The aim of this study was to examine an in vivo and in vitro possibility for alkaline- and acid-DNases to be activated by SNP and peroxynitrite.It has been documented that nitric oxide (NO) donor sodium nitroprusside (SNP) and authentic peroxynitrite are capable of promoting apoptosis in a number of different cell types. Various endonucleases have been proposed as candidates responsible for the internucleosomal cleavage of the genomic DNA observed during apoptosis, but the main effect is attributed to the alkaline-DNases (Mgin vivo treatment with either SNP or peroxynitrite. A difference observed between the in vivo and in vitro effect of oxide donor (i.e., SNP) or peroxynitrite upon alkaline DNase activity existed, and it may be due to the existence of the "inducible" endonuclease. After a spectrophotometric scan analysis of purified DNA, it was documented that both SNP and peroxynitrite induce various DNA modifications (nitroguanine formation being the most important one) whereas DNA fragmentation was not significantly increased.The effect on liver tissue alkaline and acid DNase activity together with the markers of tissue and plasma oxidative and nitrosative stress (lipid peroxidation, SH group content, carbonyl groups and nitrotyrosine formation) was investigated in plasma and liver tissue. The activity of liver alkaline DNase increased and that of acid DNase decreased after Alkaline DNase activation seems to be associated with the programmed destruction of the genome, leading to the fragmentation of damaged DNA sites. Thus, the elimination of damaged cells appears to be a likely factor in prevention against mutation and carcinogenesis. Potentially toxic levels of peroxynitrite can be achieved whenever NO• and O2.- production is stimulated, due to the fact that a 100-fold increase in the rate of peroxynitrite formation occurs for every 10-fold increase in NO• and O2.- concentration [In its response to tissue damage and inflammation induced by a variety of xenobiotics, endotoxins and disease states , post-ischemic and regenerative injury, the liver produces a large quantity of nitric oxide (NO). Nearly all cell types in liver tissue, including hepatocytes, Kupffer cells, stellate cells and endothelial cells, have the capacity for generating NO. It has been documented that NO is capable of promoting apoptosis in a number of different cell types, generally classified as cGMP-dependent or cGMP-independent -4. The pin vitro . The facin vitro ,7. The rntration .1 phase of programmed cell death, the result of which is the activation of double-stranded DNA fragmentation in the F phase during which the nuclear morphology dramatically changes [Apoptosis, frequently termed "programmed cell death", is the form of cell death that occurs in normal liver in the course of its development and organogenesis, and in adult liver during the renewal of hepatocytes. In addition, apoptosis can be triggered by several hepatotropic viruses and toxic drugs, as well as in various liver diseases and experimental liver conditions such as hepatic allograft rejection. Degradation of the nuclear DNA, a common phenomenon observed in many organisms throughout the evolutionary scale, is one of the best-characterized biochemical features of apoptotic cell death. It has been established that the cell undergoes epigenetic reprogramming in the D changes . The cle changes . The mos changes ,13. Chan changes ,15.in vivo administration and in vitro treatment of isolated rat hepatocytes or purified commercial enzymes either with SNP or authentic peroxynitrite.On the basis of the pharmacological data supporting the critical role of NO and peroxynitrite in apoptosis, current research studies have evaluated the activity of alkaline and acid DNase during the administration of SNP or of peroxynitrite, as well as the changes in numerous susceptible parameters of nitrosative stress, including SH group oxidation, carbonyl group formation, lipid peroxidation and DNA modification. An assay of enzyme activity was performed using liver tissue after in vivo susceptibility of liver tissue to NO donor SNP and authentic peroxynitrite. A considerable attention has been paid to the establishment of in vivo tolerability and to the markers of apoptotic effects. Both NO and peroxynitrite can directly react with aromatic and sulfhydryl nucleophiles and nitrate aromatic residues. Sulfhydryl groups oxidation was documented in the plasma and, almost equally, in liver tissue. Peroxynitrite administration led to a more pronounced decrease in the concentration of other plasma free radical scavengers such as uric acid, and was followed by an increase in plasma nitrate concentration ] may play a significant role in liver cell toxicity. Neither plasma nor liver protein carbonyls showed any significant increase. This may be a likely consequence of the significant increase in aromatic amino acids nitration, presumably tyrosine the spectral contribution of which was substracted from the samples treated with 2,4-dinitrophenylhidrazine. Plasma and liver MDA concentrations were not significantly changed, either. The obtained results do not support the data suggesting that oxygen radicals, probably generated during cellular SNP metabolism, may mediate cell toxicity and apoptosis, but do confirm previous in vitro observations [in vivo treatment with either SNP or peroxynitrite  and by different NO donors, including SNP. NO has been shown to play an unprecedented range of roles in biological systems, acting as a universal intracellular and transcellular signaling molecule and the regulator of vascular tone, cell proliferation and apoptosis [2+-dependent endonuclease activity in the culture of neuronal cells. In our in vivo study  and inducible (NO-dependent) endonuclease are similar, as well as their optimum pH range (7.5–8.0). A likely conclusion is that Mg2+-dependent endonuclease seems to be a result of de novo synthesized or the pre-existing Ca2+/Mg2+-dependent endonuclease activation. Up to now, several Mg2+- or Ca2+/Mg2+-dependent alkaline DNases (DNase I) with an optimum activity within the range of 7.5–9.5 have been purified. Some of them, including specific caspase3-activated DNase (CAD), are active upon release of the specific inhibitor ICAD [N-nitrosylation sites are important for DNase II regulation [NOpoptosis -20. Peropoptosis ,21. Cellpoptosis -24. In cpoptosis ,26. The poptosis  indicatetor ICAD ,28. DNastor ICAD . The alktor ICAD ,31. Besitor ICAD . The spegulation ,33. SincThe formation of 8-nitroguanine, 8-oxo-deoxyguanine and oxazolone and the oxidative modification of 2'-deoxyribose into TBA-responsive compounds are the most prominent nucleotide modifications after reactive nitrogen species attack ,35. A hiIn vivo administrated SNP and peroxynitrite increase the activity of alkaline DNase. They also induced DNA modifications, such as nitroguanine formation. The obtained DNase activation seems to be associated with the programmed destruction of the genome and cell death. Given the above results and observations, the elimination of damaged hepatic cells appears to be a likely factor in prevention against mutation and carcinogenesis.SNP, DNA, DNase I (E.C. 3.1.21.1.) and DNase II (E.C. 3.1.22.1) were obtained from Sigma-Aldrich Company. RPMI-1640, fetal calf serum (FCS) and collagenase were purchased from ICN (Costa Mesa, CA). Authentic peroxynitrite was freshly synthesized by the quench-flow technique  and its in bolus in systemic circulation by intraventricular injection under penthobarbital sodium anesthesia. The concentrations were calculated according to the literature data concerning their in vivo tolerability and in vitro ability to induce apoptotic effects [Twenty-four male Sprague-Dawley rats, three months old, were divided into three different groups, each comprising 8 animals. Either SNP (10 mg/kg BW) or peroxynitrite (0.5 ml/kg BW of 30 mmol solution) in a volume of 100 μl were administrated  effects ,40. The  effects . The cor8 cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given in vitro concentrations were calculated according to the literature data [in vitro NO release [The isolation of hepatocytes was done according to a method already published , by usinure data . Purifie release .The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al.  and acid-1). The difference between the spectrum of the DNPH-treated sample and that of the HCl control was determined and expressed as μmol DNPH/g protein. As nitrotyrosine also absorbs at 370 nm, it was measured according to its spectral contribution at 370 nm. Plasma and tissue SH groups were measured by using DTMB according to the Elman method [The extraction of tissue DNA and proteins was performed according to the method of Wannemacher et al.  modifiedn method . Plasma n method . Nitraten method . Plasma Statistical analysis was made with the software SPSS. The effect of treatments was firstly evaluated by one-way ANOVA. If there was a significant effect, experimental data sets were compared against the control group by the Dunnett post hoc test. Significance level was set at α = 0.05. Data were normally distributed with equal variances among groups.in vivo and in vitro experiments, culture experiments and wrote the paper. RP and GN carried out DNA spectral analysis. TC performed measurement of SH groups and lipid peroxides. IS performed the measurement of nitrates and nitrites. TJ assisted during in vivo and in vitro experiments and did the graphical presentation. DS performed statistical analysis and assisted during in vivo experiments. DP and RK assisted during in vitro experiments and participated in the design of the study. All the authors read and approved the final manuscript.GK carried out the


The Steno hypothesis states that changes in basement membrane anionic charge leads to diabetic microvascular complications. In diabetic nephropathy, loss of basement membrane glycosaminoglycans and the association between glomerular basement membrane heparan sulphate and proteinuria has been documented. A correlation between erythrocyte surface and the glomerular capillary wall charges has also been observed.The aim of this study is to evaluate the relationship between retinopathy and erythrocyte anionic charge and urinary glycosaminoglycan excretion in type 2 diabetic patients.49 subjects (58 ± 7 yrs, M/F 27/22) with type 2 diabetes with proliferative retinopathy (n = 13), nonproliferative retinopathy (n = 13) and without retinopathy (n = 23) were included in the study. 38 healthy subjects were selected as control group (57 ± 5 yrs, M/F 19/19). Erythrocyte anionic charge (EAC) was determined by the binding of the cationic dye, alcian blue. Urinary glycosaminoglycan and microalbumin excretion were measured.6 RBC, 312 ± 30 ng alcian blue/106 RBC for diabetic and control groups respectively, p < 0.001). We did not observe an association between urinary GAG and microalbumin excretion and diabetic retinopathy. EAC is found to be negatively corralated with microalbuminuria in all groups.EAC was significantly decreased in diabetic patients with retinopathy (255 ± 30 ng alcian blue/10We conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients. Durin1c .Diabetic patients with evidence of nephropathy are characterized by a 5 to 10 times higher incidence of proliferative retinopathy . Thus, tThe Steno hypothesis proposed that increased loss of anionic charge from basement membranes leads to diabetic microangiopathy . This hyAnionic content of basement membranes predominantly produced by glycosaminoglycan molecules (GAG). They constitute the charge selective barrier of the glomerul basement membrane . SeveralAlthough abnormal GAG metabolism is a well known phenomen in diabetic nephropathy, it is still debated whether it has a pathogenic role in diabetic retinopathy. The aim of this study was to evaluate the relationship between diabetic retinopathy and erythrocyte anionic charge as well as the urinary glycosaminoglycan excretion in type 2 diabetic patients.49 outpatients  with type 2 diabetes mellitus diagnosed after the age of 30, were included and divided into 3 subgroups according to severety of retinopathy. Diabetes was diagnosed according to American Diabetes Association criteria. The onset of diabetes was after the age of 30 in these patients.The study was approved by the institutional ethics committee and patients gave written informed consent. Exclusion criteria included hypertension, myocardial infarction, cerebrovascular disease, heart failure, treatment with antiaggregants, steroids or other drugs that effect blood pressure and glucose, serum creatinine > 200 μmol/l, connective tissue disorders and other systemic disease.Renal and bladder infection diseases of patients and controls were excluded biochemically and microbiologically.0). Group 2 consisted of 13 patients with nonproliferative retinopathy (R1-hemorrhages, microaneurysms, cotton-wool exudates). The 13 patients in group 3 (R2) had proliferative retinopathy (R2-neovascularisations, vitreous hemorrhages, ablatio of the retina).The ocular fundus was examined by an ophthalmologist after dilation of the pupils and classified. The patients were divided into three groups according to the severity of retinopathy. Group 1 consisted of 23 patients without retinopathy  was measured by means of cationic dye alcian blue 8GX  according to the method of Levin with minor modifications as follows: . From ciGAG) excretion was determined in 24 hr urine samples spectrophotometrically at 520 nm by the addition of dimethylmethylene blue (Aldrich Chem Co., USA) and bovine kidney heparan sulfate as standard  [Urinary glycosaminoglycan (U H 7640) . In our Serum sialic acid was measured in serum with a colorimetric method described by Svennerholm with a Shimadzu UV-1201 spectrophotometer (Shimadzu, Japan) with a wavelength of 525 nm. Using N-acetyl neuraminic acid  as standard . Intra aUrinary albumin excretion was measured by an immunoturbidemetric assay (Roche diagnostics, USA) on automated clinical chemistry analyzer (Hitachi 902). In all patients, the annual level was determined as the mean of urinary albumin excretions in three 24 h urine collections taken at home during normal physical activity. The intraassay and interassay coefficients of variation were 1.3 and 4.3 % for a mean value of 25 mg/l albumin concentration.Serum glucose, cholesterol, triglyceride and HDL (Dade Behring, USA) were evaluated with enzymatic methods (Dade Behring, USA) on an automated clinical chemistry analyzer (Dimension RxL). Creatinin was assayed by the Jaffe reaction on an automated clinical chemistry analyzer (Dimension RxL).1C (reference range 4.4–6.0 %) was measured by a turbidimetric inhibition immunoassay technique (Roche Diagnostics, USA) on automated clinical chemistry analyzer (Hitachi 902). The intraassay and interassay coefficients of variation were 1.8 and 3.0 % for a mean value of 10.5 % HbA1C concentration.HbAThe analysis of the data was performed with a PC compatible Instat-II programme. Paired t test and ANOVA were used where appropriate. Correlation analysis was performed with Spearman rank test. The differences were considered significant when the probability was p < 0.05. The results were given as mean ± SD.The clinical characteristics of the study groups are shown in Table 1C levels were not different in subgroups of our diabetic population.Results of laboratory parameters are shown in Table 6 RBC in healthy controls. Diabetic patients both with nonproliferative and proliferative retinopathy had a significantly lower alcian blue binding to RBCs compared with diabetic patients without retinopathy (p < 0.001).Distribution of erythrocyte anionic charges of all study groups are demonstrated in figure A marked (p < 0.05) difference was found between type 2 diabetic patients (8.3 ± 4.1, 8.7 ± 4.8, 8.4 ± 3.3 mg/24 h, Table A significant difference (p < 0.05) was observed in serum sialic acid values in diabetic patients compared with healthy controls. Serum sialic acid level was higher in the nonproliferative retinopathy group, but the difference was statistically insignificant (p > 0.05).Microalbumin level was significantly p < 0.05 higher in diabetic patients (73 ± 40 35–250], 75 ± 21 [50–202], 85 ± 45 [56–224] mg/24 h, Table 50, 75 ± Results of the Spearman rank correlation test are shown on table Our study demonstrates a statistically significant quantitative reduction of alcian blue binding to RBCs in diabetic patients than healthy subjects, which is in accordance with literature ,16. PrevErythrocyte anionic charge by itself is unlikely to be important in the pathogenesis of diabetic retinopathy but reduced RBC alcian blue binding was found to be associated with the loss of glomerular basement membrane anionic charges in diabetic rats and patients . ChangesSeveral authors have questioned the validity of the alcian blue binding assay. The major objections concern the incomplete alcian blue dissolution in PBS buffer and its tendency to precipitate with time ,23. We pDiabetic retinopathy is characterized by gradually progressive alterations in the retinal microvasculature leading to vascular hyperpermeability, capillary occlusion and ultimately neovascularization. Thickening of the retinal microvascular basement membrane and loss of anionic content are well documented morphological features of diabetic retinopathy ,21,24. TGlycosaminoglycan primarily heparan sulfate has been implicated in permeability properties of basement membrane of glomerular basement membrane as well as the retinal basement membrane . Kahaly Systemic treatment of IDDM patients with proteinuria with danaparoid sodium, a glycosaminoglycan, not only reduced proteinuria, but also decreased the number of hard exudates in the retina . StudiesIn this study we found that urinary GAG excretion is increased in diabetic patients than healthy controls, but there was no difference in GAG excretion between nonproliferative and proliferative retinopathy patients. We couldn't find a correlation between GAG excretion and EAC but we found a correlation between albumin excretion and EAC. Although our working hypothesis is that the reduction in anionic charge in basement membranes may be due to an abnormal GAG metabolism, we couldn't confirm it with this study.Our study did not directly address the pathophysiology of diabetic retinopathy but a reduction in erythrocyte charge perhaps connected with the properties of retinal basement membrane.Several reports suggest that elevated serum sialic acid levels in type 1 and type 2 diabetic patients indicate microvascular damage ,33. In oWe conclude that type 2 diabetic patients with low erythrocyte anionic charge are associated with diabetic retinopathy. Reduction of negative charge of basement membranes may indicate general changes in microvasculature rather than retinopathy. More prospective and large studies needs to clarify the role of glycosaminoglycans on progression of retinopathy in type 2 diabetic patients.BMI: Body mass indexEAC: Erythrocyte anionic chargeGAG: GlycosaminoglycanPBS: Phosphate buffered salineRBC: ErythrocytesGAG : Urinary glycosaminoglycanUThe authors declare that they have no competing interests.YB : Evaluated laboratory parameters. Designed the study. Wrote the paper.HD: Diagnosed outpatients according to American Diabetes Association criteria.MA: Examined ocular fundus after dilation of the pupils and classified according to the severity of retinopathy.DY: Performed the analysis of the data with a PC compatible Instat-II programme. Improved the manuscript.The pre-publication history for this paper can be accessed here:


A new study settles a long-standing dispute about the genesis of an endangered species. With scant fossil evidence supporting a prehistoric presence, scientists could not say for sure where Borneo's elephants came from. Did they descend from ancient prototypes of the Pleistocene era or from modern relatives introduced just 300–500 years ago? That question, as Fernando et al. report in this issue, is no longer subject to debate.Applying DNA analysis and dating techniques to investigate the elephants' evolutionary path, researchers from the United States, India, and Malaysia, led by Don Melnick of the Center for Environmental Research and Conservation at Columbia, demonstrate that Borneo's elephants are not recent arrivals. They are genetically distinct from other Asian elephants and may have parted ways with their closest Asian cousins when Borneo separated from the mainland, effectively isolating the Borneo elephants some 300,000 years ago.In the 1950s, Borneo elephants had been classified as a subspecies of Asian elephants (either Indian or Sumatran) based on anatomical differences, such as smaller skull size and tusk variations. This classification was later changed, partly because of the popular view that these animals had descended from imported domesticated elephants. Until now, there was no solid evidence to refute this belief and no reason to prioritize the conservation of Borneo elephants.Their new status, as revealed by this study, has profound implications for the fate of Borneo's largest mammals. Wild Asian elephant populations are disappearing as expanding human development disrupts their migration routes, depletes their food sources, and destroys their habitat. Recognizing these elephants as native to Borneo makes their conservation a high priority and gives biologists important clues about how to manage them.


There is a much-quoted saying, attributed to the epidemiologist Geoffrey Rose: “A large number of people exposed to a small risk may generate many more cases than a small number exposed to a very high risk.” This is true for many individual risk factors such as salt intake (linked to high blood pressure and cardiovascular disease) and speeding on the highway (linked to injuries and accidents). Does it apply to many other global health risks? The study by Anthony Rodgers and colleagues suggests that it does.To develop effective health policies, one must understand the existing health risks and disease burdens. On a worldwide scale, this is a tough challenge. The Global Burden of Disease Database, maintained by the World Health Organization (WHO), collects data from countries around the world on risk factors such as tobacco, malnutrition, childhood abuse, unsafe sex, childbirth, and cholesterol levels, as well as on disease burdens, for example depression, blindness, and diarrhea. A large group of scientists from all over the world has developed a framework to analyze these data. To compare different risks or burdens, they calculate disability-adjusted life-years, or DALYs—the number of healthy life years lost because of a particular disease or risk factor.Rodgers and colleagues used data from the WHO database for 26 risk factors and from 14 epidemiological subregions of the world to calculate the proportion of risk-factor-attributable disease burden in different population subgroups defined by age, sex, and exposure level. For being underweight in childhood, for example—the leading risk factor for global loss of healthy life—they found that only 35% of the disease burden occurred in severely underweight children, the rest occurred in those only moderately underweight. The relative risks for the moderately underweight are much lower, but the number of children in that category is so large that the total attributable burden amounted to almost two-thirds of the total global burden of disease for that risk factor.The analysis confirms—and extends to a global level—previous research showing that many major health risks are important across the range of exposure levels, not just among individuals exposed to high levels of risk. It also points to risk factors that are particularly prevalent among specific populations and age groups, and for which highly targeted interventions could be effective.Despite numerous caveats and limitations of studies like this one, such analyses are essential aids in guiding the distribution of limited funds to lower the burden of life years lost to premature death and disability.


In this study, we examined the correlation between excitation-contraction coupling characteristics and skeletal muscle fibre type by (1) localizing the distribution of dihydropyridine receptor (DHPR) protein and (2) comparing the effect of DHPR blocker on muscles with different fibre type composition, in order to better understand the differences between contractile phenotypes of fibres and to explain the contradictory reports to date on the interaction of dihydropyridines with skeletal muscle isoform of DHPR.vs. 0.052 ± 0.011 N, femoris 0.045 ± 0.005 N vs. 0.033 ± 0.005 N and gastrocnemius 0.089 ± 0.016 N vs. 0.075 ± 0.014 N, respectively. The attenuation of contraction force proportional to the cross-sectional area of the muscle was significantly (P = 0.023) higher in gluteus (28.3 ± 3.5 %) and femoris (27.6 ± 3.2 %) as compared to gastrocnemius (16.1 ± 2.5 %). However, no significant change in the control measurements was observed ruling out the possibility of fatigue.Histochemical experiments revealed that fluorophore conjugated dihydropyridines stain selectively the membranes of muscle fibres. The staining was most evident in type IIA fibres. The major fibre type in gluteus and femoris, revealed by mATPase staining, was IIA (45.0 and 38.1 %, respectively). In gastrocnemius the content of IIA fibres was 22.7 %. Contraction forces before and after the addition of blocker for the three muscles investigated were: gluteus 0.075 ± 0.017 N The results indicate that the attenuation of the contraction force was largest in muscles with a high percentage of type IIA fibres. This supports our finding that the abundance of dihydropyridine receptors of IIA fibres outnumbers that in the other fibre types. The present data show that the correlation of density of dihydropyridine receptors can be one of the important factors influencing the overall contractile properties of the muscle and for its part explain the contradictory results of previous studies on coupling process. One of the features characterizing mammalian skeletal muscle tissue is the structural variability of muscle fibres. The inclusive properties of skeletal muscle lead to functional diversity of muscle fibres, which has been related to differences in relative proportions of membrane structures and to different amounts of contractile proteins . Althoug2+-release via the ryanodine receptor [1 (170 kDa), α2 (140 kDa), β (55 kDa), γ (33 kDa) and δ (24–33 kDa) [1 subunit contains the receptor for dihydropyridines (DHPs) and other calcium antagonists. DHPs, such as nifedipine, nitrendipine, and nimodipine, bind to DHP receptor in a specific, saturable and reversible fashion. In addition, receptors show high affinity for DHP ligands. The dissociation constant (KD) for skeletal muscle membranes is usually in the range of 1–10 nM [Voltage-dependent dihydropyridine receptors play an important role in the function of skeletal muscle. As well known, contraction is initiated by a depolarization of the transverse tubular (T-tubular) membrane, which in turn causes calcium to be released from the stores of sarcoplasmic reticulum (SR). The DHPR is located in the T-tubule membrane acting as a voltage sensor. Linked to the ryanodine receptor located in the sarcoplasmic reticulum membrane, this channel protein triggers the intracellular Careceptor . The DHP–33 kDa) ,7. The p 1–10 nM . Dihydro 1–10 nM .2+ release needed in E-C coupling, the α1-subunit of the molecule functions also as a calcium channel [Besides being the voltage sensor for Ca channel  giving r channel . However channel . Firstly channel  reportedIn the present study we examined the E-C coupling characteristics of muscles with different dihydropyridine receptor expression defined by fibre type composition. To test the hypothesis that muscles with different cell type compositions, i.e. DHPR quantities, react differently to calcium channel blocker, we measured contraction forces of three muscles before and after adding the blocker solution. We found variation in the activities of muscles depending on the cell type composition of the muscles. The findings confirm the previously observed pattern and elucidate the role of dihydropyridine receptor in muscle cell contraction.In order to analyse the distribution of cell types in the gluteus maximus (GLU), rectus femoris (RF) and gastrocnemius (GAS) muscles, tissue samples were collected and subjected to SDS-PAGE. The different muscles of mouse contained four myosin heavy chain (MHC) isoforms in the increasing electrophoretic mobility, MHCIIa, MHCIId, MHCIIb and MHCI Fig.  as compaWe have previously shown that the density of dihydropyridine receptors  in T-tubule membranes increases markedly during the postnatal development of mouse skeletal muscle . Furtherin vitro nifedipine effect on the muscle force production was determined as the percentage of the remaining channel mediated contraction force. The weakening of contraction force proportional to the cross-sectional areas of the muscles was significantly (P = 0.023) higher in GLU (28.3 ± 3.5 %) and RF (27.6 ± 3.2 %) in comparison to GAS (16.1 ± 2.5 %). Experiments performed in increasing concentrations of nifedipine resulted in a clear dose-response curve , m. gastrocnemius (N = 6) and m. gluteus maximus (N = 6) from adult mice (strain CD-1) were removed after the animals were killed by paracervical dislocation in accordance with the Animal Ethics Committee of the University of Oulu (licence no. 046/03). The muscles were homogenized in 6 vol of homogenization buffer [62.5 mM Tris-HCl, pH 6.8] and boiled in sample buffer as previously described for 5 min at a final protein concentration of 0.5 mg ml-1 . Total p-1 . Myosin 2 (18 mM) and Tris-HCl (199 mM) the sections were incubated in substrate solution  at room temperature for 45 min. After incubations in 11 mM CaCl2, 2% CoCl2, and 10 mM sodium barbital, the colour was developed in 2% (v/v) ammonium sulphide for 45 s. After washing with distilled water, the sections were dehydrated in ethanol, cleared in xylene and mounted with DPX. An alkaline preincubation in a solution containing 34 mM 2-amino-2-methyl-1-propanol, 120 mM CaCl2, adjusted to pH 10.3 with HCl was also used as previously described by Guth and Samaha [Serial cross-sections, 8 μm thick, were cut on a cryostat microtome at -25°C, mounted on cover slips and stained for myosinATPase (mATPase) with acid preincubation according to Hämäläinen and Pette [d Samaha . After pFor fluorescence labelling, RF muscle from mouse was cut into 8 μm cryostat sections, dried for 30 min and treated as previously described by Mänttäri et al . The con2 – 5% CO2) physiological saline solution (+37°C, pH 7.4) containing 137.0 mM NaCl, 2.7 mM KCl, 1.0 mM MgSO4· 7H2O, 1.8 mM CaCl2, 0.4 mM NaH2PO4, 12 mM NaHCO3 and 5.5 mM glucose. After an equilibration period of 2 min, the muscles were supramaximally stimulated using steel electrode, pulses of 1 ms duration at 80 V (model S44, Grass Instrument Co.) and muscle length selected to elicit maximal twitch force. The maximum force of the muscle was measured in series of three stimulus impulses with one minute equilibration time between the stimulations. Recordings of transducer output were A/D converted and collected on a computer at a sample frequency of 1000 Hz.Adult mice (weighing 23 – 45 g) were killed by paracervical dislocation, and the pelvic region and right hind limb were skinned. The muscle of interest (m. rectus femoris, m. gastrocnemius or m. gluteus maximus) was dissected out and suspended between a fixed clamp at the base of an organ bath and a Grass FTO3C (USA) force-displacement transducer. Muscles were maintained in an oxygenated -3,5-pyridinedicarboxylic acid dimethyl ester], equilibrated for 2 min, and assessed as described above i.e. the muscle was a control in itself. In addition, series of measurements were made with m. gastrocnemius (N = 6) and m. gluteus (N = 6) using nifedipine concentration range of 2 – 30 μM. The concentration of the drug was progressively increased by adding the nifedipine solution to the saline solution bath. During the experiment, the nifedipine solutions were maintained in dark to prevent photo-bleaching. To exclude the possibility of fatigue caused by successive stimulations, the protocol control measurement was performed similarly devoid of nifedipine.The statistical significance of differences between means of contraction forces before and after the addition of calcium channel blocker was evaluated by paired samples t-test. The difference in parameters between the different types of muscle were analysed with independent samples t-test for equality of means. A P value of < 0.05 was accepted as indicative of a significant difference between the two sets of observations. The significance of differences between the muscles was evaluated with one way analysis of variance . The significant difference was stated as mentioned above. The values are presented as the means ± SE. All the statistical analyses were performed with the SPSS for Windows software.DHP; dihydropyridineDHPR; dihydropyridine receptorE-C coupling; excitation-contraction couplingFOG; fast oxidative glycolyticmusculus gastrocnemiusGAS; musculus gluteus maximusGLU; mATPase; myosin adenosine triphosphataseMHC; myosin heavy chainmusculus rectus femorisRF; SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresisSR; sarcoplasmatic reticulumT-tubule; transverse tubuleIIa, IIb, IId; myosin heavy chain typeIIA, IIB, IID; fibre typeSM carried out most of the experiments, assisted in interpretation of results, and participated in writing the manuscript. MJ conceived of the study, participated in its design and coordination, and participated in writing the manuscript.


Enterobacter aerogenes and Klebsiella pneumoniae are common isolates in clinical microbiology and important as producers of extended spectrum β-lactamases (ESBL). The discrimination between both species, which is routinely based on biochemical characteristics, is generally accepted to be straightforward. Here we report that genotypically unrelated strains of E. aerogenes can be misidentified as K. pneumoniae by routine laboratories using standard biochemical identification and using identification automates.K. pneumoniae or K. terrigena with the routinely used biochemical tests and with API-20E, were identified as E. aerogenes by tDNA-PCR – an identification that was confirmed by 16S rRNA gene sequencing for five of these isolates. Misidentification also occurred when using the automated identification systems Vitek 2 and Phoenix, and was due to delayed positivity for ornithine decarboxylase and motility. Subculture and prolonged incubation resulted in positive results for ornithine decarboxylase and for motility. It could be shown by RAPD-analysis that the E. aerogenes strains belonged to different genotypes.Ten clinical isolates, identified as E. aerogenes isolates can be easily misidentified as Klebsiella due to delayed positivity for ornithine decarboxylase and motility. The phenomenon may be widespread, since it was shown to occur among genotypically unrelated strains from different hospitals and different isolation dates. A useful clue for correct identification is the presence of an inducible β-lactamase, which is highly unusual for K. pneumoniae. In several instances, the use of genotypic techniques like tDNA-PCR may circumvent problems of phenotypic identification.Clinical  Klebsiella pneumoniae isolates as the predominant ESBL-producers [Enterobacter aerogenes. It is also known that most of the E. aerogenes isolates in the Belgian hospitals belong to one of two predominant Belgian clones (BEI and BEII) [K. pneumoniae or K. terrigena by conventional biochemical testing were in fact E. aerogenes as could be shown by the use of genotypic methods, i.e. tDNA-PCR, validated by 16S rRNA gene sequencing, and by extensive phenotypic testing (including subculture and prolonged incubation).Enterobacteriaceae with β-lactam resistance due to the production of Extended-Spectrum β-Lactamases (ESBL) were discovered in the eighties and since that time became epidemic and endemic in hospitals worldwide . Since troducers  to predond BEII) , a situand BEII) . During E. aerogenes, K. pneumoniae or Raoultella (Klebsiella) planticola, the second code did not yield any identification, and the third code resulted in a weak but acceptable identification as K. terrigena. The isolates with the codes 5205773 and 5205753 were identified as K. pneumoniae by additional biochemical testing due to negative reactions for motility (tested in semi-solid agar) and ornithine decarboxylase. However, these isolates all possessed an inducible cefalosporinase, as detected on the antibiogram using a disk approximation test, a finding which strongly contradicts an identification as K. pneumoniae or Klebsiella sp.For ten clinical isolates, i.e. seven ESBL-producing clinical isolates collected during 2001 and three more recently collected isolates  and K. pneumoniae (BG) isolates, were tested in two different systems, i.e. Vitek 2 (bioMérieux, Marcy l'Etoile, France) and Phoenix (BD Biosciences, Sparks, Md.). Both automated systems yielded the same results as the API20E, i.e. that the E. aerogenes isolates, aberrant due to a slow reaction for motility and ornithine decarboxylase, were misidentified as K. pneumoniae  and antagonism (flattening of the inhibition zone), which is suggestive for a combination of an ESBL and an inducible β-lactamase. Again, inducible β-lactamases are very rare in Klebsiella spp. but typical for Enterobacter spp. It should be noticed that this phenomenon will not be detected by automated MIC-determination systems like Vitek 2 and Phoenix. Using PCR and sequencing as described previously [Disk diffusion antibiotic susceptibility testing, carried out according the NCCLS guidelines, revealed basically the same resistotype for all isolates, characterized by resistance to ceftazidime and susceptibility to ceftriaxone. Additional resistance to aztreonam was observed for some isolates, reflecting the most dominant resistance patterns for the E. aerogenes.The genotypic relationship of the phenotypically aberrant isolates was investigated with AP-PCR. Isolates GA1, GA2, GA3, MN2, MN3 and DBH were corresponding to Belgian clone BEI . species  which sh species  confirm mobilis" , which iKlebsiella spp., and an identification as Klebsiella sp. should warrant further investigation.However, the combination of an inducible β-lactamase and/or high-level cefoxitin resistance, which are rare in E. aerogenes misidentified as K. pneumoniae or K. terrigena due to delayed or negative ornithine decarboxylase and motility was reported previously, and was also discovered because of unexpected imipenem resistance of the so-called K. pneumoniae isolates [The phenomenon of isolates . Also inE. aerogenes as K. pneumoniae (or even K. terrigena) is probably not uncommon and probably also geographically widespread. This can be deduced from the following considerations: i) this phenomenon of misidentification of E. aerogenes was already reported in 1993 [The problem of misidentification of  in 1993 , ii) theKlebsiella species and of Enterobacter aerogenes, but do not mention the problem of possible delayed activity, possibly also because the study started from validated strains of each species.On the other hand, the problem seems to be largely unknown. In a recent study, Hansen and colleagues  carried E. aerogenes and K. pneumoniae. Here we showed that E. aerogenes isolates exist for which ornithine decarboxylase and motility are negative or delayed positive, and that as such these isolates can be misidentified as K. pneumoniae. This phenomenon may be quite frequent and geographically widespread. Genotypic identification techniques like tDNA-PCR, which moreover are cheaper than phenotypic testing for many bacterial species, can be semi-automatized, are faster and mostly have a higher discriminatory power, which is also reflected in this study.Identification in a routine clinical microbiology laboratory of the most commonly encountered Enterobacteriaceae is usually considered to be fast and straightforward, but apparently identification problems may occur due to diminished or delayed expression of some characteristics, even for well-established species like tDNA-PCR was carried out using the outwardly directed tRNA-gene consensus primers T5A (5'AGTCCGGTGCTCTAACCAACTGAG) and T3B (5'AGGTCGCGGGTTCGAATCC), thus amplifying the intergenic tRNA-spacers, as described previously ,11. Elec).For five of the phenotypically aberrant isolates, the complete 16S rRNA sequence was determined by amplification of the 16S rRNA-gene with the primers 5'-AGTTTGATCCTGGCTCAG and 5'-TACCTTGTTACGACTTCGTCCCA , and seqThe genotypic relationship of the isolates was investigated using arbitrarily primed PCR with RAPD Ready-to-Go beads  and the ERIC II primer 5'-AAGTAAGTGACTGGGGTGAGCG . AnalysiGC, GV and AM were responsible for sample collection and initial biochemical identification. PVD and GW carried out automated biochemical identification. GW in addition carried out extended biochemical characterization. TDB and MV carried out the molecular analysis. GC, GV, TDB and MV drafted the manuscript. All authors read and approved the final manuscript.


Over recent years increased emphasis has been given to performance monitoring of NHS hospitals, including overall number of hospital readmissions, which however are often sub-optimally adjusted for case-mix. We therefore conducted a study to examine the effect of various patient and disease factors on the risk of emergency medical readmission.The study setting was a District General Hospital in Greater Manchester and the study period was 4.5-years. All index emergency medical admission during the study period leading to a live discharge were included in the study (n = 20,209). A multivariable proportional hazards modelling was used, based on Hospital Episodes Statistics data, to examine the influence of various baseline factors on readmission risk. Deprivation status was measured with the Townsend deprivation index score. Hazard ratios (HR) and associated 95% confidence intervals (CI) of unplanned emergency medical admission by sex, age group, admission method, diagnostic group, number of coded co-morbidities, length of stay and patient's deprivation status quartile, were calculated.Significant independent predictors of readmission risk at 12 months were male sex (HR 1.13, CI: 1.07–1.2), age (age >75 (HR 1.57, CI 1.45–1.7), number of coded co-morbidities (HR for >4 coded co-morbidities: 1.49 CI: 1.26–1.76), admission via GP referral (HR 0.93, CI 0.88–0.99) and primary diagnosis of heart failure (HR 1.33, CI: 1.16–1.53) and chronic obstructive pulmonary disease/asthma (HR 1.34, CI: 1.21–1.48). Higher level of deprivation was also significantly and independently associated and with increased emergency medical readmission risk at three (HR for the most deprived quartile 1.21, CI: 1.08–1.35), six (HR 1.21, CI: 1.1–1.33) and twelve months (HR 1.25, CI: 1.16–1.36).There is a potential for improving health and reducing demand for emergency medical admissions with more effective management of patients with heart failure and chronic obstructive airways disease/asthma. There is also a potential for improving health and reducing demand if reasons for increased readmission risk in more deprived patients are understood. The potential influence of deprivation status on readmission risk should be acknowledged, and NHS performance indicators adjustment for deprivation case-mix would be prudent. The 200Unplanned hospital re-admissions may represent adverse events and could therefore indicate poor quality of care,4. The iAlthough current NHS performance indicators use readmission rates at 28-days,2, thereIt can be hypothesised that factors such as patient sex, length of stay, number of coded co-morbidities, method of admission , as well as patient deprivation, independently influence the probability of hospital readmission. Lower socioeconomic status in particular is independently associated with increased risk for many adverse health outcomes, includiThis work was carried out as part of the routine function of Stockport NHS Trust Clinical Effectiveness Unit, and in relation to work commissioned by the then "Emergency Demand Management Group" of the Stockport Primary Care Trust. The objective was to accurately describe the epidemiology of emergency medical readmissions so that demand management strategies (e.g. appropriate targeting of resources to patients with certain conditions, or certain types of presentation) could be informed.Stockport NHS Trust is a district general hospital in Greater Manchester, serving a reference population of about 300,000. About 85% of all patients emergency medical admissions are from Stockport, a population with a slightly lower, compared to the England and Wales average, Standardised Mortality Ratio from all causes  of 96 (95% CI 94–98).Hospital Episodes Statistics Data from April 1997 to September 2001 for Stockport NHS Trust were analysed and all emergency medical admissions in Stockport residents leading to live discharge were identified. An emergency medical admission was defined as an emergency hospital admission to any medical specialty in person over 18 years of age. Some persons had more than one emergency medical admission during the study period, and emergency medical admissions other than the index admission  were excluded. This was because not restricting analysis to index admissions would have meant that any deprivation gradients in readmission rates would have been confounded by deprivation gradients in index admissions, as previously described. This isAn emergency medical re-admission was defined as the first subsequent emergency medical admission during a follow-up period of either 28-days, or 3, 6 and 12 months respectively, following a first (index) emergency medical admission that led to a live discharge, and through the use of a single patient identifier. Observations were censored at the end of the chosen follow-up periods (as above) or at the time of intervening death unrelated to readmission to the study hospital. The latter was ascertained by data-linking to the Stockport Health Authority Public Health Mortality File produced by the Office for National Statistics.Index admission data were originally available on: sex; age; length of stay of index admission; International Classification of Diseases (ICD)-10 coded primary diagnosis; number (up to four) of coded co-morbidities; patient post code and admission method . Information on primary diagnosis was aggregated into five categorical groups comprising chronic obstructive pulmonary disease / asthma (ICD codes J44.0–45.9 respectively), heart failure (I50.0–50.9), acute coronary syndrome (I20.0 [unstable angina] and I21.0–9 [acute MI]), stroke (I60.0–I67.0) and all other conditions . Length of stay was divided into quartiles (<2, 2–5, 6–11 and >11 days). Deprivation status was subsequently ascribed with an area-based measure, using the 1991 Census enumeration district (ED) of patient's post-code, and by the use of Townsend multiple deprivation index score. Four deprivation groups were defined, using quartiles of the range of the Townsend scores between Stockport EDs.sex, age group, diagnostic group (defined as above), admission method, number of coded co-morbidities (0–4), length of stay group (quartile) and deprivation group (quartile). Statistical significance for each of the above variables was assessed by the log rank test. A series of proportional hazards models with follow-up at 28 days, 3, 6 and 12 months were subsequently constructed to examine the adjusted hazard (risk) ratio of emergency medical readmission. Each model included all variables found to be significant in the uni-variable analysis at the 0.05 probability level. The proportional hazards assumption was tested using the Schoenfeld residuals as per the stphtest command in STATA. This showed that a time varying co-variate should be included in all four models, in relation to the length of stay variable (i.e. that an interaction term between length of stay and time of follow-up should be included), and this was hence included in the models.Kaplan-Meier readmission-free curves at 28 days, and 3, 6 and 12 months were constructed for each of the following variables: Additionally, for the number of coded co-morbidities and deprivation group variables, a test for trend was performed, entering the actual values as continuous variables. In this context, the test for trend value indicates the proportional change in the risk of readmission associated with one unit change in the exposure variable (i.e. number of coded co-morbidities, Townsend deprivation score).There were 21,118 index emergency medical admissions corresponding to an equal number of patients leading to a live discharge during the study period, but primary diagnosis information was only available for 20,209 index emergency medical admissions . Test for trend confirmed the strong and statistically significant effect of deprivation at 3–12 months but there was no effect at 28 days.Admission method via a GP referral was significantly associated with a lower readmission risk at one year, but not at any other time intervals.Length of stay of the index admission influences readmission risk differently, depending on the length of follow-up as there was a highly significant interaction between length of stay group and time of follow-up see . Taking The findings indicate that in the study hospital about a quarter of patients with an index emergency medical admission will be readmitted in the same hospital during the subsequent year. Male sex and older age were strongly and independently associated with higher readmission risk, along with diagnosis of heart failure and chronic obstructive pulmonary disease or asthma. Effective measures to reduce readmission rates for patients suffering from these two conditions in particular are available,14 but nAs originally hypothesised, patient deprivation status exerted a significant independent effect on the risk of emergency medical readmission at 3–12 months of follow-up, with more deprived patients having had a higher readmission risk. There are several theoretical reasons why deprived patients may be at higher readmission risk, including: disease factors, such as greater disease severity in deprived patients; patientAlthough this is a single-centre study, the results may also have implications for the way current and future NHS performance indicators relating to readmission rates are both constructed and interpreted. NHS hospitals serving pre-dominantly deprived populations might in principle be disadvantaged if indicators are not adjusted for the impact of deprivation on case-mix. Although this study showed no significant effect of deprivation status on readmission risk at 28-days, which is the follow-up period currently used by the performance indicators,2, care The Department of Health includes performance indicators in the calculations of award of "three-star" status, which in turn is the "gateway" to "Foundation" status". StandarAll studies using administrative data are sensitive to the quality of routine data collection. The validity of HES data in relation to age, sex, length of stay, admission method, and area of residence is generally good, but misclassification errors may occur in relation to diagnostic codes and the extent to which co-morbidities are recorded and coded. CurrentA limitation of the study is that, besides the very large sample size, the findings are based on one single hospital in an urban English setting, and in principle the results are not generalisable. Similarly, the study was not population-based, so readmissions that may have occurred to other hospitals  were not ascertained. In theory such readmissions may have occurred at a differential rate between different deprivation groups. However this factor is unlikely to have biased the results in any considerable way for two reasons. First, the emergency (as opposed to elective) nature of the studied condition  makes it unlikely that either patients or doctors exercise an important degree of choice on which hospital a patient is admitted or readmitted, independently of patient deprivation status. Second, due to local geography and service configuration, 85% of the total medical admissions in Stockport residents occur at Stockport NHS (unpublished data). Similarly, by the nature of the hospital-centred nature of the study, admissions to private hospitals could not have been accounted. However, most admissions to private hospitals are for elective surgical procedures  for which we believe this is unlikely to have introduced considerable degree of bias. Lastly it is worth remembering that current NHS performance indicators for hospital Trusts are not population-based.Our study suggests that there is an important potential for both managing emergency demand and improving individual patient experience by focusing on the effective management of heart failure and chronic obstructive pulmonary disease. Although there is a similar potential by reducing differentials in readmission risk between deprivation groups, more research is required in order to understand reasons for such differentials in order to inform relevant policy measures. In the mean time, standardisation or other adjustment of hospital readmission indicators for patient socio-economic status in the future would be prudent. Failure to do so may disadvantage hospitals serving primarily deprived communities.The author(s) declare that they have no competing interests.The study was conceived and designed by GL and GC. GL, DH and IG analysed data. All authors contributed in the interpretation of findings and in the writing of the paper. GL and GC are guarantors.The pre-publication history for this paper can be accessed here:Length of stay group and readmission free Kaplan-Meier curves (0–28 days). This file demonstrates that during follow-up of 0–28 days, length of stay is not proportional to readmission risk, reason for which a time varying co-variate was included in the Cox regression model (see main article Text).Click here for file


Human sapovirus (SaV), an agent of human gastroenteritis, cannot be grown in cell culture, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the formation of virus-like particles (VLPs). In this study we compared the time-course expression of two different SaV rVP1 constructs. One construct had the native sequence (Wt construct), whereas the other had two nucleotide point mutations in which one mutation caused an amino acid substitution and one was silent (MEG-1076 construct). While both constructs formed VLPs morphologically similar to native SaV, Northern blot analysis indicated that the MEG-1076 rVP1 mRNA had increased steady-state levels. Furthermore, Western blot analysis and an antigen enzyme-linked immunosorbent assay showed that the MEG-1076 construct had increased expression levels of rVP1 and yields of VLPs. Interestingly, the position of the mutated residue was strictly conserved residue among other human SaV strains, suggesting an important role for rVP1 expression. Caliciviridae is made up of four genera, Sapovirus, Norovirus, Lagovirus, and Vesivirus, which contain sapovirus (SaV), norovirus (NoV), rabbit hemorrhagic disease virus, and feline calicivirus strains, respectively. Human SaV and NoV strains are agents of gastroenteritis. The prototype strain of human SaV, the Sapporo virus, was originally discovered from an outbreak of gastroenteritis in an orphanage in Sapporo, Japan, in 1977 [The family  in 1977 . The SaVCaliciviridae.Human SaV and NoV strains are noncultivable, but expression of the recombinant VP1 (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV ,4 In a rThe purpose of this study was to compare the time-course expression of two different SaV rVP1 constructs in a baculovirus expression system by Northern blotting, Western blotting, enzyme-linked immunosorbent assay (ELISA), and EM. Our novel results have indicated that nucleotide point mutations increased the yields of SaV VLPs in insect cells, offering an alternative explanation for the increased expression levels of rVP1 and yield of VLPs.6 confluent Tn5 cells in 1.5 ml of Ex-Cell 405 medium followed by incubation at 26°C. RNA transcription and rVP1 expression experiments were run in parallel for the Wt and MEG-1076 constructs.Expression of SaV rVP1 in a baculovirus expression system results in the self-assembly of VLPs . HoweverTotal RNA was extracted from the cells at 1, 2, 3, 4, 5, 6, 7, and 8 days postinfection (dpi) for Wt and MEG-1076 constructs. Equal amounts (500 ng) of total RNA were added to a 2% agarose gel containing formaldehyde and stained with SYBR Gold Fig. . The rVPWestern blot analysis was used to compare the expression levels of Wt and MEG-1076 rVP1. The culture medium was separated from the cell lysate 1, 2, 3, 4, 5, 6, 7, and 8 dpi as described in the Materials and Methods. Equal volumes of culture medium and cell lysate at each dpi were used for both constructs. Proteins were separated by SDS-PAGE, electrotransferred to PVDF, and detected with a 1:3000 dilution of hyperimmune rabbit Mc114 VLP antiserum. A band at the predicted rVP1 size (60 K) was first detected in the culture medium at 2 and 4 dpi for MEG-1076 and Wt constructs, respectively, which increased each day thereafter as evidenced by an increase in band intensity Fig. . As indiA thin band of approximately 55 K was also detected in the culture medium that appeared at 4 and 5 dpi for Wt and MEG-1076 constructs, respectively, and increased each day thereafter. In a different experiment, we determined the amino acid sequence of the MQG-1076 upper and lower bands by an Edman's degradation method. We discovered that the first three amino acid residues were MQG for both the upper and lower bands. This result indicated that the 55 K bands for these constructs were likely truncated or C-terminal deleted forms of rVP1. A thin band of 60 K was detected at every dpi in the cell lysate for the MEG-1076 construct Fig. , howeverE. coli expressed VP2 (data not shown).The VP2 amino acid sequence was the same in all constructs. We did not detect rVP2 during the time-course expression of the MQG-1076 construct using the antiserum raised against An antigen ELISA system was used to compare the yields of Wt and MEG-1076 VLPs at 1, 2, 3, 4, 5, 6, 7, and 8 dpi. The ELISA incorporated hyperimmune rabbit (capture) and guinea pig (detector) antisera raised against purified Mc114 VLPs [The MEG-1076 construct contained a nucleotide point mutation in which resulted in an amino acid substitution at position 358 in VP1. We aligned 21 different VP1 amino acid sequences of SaV GI, GII, and GV strains and found this residue was strictly conserved, but more importantly, there was a strictly conserved amino acid motif at this site, NGDV (data not shown). However, when we included a porcine SaV GIII strain and a recently identified SaV GIV strain (PEC and Hou-7, respectively), only the GD site was strictly conserved, though several other amino acids nearby were also strictly conserved  had decreased levels of rVP1 mRNA at 36 hours post-infection, by approximately 50%, when compared to a construct without the mutation (ORF2+ORF3+3' UTR construct). Bertolotti-Ciarlet suggested that the RNA secondary structure or changes in the mRNA stability could be responsible for the different steady-state levels, but this was not proven.In this study, we compared the time-course expression of two different Mc114 SaV rVP1 constructs in a baculovirus expression system . Another explanation may have been differences in the extents to which these baculoviruses induce apoptosis and all these may result from features in the baculovirus skeleton rather than from the inserted SaV sequence. Such effects might for instance affect the number of adherent cells harvested or the degradation rates of both proteins and RNAs. However, we found that the MQG-1076 construct, developed from a separate experiment, had similar expression levels to that of the MEG-1076 construct (data not shown), which may eliminate the possibility that the baculovirus skeleton played a role in the increased yields of VLPs. On the other hand, we could not demonstrate whether the nucleotide mutations in VP1 and/or in ORF2 affected the transcription, a construct with only one of these mutations would be needed. Nevertheless, our results indicate that translation was exclusively affected by the single amino acid substitution in VP1. Therefore, the final increase in yields of VLPs may have been coupled at multiple levels, involving one or both of the nucleotide mutations in VP1 and VP2.We did not detect rVP2 during the time-course expression of the MQG-1076 construct (data not shown). The Wt and MEG-1076 constructs had an identical amino acid sequence, which would suggest a similar negative-result. NoV studies have found that inclusion of VP2 increases the stability of VLPs, though the expression level of NoV rVP2 was low [The amino acid substitution (N → S) for the MEG-1076 construct occurred in the VP1 gene at residue 358. This asparagine residue was recently identified as a moderately conserved residue among the caliciviruses capsid proteins [Caliciviridae and identified strictly and moderately conserved residues, and predicted the P1 and P2 domains of SaV VP1 based on NoV X-ray crystallography structure. Based on these predictions, the residue at position 358 (amino acid sequence) was found as a moderately conserved residue among the caliciviruses. This arginine residue was predicated to be in the P2 domain, which is defined as the outer most protruding domain for NoV and thought to provide strain diversity [Recently, the cryo-EM analysis of SaV was determined and compared to NoV X-ray crystallography structure . Chen etiversity . FurtherSaV GI Mc114 strain (GenBank accession number, AY237422) was isolated from a male infant seven months of age from the McCormic Hospital, Chiang Mai, Thailand on the 7th May 2001 . RNA extGAG → CAG) and 1076 (AAT → AGT) and one nucleotide point mutation in ORF2 at position 1895 (GTG → GTA) (relative to the VP1 start and represented in bold). Primer and PCR errors likely introduced these mutations. These three nucleotide point mutations resulted in two amino acid substitutions in the VP1 gene, one at the second residue, where glutamic acid (E) → glutamine (Q), and one at residue 358, where asparagine (N) → serine (S). The nucleotide point mutation in ORF2 did not result in an amino substitution. Despite the two amino acid substitutions, the MQG-1076 construct formed VLPs. We designed another construct (MEG-1076) using the pDEST8-MQG-1076 as template but with a new sense primer and used KOD-plus DNA polymerase according to the manufacture's instructions (Toyobo, Japan). The MEG-1076 construct had the same nucleotide point mutations at positions 1076 in VP1 and 1895 in VP2 as the MQG-1076 construct but not at nucleotide 4 in VP1  was amplified with ExTaq DNA polymerase. However, this construct was later found to have two nucleotide point mutations in ORF1 at positions 4 , and then resuspended in 30 μl of Grace's medium. The cell lysate from the first centrifuge was resuspended in 200 μl of Grace's medium and stored at 4°C.Recombinant bacmids were transfected into Sf9 cells (Riken Cell Bank, Japan) and the recombinant baculoviruses was collected as previously described . The expg, the supernatant removed, and then the cells were dissolved with 1 ml of Isogen. The cells were stored at -80°C. RNA was purified by a chloroform/ ethanol method (Nippon Gene, Japan). Briefly, RNA was mixed with chloroform, centrifuged at 12,000 × g for 15 min at 4°C, and the aqueous layer collected. This was repeated once, and then the aqueous layer collected and mixed with isopropanol and stored overnight at -20°C. The solution was mixed, centrifuged at 12,000 × g for 15 min at 4°C, and the supernatant discarded. The pellet was resuspended in 80% ethanol, centrifuged at 12,000 × g for 15 min at 4°C. This was repeated once, and then the pellet air-dried and resuspended in 25 μl of TE, and stored at -80°C. The amounts of purified RNA were determined spectrophotometrically (Bio-Rad, USA). The same amounts (500 ng) of total RNA were loaded for each construct and each dpi onto a 2% denaturing agarose gel containing formaldehyde. The amounts of total RNA were compared using SYBR Gold staining (Invitrogen, USA). RNA was transferred to a positively charged nylon transfer membrane  under vacuum  and analyzed by Northern blotting according to the DIG Northern Starter Kit (Roche, USA), except for a minor modification. Briefly, a RNA probe corresponding to Mc114 VP1 position 157 to 1283 (anti-VP1) was generated from a PCR fragment (native sequence) according to the manufacture's instructions (Roche, USA). Hybridization was performed overnight at 68°C with anti-VP1 in 10 ml of ultrasensitive hybridization buffer (Ambion, Canada). After hybridization, immunological detection was performed according to the manufacture's instructions (Roche, USA).Total RNA was prepared from the attached cells at 1, 2, 3, 4, 5, and 6 dpi with 1 ml of Isogen (Nippon Gene, Japan). For 7 and 8 dpi, the cell culture medium (containing unattached cells) was collected and centrifuged for 5 min at 3,000 × Western blotting, ELISA, and EM were used to examine rVP1 expression as previously described . However. In total, we aligned different 21 SaV GI, GII, GIII, GIV, and GV sequences, and included: Arg39, AY289803; Bristol, AJ249939; C12, AY603425; Cruise ship/00, AY289804; PEC, AF182760; Dresden, AY694184; Hou-7, AF435814; Houston/86/US, U95643; Houston/27/90/US, U95644; London/29845/92/UK, U95645; Lyon/598/97/F, AJ271056; Manchester, X86560; Mc2, AY237419; Mc10, AY237420; Mex340/1990, AF435812; Mex14917/00, AF435813; NK24, AY646856; Parkville, U73124; Potsdam, AAG01042; Plymouth, X86559; Sapporo/82/Japan, U65427; and Sakaeo-15, AY646855.VP1 nucleotide sequences were translated using Genetyx software (software development Co. Version 11.2.2) and submitted to online ClustalW at DDBJ The author(s) declare that they have no competing interests.GH carried out the study and wrote the manuscript. KK, TO, KN, and NT participated in the design of the study and helped to draft the manuscript.


In the hypothesis that the increased collagen metabolism in the intestinal wall of patients affected by inflammatory bowel disease (IBD) is reflected in the systemic circulation, we aimed the study to evaluate serum level of procollagen III peptide (PIIIP) in peripheral and splanchnic circulation by a commercial radioimmunoassay of patients with different histories of disease.Twenty-seven patients, 17 with Crohn and 10 with ulcerative colitis submitted to surgery were studied. Blood samples were obtained before surgery from a peripheral vein and during surgery from the mesenteric vein draining the affected intestinal segment. Fifteen healthy age and sex matched subjects were studied to determine normal range for peripheral PIIIP.In IBD patients peripheral PIIIP level was significantly higher if compared with controls ; splanchnic PIIIP level was 5.5 ± 2.6 μg/l showing a positive gradient between splanchnic and peripheral concentrations of PIIIP. No significant differences between groups nor correlations with patients' age and duration of disease were found.We provide evidence that the increased local collagen metabolism in active IBD is reflected also in the systemic circulation irrespective of the history of the disease, suggesting that PIIIP should be considered more appropiately as a marker of the activity phases of IBD. Crohn's disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of unknown origin of adolescent and young adulthood  where geCurrent opinions suggest that an increased synthesis of collagen type I, III, and V may play an important role in the pathophysiological mechanism leading to intestinal fibrosis -15. An iHowever, not all authors are in agreement regarding the increased serum levels of the aminoterminal propeptide (PIIIP) of collagen in peripheral and splanchnic circulation of patients with active IBD ,14. BeloTwenty-seven patients affected by active IBD, 17 with CD (age 40.2 ± 13.1, yrs from diagnosis 9.2 ± 5.5), and 10 with UC (age 50.3 ± 15.6, yrs from diagnosis 9.8 ± 7.4) submitted to surgery, were enrolled in the study in a double blind fashion. The protocol was approved by local Ethical Committee and informed consent was obtained from all participants to the study. Three patients had CD in small bowel only, two in large bowel only, 12 had ileocolonic disease. Disease activity was assessed according to the Crohn's Disease Activity Index (CDAI)  and the Patients affected by CD were operated on for recurrent obstruction, whereas patients with UC were submitted to surgery because of refractory to medical therapy.The clinical diagnosis was confirmed by histology Fig. ; all cas- for CD: deep ulcers, marked proliferation of small lymphoid nodules involving all layers of intestinal wall sometime with sarcoid-type granulomas and serosal inflammation;- for UC: mucosal erosions and superficial ulcerations usually limited to the upper submucosa with cryptic abscesses and glandular destruction.The clinical profile of the studied patients is reported Table . Two patDifferent kinds of collagen have been identified in humans. All of them derive from longer precursor molecules (procollagens). They are synthesized intracellularly and secreted in extracellular space where they are cleaved by aminoproteases -23. AmonData were analyzed using a computer statistical software . All the quantitative variables were tested for Gaussian distribution with the Kolmogorov-Smirnov test. All that followed this distribution were presented as mean ± standard deviation.Differences at baseline in collagen parameters between IBD patients and controls were tested for significance using the analysis of variance with the Bonferroni correction. The relation between collagen parameters and the estimated duration of the disease and indices of disease were tested with regression analysis. In all cases, a p value less than 0.05 was considered significant.At baseline, before surgery, serum p-PIIIP in IBD patients were significantly higher if compared with healthy controls  fig . No signDuring surgery, serum s-PIIIP in IBD patients was 5.5 ± 2.6 μg/l. No significant differences were found when comparing CD and UC subgroups . A positive gradient was found in IBD patients between splanchnic and peripheral serum concentrations of PIIIP (0.7 ± 1.9 μg/l). This gradient was confirmed when separately considering each disease, without significant differences between the two subgroups  fig .No significant correlation was found between peripheral and splanchnic levels of PIIIP and the age of the patients and the estimated duration of the disease. Regarding the activity indices, the number of patients belonging to each class was not enough to perform a statistical analysis. Notwithstanding, for the TWI in UC patients a significant difference in PIIIP levels was found between mild and severe form of the disease Table . FinallyCD and UC are chronic pathologies characterized by an early onset followed by sporadic episodes of acute symptoms during lifetime, debilitating the affected patients to perform their daily functions . Until nIn the present study we have found that intestinal collagen metabolism in IBD patients was increased and that it is reflected in local and systemic circulation. Differently from some experiences ,13, we hWhen comparing the mild with the severe form of the disease, a significant difference in PIIIP levels was found only in patients affected by UC. This data will probably be confirmed when the number of patients enrolled in each disease-related activity categories is extended as presently in our series the majority of the patients were classified as severe.The effect of glucocorticoids on collagen synthesis, collagenase, and collagen degradation has not yet fully been clarified . In our In conclusion we provide evidence that collagen metabolism in IBD is reflected in the systemic and local circulation, without any differences between UC and CD, irrespective of the age of the patients and the duration of the disease. Therefore, this marker may give further information on the activity phases rather than on the entire history of the disease. Further data on the possible use of PIIIP as useful marker of choice for surgical option are attended from the follow-up at 6 and 12 months, which is still on-going .Inflammatory bowel diseases = IBDProcollagen III propeptide = PIIIPPeripheral Procollagen III propeptide = p-PIIIPSplanchnic Procollagen III propeptide = s-PIIIPCrohn's disease = CDUlcerative Colitis = UCCrohn's Disease Activity Index = CDAITruelove-Witts index = TWIThe author(s) declare that they have no competing interests.UC conception and design, interpretation of data, drafting the articleMDS conception and design, interpretation of data, drafting the articleECA performed surgical operations, critical revision of the article, final approval of the versionBO patients' enrollement, blood samples collectionRP statistical analysis, interpretation of data, drafting the articleAP echocardiographic studiesGB radioimmunoassaysEO radioimmunoassaysSF histological examinationsFM interpretation of data, critical revision of the article, final approval of the versionMMC conception and design, interpretation of data, drafting the articleAll Authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Balanced decisions about health care interventions require reliable evidence on harms as well as benefits. Most systematic reviews focus on efficacy and randomised trials, for which the methodology is well established. Methods to systematically review harmful effects are less well developed and there are few sources of guidance for researchers. We present our own recent experience of conducting systematic reviews of harmful effects and make suggestions for future practice and further research.We described and compared the methods used in three systematic reviews. Our evaluation focused on the review question, study designs and quality assessment.One review question focused on providing information on specific harmful effects to furnish an economic model, the other two addressed much broader questions. All three reviews included randomised and observational data, although each defined the inclusion criteria differently. Standard methods were used to assess study quality. Various practical problems were encountered in applying the study design inclusion criteria and assessing quality, mainly because of poor study design, inadequate reporting and the limitations of existing tools. All three reviews generated a large volume of work that did not yield much useful information for health care decision makers. The key areas for improvement we identified were focusing the review question and developing methods for quality assessment of studies of harmful effects.Systematic reviews of harmful effects are more likely to yield information pertinent to clinical decision-making if they address a focused question. This will enable clear decisions to be made about the type of research to include in the review. The methodology for assessing the quality of harmful effects data in systematic reviews requires further development. Systematic reviews are important tools for evidence-based health care. They are certainly one of the reasons for the progress that has been made in obtaining reliable evidence on the beneficial effects of interventions. A recent study of the medical literature, using Medline and the Cochrane Library, showed that the number of systematic reviews published has increased dramatically, from a single publication in the years 1966 to 1970, to 23 in 1981 to 1985, and 2467 in 1996 to 2000 [The methodology for conducting systematic reviews of beneficial effects from RCTs is well established, whereas the methods for systematically reviewing randomised or observational data on harmful effects are less well developed and less often used. Only 1.25% of 3604 publications cited in the 2001 edition of Side Effects of Drugs (SEDA-24) were systematic reviews [It is not uncommon, even among experienced reviewers, to assume that the objective of a systematic review of harmful effects should encompass all known and previously unrecognised harmful effects and that data from all types of study design should be sought. We have re-visited three systematic reviews of drug interventions in which we had reviewed harmful effects, to evaluate our own recent experience, identify areas for improvement and to share our ideas with other researchers undertaking reviews.We used three reviews for this study on the basis that they had been completed recently (between 2001 and 2003) and that one of us had been the lead reviewer of harmful effects in each review. The reviews were conducted as Health Technology Assessments for the National Coordinating Centre for Health Technology Assessment (NCCHTA) on behalf of the National Institute for Clinical Excellence (NICE). The reviews, in order of completion, were: nicotine replacement therapy (NRT) and bupropion sustained release (SR) for aiding smoking cessation , atypicaWe described and compared the methods used in each review and the problems we encountered in applying those methods. We focused our evaluation on the review objectives, the inclusion criteria for study design and the quality assessment of the primary studies. We do not report on the matter of searching for studies about harmful effects which presents another challenge to those who conduct systematic reviews ,11, becaThe main components of the three systematic reviews of harmful effects are described in Table The schizophrenia review objective appeared to be appropriate in seeking to determine the incidence of named outcomes that were considered by health economists to be most likely to lead to a change in prescribed treatment . The objAll three reviews included study designs other than RCTs to assess harmful effects. The types of non-randomised studies included for each review reflected differences in the reviews' objectives, our judgment as reviewers as to where the most useful data were likely to be found, and was to some extent pragmatic in terms of the time available to complete the reviews. The reviews with the broad objectives included more non-randomised studies and more diverse study designs. The schizophrenia and epilepsy reviews specified a minimum size and duration of study to be included (see table) in an attempt to add data over and above what was available from the largest and longest RCTs. Doing this did involve some indeterminable risk of missing important information.The review of observational studies carried out in the schizophrenia review was necessary because the pre-determined harmful effects of interest were known to be under-reported in RCTs . The incOnce the inclusion criteria for study design had been defined, applying them was problematic. Reports of primary studies rarely described the study design in sufficient detail. Many of the studies included in the schizophrenia review purported to be cohort studies but on closer examination were in fact large case series involving more than one intervention. Some of the 'cohort study' data on bupropion SR included in the smoking cessation review had actually been derived retrospectively from RCTs. How exactly the 'cohorts' had been established in studies of epilepsy was often unclear in terms of the source population, eligibility criteria, and selection, or was simply not reported. Had we, in all three reviews, only included reports of studies fitting textbook definitions of particular study designs, virtually all of the primary study reports we identified would have been excluded. The inclusive approach we took turned out to be unrewarding.In the smoking cessation review, in addition to difficulties with the study design inclusion criteria, application of the criterion to only include studies in which assessment of adverse events was the primary objective was problematic because it involved a high degree of subjective judgment.We encountered problems when applying published checklists in our reviews of harmful effects. The response to some questions depended on the outcome of interest, for example, follow-up may have been adequate for the assessment of the primary  outcome of the study but not for the collection of data on harmful effects. We also found that published checklists omit key features such as how harmful effects data were recorded. In the epilepsy review we were in a position to learn from the earlier reviews and spent time clarifying the questions in the checklists so that they would provide information relevant to the reliability of the harmful effects data. We also added items pertinent to reports of harmful effects such as how and when events were recorded and whether the time at which they occurred during the study was reported. Although this informed approach was a step in the right direction, the major hindrance to applying checklists in all three reviews was inadequate reporting of the basic design features of the primary studies.Once the quality criteria had been applied there remained the challenge of interpreting the results. In our reviews we described the evidence identified and tabulated the response to each checklist question for each primary study. This generated lengthy summaries that had limited utility. Even comparing validity within study designs (not across them) we found it impossible to synthesise the information as all the included studies had methodological flaws and features that could not be assessed due to inadequate reporting. Reaching a decision about which studies were likely to give the most reliable results was not straightforward.Our experience of reviewing harmful effects mirrors that of other researchers in that a significant investment of effort failed to yield significant new information ,17.A focused review question is standard practice for assessing beneficial outcomes in systematic reviews and should also be so when reviewing harms. Researchers conducting reviews need to make sure that they address a well-formulated question about harms that are likely to impact on clinical decisions. Focusing a review question about harmful effects will not necessarily mean restricting it to specific adverse events but may mean, for example, addressing a particular issue such as long-term effects, drug interactions, or the incidence of mild effects of importance to patients. If the aim of the research is to look for previously unrecognised harmful effects, analysis of primary surveillance data may be more appropriate than a systematic review . ResearcIt is important to realise that an unquestioning belief that observational studies are the best source of harmful effects data simply because they are not RCTs can be a pitfall. It is essential to think carefully about the review question before widening the inclusion criteria to include non-randomised study designs. Some harmful effects, such as very rare events or those emerging in the long-term, are unlikely to be addressed adequately in RCTs. But, even if observational studies are appropriate to the review question researchers should be prepared for the difficulty of interpreting observational study data outweighing the anticipated benefits.The importance of quality assessment of RCTs in systematic reviews of effectiveness is well established , but deba priori what exactly they need to glean from their quality assessment of the primary studies in their own review of harmful effects and it may be necessary to differentiate clearly between internal and external validity.It is essential that quality assessment is able to discriminate poor from better quality studies of harmful effects. Levels of evidence hierarchies have several shortcomings. The hierarchy of evidence is not always the same for all harmful or beneficial outcomes. For example, an RCT with adequate internal validity but limited sample size or follow-up may be a less reliable source of information about relatively uncommon harmful effects emerging in the long-term than a large well-conducted cohort study with many years of follow-up. Another problem with ranking evidence in a hierarchy is that different dimensions of quality get condensed into a single grade, resulting in a loss of information. Furthermore, the dimensions included in current hierarchies may not be the most important in terms of reflecting the reliability of a particular study's findings . ResearcWe suggest that further research is needed to collate, assimilate and build on the existing information relevant to systematically reviewing primary studies for harmful effects of health care interventions. This should include a review of the literature pertinent to the methodology of incorporating evidence of harmful effects in systematic reviews; a description and categorisation of the methods used in systematic reviews published to date, and any evidence from methodological research on which they are based; and the development of quality assessment methods.Appraisal of our recent experience highlighted some of the problems inherent in conducting systematic reviews of harmful effects of health care interventions. Such reviews need to address a well-formulated question to facilitate clear decisions about the type of research to include and how best to summarise it, and to avoid repeating what is already known. The review question about harmful effects needs to be relevant to clinical decision-making. A systematic review of the methodology pertinent to systematic reviews of harmful effects is warranted.None declared.AMB, HMM and NFW conducted the review work described. NFW conceived of the study. NFW and HMM drafted the manuscript. All authors contributed to, read and approved the final manuscript.The pre-publication history for this paper can be accessed here: