high level of polyphenols (tannin) is suspected. Modifications of the digestion buffer by the addition of polyvinyl pyrrolidone , or polone (PVP ) or polyone (PVP ) in ordeIn summary, the protocol is simple and reliable, does not require grinding, centrifuging, or the use of hazardous chemicals. A large number of samples can be processed simultaneously, and full automation of the protocol is possible with existing workstations. Many different species were successfully tested. The method can be adapted to each species by modification of the digestion buffer, of the amount of the enzymatic cocktail added during the digestion or of the digestion time.The method is perfectly adapted to situations when high-throughput isolation of PCR-ready genomic DNA is required. Moreover, because of the high-yield and high-molecular weight DNA reliably obtained, sensitive PCR-based techniques could be applied: AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA), SSR (Simple Sequence Repeat polymorphism).Trichoderma longibrachiatum Rifai . The fermenting medium (7 liters) consisted of wheat bran (25 g/L), solid corn steep (25 g/L), hydrolyzed starch (45 g/L), mineral salts and fed by lactose (25% solution at feeding rate of 50 ml/h) after the first 48 hours of fermentation. The fermentation was carried out at 32°C for 144 h. Extracellular secreted enzymes were then isolated by centrifugation (5000 g for 30 min.) and concentrated by ultrafiltration (molecular weight cut-off 10 kD) at 200–250 mg/ml of protein. The obtained enzymatic cocktail was used directly for DNA isolation from plant tissues. It contains, among others, cellulases, beta-glucanases, xylanases, mannanases, xyloglucanases, pectinases, glycosidases . Trichoderma longibrachiatum was not detected by PCR in the enzymatic cocktail, and cellulase from this organism is in the GRAS list  of the US Food and Drug Administration  under the number §184.1250.A mixture of cell wall degrading enzymes was isolated from The enzymatic cocktail remains stable at least for two years at 4°C. Substantial aliquots of the enzymatic preparation can be obtained from the first author.One hundred and fifty six plant species from the Botanical Garden of Geneva were tested with the described enzymatic method of DNA isolation . Leaf ti® DNA DIRECT™ Universal  was added. The protocol of DNA isolation was then conducted according the manufacturer's instructions in 1.5 ml microtubes.Leaf disks (5 mm in diameter) were incubated in 50 μl of digestion buffer  and 5 μl of the enzymatic cocktail. After digestion , 30 μl of liquid containing cell debris were drawn up and 200 μl of Dynabeads® Magnetic 96 Plant System  and the MagnaBot® 96 Magnetic Separation Device, according to the manufacturer's instructions. For both protocols, DNA was eluted in 40 μl of TE8 . Fresh leaves were generally used, but silica gel-dried leaf tissue can also be digested. As well as leaf tissues, seed tissues were tested. To allow the enzyme solution to penetrate the seed tissue, seeds were broken into pieces of 1–3 mm in side, and one piece was used for DNA isolation. To examine the amount of DNA isolated, 10 μl of the eluted DNA was loaded on a 1% agarose gel containing ethidium bromide and the DNA band was compared with a known amount of lambda DNA /Hind III loaded into the gel.Alternatively, leaf disks of 48 randomly chosen species were digested simultaneously overnight in the same conditions on a sealed flat bottom microtitration plate  and one fifth of this DNA was loaded for agarose gel electrophoresis.One μg of lambda DNA/Hind III was added to the digestion mixture alone or in the presence of a leaf disk of Ilex aquifolium. Known amounts (from 14 to 53 mg) of liquid nitrogen-ground leaf tissue of Ilex were conventionally extracted and the isolated DNA was re-dissolved in the proportion of 50 μl of TE buffer per leaf disk (approximately 3 mg), the proportion used in the enzymatic method. The amounts of DNA were then compared by agarose gel electrophoresis.The amount of DNA isolated by a method of DNA extraction based on CTAB  was comatpB-rbcL spacer, [Ilex aquifolium, Aster amellus and Solanum tuberosum. One μl of isolated DNA were used in 25 μl of standard PCR reactions . RAPD amplifications were tested with primer 5' CGGCCCCTGT using 1 μl of the isolated DNA was added to 25 μl of standard PCR reaction .PCR amplifications of a plastid fragment , a mult spacer, ) and a s spacer, ) were teJFM conceived of the method, carried out preliminary experiments and drafted the manuscript. OS and AVM checked and analyzed the biological and chemical activities of the enzymatic cocktail. LA accumulated and interpreted the data. AS designed the enzymatic cocktail.List of species investigated.Click here for fileTypical enzymatic activities and properties of the cocktail used for plant DNA isolation.Click here for file


Atrial fibrillation (AF), the most common and rising disorder of cardiac rhythm, is quite difficult to control and/or to treat. Non pharmacological therapies for AF may involve the use of dedicated pacing algorithms to detect and prevent atrial arrhythmia that could be a trigger for AF onset. Selection 900E/AF2.0 Vitatron DDDRP pacemaker (1) keeps an atrial arrhythmia diary thus providing detailed onset reports of arrhythmias of interest, (2) provides us data about the number of premature atrial contractions (PACs) and (3) plots heart rate in the 5 minutes preceding the detection of an atrial arrhythmia. Moreover, this device applies four dedicated pacing therapies to reduce the incidence of atrial arrhythmia and AF events.To analyze the reliability to record atrial arrhythmias and evaluate effectiveness of its AF preventive pacing therapies.We enrolled 15 patients , with a DDDRP pacemaker implanted for a “bradycardia-tachycardia” syndrome, with advanced atrioventricular conduction disturbances. We compared the number and duration of AF episodes’ stored in the device with a contemporaneous 24h Holter monitoring. After that, we switched on the atrial arrhythmias detecting algorithms, starting from an atrial rate over 180 beats per minute for at least 6 ventricular cycles, and ending with at least 10 ventricular cycles in sinus rhythm. Thereafter, in order to evaluate the possible reduction in PACs number and in number and duration of AF episodes, we tailored all the four pacing preventive algorithms. Patients were followed for 24±8 months (from 20 to 32 months).All 59 atrial arrhythmia episodes occurred in the first part of this trial, were correctly recorded by both systems, with a correlation coefficient (r) of 0.96. During the follow-up, we observed a significant reduction not only in PACs number (from 83±12/day to 2.3±0.8/day) but also in AF episodes (from 46±7/day to 0.12±0.03/day) and AF burden (from 93%±6% to 0.3%±0.06%). An increase in atrial pacing percentages (from 3%±0.5% to 97%±3%) was also contemporaneously observed.In this pacemaker, detection of atrial arrhythmia episodes is highly reliable, thus making available an appropriate monitoring of heart rhythm, mainly suitable in AF asymptomatic patients. Moreover, the significant reduction of atrial arrhythmia episodes indicates that this might represent a suitable therapeutic option for an effective preventive therapy of AF in paced brady-tachy patients. Atrial fibrillation (AF) represents one of the major clinical, social and economical medical challenge ,2. ProgrDifferent therapeutic strategies, both pharmacological and non pharmacological, are available to restore sinus rhythm, prevent arrhythmia recurrences and cardio-embolic events and control the mean ventricular rate ,2,5. WhaIt is an affirming concept that AF should not be considered as a simple cardiac rhythm disturbance, but rather as a syndrome with a variety of clinical presentations and two frequently negative hemodynamic feature: 1) the lack of atrial contribution to ventricular filling and 2) the irregularity and/or the fast shortening of cardiac cycle lenght [ the lack the irreIn 1995, Wijffels and coll.  showed, to reduce the arrhythmogenetic effects of bradycardia and, in particular, the atrial refractory periods dispersion;to override atrial premature contractions (PACs), as a possible cause of AF onset;to reduce the compensatory pauses after an atrial or a ventricular premature contraction, thus reducing the dispersion of atrial refractoriness induced by the “short cycle – long cycle” mechanism.There are three main electrophysiological assumptions -24 for pA prospective randomised study from Andersen and coll.  on 225 pTo improve the capability to prevent the onset of AF, other several approaches have been tested. In patients with marked intraatrial conduction period delays, Saksena  and DaubIt’s also well known that variability in the origin of PACs should increase the probability of a re-entry mechanism, mainly in presence of an anomalous substrate. On this account, it has been proposed to reduce atrial rate variability using an overdrive atrial steady stimulation alone, although this approach did not obtain significant results. Alternatively, other pacing techniques such as bursts and drives algorithms have been proposed. Several trials -24 have In particular, the Atrial Dynamic Overdrive Pacing Trial (ADOPT)  evaluateMoreover, in the ATTEST study  was eval® 900E/AF2.0 Vitatron™, and to reduce the trigger mechanisms, possibly responsible for AF onset.Based on the hypothesis of the effectiveness of a non-pharmacological antiarrhythmic electrical therapy, our study was aimed at evaluate the reliability of the four specific pacing algorithms of the DDDRP pacemaker, the SelectionWe initially evaluated the atrial arrhythmia recording reliability in 15 patients, implanted with a Selection™ 900E/AF2.0 Vitatron® device, for a “brady-tachy” syndrome, with advanced atrioventricular conduction disturbances. Patients were enrolled in a period of 10 months, starting from January 2000 and ending in October 2000. Patients were 9 males and 6 females, with a mean age of 71±5 years, without any previous history of myocardial infarction, angina, diabetes and other known risk factors and in I NYHA functional Class. All patients were initially under oral anticoagulant therapy (INR 2.0-3.0) and IC antiarrhythmic drug therapy (Acetate Flecainide 200 mg per day) for at least 6 months prior to implant. Therapies were not discontinued during the follow-up.In order to evaluate the Selection™ 900E/AF2.0 Vitatron® atrial arrhythmia recording reliability, we compared the number of atrial arrhythmic episodes and their onset and duration,properly stored in the pacemaker, with a contemporaneous 24h standard Holter cassette recording . A statistical correlation analysis was performed. The pacemaker parameters were settled in order to record atrial arrhythmic episodes lasting at least 6 ventricular cycles for their onset and 10 ventricular cycle for their ending. The atrial cut-off detection rate was of 180 atrial waves per minute. Clinical and follow-up features of our population are summarized in The second aim was a middle-term efficacy evaluation  of all the four Selection™ 900E/AF2.0 Vitatron® available pacing algorithms, in order to prevent the trigger mechanisms, possibly responsible for AF onset. Each pacing algorithm was tailored according to the clinical and electrophysiological features of each patient, according to the previously recorded atrial arrhythmic events.™Pace Conditioning this algorithm consist on a permanent overdrive atrial pacing with an atrial pacing rate of about 15 b.p.m. higher than the beneath intrinsic effective atrial rate.™PAC Suppression this algorithm is designed to reduce the incidence of atrial tachyarrhythmia by a temporary stable atrial overdrive pacing following a PAC. This algorithm provides a temporary atrial overdrive pacing lasting for 600 ventricular cycles after a sensed PAC. At the end of this 600 ventricular cycles, the pacing rate progressively reduces till the lower rate limit or till the emergence of a stable sinus rhythm.™Post-PAC Response this algorithm is designed to reduce the post-extrasystolic pauses by controlling the atrial rate in the 2 beats after a PAC. The first atrial paced beat’s rate is determined by an averaging between the previous physiologic RR interval and the PAC pairing rate. From the second beat onwards, the atrial fleeing rate returns to the atrial physiologic rate.™Post-Exercise Rate Control the post-exercise rate control has been specifically designed to prevent a too fast lowering of heart rate after a physical activity. The post-exercise rate increases proportionally to the difference between the physiological heart rate and the target heart rate, i.e. 90% of the physiological heart rate.The four used atrial pacing algorithms were the following four:During the first part of this trial, i.e. that comparing Holter recordings and pacemaker storage data, we observed 59 episodes of paroxysmal AF, lasting from some seconds to some minutes, with a highly significant correlation coefficient between Holter and pacemaker recordings (r=0.96) . The AF As shown in However, it was unfeasible to perform a reliable multivariate analysis because of the reduced number of patients, because of the lack of multiple risk factors, because of the contemporaneous usage in the same patients of acetate flecainide and of pacing prevention algorithms.Presently, very few clinical randomised trials. -32 have ™ 900E/AF2.0 Vitatron® algorithms for detecting and monitoring AF. Moreover, we recorded a significant number of supraventricular tachyarrhythmia’s episodes, thus making stronger the statistical reliability of these data. Subsequently, we detected a progressive and significant reduction in AF burden and episodes. This decreasing in AF episodes and burden may be mainly related to the increase of atrial pacing, obtained by using a newly designed pacing overdrive algorithm responsible of atrial ectopic beats’ suppression in keeping with several trials [Our data confirm the significant reliability of Selectionl trials -28.® 900E/AF2.0® Vitatron™ pacemaker). Alternatively, as the data shown in Most of the discussed trials showed a lower reduction in AF episodes and burden if compared with our study . As showed in our recordings, the exceeding share of AF episodes and AF burden reduction is more likely to be entrusted to the newly designed four preventive pacing algorithms stored in the tested device (SelectionThe AF antiarrhythmic drugs therapy represents the first and more effective therapy, although it is well known that it is not always effective and free from side effects. In our patients, implanted for a sick sinus syndrome and at least partially resistant to AF pharmacological treatment, new interventional non-pharmacological solutions, such as tailored antiarrhythmic pacing algorithms, may represent a further effective therapeutical option. Recent analysis of AFFIRM  and RACEIn conclusion, our experience suggests the reliability of well tailored pacing algorithms on AF control in selected patients with brady-tachy syndrome, refractory to pharmacological rhythm control. However, we should say that this study is partially limited by the small and highly selected sample used, that reduces the possibility of translating theses results to general population. Moreover, this was not a truly randomized, controlled study, and so interpretation of the clinical significance of the reduction of AF episodes, as also shown however by several other trials in International Literature, is uncertain, thus not allowing us for making really definitive conclusions. The main information, however, we can derive from this trial is that we should better evaluate, before every pacemaker implantation, the different AF onsets of each patient eligible for such a procedure, because only a really profound knowledge of the different onset mechanisms of atrial tachyarrhythmias in each subject may give us the possibility of choosing a really effective implantable device for these sometimes very different patients, thus obtaining the maximum effective clinical results from these pacing algorithms in terms of reduction of AF episodes and burden.


A comparison of the amount of and the kinetics of induction of procalcitonin (PCT) with that of C-reactive protein (CRP) during various types of and severities of multiple trauma, and their relation to trauma-related complications, was performed.Ninety adult trauma patients admitted to the intensive care unit of our tertiary care hospital were evaluated in a prospective case study. During the initial 24 hours after trauma the Injury Severity Score, the Sepsis-related Organ Failure Assessment score, and the Acute Physiology and Chronic Health Evaluation II score were evaluated. PCT, CRP, the sepsis criteria , and the Sepsis-related Organ Failure Assessment score were measured at days 1–7, as well as at days 14 and 21, concluding the observation period with the 28-day survival.The induction of PCT and CRP varied in patients suffering from trauma. PCT increased only moderately in most patients and peaked at day 1–2 after trauma, the concentrations rapidly declining thereafter. CRP ubiquitously increased and its kinetics were much slower. Complications such as sepsis, infection, blood transfusion, prolonged intensive care unit treatment, and poor outcome were more frequent in patients with initially high PCT (>1 ng/ml), whereas increases of CRP showed no positive correlation.In patients with multiple trauma due to an accident, the PCT level provides more information than the CRP level since only moderate amounts of PCT are induced, and higher concentrations correlate with more severe trauma and a higher frequency of various complications, including sepsis and infection. Most importantly, the moderate trauma-related increase of PCT and the rapidly declining concentrations provide a baseline value near to the normal range at an earlier time frame than for CRP, thus allowing a faster and more valid prediction of sepsis during the early period after trauma. Multiple-trauma patients are especially prone to develop complications such as infections and sepsis. Since clinical symptoms and conventional markers are not always reliable signs for the diagnosis of sepsis and infection, biomarkers such as procalcitonin (PCT) or C-reactive protein (CRP) are often used as a diagnostic tool in these patients. Multiple-trauma patients, however, similar to patients undergoing elective surgery, may show an increase of PCT, CRP, and other biomolecules, indicating inflammation, during the early postoperative or post-traumatic period independent of the diagnosis of sepsis or infection -4.Several studies previously described the kinetics and the amount of PCT induced after elective surgery and trauma ,3-8. TheThe aim of this study was to describe the amount of and the time course of PCT and CRP induction in patients with various types of and severities of high-velocity trauma. We further registered trauma-related complications , as described by the Sepsis-related Organ Failure Assessment (SOFA) score, the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, the duration of stay in the intensive care unit (ICU), and the overall outcome.After approval by the local ethics committee, all patients with physical trauma due to an accident admitted to the ICU of our tertiary health care institution between May 1998 and April 2000 were prospectively included in the study. Inclusion criteria included age older than 16 years and survival for at least 12 hours. No chemical or burn trauma patients were included. Patients underwent surgical treatment when necessary for blood loss, wound treatment, or bone fractures according to accepted standards of care. PCT, CRP, all clinical, microbiological, and laboratory data, and all diagnostic and therapeutic options were registered. The data analyzed included data collected once during admission: age, gender, chronic conditions, severity of trauma according to the Injury Severity Score (ISS) , the APA9/l or <4,000 × 109/l, or body temperature >38°C or <36°C. Blood transfusions were given until the patient was hemodynamically stable or until hemoglobin values exceeded 8.0 g/dl, according to local guidelines.The type of and severity of trauma was classified according to the ISS. Severe trauma was assumed at ISS ≥20 according to the consensus of previous publications ,14. Infe®PCT luminometric assay  and the CRP level was measured using a nephelometric assay  [®PCT is 0.3 ng/ml. PCT concentrations were expressed as initial peak levels according to their maximum concentrations on day 1 or day 2 after trauma, and the CRP concentrations were from day 1 to day 3 after trauma due to the slower CRP induction kinetics.The PCT level was measured by the LumitestGermany) . The funThe initial ISS categorizes the type of and severity of trauma according to a system of points based on injury to six regions of the body . The ISSU test was used to compare the groups. Correlations were calculated by the Spearman rank correlation. Increased risk was calculated by the odds ratio, and significance was tested by the chi-square test. The area under the curve of the receiver operating characteristic was calculated and plotted by SPSS 10.0. Statistical comparison between the area under the curve of the various parameters was calculated using the method developed by Hanley and McNeil [z > 1.96 indicates a level of significance or an alpha error less than 5%. The McNemar test was used for comparison of sensitivity and specificity among parameters and scores at a given cutoff point. Statistical significance was accepted for P < 0.05. A Bonferroni correction was calculated for each group of comparisons.Statistical evaluation was carried out using the program SPSS 10.0 for Windows. Variables were defined as the median and upper and lower quartiles. When Kruskal–Wallis analysis indicated a significant difference among groups, the Mann–Whitney d McNeil , in whicOut of 102 patients with accidental high-velocity multiple trauma, 90 met the inclusion criteria during the study period. Twelve of the initially evaluated patients could not be followed up because of a fatal outcome within 12 hours. The median age was 34 years . Eighty-four patients (93%) were traumatized due to motor vehicle accidents, whereas six patients (7%) suffered from trauma following a fall from a greater height. The ISS ranged between 5 and 50 , and the duration of ICU treatment averaged 12 days . Further baseline characteristics for the patients are presented in Table The PCT concentration was increased above the normal levels of 0.5 ng/ml in 71% of the patients on day 1 or day 2 after trauma. Peak concentrations occurred on day 1 in 56% of the patients and on day 2 in another 26% . Eighty-five percent of the patients underwent surgical procedures during the initial observation period (days 1–2 after trauma). Initial PCT and CRP levels were similar in patients undergoing early surgery and those with no or late surgery. PCT concentrations, but not CRP levels, correlated with the number of blood units given on the initial day of trauma . Accordingly, PCT concentrations were significantly lower in patients who had moderate blood loss  as compared with those with major blood loss .The majority of the patients presented with multiple trauma of various regions of the body. Seven patients were injured in only one or two regions, 25 patients in three regions, 35 patients in four regions, and 23 patients in five or more regions. PCT and CRP concentrations according to the region of injury are summarized in Table P < 0.001 and P <0.177, respectively) , and the CRP concentrations (day 1–3) did not correlate at all with the ISS .When patients were categorized into those with moderate or severe trauma (ISS <20 or ≥20), the initial PCT but not CRP was significantly higher in patients with severe trauma (P < 0.001). However, there was no distinct arithmetical correlation between both parameters .Patients with high PCT levels early after trauma were treated for a longer period of time in the ICU than those who initially presented with low PCT values. In the case of PCT levels greater than 2.5 ng/ml (day 1 or 2 after trauma) the average duration of treatment in the ICU was 17 (± 13) days, as compared with 5 (± 4) days in those with PCT < 0.5 ng/ml (P = 0.49). The median ISS were 41 and 50, respectively. In contrast, CRP concentrations during the first week showed no differences between survivors and nonsurvivors.Fifteen of the 90 patients analyzed died within 28 days after trauma, all following severe trauma according to an ISS of 27–50 median, 41). The PCT, but not CRP, concentrations during the first week after trauma were significantly higher in nonsurvivors as compared with those in survivors Figures  and 4. D1. The PCP < 0.001) for suspected infection versus no suspected infection, and 3.01 ng/ml versus 0.57 ng/ml for proven infection versus no proven infection (P < 0.001). Initial CRP concentrations (days 1–3) did not significantly differ in patients developing a proven infection  or in patients in whom infection was suspected . For a PCT value ≥1 ng/ml the odds ratio for the development of an infection was 6.1 .In the present study, infection was suspected in 49 of the 90 patients and was proven in 40 patients during the 21-day observation period. Positive microbial findings were derived from pneumonia in 31 cases, from positive blood cultures in 10 patients, from colitis in 10 patients, and from wound and fungal infections in four patients. Five patients had urinary tract infections . On average, infections occurred 6 ± 3 days (mean ± standard deviation) after the trauma. Initial PCT levels (days 1 and 2 after trauma) were significantly higher in patients who subsequently developed infections: 2.69 ng/ml versus 0.54 ng/ml . Among patients without an infection only 16 out of 50 patients (30%) were treated on the ICU for more than 7 days, as compared with 38 out of 40 patients (95%) in whom an infection had developed. Similarly, PCT concentrations on day 7 were significantly higher in patients who had an infection, compared with those without an infection: 0.64 ng/ml  versus <0.3 ng/ml  . CRP concentrations did not significantly differ at this time point .The duration of treatment on the ICU was also different in patients with diagnosis of infection as compared with in those without. On average, patients without an infection were treated in the ICU for 6 ± 4 days (mean ± standard deviation), as compared with 20 ± 12 days for those who had an infection (P = 0.003), severe sepsis  or septic shock . PCT levels of patients who had either SIRS or sepsis were not significantly different . PCT concentrations remained elevated in patients with sepsis, severe sepsis, or septic shock, but rapidly dropped back to near-normal values in patients without sepsis  (Table The odds ratio for the development of sepsis/severe sepsis or septic shock was increased for PCT (≥1 ng/ml), for ISS (≥20), and for SOFA score (≥12), but not for the other parameters (t) Table .r = 0.438, P < 0.01; not significant for multiple comparisons). Moreover, PCT levels measured daily only weakly correlated with the corresponding SOFA score at this time point . Similarly, the initial PCT values did not correlate with the APACHE II score, measured 1 day after the trauma had occurred .Although high PCT levels related to a higher frequency of developing sepsis, the initial PCT and CRP concentrations did not correlate with the severity of organ dysfunction, measured as maximum SOFA score values during the study period  ,3,17,18.Typically, a cutoff PCT-level of 1.5–2 ng/ml has been described as an indicator of increased risk after various types of elective surgery  ,24.To compare the increase of PCT in this study in patients who later developed complications with the data of previous studies, we calculated a cutoff value for the diagnosis of sepsis. Contrary to the previous studies, however, this cutoff value only indicates the risk for the development of such complications during the further course of the disease, but not the actual sensitivity or specificity for the diagnosis of sepsis or the respective complications at this specific time point. Nevertheless, PCT with a cutoff value of 1.5 ng/ml had a specificity of 70% for the diagnosis of sepsis in this study. These data were similar to the results of PCT measurements of a study published previously by Wanner and colleagues. They reported a cutoff value of 1.5 ng/ml PCT on day 1 or day 3 after trauma and a sensitivity and specificity of 75.6% and 77.3%, respectively, for the diagnosis of sepsis as compared with the noninfection-related severe SIRS [Also in our study the amount of PCT and CRP typically induced after accidental trauma did not substantially exceed those concentrations reported previously after elective surgery, especially if major surgical procedures were compared ,7,20-22.PCT concentrations of approximately 1–1.5 ng/ml are also reported in various studies to be the best cutoff level for the diagnosis of sepsis as compared with SIRS -28. VeryIn the present study, PCT levels were not different in patients who underwent surgical procedures early after trauma as compared with those without surgery. We found a tendency to higher PCT levels following abdominal trauma. This is in agreement with former observations in patients undergoing elective surgical procedures at different regions of the organism, where we found higher PCT and CRP levels after surgery of the intestine  than after minor and less elective traumatic surgery  [in vivo and in vitro studies have indicated that not only bacterial endotoxins (lipopolysaccharide), but also various other mediators are capable of inducing PCT. In clinical and experimental studies PCT was induced by tumor necrosis factor alpha, IL-2, IL-6, phytohemagglutinin, and other proinflammatory stimuli [in vitro experiments provide further data that better explain the trauma-related and infection-related induction and function of PCT. The induction of and action of PCT is a multifactorial process, which involves different cell–cell interactions, a time-dependent pattern of cellular activation, and different cellular behavior when cells are native or pre-exposed to proinflammatory stimuli. In short, only adherent monocytes produce a significant amount of PCT. Circulating monocytic cells do not produce a quantitative amount of PCT. This is the first key for the understanding of PCT induction after trauma, since the expression of adhesion molecules and receptors also occurs in traumatized tissue.The cause of the induction of PCT after trauma is not completely understood. Experimental data from both  stimuli -31. HoweOnce induced, PCT acts as a chemokine in this area, attracting further monocytes; however, this property is lost after a few hours, when cells are preincubated or are in contact with PCT, thus limiting the local action to the acute phase response . FurtherShould systemic inflammation, sepsis, or organ dysfunction occur, there is a continuous and systemic stimulus for the production of PCT, whereas the trauma-related local induction of PCT soon declines, if the inflammation in the traumatized tissue disappears. Consequently, local tissue injury in combination with the activation of the immune system is a major cause for the induction of PCT after trauma. During a systemic inflammatory response such as sepsis there exist additional major sources for PCT . Increased levels of PCT, compared with lower circulating plasma levels, have been measured in hepatic venous blood , and a sOther complications of trauma that are associated with severe tissue trauma  and transfusion of allogenous blood cells may also contribute to the induction of PCT and may promote the development of organ dysfunction and poor outcome in these patients. PCT induction occurred during hemorrhagic shock in a baboon shock model, but it was weaker than PCT induction during endotoxin shock .In vitro, PCT augmented the induction of inducible nitric oxide synthase in cultured smooth muscle cells preincubated with proinflammatory stimuli [The biological action of PCT obviously has a significant influence on the course of the systemic inflammation and sepsis. The mortality rate of hamsters decreased when calcitonin precursors where neutralized , and the stimuli . A negatPCT and CRP levels measured early after trauma increase in patients with severe trauma. In addition to previous studies, we have analyzed the various types of trauma and have compared PCT with CRP and various clinical score systems. Unlike CRP, PCT values typically declined rapidly after trauma. The rapid decline of the trauma-induced response of PCT towards its normal range compared with the long-lasting increase of CRP promises an earlier diagnostic use of PCT as a marker of sepsis and infection than of CRP, since sepsis and infection can be diagnosed with high specificity only if there is no or only minor unspecific induction ,41. Our • 	The inflammation-related markers PCT and CRP increase early after trauma.• 	The increase for PCT, but not for CRP, is somewhat related to the severity of trauma but remains within moderate levels (<1 to 2 ng/ml), whereas CRP levels increase during even minor trauma (>100 mg/l).• 	The kinetics of PCT increase and its decline after trauma is faster than that seen for CRP .• 	PCT levels, but not CRP levels, elevated early after trauma are related to a higher rate of posttraumatic complications , more frequent infection, a worse outcome, and longer duration of treatment on the ICU.• 	The more rapid decline of PCT levels towards its normal range allows an earlier use of this marker for its usual indications than CRP, whose levels are elevated for a long time.APACHE II = Acute Physiology and Chronic Health Evaluation II; CRP = C-reactive protein; ICU = intensive care unit; IL = interleukin; ISS = Injury Severity Score; PCT = procalcitonin; SIRS = systemic inflammatory response syndrome; SOFA = Sepsis-related Organ Failure Assessment.MM received support for holding lectures on inflammation markers from BRAHMS Diagnostica AG, Germany.MM developed the study design and coordinated its implementation. MM and JS participated in interpretation/discussion of results and drafted and revised the manuscript. MM and HA were responsible for patient recruitment as well as data collection and they carried out the statistical analysis. All authors read and approved the final manuscript.


We report the identification of a novel polyomavirus present in respiratory secretions from human patients with symptoms of acute respiratory tract infection. The virus was initially detected in a nasopharyngeal aspirate from a 3-year-old child from Australia diagnosed with pneumonia. A random library was generated from nucleic acids extracted from the nasopharyngeal aspirate and analyzed by high throughput DNA sequencing. Multiple DNA fragments were cloned that possessed limited homology to known polyomaviruses. We subsequently sequenced the entire virus genome of 5,229 bp, henceforth referred to as WU virus, and found it to have genomic features characteristic of the family Polyomaviridae. The genome was predicted to encode small T antigen, large T antigen, and three capsid proteins: VP1, VP2, and VP3. Phylogenetic analysis clearly revealed that the WU virus was divergent from all known polyomaviruses. Screening of 2,135 patients with acute respiratory tract infections in Brisbane, Queensland, Australia, and St. Louis, Missouri, United States, using WU virus–specific PCR primers resulted in the detection of 43 additional specimens that contained WU virus. The presence of multiple instances of the virus in two continents suggests that this virus is geographically widespread in the human population and raises the possibility that the WU virus may be a human pathogen. We have identified a novel virus, referred to as WU virus, in the family Polyomaviridae by screening of human respiratory secretions. Two human polyomaviruses, BK and JC, were identified in 1971 and infect the majority of humans around the world. These two viruses are closely related to each other and are both are pathogenic in immunocompromised individuals. Earlier this year, a third polyomavirus, KI, was described in human clinical specimens, although its pathogenicity and prevalence in humans has not yet been established. The discovery of WU virus brings the number of polyomaviruses detected in humans to four. WU differs from BK and JC significantly in its genome sequence and in its relative tissue tropism, suggesting that it is likely to have unique biological properties. This discovery raises many questions for further investigation, such as, Is WU virus a human pathogen? If so, what kind of disease does it cause? Where in the body does WU virus reside? At what age does infection typically occur? Perhaps most importantly, there are likely to be many more as of yet unidentified viruses infecting the human body. Viral infections of the respiratory tract are responsible for significant mortality and morbidity worldwide . DespiteViruses in the family Polyomaviridae possess double-stranded DNA genomes and infect a variety of avian, rodent, and primate species. To date, two polyomaviruses, BK virus and JC virus, have been unambiguously described as human pathogens. BK and JC viruses are ubiquitous worldwide, and in adult populations, seroprevalence rates approaching 75% and 100%, respectively, have been reported ,12. Inferas transforms primary human cells [Much of the interest in polyomaviruses and SV40 in particular derives from the transforming properties carried by the early transcriptional region of the viral genome that encodes for the small T antigen (STAg) and and large T antigen (LTAg). T antigen is capable of binding both p53 and Rb proteins and interfering with their tumor suppressor functions. The early region alone is sufficient to transform established primary rodent cell lines  and in can cells . This haan cells .We describe the identification and characterization of a novel polyomavirus initially detected by high throughput sequencing of respiratory secretions from a patient suffering acute respiratory disease of unknown etiology. The virus was detected in the respiratory secretions from an additional 43 patients in two continents, and the complete genomes of multiple isolates were sequenced.Haemophilus influenzae (three reads), Streptococcus pneumoniae, Corynebacterium pseudodiphthericum, and Leifsonia xyli (unpublished data). Upon further examination, the six viral reads were collapsed into three unique regions, each of which possessed only limited homology to known polyomavirus proteins  from a 3-year-old patient admitted to the pediatric ward of the Royal Children's Hospital in Brisbane with pneumonia was collected in October 2003. The patient had no other remarkable clinical traits other than the respiratory features of pneumonia. Testing of nucleic acid extracted from the NPA using a panel of 17 PCR assays for known respiratory viruses as described . One 384The complete genome of WU was sequenced to 3× coverage using cloned fragments of the viral genome generated by a series of PCR primers. Analysis of the DNA sequence revealed genomic features characteristic of polyomaviruses. First, the WU genome size of 5,229 base pairs (bp) was quite comparable to those of the primate polyomaviruses BK , JC , and SV40 . In addition, the overall GC content of the WU genome was 39%, which is quite similar to the GC content of BK (39%), JC (40%), and SV40 (40%). The genome organization included an early region coding on one strand for STAg and LTAg, and a late region coding on the opposite strand for the capsid proteins VP1, VP2, and VP3 . These t5CX7–8CXCX2CX21–22CSCX2CX3WF, that was present in both of the predicted isoforms of WU STAg. This motif, which is present in all STAgs, was perfectly conserved in WU virus with the exception of the initial cysteine residue.In the early region, an unspliced open reading frame of 194 amino acids was detected that possibly encodes for the STAg. As the paradigm in other polyomaviruses is that STAg is expressed from a spliced message, analysis of potential splice sites revealed the presence of a putative splice donor sequence just one nucleotide 5′ of the initially predicted stop codon. Splicing to a downstream putative splice acceptor site would excise an intron of 70 nucleotides and generate a slightly larger STAg of 217 amino acids . While tIn all polyomaviruses, the initial ∼80 amino acids of the N-terminus of the STAg and LTAg are identical; the LTAg is generated by alternative splicing of the early mRNA transcript. In WU virus, a conserved splice donor site was identified immediately after amino acid 84 of the early open reading frame. The position of the splice site is similar to that found in SV40, BK, and JC virus, which occur after amino acids 82, 81, and 81, respectively. Splicing to a conserved splice acceptor site would generate a predicted protein of 648 amino acids . The preBased on comparative sequence analysis of LTAgs, the polyomaviruses are classified into two subclasses: a primate-like group exemplified by SV40, and a mouse polyoma-like group exemplified by murine polyoma virus . Using tIn addition to encoding LTAg and STAg, murine and hamster polyomaviruses utilize alternative splicing to generate an intermediate-sized protein referred to as middle T antigen. The WU virus early region was scanned for splicing motifs similar to known murine and hamster polyomavirus splice donor and acceptor sequences, but no obvious combination of splice sites was detected that would yield a middle T antigen sequence in the size range of known middle T antigens. In addition, SV40, JC, BK, and baboon polyomavirus all encode a fourth late protein termed the agnoprotein. There was no open reading frame present in WU with any detectable homology to the known agnoproteins. Thus, our sequence analysis suggests that neither middle T antigen nor agnoprotein are encoded by WU virus, although it is possible that the sequences have diverged beyond our ability to recognize the appropriate splice sites or protein products.Multiple sequence alignments of the predicted STAg, LTAg, VP1, and VP2 open reading frames revealed that WU virus was clearly a novel virus that is most closely related to KI virus . NeighboPCR primers were designed to specifically amplify WU. The initial screen used primers targeting the VP2 region, which possessed less than 20% amino acid homology to JC and BK virus to minimize the possibility of cross reactivity with the known human polyomaviruses. Empirical testing of the primers on samples known to contain BK and JC confirmed that the primers did not cross react with either of these genomes (unpublished data). Positives in the initial screen for WU virus were sequenced and then further confirmed by a second PCR reaction using primers targeting the 3′ end of the WU virus LTAg coding sequence. All 43 positive samples in the initial screen were confirmed using the second pair of PCR primers. A subset of samples that tested negative in the initial screen was also tested with the second PCR primer pair, and none of those samples were positive.In order to assess the prevalence of WU polyomavirus, a cohort of 1,245 respiratory specimens collected in 2003 in Brisbane was examined. Thirty-seven out of the 1,245 (3.0%) samples tested were positive for the virus . In thisIn addition, we examined two cohorts of patients from St. Louis, Missouri, United States. In one set of upper respiratory specimens collected in 2006, five out of 410 were positive for WU virus in the PCR assay. In addition, 480 bronchoalveolar lavage samples from patients (mostly adults) with severe acute respiratory illness were tested, yielding one positive. Of the positive samples, all six were co-infected with other viruses . The ageTo assess the sequence variation within different isolates, we analyzed the 250-bp region encompassed by the initial screening primers for all 43 cases . SeveralBecause BK and JC virus are frequently excreted in urine, we examined urine samples from patient cohorts in both St. Louis and Brisbane for the presence of WU virus by PCR. In the St. Louis cohort, urine samples from 200 adult patients participating in a study of polyomavirus infections in kidney transplant recipients were tested . For mosWe used a high throughput sequencing strategy to search for novel agents that were present in respiratory tract infections of unknown etiology. The focus of this study was on individual clinical specimens that still lacked a diagnosis after analysis with an extensive panel of diagnostic assays for known respiratory viruses. In one such patient sample, novel sequences with limited homology to known polyomaviruses were detected. Complete genome sequencing and phylogenetic analysis revealed that the new virus clearly had the genomic organization typical of polyomaviruses but was divergent from all previously described polyomaviruses. In keeping with the two-letter virus names for human polyomaviruses, we have named this novel polyomavirus WU virus ,26. OverDetailed analysis of the viral DNA sequence and genomic organization confirmed the novelty of WU virus. At all loci, WU virus was most similar to KI virus, but the degree of divergence between WU and KI was greater than the divergence between SV40 and BK, indicating that WU and KI are clearly distinct viruses . Based oWe detected WU in 37 out of 1,245 (3.0%) patient specimens in Brisbane  and in six out of 890 (0.7%) patient specimens tested in St. Louis. As the positive specimens were all collected from 2003 through 2006, it appears that WU is currently circulating, and its presence in both North America and Australia suggests that the virus is geographically widespread in the human population. The age range of patients that tested positive for WU virus spanned from 4 months to 53 years. The majority (86%) of the cases were found in children 3 years of age and under. Of the four positive specimens from adult patients  causes interstitial pneumonia and significant mortality. MPPV also differs from other polyomaviruses in that besides the kidneys, it can also be detected in the lungs, liver, spleen, and blood of suckling mice . Thus, tOne striking observation from these studies is the relatively high frequency of co-infection detected in the respiratory secretions: 72% overall (100% in the St. Louis cohort and 68% in the Brisbane cohort). Although more extensive studies are necessary to confirm the generality of this observation, this raises several intriguing non-mutually exclusive possibilities to consider: 1) WU may be an opportunistic pathogen; 2) WU infection may predispose or facilitate secondary infection by other respiratory viruses; and 3) WU may be a part of the endogenous viral flora that is reactivated by inflammation or some other aspect of viral infection. Recent studies of the prevalence of the newly identified human bocavirus have also reported higher levels of co-infection than previously described for other viruses found in the respiratory tract, with co-infection rates as high as 50% reported ,29. In aExtremely high sequence divergence was observed in the capsid proteins VP1 and VP2 of WU virus and KI virus as compared to the other known polyomaviruses. This divergence may reflect a different “lifestyle” for the WU/KI branch as compared to known polyomaviruses. Our data demonstrating the presence of WU in respiratory secretions and its absence in urine samples suggest that the mode of transmission or the sites of persistence of WU may be distinct from the other human polyomaviruses. As such, the structure of the virion must be optimized to enable the virus to survive dramatically distinct physiological and environmental conditions. This may partially explain the observed sequence divergence in the capsid proteins.Another question raised by this study relates to the potential antigenic cross reactivity of the WU capsid proteins. In terms of establishing the seroprevalence of WU itself and determining whether seroconversion accompanies acute infection with WU, it will be essential to conduct these studies with consideration for potential cross reactivity to KI, BK, JC, and SV40 antibodies. In addition, it is tantalizing to speculate whether serum antibodies to WU have the potential to cross react to SV40-derived antigens, and if so, whether they may at least partially account for some of the studies that report the presence of SV40 antibodies in the human population that is too young to have suffered exposure from contaminated polio vaccination –32.In conclusion, we have identified and completely sequenced the genome of a novel polyomavirus. This virus appears to be geographically widespread in the human population as evidenced by the detection of 44 distinct cases in two continents. Based on preliminary analysis, WU and KI virus share some strikingly similar properties, including their complement of genes, phylogenetic relationship, and physical sites of detection in the human body. These data suggest that WU virus and KI virus define a novel branch within the Polyomaviridae family with unexplored biology and pathogenicity. Another implication of these results is that the diversity of viruses in this family may be far greater than currently realized. Further experimentation is now underway to determine the relative pathogenicity of WU virus in humans and to understand the molecular properties of the virus. Since the T antigen of WU is predicted to have transforming properties by analogy to other polyomavirus T antigens, one question currently under investigation is whether a subset of human tumors may be associated with WU.Brisbane cohort. A total of 1,245 specimens (predominantly NPAs) were collected between January 1, 2003, and December 22, 2003, from patients presenting to the Royal Children's Hospital in Brisbane, Queensland, Australia, with symptoms consistent with acute lower respiratory tract infection.St. Louis cohort #1. A total of 480 BAL specimens were tested. These included samples from a retrospective and a prospective collection. The retrospective specimens were from a sequential collection of BAL specimens submitted routinely to the Virology Laboratory at St. Louis Children's Hospital between December 2002 and August 2003 . For theSt. Louis cohort #2. This collection was made up of respiratory specimens, mostly nasopharyngeal swabs, submitted for routine virologic testing to the Virology Laboratory at St. Louis Children's Hospital between September 2005 and June 2006. The majority of these specimens were from children. Of the 410 specimens in this collection, 200 were selected because they had been found to be positive by fluorescent antibody staining or culture for influenzavirus A or B, respiratory syncytial virus, parainfluenza virus, rhinovirus, or adenovirus.n = 50), (ii) pediatric clinic (n = 52), (iii) antenatal clinic (n = 33), (iv) indigenous health clinic (n = 36), and (v) bone marrow transplant patients (n = 55).Brisbane cohort. Urine specimens (226) that were submitted during 2003 to the diagnostic laboratory for routine investigation were collected. These represented a diverse mixture of donors, including those from (i) sexual health clinic  according to the manufacturer's instructions. PCR assays for 17 known respiratory viruses were performed as described [Brisbane cohort. Nucleic acids were extracted from 0.2 ml of each specimen using the High Pure Viral Nucleic Acid kit . Specimens that were negative were also cultured using cell culture systems that could detect the same group of viruses plus rhinoviruses, cytomegalovirus, and herpes simplex virus. Total nucleic acid extracts were purified using a Qiagen M48 instrument (http://www.qiagen.com). Nucleic acid extracts were tested for a panel of respiratory viruses using the EraGen MultiCode-PLx respiratory virus panel , a multiplex PCR assay that detects the following viruses: influenza A and B, respiratory syncytial virus A and B, parainfluenza 1–4, human meatpneumovirus, adenovirus subgroups B, C, and E, rhinoviruses, and coronaviruses OC43, 229E, and NL63.St. Louis cohort. All respiratory specimens were tested originally by fluorescent antibody staining using a panel of monclonal antibodies directed against influenza A and B, respiratory syncytial, parainfluenza 1–3, and adenoviruses  for 60 min at 37 °C. Total nucleic acid was extracted using the Masterpure Complete DNA and RNA Purification Kit . Then, 100 ng of total nucleic acid was randomly amplified using the RdAB protocol exactly as described [http://www.usbweb.com) enabled second strand cDNA synthesis as well as random priming of DNA originally present in the total nucleic acid sample. Amplicons were then generated via 40 cycles of PCR using primer-B (5′ GTTTCCCAGTCACGATA) with a cycling profile of: 94 °C 30 s; 40 °C 30 s; 50 °C 30 s; 72 °C 60 s. The primer-B–amplified material was TOPO cloned into pCR4.0  and transformed into bacteria, and white colonies were picked into 384-well plates. DNA was purified by magnetic bead isolation and sequenced using standard Big Dye terminator (v3.1) sequencing chemistry. Reaction products were ethanol precipitated, resuspended in 25 ul of water, and loaded onto the ABI 3730xl sequencer.Samples were prepared in the following manner for high throughput sequencing analysis. A total of 200 ul of neat NPA sample was thawed and directly treated with DNase I  remaining reads were aligned to a bacterial database using BLASTn with an e−10 cutoff; and 3) remaining reads were aligned to the viral RefSeq protein database using BLASTx with an e−2 cutoff [Sequences were assessed for quality using Phred , and rea2 cutoff .The WU genome derived from the index case was sequenced to 3× coverage using six unique pairs of PCR primers for the amplification. Amplicons were cloned into pCR4.0 and sequenced using standard sequencing technology. All primers used for amplification and sequencing are listed in Protein sequences associated with the following reference virus genomes were obtained from GenBank: BK virus, JC virus, bovine polyomavirus, SV40, baboon polyomavirus (simian agent 12), finch polyomavirus, crow polyomavirus, goose hemorrhagic polyomavirus, African green monkey polyomavirus, budgerigar fledgling polyomavirus, murine pneumotropic virus, hamster polyomavirus, and murine polyomavirus (see Supporting Information for accession numbers). For WU virus, predicted open reading frames were used. For STAg, the predicted open reading frame of 194 amino acids was used for analysis. Multiple sequence alignment was performed using ClustalX (1.83). Neighbor-joining trees were generated using 1,000 bootstrap replicates.For all PCR assays, standard precautions to avoid end product contamination were taken, including the use of PCR hoods and maintaining separate areas for PCR set up and analysis. For initial screening of WU virus, PCR primers AG0044 5′ tgttacaaatagctgcaggtcaa and AG0045 5′ gctgcataatggggagtacc were used with Accuprime hot start Taq (Invitrogen) to amplify 1 ul of template using the following program: 40 cycles of 94 °C 30 s; 56 °C 30 s; 72 °C 60 s. For every 88 samples tested, seven no-template negative controls were interspersed between the actual samples. Products were visualized following electrophoresis on 1% agarose gels. The resulting 250-bp amplicon was sequenced directly in both directions using primer AG0044 and AG0045. These sequences have been deposited in GenBank (see Supporting Information for accession numbers). Secondary confirmation was performed using primers AG0048 5′ TGTTTTTCAAGTATGTTGCATCC and AG0049 5′ CACCCAAAAGACACTTAAAAGAAA that generate a 244-bp amplicon in the 3′ end of the LTAg coding region. The same cycling profile of 40 cycles of 94 °C 30 s; 56 °C 30 s; 72 °C 60 s was used. For detection of both BK and JC viruses, primers AG0068 5′ AGTCTTTAGGGTCTTCTACC and AG0069 5′ GGTGCCAACCTATGGAACAG were used with a profile of 40 cycles of 94 °C 30 s; 56 °C 30 s; 72 °C 60 s.Figure S1A) The initial six shotgun reads with homology to polyomaviruses. B) The three contigs derived from the six reads.(38 KB PDF)Click here for additional data file.Figure S2The consensus TAg binding motif is GAGGC. The known primate polyomaviruses SV40, JC, BK, and baboon polyomavirus all have four copies of the copies of the binding site oriented as shown above for SV40 (NC_001669). The first nucleotide of the third copy of the consensus TAg binding site is defined as nucleotide 1 for WU and SV40. Differences between SV40 and WU Virus are 1) one of the TAg binding sites in WU virus appears to be a non-canonical TAGGC; 2) the second and third consensus TAg binding sites in WU virus overlap; and 3) the nucletoide spacing between the TAg binding sites in WU virus varies from the prototype SV40 as shown. Shown in blue is the polyA/T tract that is commonly found to the late side of the origin in polyomaviruses(462 KB PDF)Click here for additional data file.Figure S3A consensus LTAg donor site was detected. Splicing to the consensus downstream acceptor would generate a LTAg of 648 amino acids. For STAg, an unspliced open reading frame of 194 amino acids was identified. A predicted slice donor site was also detected that would result in excision of a 70-nucleotide intron and production of a 217–amino acid open reading frame.(542 KB PDF)Click here for additional data file.Figure S4Multiple sequence alignment of WU virus LTAg with 13 other reference sequences reveal the presence of carboxyl terminus extensions in baboon polyoma, BK, JC, and SV40. WU virus does not appear to encode such a region.(5.4 MB PDF)Click here for additional data file.Figure S5Locations of original shotgun reads are depicted as indicated. Locations of all sequencing primers are mapped to the complete genome. Primers used for amplification are shown in red.(551 KB PDF)Click here for additional data file.Table S1(35 KB PDF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) protein sequences used in this paper are as follows:The GenBank (LTAg: African green monkey (NP_848008); baboon polyomavirus 1 (YP_406555); BK (YP_717940); bovine (NP_040788); budgerigar (NP_848014); crow (YP_529828); finch (YP_529834); goose (NP_849170); hamster (NP_056730); JC (NP_043512); KI Stockholm 60 (ABN09921); murine (NP_041264); murine pneumotropic (NP_041232); SV40 (NP_043127).STAg: African green monkey (NP_848009); baboon polyomavirus 1 (YP_406556); BK (YP_717941); bovine (NP_040789); budgerigar (NP_848015); crow (YP_529829); finch (YP_529835); goose (NP_849171); hamster (NP_056732); JC (NP_043513); KI Stockholm 60 (ABN09920); murine (NP_041266); murine pneumotropic (NP_041233); SV40 (NP_043128).VP1: African green monkey (NP_848007); baboon polyomavirus 1 (YP_406554); BK (YP_717939); bovine (NP_040787); budgerigar (NP_848013); crow (YP_529827); finch (YP_529833); goose (NP_849169); hamster (NP_056733); JC (NP_043511); KI Stockholm 60 (ABN09917); murine (NP_041267); murine pneumotropic (NP_041234); SV40 (NP_043126).VP2: African green monkey (NP_848005); baboon polyomavirus 1 (YP_406552); BK (YP_717937); bovine (NP_040785); budgerigar (NP_848011); crow (YP_529825); finch (YP_529831); goose (NP_849167); hamster (NP_056734); JC (NP_043509); KI Stockholm 60 (ABN09918); murine (NP_041268); murine pneumotropic (NP_041235); SV40 (NP_043124).WU complete genome sequences have been deposited under accession numbers EF444549–EF444554. VP2 partial sequences have been deposited under accession numbers EF444555–EF444593.


These genes were tested for association with serum lipoprotein levels and particle sizes, apolipoprotein A1, B, and C-3 levels and with outcomes of hypertension, insulin resistance, and mortality. The prevalence of homozygosity for the −641C allele in theAPOC3 promoter (rs2542052) was higher in centenarians (25%) and their offspring (20%) than in controls (10%) . This genotype was associated with significantly lower serum levels of APOC3 and a favorable pattern of lipoprotein levels and sizes. We found a lower prevalence of hypertension and greater insulin sensitivity in the −641C homozygotes, suggesting a protective effect against CVD and the metabolic syndrome. Finally, in a prospectively studied cohort, a significant survival advantage was demonstrated in those with the favorable −641C homozygote (p < 0.0001). Homozygosity for theAPOC3 −641C allele is associated with a favorable lipoprotein profile, cardiovascular health, insulin sensitivity, and longevity. Because modulation of lipoproteins is also seen in genetically altered longevity models, it may be a common pathway influencing lifespan from nematodes to humans.Alteration of single genes involved in nutrient and lipoprotein metabolism increases longevity in several animal models. Because exceptional longevity in humans is familial, it is likely that polymorphisms in genes favorably influence certain phenotypes and increase the likelihood of exceptional longevity. A group of Ashkenazi Jewish centenarians  is more prevalent in centenarian humans than controls. This genotype is associated with a favorable lipoprotein profile, cardiovascular health, insulin sensitivity, and longevity. Manipulation of single genes can confer longevity in lower organisms, supporting the notion that understanding pathways for exceptional longevity may be possible in humans. Because the contribution of genetics to lifespan is estimated to be only ˜20%, a way to enhance the odds of identifying genetic factors is to study those with very exceptional survival . FavorabBecause of the strong heritability of this survival advantage, we began a search for gene variants that could explain the extended life of centenarians and their families among a genetically homogeneous population of Ashkenazi Jews. Crucial to our approach is the knowledge that only 1/10,000 survives to 100 y, while the average lifespan in the US is ˜77 y. Thus, enrichment in the frequency of certain gene variants at extreme old age probably reflects a selection effect that increases the likelihood of survival. Here we test if polymorphisms in certain candidate genes are significantly more frequent in centenarians than in younger participants. Given that the extreme old age of the probands precludes the use of a proper age-matched control group for analysis of traits that may be related to exceptional longevity, , we also recruited their offspring, many of whom presumably inherited longevity gene variants and thus should also manifest the longevity phenotype. We reasoned that this approach would permit comparison of the offspring to age-matched controls who would not be expected to be enriched with longevity gene variants, thereby accounting for period and cohort effects and phenotypic variation that may occur at older age .(CETP) gene, and decreased CETP levels. Further, through a genome-wide scan, Geesaman and coworkers found increased frequency of polymorphisms in the microsomal transfer protein in centenarian sibling pairs but not in other populations . In. InCETP APOC3 andCETP encode gene products involved in lipoprotein metabolism and thus the favorable alleles showing monotonic increases in allele frequencies among older age strata would also be expected to be associated with a favorable lipoprotein profile. Indeed, participants of all groups carrying theAPOC3 −641 CC genotype had lower serum levels of APOC3 compared with those carrying either CA or AA (CA/AA) genotype . Given the higher prevalence of this genotype in centenarians, it is therefore not surprising that serum levels of APOC3 were lower in centenarians and their offspring than in controls  (tively) B.APOC3 −641 CC genotype are shown inn = 131) and controls (n = 126) not using lipid lowering drugs in this analysis. In females, triglycerides (TG), high density lipoprotein cholesterol (HDL), and their ratio, as well as low density lipoprotein cholesterol (LDL) lipoprotein particle size were significantly more favorable among those with the CC genotype compared to the CA/AA genotypes. The same trends were observed in most of the associations in men. Since there was no significant interaction between gender and genotypes, we present also the combined group of females and males and their significance. As shown inAPOC3 CC genotype.Relationships between other lipoprotein traits andAPOC3 −641 CC genotype was associated with greater insulin sensitivity in the combined offspring/control group   A.APOC3 −641 CC genotype than among those with CA/AA genotypes (p = 0.04) . Other known variants of theAPOC3 gene: C(−482)T, T(−455)C, C1100T, C3175G, and T3206G,  were not associated with TG levels, insulin sensitivity, or blood pressure.The most prevalent CVD marker in an aging population is progressive increase in blood pressure. Therefore, this trait was examined in our participants as a surrogate for vascular aging. The prevalence of hypertension   was sign= 0.04) B. Since APOC3 genotype (p = 0.0007).Of the 381 participants we have genotyped since 1998, 64 had the CC genotype. To describe the relationship between genotype and death, we plotted the Kaplan-Meier survival function estimates of study participants byenotype . SurvivaAPOC3 gene either within the promoter region, in particular the T-455C variant polymorphism in the insulin response element, or in the gene, are associated with elevated TG levels but not with significant change in APOC3 levels , range 95–107 y; 48% over the age of 100 y) were recruited to participate in the study. The participants' ages were defined by birth certificates or dates of birth as stated on passports. Probands were required to have been living independently when they were 95 y of age as a reflection of good health, although at the time of recruitment they could be at any level of dependency. In addition, probands were required to have a child who was willing to participate in the study. The offspring group consisted of 122 females and 94 males . The control group included 258 participants derived from two sources: Ashkenazi Jews from the general population recruited by the Einstein Aging Study and spouses of the recruited offspring. The study sample included 183 participants with mean age 71.3 (0.67) y, 57% female . SpousesA research nurse visited the research participants in the morning to obtain a medical history, perform a physical examination, and draw a fasting venous blood sample. Standardized health histories were obtained using a questionnaire. At that visit, the offspring and the participating spouses underwent similar evaluations, as previously described ,6. SystTotal plasma cholesterol, TG, HDL, LDL, APOA1, and apolipoprotein B concentrations for study participants were measured by standard automated methods at the clinical laboratories of Montefiore Medical Center (coefficient of variance [CV] ranged 2.5%–3.5%). APOC3 concentrations in human serum were measured by ELISA using a commercially available kit  (CV ranged 2%–5%).LDL and HDL subclass levels and mean particle sizes were determined for all participants by proton nuclear magnetic resonance spectroscopy at LipoScience. Each nuclear magnetic resonance measurement produces the concentrations of three LDL subclasses and five HDL subclasses of varying size, as previously described  (CV rangAPOC3 on Chromosome 11q: C(−641)A, C(−482)T, T(−455)C, C1100T, C3175G, T3206G. Briefly, DNA was amplified using multiplex reaction containing biotinylated primer pairs. Amplified fragments within each polymerase chain reaction product pool were then detected colorimetrically with sequence-specific oligonucleotide probes immobilized in a linear array on nylon membranes strips. Probe specificities had previously been confirmed by sequencing and by use of DNA genotyped independently through other methods such as restriction length polymorphism analysis [A multilocus polymerase chain reaction–based assay was utilized to genotype known polymorphisms of641)A, C−2T, T and Stata version 8.2SE . Analyses of lipid parameters were stratified by sex, given that there are known differences between female and male lipid profiles and cardiovascular risks ,50. Resp = 0.05/66 = 0.0007.In order to take into consideration the false positive associations resulting from multiple tests of the 66 SNP , a BonfeAPOC3 C(−641)A genotype frequencies between the groups, the chi-square test was performed. Genotype frequencies of all SNP were found to be in Hardy-Weinberg equilibrium. A (corrected)p-value less than 0.05 was considered the threshold for statistical significance. Given that this is a relatively homogeneous population, stratification was not applied, except to control for gender effects as noted above. The haplotype association test between cases (probands) and controls was performed using Haploview 3.2 software  [For a comparison of the difference inex.php) .The participants' survival distribution was estimated by the Kaplan-Meier method, and the significance of the difference in survival distribution among the groups was tested by means of a log rank test. Wilcoxon statistics were calculated to test homogeneity between the groups.Table S1p-Value).The reported values are percent in Control/Proband and its significance of the change (CETP-VV andAPOC-3 CC remain significant (p < 0.0066).After Bonferroni correction only(135 KB DOC)Click here for additional data file.


Organophosphate developmental neurotoxicity involves multiple mechanisms converging on neural cell replication and differentiation.We evaluated mechanisms contributing to the adverse effects of chlorpyrifos (CPF) on DNA synthesis, cell number and size, and cell signaling mediated by adenylyl cyclase (AC) in PC12 cells, a neuronotypic cell line that recapitulates the essential features of developing mammalian neurons.In undifferentiated cells, cholinergic receptor antagonists had little or no protective effect against the antimitotic actions of CPF; however, when nerve growth factor was used to evoke differentiation, the antagonists showed partial protection against deficits in cell loss and alteration in cell size elicited by CPF, but were ineffective in preventing the deterioration of AC signaling. Nicotine, which stimulates nicotinic acetylcholine receptors but also possesses a mixture of prooxidant/antioxidant activity, had adverse effects by itself but also protected undifferentiated cells from the actions of CPF and had mixed additive/protective effects on cell number in differentiating cells. The antioxidant vitamin E also protected both undifferentiated and differentiating cells from many of the adverse effects of CPF but worsened the impact on AC signaling. Theophylline, which prevents the breakdown of cyclic AMP, was the only agent that restored AC signaling to normal or supranormal levels but did so at further cost to cell replication.Our results show definitive contributions of cholinergic hyperstimulation, oxidative stress, and interference with AC signaling in the developmental neurotoxicity of CPF and point to the potential use of this information to design treatments to ameliorate these adverse effects. The nearly ubiquitous exposure of the human population to organophosphate pesticides has raised increasing concern about their propensity to elicit developmental neurotoxicity at exposures that go undetected because of the absence of systemic signs of intoxication . At highThe diversity of mechanisms underlying the developmental neurotoxicity of CPF and the organophosphates is one of the main reasons for the wide developmental window over which the immature brain remains vulnerable to these agents, ranging from the earliest stages of brain formation through the postnatal consolidation of synaptic connections , 2005. Fin vivo  were seeded onto 100-mm poly-d-lysine–coated plates in RPMI-1640 medium  supplemented with 10% inactivated horse serum , 5% fetal bovine serum , and 50 μg/mL penicillin/streptomycin (Invitrogen). Cells were incubated with 7.5% CO2 at 37°C and the medium was changed every 2 days. For studies in the undifferentiated state, the medium was changed 24 hr after seeding to include the various test substances: CPF , atropine plus mecamylamine, nicotine, vitamin E, or theophylline . Because of poor water solubility, CPF was dissolved in dimethyl sulfoxide  and vitamin E was dissolved in 95% ethanol, achieving final respective concentrations of 0.1% and 0.05%, respectively, in the culture medium; accordingly, all control cultures also included the appropriate vehicles, which had no effect on the PC12 cells ; each culture was examined under a microscope to verify the subsequent outgrowth of neurites. The test agents were added concurrently with the start of NGF treatment, and cultures were maintained for 6 days, with the indicated agents included with every medium change.Because of the clonal instability of the PC12 cell line , we perf12 cells . For stuWe chose the concentrations of test agents on the basis of previous work. Given our main objective of trying to prevent the adverse effects of CPF, our strategy was to elicit a robust response for each of the effects to be evaluated. Accordingly, we used 30 or 50 μM CPF, concentrations high on the dose–response curves for oxidative stress, inhibition of DNA synthesis, and interference with cell acquisition, but below the threshold for outright cytotoxicity . Atropin3H]thymidine  along with the continued inclusion of the test substances. After 1 hr, the medium was aspirated and cells were harvested in ice-cold water. Duplicate aliquots of each sample were treated with 10% trichloroacetic acid and sedimented at 1,000 × g for 15 min to precipitate macromolecules. The resulting pellet was washed once with additional trichloro-acetic acid and then with 75% ethanol. The final pellet was hydrolyzed with 1 M potassium hydroxide overnight at 37°C and neutralized with 6 M hydrochloric acid, and the DNA was precipitated with ice-cold 5% trichloroacetic acid and resedimented. The supernatant solution, comprising solubilized RNA and protein, was discarded. The DNA-containing pellet was hydrolyzed in 5% trichloroacetic acid for 15 min at 90°C and resedimented, and an aliquot of the supernatant solution was counted for radiolabel. Another aliquot was assayed for DNA spectrophotometrically by absorbance at 260 nm. Previous work has demonstrated quantitative recovery of DNA by these techniques . Cells were harvested in ice-cold buffer and homogenized , and aliquots were withdrawn for measurements of DNA and protein using dye-binding methods .g for 10 min and the pellet was washed and resedimented. The membrane pellets were resuspended using a smooth glass homogenizer fitted with a Teflon pestle, in a buffer consisting of 250 mM sucrose, 2 mM magnesium chloride, and 50 mM Tris (pH 7.5). Incubations contained 50 μg of membrane protein , in a total volume of 250 μL. The enzymatic reaction was stopped by placing the samples in a 90–100°C water bath for 5 min, followed by sedimentation at 3,000 × g for 15 min, and the supernatant solution was assayed for cyclic AMP by radioimmunoassay . AC activity was evaluated under three different conditions: basal activity, the response to 10 mM NaF, and the response to 100 μM forskolin .The AC assay procedures and stimulant concentration profiles have been described in detail previously . To prep protein  in a medp < 0.05. To facilitate visual comparisons among the different studies, all experiments were normalized so as to share a standard set of control values, which were calculated as the average across all the experiments. The actual values varied by as much as 50% among different platings of cells. However, all statistical comparisons were conducted on the original data.Results are reported as means and SEs. Because of the multiple treatments in each experiment, significant differences were first established by a global analysis of variance (ANOVA) incorporating all treatments, followed by Fisher’s protected least-significant difference to evaluate differences between specific treatment groups. Significance was assumed at the level of In earlier work, we showed that the combination of atropine plus mecamylamine failed to prevent the decline in DNA synthesis evoked by CPF exposure in undifferentiated PC12 cells . In diffCPF exposure also impaired AC signaling in differentiating PC12 cells. For basal AC activity, 30 μM CPF reduced activity by about one-third ; the comWe previously showed partial protection by nicotine against the antimitotic effects of CPF in undifferentiated PC12 cells, despite the fact that nicotine, by itself, had a slight inhibitory effect . In diffBecause we had not previously evaluated the effects of vitamin E on DNA synthesis in undifferentiated PC12 cells, we compared its effects alone or in combination with CPF . At a coSurprisingly, vitamin E elicited deficits in basal AC activity quite similar to those seen with CPF . Here th3H]thymidine into DNA: 8,863 ± 278 dpm/μg DNA for controls (n = 8), 3,638 ± 133 for 1 mM theophylline , and 753 ± 43 for 10 mM theophylline . Accordingly, we were unable to detect any potential ability of theophylline to prevent the much smaller decrement in DNA synthesis caused by CPF, but this likely represents the technical limitation imposed by the large effect of theophylline by itself. In differentiating cells, theophylline also had a significant adverse impact on cell number as monitored by DNA content  and inhibitory (Gi) G-proteins; this result implies that the organophosphate treatment produces a shift in the relative expression and/or function of Gs and Gi, which would likely influence the activities of receptors that stimulate or inhibit AC. Although we did not pursue this issue in the present study, it is clearly an important consideration for future work, both in terms of additional mechanisms for CPF-induced developmental neurotoxicity and for contributions to subsequent neurotransmitter mechanisms underlying behavioral anomalies.The third target we examined was AC activity. Consistent with its effects in the developing brain , CPF eliin vivo as brain development progresses (in vivo than seen here. Similar to nicotine, vitamin E had complex effects after addition of NGF because, although it protected the cells from oxidative stress caused by CPF, it also interferes with the role of endogenous oxidative factors in neural cell differentiation. Finally, theophylline was the only agent that was able to fully restore the impairment of AC activity caused by CPF, but it did so only because it elevated AC by itself, not because it interfered directly with the effect of CPF. Here again, although theophylline by itself had adverse effects on DNA synthesis and indices of cell number, the possibility remains that analogs may prove better than the parent compound. Theophylline is relatively nonspecific as a phosphodiesterase inhibitor, and we recently found that CPF targets only a few phosphodiesterase and AC subtypes (In the developing brain, the neurotoxicity of CPF undergoes distinct transitions in mechanisms and cellular targets, so that although adverse effects are elicited at virtually any maturational stage, the regions, neurotransmitters, and behaviors that are affected shift according to the period in which exposure occurs , 2005. Hogresses . Nicotinogresses , so thatogresses , 2004; togresses , an effesubtypes . Thus, iin vitro models are not a substitute for animal studies of protection against organophosphate-induced developmental neurotoxicity, our results provide proof that the proposed divers mechanisms by which CPF damages developing neurons are indeed exerted directly by this agent and not indirectly through complex effects on the maternal–fetal unit, and further, provide a “proof of principle” that interventions aimed at these mechanisms may indeed prove valuable for amelioration of the adverse effects. However, the importance of our findings transcends these issues. Organophosphates represent 50% of all insecticides used worldwide (Although orldwide , and the