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It has a single polar flagellum and is motile when grown in liquid medium (broberg et al . V. parahaemolyticus is a marine bacterium easily recovered from estuarine and coastal waters worldwide, as well as from seawater (hayat et al . V. parahaemolyticus requires presence of salinity with optimum levels of 13% to survive and multiply, which is in the range of 0.83% levels commonly found in marine environments (yeung and boor 2004). V. parahaemolyticus causes three major syndromes of clinical illness, i.e., gastroenteritis, wound infections, and septicemia . Gastrointestinal illness caused by v. parahaemolyticus is typically accompanied by symptoms including vomiting, diarrhea, headache, nausea, low - grade fever, and abdominal cramps, and is commonly self - limiting . The mean incubation period for v. parahaemolyticus infection is 15 h (nair et al . 2007). Clinical isolates of v. parahaemolyticus most often produce either the thermostable direct hemolysin (tdh) or tdh - related hemolysin (trh) encoded by tdh and trh genes, respectively . However, only bacteria producing virulence factors, i.e., tdh and/or trh, are considered to be pathogenic and can cause acute gastroenteritis (or, more rarely, invasive septicemia) (bisha et al . Tdh is capable of producing hemolysis on wagatsuma agar which is called kanagawa phenomenon (kp). Most of the strains (90%) isolated from clinical cases show this type of hemolysis, while only 12% of the strains of environmental origin are kp positive (drake et al . Trh is another virulence factor that has been discovered in clinical strains lacking tdh gene . Trh is a 23-kda protein, immunologically related to tdh and environmental strains producing this gene, and produces urease (pal and das 2010). Epidemiological studies have revealed an association between the kanagawa phenomenon and gastroenteritis (okuda et al . The objectives of the present study are, first, to determine the prevalence of total and pathogenic v. parahaemolyticus in seawater and sediment in the southern coast of the caspian sea and, second, to investigate the relationships with water temperature and salinity . The strains isolated were screened for hemolytic activity and for the presence of the tdh and trh genes . A limitation of the research was the long distance between the sampling sites and the laboratory . The seawater samples and sediments were collected at a depth of 50 and 1020 cm, respectively, by aseptic techniques . Of the 300 total samples, 150 seawater and 150 sediment samples were collected from seven different sites in the caspian sea in april, may, june, july, and august (fig . 1area of study and locations of the sampling sites in the southern coast of the caspian sea (northern iran). Site 1 bandar torkaman, site 2 sari, site 3 noor, site 4 chalus, site 5 ramsar, site 6 chamkhaleh, site 7 bandar anzali area of study and locations of the sampling sites in the southern coast of the caspian sea (northern iran). Site 1 bandar torkaman, site 2 sari, site 3 noor, site 4 chalus, site 5 ramsar, site 6 chamkhaleh, site 7 bandar anzali the samples were placed in sealed containers with dry ice and transported frozen to the laboratory for analysis . The time between sample collection and analysis was approximately 24 h, while sampling, water temperature, and salinity were measured . Twenty - five milliliters of seawater samples and 25 g of sediment samples were added to 225 ml of alkaline peptone water (apw) containing 1% nacl (ph 8.6), and 10 and 10 apw dilutions were prepared in duplicate and incubated at 37 c for 624 h. enrichment broth was streaked onto thiosulfate - citrate - bile salts - sucrose agar plates and incubated at 37 c for 1824 h. the green or blue - green colonies were presumptively selected as v. parahaemolyticus colonies and transferred to trypticase soy agar plate containing 3% nacl . After incubation at 37 c for 24 h, the isolates were tested using conventional bacterial methods, including gram's staining, culture sulfide indole motility and triple sugar iron tests media, cytochrome oxidase activity tests, lysine iron agar tests, urea tests, and tests for arabinose, lactose, mannitol, mannose, and sucrose fermentation (colakoglu et al . Positive reactions were recorded as a zone of hemolysis surrounding the spot of growth on the human blood plate . The interpretation times for the test were 24, 48, and 72 h (canizalez - roman et al . V. parahaemolyticus strains were seeded in luria bertani agar supplemented with 1% nacl and incubated at 37 c for 24 h. five colonies were selected, resuspended in 100 l of filtered sterile distilled water, and boiled for 1520 min to liberate the nucleic acid (cabrera - garcia et al . The tdh and trh genes were amplified with the following primer sets: 5-gtaaaggtctctgacttttggac-3 and 5-tggaatagaaccttcatcttcacc-3 for tdh, and 5-ttggcttcgatattttcagtatct-3 and 5-cataacaaacatatgcccatttccc-3 for trh (tada et al . These primer sets produced 251- and 373-bp amplicons, respectively . The reaction mixtures (final volume, 25 l) contained 3 l of the solution containing dna, 2.5 l of 10 reaction buffer, 4 l of 25 mm mgcl2, 1 l of taq polymerase (5 u/l), 4 l of deoxynucleoside triphosphates (1 mmol), 1 l of each primer (20 pmol), and 8.5 l of distilled water . The reactions were performed as follows: initial denaturation at 94 c for 1 min, followed by 35 cycles of denaturation at 94 c for 1 min, annealing at 55 c for 1 min, extension at 72 c for 1 min, and a final extension at 72 c for 7 min . The amplified products were separated by electrophoresis in ethidium bromide - stained 2% agarose gels in tris twenty - five milliliters of seawater samples and 25 g of sediment samples were added to 225 ml of alkaline peptone water (apw) containing 1% nacl (ph 8.6), and 10 and 10 apw dilutions were prepared in duplicate and incubated at 37 c for 624 h. enrichment broth was streaked onto thiosulfate - citrate - bile salts - sucrose agar plates and incubated at 37 c for 1824 h. the green or blue - green colonies were presumptively selected as v. parahaemolyticus colonies and transferred to trypticase soy agar plate containing 3% nacl . After incubation at 37 c for 24 h, the isolates were tested using conventional bacterial methods, including gram's staining, culture sulfide indole motility and triple sugar iron tests media, cytochrome oxidase activity tests, lysine iron agar tests, urea tests, and tests for arabinose, lactose, mannitol, mannose, and sucrose fermentation (colakoglu et al . Positive reactions were recorded as a zone of hemolysis surrounding the spot of growth on the human blood plate . The interpretation times for the test were 24, 48, and 72 h (canizalez - roman et al . Bertani agar supplemented with 1% nacl and incubated at 37 c for 24 h. five colonies were selected, resuspended in 100 l of filtered sterile distilled water, and boiled for 1520 min to liberate the nucleic acid (cabrera - garcia et al . The tdh and trh genes were amplified with the following primer sets: 5-gtaaaggtctctgacttttggac-3 and 5-tggaatagaaccttcatcttcacc-3 for tdh, and 5-ttggcttcgatattttcagtatct-3 and 5-cataacaaacatatgcccatttccc-3 for trh (tada et al . These primer sets produced 251- and 373-bp amplicons, respectively . The reaction mixtures (final volume, 25 l) contained 3 l of the solution containing dna, 2.5 l of 10 reaction buffer, 4 l of 25 mm mgcl2, 1 l of taq polymerase (5 u/l), 4 l of deoxynucleoside triphosphates (1 mmol), 1 l of each primer (20 pmol), and 8.5 l of distilled water . The reactions were performed as follows: initial denaturation at 94 c for 1 min, followed by 35 cycles of denaturation at 94 c for 1 min, annealing at 55 c for 1 min, extension at 72 c for 1 min, and a final extension at 72 c for 7 min . The amplified products were separated by electrophoresis in ethidium bromide - stained 2% agarose gels in tris borate a total of 300 samples were analyzed; these samples included 150 seawater and 150 sediment samples . Overall, v. parahaemolyticus strains were isolated from 20.3% of the samples (table 1). The highest isolation rate of v. parahaemolyticus was observed in august, with a total of 23 positive samples (28.3%). The temperatures ranged between 34 and 39 c during the sampling period . By contrast, the lowest isolation rate of v. parahaemolyticus was observed in april, with a total of 3 positive samples (5%). The temperature of the seawater ranged from 10 to 15 c during the sampling period . Salinity was highest in summer (19 ppt), whereas the minimum levels were recorded during the spring (2 ppt). The average salinity was 10.5 ppt during the sampling period.table 1occurrence and the frequency of v. parahaemolyticus in the seawater and sedimentmonthaprilmayjunejulyaugusttotal frequencysourcewsw + swsw + swsw + swsw + swsw + ssitesfrequencyn%n%n%n%n%n%n%n%n%n%n%n%n%n%n%n%site 111.611.611.611.623.323.623.611.63546.693site 211.611.611.611.623.311.611.623.623.662site 311.611.623.323.311.611.623.623.623.672.3site 411.611.611.611.623.311.63523.658.323.623.646.6134.3site 511.611.611.611.623.33511.646.646.623.6610134.3site 611.611.623.311.611.623.641.3site 711.611.623.323.311.63523.623.623.611.635103.3total11.623.3353558.3813.358.3711.61118.3711.69151626.61016.61321.62328.36220.3 w water, s sediment occurrence and the frequency of v. parahaemolyticus in the seawater and sediment the highest prevalence of v. parahaemolyticus was detected at sampling sites 4 (4.3%) and 5 (4.3%). Of the 62 strains isolated, 26 (8.3%) were obtained from seawater samples and 36 (12%) from sediments . Only two of the 62 strains (2.53%) amplified the 250-bp tdh gene fragment, which correlated with positive hemolytic activity . The trh gene fragment was amplified in four (5.06%) of the strains analyzed . In the present study, we demonstrated the presence of v. parahaemolyticus in 20.3% of the seawater and sediment samples analyzed . This study was the first research to investigate the isolation and distribution of this pathogen in the southern coast of the caspian sea . Cabrera - garcia et al . (2004) reported that 15% of the seawater samples contained v. parahaemolyticus . This pathogen has also been isolated in canada (kelly and stroh 1988), france (hervio - heath et al . 2002), asia (alam et al . 2002), and the usa (depaola et al ., 26 (8.3%) were obtained from seawater samples and 36 (12%) from sediments . (2012) have reported that of the 144 strains isolated, 35% was obtained from seawater samples and 16% from sediment . Thus, it is speculated that the observed prevalence may also be produced by seasonal variation of the v. parahaemolyticus population due to salt and oxygen concentrations, interactions with the plankton, the presence of sediment, and the organic matter in the suspension, fish, and seafood . In one study, it was observed that the maximum probability of v. parahaemolyticus detection was around 25 ppt salinity (martinez - urtaza et al . 2008). The other reason for the low prevalence of v. parahaemolyticus in the southern coast of the caspian sea (the average salinity is 10.5 ppt) is its lower salinity than other seas . The distribution of v. parahaemolyticus in the marine environments is known to be related to the water temperatures . Studies have shown that the organism was rarely detected in seawater until water temperatures rose to 15 c or higher . Ecological study of v. parahaemolyticus in the chesapeake bay of maryland (usa) found that the organism survived in sediment during the winter and was released from sediment into water column when water temperatures rose to 14 c in late spring or early summer (kaneko and colwell 1978). The isolation rate of v. parahaemolyticus was high (28.3%) in august because a higher rate of evaporation results in high salinity . In one study, it was observed that only two pathogenic v. parahaemolyticus were confirmed in environmental samples . The extremely low presence of pathogenic populations of v. parahaemolyticus in environmental samples is a constant characteristic in most of the investigations carried out in different regions of the world (martinez - urtaza et al . We observed positive tdh gene amplification in two of the 62 strains analyzed, which was correlated with the positive hemolytic activity in wagatsuma agar (kp). Virtually, all clinical isolates of v. parahaemolyticus are kp, whereas only 12% of environmental strains are kp (drake et al . 2007). This implies that there is a source of human fecal contamination in the seawater and sediment of the southern coast of the caspian sea . This information may be important for preventing sanitary problems that may affect the health of the population . In conclusion, this study provided new information on the abundance of v. parahaemolyticus in seawater and sediment samples in the southern coast of the caspian sea . The data presented in the present study show the presence of pathogenic v. parahaemolyticus and suggest the need for a study of such organism in seafood in the caspian sea.
Imbalances in the levels of excitatory and inhibitory neurotransmitters, such as serotonin, dopamine, and gaba, can lead to severe cns disorders like epilepsy, schizophrenia, anxiety, and depression . Tackling cns diseases related to the gabaergic system is most commonly achieved by using drugs of the benzodiazepine family (e.g., diazepam), which allosterically modulates the pentameric gabaa receptor (gabaa - r). However, an alternative way of enhancing gaba action is inhibition of the corresponding neurotransmitter uptake system . In the case of the gaba transporter (gat) family, four gaba reuptake transporter subtypes (gat13, bgt1) and one vesicular carrier exist in mammalian organisms . The gat family belongs to the neurotransmitter: sodium symporters (nss) which is organized as oligomers at the plasma membrane while, in contrast to the gabaa - r, functions as a monomer . Usually, nss transporters use a sodium gradient for uphill transport of neurotransmitters out of the synaptic cleft . In certain cases, a reverse transport mode is also known, releasing neurotransmitter in a nonvesicular way . At present, only one drug targeting this receptor, the anticonvulsant tiagabine, is on the market . Tiagabine selectively inhibits gat1, the most abundant gat subtype in the human brain . An x - ray crystallographic structure is not yet available for any member of the gat family, but a number of homology models have been constructed . The molecular basis of tiagabine action, however, remains elusive, as experimental evidence for proposed binding modes is still lacking . Furthermore, ligand - based exploration of inhibitor scaffolds is limited by the low tolerance of this transporter for inhibitor modification . On the basis of a set of tiagabine analogs from literature sources, we recently investigated ligand - based structure activity relationships of the compound class . Briefly, binary qsar allowed classification of gaba uptake inhibitors into active and inactive bins by using the degree of rigidity and polarity distribution as main descriptors . With the increasing knowledge provided by the x - ray structures of analogous transport proteins, structure - based approaches for elucidating the molecular basis of drug, we describe a binding hypothesis of tiagabine in gat1 and its successful validation by in silico screening . The closest transporter proteins related to hgat1 for which structures are available are the bacterial leucine amino acid transporter protein, leutaa, and the drosophila dopamine transporter, ddat . Despite its lower overall sequence identity, closer substrate relationship and significantly higher resolution of 2.00 vs 2.95 several sequence alignments between hgat1 and leutaa have been published, and all alignments are almost identical within the conserved central substrate binding cavity . Both template candidates were available in an open - to - out conformation, thus granting access to bulky inhibitor molecules . Suitable templates for the intracellular n- and c - terminal domains of hgat1 are not available and thus were not included in the final homology model . Because of the differing stoichiometry of eukaryotic nss family members for cl, the leut structure (pdb code: 3f3a) was modified by engineering a chloride binding site using structural information from crystal structure of the ddat and topological information from the literature . On the basis of a combination of low b - values and proximity to the binding site or stabilization of adjacent domains, several water molecules were selected and kept in the template file . Finally, a known disulfide bridge between c164 and c173 of el-2 was defined . Modeller was used to generate 100 models, which were ranked according to their respective discrete optimized protein energy (dope) score for estimating the geometric quality . For the 10 highest ranked models, additional quality checks were performed using the model assessment tools of the swiss - model server . Models with core residues showing disallowed geometry according to the ramachandran plot were omitted . The remaining models were visually inspected for their ability to reflect residue proximity and accessibility data from literature . In addition, the models were evaluated regarding the orientation of nonconserved polar residues in tm regions . Subsequently, the best structure was selected according to aforementioned criteria and subjected to a soft minimization protocol for relaxation of the system . Focused sampling of the conformational space in the putative tiagabine interaction site tiagabine cannot be accommodated in the occluded state of the transporter, as both the extracellular gate between r69 and d451 as well as the upper lid of the binding side, formed by the bulky f294 side chain, are limiting the available space . In addition, preliminary md simulations of the transporter model in the apo open - to - out state led to rearrangement of the gating residues impeding subsequent placement of compounds larger than substrates like gaba, guvacine, or nipecotic acid . Thus, tiagabine was placed into the central cavity using glide prior to 30 ns of molecular dynamics simulations, which was used for validating and equilibrating the model . Subsequently, 10 representative snapshots for the last 10 nanoseconds of the run were extracted based on maximum rmsd diversity of binding pocket residues . Thus, focused sampling of the conformational space in the binding site could be achieved, using the snapshots as input structures for subsequent docking experiments . The constrained gaba analogs, nipecotic acid and guvacine (2, 3), are potent uptake inhibitors in vitro but are unable to penetrate the blood brain barrier . In contrast, tiagabine and the selective gat-1 inhibitor sk&f 89976-a (4, 5) that contain bulky aromatic substituents are pure inhibitors that are not transported (figure 1). A large number of systematically modified derivatives of the basic tiagabine scaffold have since been synthesized and tested . These derivatives contain a conformationally restricted gaba - mimetic nipecotic acid or guvacine moiety, a 48 atom linker, and a large, mostly diaromatic, hydrophobic moiety . A total of 162 compounds were extracted from the literature, spanning an activity range from low nanomolar to millimolar ic50 values, each of them tested under comparable assay conditions . Ligands exhibiting substantial activity differences linked to distinct structural changes in the key regions shown in figure 2 were selected for subsequent experimental data guided docking . Chemical structures and literature ic50 values of ligands with key modifications in linker length, polarity, and rigidity of the aromatic moiety . An important observation was the dramatic activity loss caused by introduction of a direct link between the two aromatic moieties (5 vs 7). In contrast, enhanced activity had been reported for introduction of a polar region in the linker . This is exemplified by compound pair 8 and 9, as well as compound 6, being the closest available derivative to reference compound 5 . In terms of activity, extending the linker length was well tolerated because compounds 5, 8, and 10 gave potent inhibitors . Finally, exchange of a benzene by a pyridine leads to a dramatic loss of activity (10 vs 11). These activity differences should also be reflected by respective differences in the ligand / protein interaction pattern and thus aid in the prioritization of the docking poses . Docking into 10 snapshots derived from the hgat-1tiagabine complex was performed in a sequential ensemble - like manner using gold, thereby allowing for minor movements of the backbone and focused sampling of the binding site side chain orientations . The binding site was defined within a 10 radius around the simulated tiagabine coordinates . Two water molecules were kept optional, as they had turned out to be stably involved in the hydrogen bonding network during the previous md simulation . However, in the subsequent docking runs, no direct contribution of these two water molecules to the binding of the selected ligands was observed . Side chain orientations of possible interaction partners for polar linker compounds 6, 8, 10, and 11 were addressed individually . Conformational sampling of the binding site had been performed with tiagabine as ligand, which lacks a corresponding electronegative moiety in the linker . Hence, the full range of conformational flexibility of the r69, y139, y140, and s452 side chains was explored using the internal rotamer library of the gold software package . For each of ligands 611, 100 docking poses per snapshot were generated and ranked by chemscore, which provides parameters for a putative interaction with sodium, in analogy to leut . However, relying on just a single scoring function bears the risk of missing relevant poses, especially when no experimentally derived complexes for redocking studies are available . Thus, all poses were reranked using rank - by - rank consensus scoring that included goldscore, chemplp, london dg, gbvi, and xscore scoring functions . Analysis of the 10 top ranked poses per ligand among the ensemble docking results clearly indicated a common binding mode for tiagabine analogs (figure 3). As already expected from literature results, the most prominent interaction was coordination of the na1 sodium cation by the negatively charged acid moiety, which fulfills an octahedral geometry, together with side chain atoms of n66, s295, and n327, as well as the backbone carbonyl oxygens of a61 and s295 . The majority of observed poses showed an interaction between the positively charged nipecotic acid nitrogen and the backbone of f294, while the carboxylate group interacting with na1 was in an equatorial conformation . In contrast, poses with the r - configured carboxy group sampled in an axial conformation tended to form an intramolecular hydrogen bond with the charged nitrogen atom . While no clear preference for one of the two carboxylate orientations could be deduced from scoring values, x - ray and nmr studies of nipecotic acid indicated a preferred equatorial configuration, which would be more pronounced by adding a bulky moiety like the biaromatic tail . In addition, skovstrup et al . Reported less stable behavior of axial - configured tiagabine poses in molecular dynamics simulations . Docking poses of tiagabine (turquoise) and analogs (orange) in 10 md snapshots of the hgat-1 model . The upper boundary of the binding pocket is formed by a bent region of el-4, extending into the hydrophobic cavity with the backbone of g360 as a ceiling beam, hence separating it into two pockets, each of which is able to accommodate a single hydrophobic aromatic rings . As it can be seen in figure 3, the ligand transporter interaction in one hydrophobic pocket is stabilized by a interaction with the side chain of y139, as well as by a cavity able to accommodate a small o - substituent as present in 5 and 6 . This cavity is confined by the side chains of i143 and y140, the latter being a residue known to be also important for ligand recognition (see figure 3). The second cavity is mainly shaped by the hydrophobic side chains of w68, f294, and a358 (not shown). Because of the relative torsion of the two pockets, poses of 7 tended to encounter an initial steric clash that was relieved after energy minimization of the complexes, whereas compounds with a terminal bis-5-methyl - thienyl (5, 6) or diphenyl (810) group were able to bind in a conformation near their global energy minimum (see figure 4), thus explaining the activity cliff between 5 and 7 . Dihedral energy landscape of 5-methyl - thiophen dihedral angles; configuration of the docking pose of 5 is marked in yellow . The positive effect of a polar atom in the linker moiety on binding seems to be the result of several factors . Transient interactions with residues in the entry path might play a significant role but are not reflected by the docking poses . Had indicated a transient interaction with the r69 side chain upon entry in the binding site . Hence, docking poses biased toward an interaction between this residue and one of the electronegative linker atoms in 6, 8, 10, and 11 were generated, turning out to be possible but rather short - lived in short md runs due to reset forces of the basic side chain (data not shown). To address the relatively low activity of compound 11, per - atom contributions to g in the binding pose an unfavorable effect of the pyridine nitrogen in the hydrophobic receptor environment was indicated (see supporting information, figure s3). In addition, repulsive forces between negative partial charges of the aromatic nitrogen atom and the oxime moiety might force the pyridine ring in a sterically unfavorable orientation . Taken together, the common orientation of the compound class was in agreement with the structure activity relationships of the ligand set and the topology of the extended substrate binding pocket . To further probe the proposed binding mode against pharmacologically relevant chemical space, the 3d orientation of the main tiagabine binding features was extracted from the docked complex and encoded in a four - feature pharmacophore model using ligandscout . Two hydrophobic features were placed in the respective cavities occupied by the thiophene moieties . Finally, a positively ionizable feature was placed on the basic nitrogen to mirror the compound s zwitterionic character (figure 5, the model is available for download at http://pharminfo.univie.ac.at). Pharmacophore model of tiagabine: hydrophobic (yellow), positive (blue), and negative (red) ionizable features in context with the gat1 substrate binding site . The sensitivity of the pharmacophore model was validated by a decoy set generated using the dud - e platform (http://dude.docking.org), retrieving just the compounds with known gat-1 activity . To test the predictive value of the model, a commercial vendor database consisting of 1.7 million compounds, as well as the drugbank index covering 1491 marketed drugs, a total of 79 and eight compounds, respectively, matched the pharmacophore query and passed the pains filter for frequent hitters . Subsequently, the virtual hits were docked into a representative md snapshot of the homology model, from which the tiagabine pharmacophore model had been derived . Ligand interaction fingerprints (plif) for the calculated poses were retrieved using moe . The interaction fingerprints were used to filter out compounds, which did not show an interaction with na1 . This reduced the hit list to 13 compounds for the enamine database available in sufficient purity, and seven compounds for drugbank, respectively (see figure 6). For the latter, tiagabine, three thyroid hormones (liothyronine, levothyroxine, dextrothyroxine), two angiotensin conversion enzyme (ace) inhibitors (ramipril, perindopril), and an antihistaminergic drug (bepotastine) were retrieved . On the basis of pharmacophoric fit and docking performance (details in supporting information, table s2), bepotastine, ramipril, and liothyronine were selected for biological testing . Compounds tested in the [h]-gaba uptake assay: enamine (red frame) and drugbank hits (blue frame), and reference compounds (black frame). Inhibitory potency of the selected compounds was evaluated by an uptake inhibition assay of radiolabeled gaba in hek cells stably expressing rgat1 . First, the ic50 value for tiagabine (5) in the test system was determined to be 0.64 0.07 m . This was about a factor of 10 higher than the value reported for the unspecific rat synaptosome assay used by andersen et al . Subsequently, compounds were measured at a concentration of 100 m against 5 as standard . Diazepam (28) and tiagabine (5) were used as negative and positive control . As illustrated in figure 7, one of the commercial screening compounds, 18, weakly reduced uptake to just below 80% of saline . One drugbank substance, 27a (liothyronine, a thyroid hormone also known as t3), turned out to significantly inhibit radioligand uptake, which prompted the acquisition and testing of other commercially available derivatives 27b d . Among those, the other bioactive hormone levothyroxine (27b) showed reduced uptake, albeit weaker than 27a . The representative of the ace inhibitors and the antihistaminergic drug bepotastine were essentially inactive . Remaining uptake of [h]-gaba in the presence of 100 m of the respective compound (n = 3). The ic50 value for liothyronine derived from a dose response curve was 13 1.7 m (figure 8), providing a direct link between reported general effects of thyroid hormones on gaba uptake and the inhibitory action of 27a on the gat-1 subtype . Inhibition curves of tiagabine (ic50 0.64 0.07 m, white squares) and liothyronine (ic50 13.0 1.7 m, black squares). As illustrated in figure 9, the pharmacophoric depiction of 27a reveals that one of the required hydrophobic features is not, as one would expect, the aromatic ring of the 3,5-diiodophenyl moiety, but rather a lipophilic iodine substituent, which could barely be deduced from chemical similarity measures . Relying on an appropriate position of the second ring relative to the interacting iodine atom, this type of interaction is also possible for 27b but not for 27d . The 3,3,5-substituted variant of the t3 layout is missing the second substituent on the proximal ring which is responsible for inducing the bioactive conformation . Activity relationships of thyroid hormone derivatives both at thyroid hormone receptors and gabaergic rat brain synaptosomes . Apparently, the steric requirements in both systems are remarkably similar, relying on correct substitution pattern, stereochemistry, and degree of lipophilicity, possibly also limiting the relative efficacy of 27c . The most remarkable difference between the observed crystallographic hormone binding mode of 27a (pdb code 3uvv) and the gat1 binding hypothesis can be found in the 4 position . The presence of the phenolic hydroxyl group is crucial for hormone receptor binding but not for interaction with the transport protein . Regarding compounds 1224, the most crucial property among those molecules seems to be the distance between the positively ionizable group and the first occurrence of lipophilic bulk, which is in close analogy to the linker in tiagabine analogs . Distal from the nitrogen atom (as seen from the negatively ionizable group, which also can be a tetrazole moiety in a reasonable distance), usually just one heavy atom separates the positive charge from the next aromatic moiety (12, 19, 20), or branching position (13, 21, 24). Alternatively, it is part of separate ring system not directly carrying the acidic moiety (1417, 22). With a slightly increased distance between the aromatic ring and the carboxylate group, 18 displays some weak activity . This observation also extends to the inactive drugbank compounds, as the nitrogen atoms of 25 and 26 are both connected to the hydrophobic part by space demanding linkers, whereas the first aromatic ring of thyroid hormones is at a distance of two heavy atoms . The presence of a pyridine moiety known to be unfavorable from the 1011 compound pair could further limit the potential of 25 despite its remarkable structural similarity to the reference compounds . Just as for the bulk of a cyclopentane moiety attached to the polar part of 26, this might considerably inhibit optimal positioning in the binding site . To further assess the degree of similarity between the compounds retrieved and the reference compound tiagabine, we calculated the tanimoto similarity values based on chemical fingerprints (maccs, fp2, fp4) derived from openbabel . The similarity between tiagabine and liothyronine as well as the slightly active 18, on the basis of maccs keys, was 25.4 and 51.5%, respectively . Inhibitory potency of the selected compounds was evaluated by an uptake inhibition assay of radiolabeled gaba in hek cells stably expressing rgat1 . First, the ic50 value for tiagabine (5) in the test system was determined to be 0.64 0.07 m . This was about a factor of 10 higher than the value reported for the unspecific rat synaptosome assay used by andersen et al . Subsequently, compounds were measured at a concentration of 100 m against 5 as standard . Diazepam (28) and tiagabine (5) were used as negative and positive control . As illustrated in figure 7, one of the commercial screening compounds, 18, weakly reduced uptake to just below 80% of saline . One drugbank substance, 27a (liothyronine, a thyroid hormone also known as t3), turned out to significantly inhibit radioligand uptake, which prompted the acquisition and testing of other commercially available derivatives 27b d . Among those, the other bioactive hormone levothyroxine (27b) showed reduced uptake, albeit weaker than 27a . The representative of the ace inhibitors and the antihistaminergic drug bepotastine were essentially inactive . Remaining uptake of [h]-gaba in the presence of 100 m of the respective compound (n = 3). The ic50 value for liothyronine derived from a dose response curve was 13 1.7 m (figure 8), providing a direct link between reported general effects of thyroid hormones on gaba uptake and the inhibitory action of 27a on the gat-1 subtype . Inhibition curves of tiagabine (ic50 0.64 0.07 m, white squares) and liothyronine (ic50 13.0 1.7 m, black squares). As illustrated in figure 9, the pharmacophoric depiction of 27a reveals that one of the required hydrophobic features is not, as one would expect, the aromatic ring of the 3,5-diiodophenyl moiety, but rather a lipophilic iodine substituent, which could barely be deduced from chemical similarity measures . Relying on an appropriate position of the second ring relative to the interacting iodine atom, this type of interaction is also possible for 27b but not for 27d . The 3,3,5-substituted variant of the t3 layout is missing the second substituent on the proximal ring which is responsible for inducing the bioactive conformation . Activity relationships of thyroid hormone derivatives both at thyroid hormone receptors and gabaergic rat brain synaptosomes . Apparently, the steric requirements in both systems are remarkably similar, relying on correct substitution pattern, stereochemistry, and degree of lipophilicity, possibly also limiting the relative efficacy of 27c . The most remarkable difference between the observed crystallographic hormone binding mode of 27a (pdb code 3uvv) and the gat1 binding hypothesis can be found in the 4 position . The presence of the phenolic hydroxyl group is crucial for hormone receptor binding but not for interaction with the transport protein ., the most crucial property among those molecules seems to be the distance between the positively ionizable group and the first occurrence of lipophilic bulk, which is in close analogy to the linker in tiagabine analogs . Distal from the nitrogen atom (as seen from the negatively ionizable group, which also can be a tetrazole moiety in a reasonable distance), usually just one heavy atom separates the positive charge from the next aromatic moiety (12, 19, 20), or branching position (13, 21, 24). Alternatively, it is part of separate ring system not directly carrying the acidic moiety (1417, 22). With a slightly increased distance between the aromatic ring and the carboxylate group, 18 displays some weak activity . This observation also extends to the inactive drugbank compounds, as the nitrogen atoms of 25 and 26 are both connected to the hydrophobic part by space demanding linkers, whereas the first aromatic ring of thyroid hormones is at a distance of two heavy atoms . The presence of a pyridine moiety known to be unfavorable from the 1011 compound pair could further limit the potential of 25 despite its remarkable structural similarity to the reference compounds . Just as for the bulk of a cyclopentane moiety attached to the polar part of 26, this might considerably inhibit optimal positioning in the binding site . To further assess the degree of similarity between the compounds retrieved and the reference compound tiagabine, we calculated the tanimoto similarity values based on chemical fingerprints (maccs, fp2, fp4) derived from openbabel . The similarity between tiagabine and liothyronine as well as the slightly active 18, on the basis of maccs keys, was 25.4 and 51.5%, respectively . With the increasing number of x - ray structures available for transmembrane transporters, structure - based computational models have provided valuable insights into the molecular basis of ligand transporter interaction . Within this article, we propose a binding mode of the antiepileptic drug tiagabine in gat1 by including knowledge from ligand - based studies into the prioritization process for docking poses . Subsequent pharmacophore - based virtual screening followed by experimental testing further confirmed the validity of the pose by identifying a commonly used drug (liothyronine) as an inhibitor of gat1 . Strikingly, liothyronine has been described long ago as potential gat inhibitor without major activity on other neurotransmitter reuptake systems (dopamine, serotonin, choline, aspartate), but final experimental confirmation for subtype gat1 since has been lacking . Compounds with significantly higher chemical similarity retrieved in a commercial vendor library all prove inactive, further implying that selective transport inhibition of the protein can only be tackled from the side of steric feature arrangement . Furthermore, the results indicate that, apart from privileged tricyclic antidepressant scaffolds known to more or less unspecifically inhibit neurotransmitter uptake, not many drugs on the market are likely to interact with gat1 . The gat1 models were constructed using the crystal structure of leut as template which shows highest available resolution in an open - to - out state of the transporter . When assessing the differences between available sequence alignments of leut and hgat1 (uniprot entries o67854 and p30531, respectively), no differences for residues in the central binding cavity were observed, except for a one - residue gap in the middle of leut - tm10, either placed over gat1-g457, s456, or a455 . As it has been optimized for gat1 and assessment of the sequence identity between hgat-1 and rgat-1, the first being the effective protein of interest, the second the one used in cell assays, stated 100% sequence identity for the observed core region and 97.9% for the whole modeled sequence (see supporting information, figure s4). The crystal structure of leut retrieved from the pdb (www.pdb.org, accession code 3f3a) was mutated in silico at position 290 using moe with a serine side chain orientation corresponding to the former glutamic acid . A chlorine ion was placed at the coordinates of the previous center of the e290 side chain, then further optimized according to interaction potential calculations, giving cl as probe . Tethering the backbone, s290 and its surrounding residues were carefully energy minimized for final optimization of the local coordinates . The models were built using modeller9v8 in the automodel class, including water molecules, the chloride ion, and two cobound sodium ions as nonprotein atoms . A disulfide bridge was defined between c164 and c173 using a modeller patch command . One hundred models were generated using very thorough vtfm (variable target function method) optimization, as provided in modeller . Output models were ranked according to dope score, and the top 10 were further assessed by the swiss - model server . According to procheck results and ramachandran plots, models with disallowed backbone geometries in transmembrane regions were omitted . Hydrogen atom assignment and soft energy minimization of the raw models was performed within moe using ligx and the charmm27 all - atom force field, otherwise with default settings . The selected complex was inserted into a pre - equilibrated and solvated popc membrane by applying the program g_membed, using the gromos 53a6 united - atom force field and periodic boundary conditions . All simulations were performed at 310 k. the system was neutralized by adding sodium and chlorine ions to a final salt concentration of 150 mm . Gradually, position restraints on the main complex were reduced from 500 kj mol nm (500 ps) to 250 kj mol nm (500 ps). After an equilibration phase without restraints, a fully stable system was achieved after 20 ns . Between 21 and 30 ns of the production run, the frames were clustered according to rmsd of residues within a radius of 7 around the ligand . One showed a stable h - bond with y60 (89% occupancy; distance 3.5; angle 60), another one was directly attached and showed an almost equally stable h - bond interaction . Protonation states were sampled according to possible states in a physiological ph range of 7.2 0.2 using ligprep . These states were cross - checked with the major microspecies calculated by the chemaxon web - tool chemicalize.org . Primary placement of tiagabine was done using glide in standard precision (sp) mode using default settings . The receptor grid was defined around binding site residues 6063, 6466, 136, 140, 294297, 300, 396, and 400 . Docking and goldscore / chemplp rescoring in the 10 md snapshots were performed with gold 5.0.1, using chemscore as primary scoring function . Early termination was disabled, keeping the 100 best solutions per ligand and snapshot . External rescoring was performed using xscore (xscore) and moe (london dg, gbvi). Consensus scores were calculated by summing up indices assigned according to respective ranks within a scoring function . Secondary docking runs for investigating side chain orientations for specific interactions with polar linker moieties were performed by (a) constraining residues y139, y140, and s456 to the internal library of allowed rotamers in gold and (b) for investigating interactions as reported by skovstrup et al ., likewise rotamer rotations of r69 and f294 were allowed but with an additional distance constraint of 1.53.5 (spring constant 5) between the r69 guanidine function and the polar linker moiety . Determination of the potential energy landscape for different dihedral angle configurations was performed with gaussian 09 . After initial geometry optimization with hf/3 - 21 g implemented in the software package, configurations with an increment of 15 were calculated using the m06 - 2x hybrid functional and the 6 - 31 g * basis set . Pharmacophore models were built using ligandscout 3.0 . For assembling the customized decoy library, 50 decoys per active compound (511) were compiled in smiles format using the dud - e platform (dude.docking.org). The nonredundant compounds with similar physicochemical properties but dissimilar 2-d topology for each input line were extracted from the zinc database . The retrieved set of decoys and the active compounds as smiles were assembled and processed to a ligandscout screening database using the maximum number of possible conformers . The drugbank database was downloaded from the web site www.drugbank.ca and consisted of 1491 entries (version of june 2013). Enamine advanced and hts screening collections were obtained from the download site at www.enamine.net (n = 1719682; version 032013). Ligandscout command line modules idbgen and iscreen were used for conformation generation and for performing the pharmacophore screening . Training set, decoy compilation, and drugbank compounds were prepared using omega - best settings (max 500 conformations), enamine advanced and hts databases were compiled with omega - fast settings (max 25 conformations) (www.eyesopen.com,). Path - based (fp2) and substructure - based (maccs, fp4) similarity fingerprinting was performed using openbabel 2.3.1 . Primary checking for pan assay interference (pains) compounds was done by uploading retrieved virtual screening hits to the pains remover web service (available at http://cbligand.org/pains), returning no suspicious compounds . Protein screening compounds were purchased from enamine (enamine ltd ., riga, latvia), fluka / sigma - aldrich (sigma - aldrich co., saint louis, mo), and avachem (avachem scientific, san antonio, tx), all with a purity 95% (see supporting information, table s4). Cloned cells (4 10 cells / well) were seeded and grown at 37 on poly(d)-lysine coated standard plasticware 24 h in advance . Uptake of [h]-gaba into was measured in the presence of 100 m of the compounds, while unspecific uptake was defined as uptake in the presence of 100 m tiagabine . For ic50 determination tested concentrations were: 5, 0.001, 0.01, 0.1, 0.3, 1, 10, 100 m; 27a, 0.01, 0.1, 0.3, 1, 3, 10, 30, 100 m . After 3 min preincubation, [h]-gaba (35 ci / mmol, perkinelmer, boston, ma) in a final concentration of 0.015 m was added . Uptake was stopped by adding ice - cold krebs - hepes buffer (10 mm hepes adjusted to ph 7.4 with 35.9 mm solid naoh, 120 mm nacl, 3 mm kcl, 2 mm cacl2, 2 mm mgso4, and 2 mm d - glucose as supplement). Cells were lysed with 1% sds (sodium dodecyl sulfate) solution, taken up in 2 ml of scintillation cocktail (rotiszint eco plus, carl roth gmbh, karlsruhe, germany) and counted in a standard liquid scintillation counter (packard tricarb 2300tr, packard instruments).
Dental erosion (de) has become more frequent during the last decades, especially in children and young people . Increased consumption of carbonated drinks but also endogenous factors are involved in de etiology . Associated esthetic, phonetic and masticatory dysfunctions are related to advanced generalized erosions which affect the entire dental arches . The important objective in the dental erosion therapy is the minimally invasive crown reconstruction . Full arch reconstructions are necessary in advanced generalized de and an esthetic project must be developed . In this case report we present a treatment plan option in a patient presenting de and lateral partial edentulous spaces . When the patient presents tooth loss and severe de, treatments involving fixed partial dentures (fpd) and all ceramic reconstructions are recommended to obtain improved esthetics and good functionality . In terms of treatment cost, advanced stages of de therefore, sustained prophylaxis has to be done to avoid de evolution: diet control has to be effective in patients with evidenced extrinsic factors . In endogenous de caused by bulimia, anorexia, gastro - esophageal reflux disease (gerd), or other gastric diseases involving vomiting, associated etiologic therapy a 47-year - old female complains of masticatory and esthetic discomfort after a mandibular bridge fracture . She also demands esthetic restorations on maxillary frontal teeth, which became progressively darker and irregular . Symptoms of gerd were related to a psychological trauma and were subsided at the time of study . After the clinical examination of the maxillary arch, we found de and composite cervical restorations on the central incisors; used composite veneers were present on the lateral incisors . De could not be evaluated on other maxillary teeth because they were all recovered by metal - ceramic fpd: an fpd on 13, 15 and 17 restoring the missing 14,16 and an extended fpd on 23 and 27 with intermediary for 24, 25, 26 (fig . A fractured mandibular fpd for the missing 35, 36, was causing masticatory and esthetic disorders; the mesial abutment tooth, 34, was extracted one year ago . A metal - ceramic fpd was found on 44, 45, 47 restoring the 46 missing tooth (fig 1). Overload abutment teeth 23, 27 show gingival recession, and x - ray examination shows important bone loss on 27 . Maxillary central incisors margins and buccal faces are affected by de, resulting in important tooth wear with shape and color modifications (fig . 1). Wear composite veneers on the lateral maxillary incisors are also non - aesthetic . Application of de questionnaire reveals few alimentary factors involved: citric fruits preference and daily carbonated drinks consumption . After clinical examination, radiologic and basic erosion wear examination (bewe), the esthetic analysis was performed . All existing fpds had to be removed and a full maxillary arch reconstruction was decided, using dental and implant supported fixed prosthesis . Eroded maxillary incisors will be restored by ceramic veneers improving esthetics and allowing good biological integration . Bonded cementation of ceramics result in strong restorations when precise fit of the veneers avoid excessive space for the cement . A metal - ceramic fpd on 13, 15 and 17 will successfully restore the missing 14 and 16 using modified saddle intermediaries (fig . 2). The prognosis of 27 as abutment tooth is uncertain because of reduced bone support . Two implants are required for an implant supported fpd restoring 24, 25, 26 edentulous space . Good health and favorable bone condition allow us to do a metal - ceramic implant supported fpd in the three - unit edentulous space of 34, 35, 36 . This treatment plan is favorable to bone maintenance, fpd resistance and avoid 33 preparation (fig . A metal - ceramic fpd with abutment teeth 44, 45, 47 was decided . Existing endodontic treatments on 44, 45 were accurate from a radiological point of view . After therapeutic decision was made, we removed the fpds and implants were inserted . After insertion, the implants were allowed to integrate . Six months later, a new radiologic control was performed, implants were uncovered and healing caps were placed . After two weeks, a first impression in irreversible hydrocolloid was taken and open custom tray was fabricated . Impression copings were adapted to implants and splinted with resin for accurate transfer of implants position to the master cast . Teeth preparations were finalized and one - step technique, maxillary and mandibular impressions were made . The metal fpd infrastructures were made in the laboratory (fig . 2, 3) and checked for correct fit on the abutment teeth in the oral cavity . The symmetry of the final metal - ceramic reconstructions adequately solves the esthetic aspects regarding color, shape, position and dimension of the new prosthetic constructions . Rose porcelain was used to make an esthetic gingival appearance on implant supported fpds (fig . The patient demanded whiter teeth; consequently we replaced the existing fillings and made a bleaching two weeks before making the veneers . Esthetic criteria was verified on ceramics; teeth visibility and correct position of incisors margins were phonetically and esthetically evaluated in front and lateral view (fig . 5, 6). Therefore, an implant - supported fpd is preferred to restore extended partial edentulous spaces when local and general conditions are favorable; crest maintenance is better when functional stimulus is transmitted to the bone by implants . In the presented case the advanced tooth wear on central maxillary incisors was due to dominant erosive mechanism caused by gerd and associated with abrasion and attrition . Good prognosis of the prosthetic treatment depends on diet education and good general condition; gerd remission is favorable for post - therapeutic maintenance . The regular six - month control was performed after treatment, to achieve good maintenance . Full arch esthetic restoration can change the patient s life by improving functional comfort and self confidence . Laborious steps are involved and long time temporizations have to be accepted by the patient before starting with the treatment plan decision.
A 70-year - old woman (height: 156 cm, body weight: 48 kg) with lower abdominal pain was admitted and associated symptoms were fever, nausea, vomiting and diaphoresis which started from the day previous to her admission . On abdominal computed tomography (ct), right ovarian cyst in size of 2.5 cm was found, and an acute appendicitis was diagnosed by an abdominal ultrasonography . An elective operation was scheduled under the collaboration of gynecology and general surgery . In the patient's past medical history, she had taken digoxin, dilatrend, nitrate, telmisartan, and thiazide for 5 years because of hypertension, congestive heart failure, af, and right coronary artery 90% stenosis on coronary angiography . An electrocardiogram before operation showed af with ventricular response 90 - 100 times / min, left ventricular hypertrophy . Cardiomegaly and pleural effusion were found on chest x - ray . On transthoracic echocardiography (tte), ejection fraction was 55% and left atrial enlargement, right atrial enlargement and eccentric hypertrophy with decreased mobility of the inferior wall of the left ventricle were shown . A moderate aortic valve insufficiency, aortic valve sclerosis, mild aortic stenosis, and severe posterior mitral valve leaflet calcification were also found and the width of mitral valve measured by pressure half - time was 1.92 cm . A chronic cerebral infarction in the right posterior cerebral artery was found on brain ct with symptoms of dysarthria . Signs of dehydration on physical examinations with prerenal azotemia of fena 0.1% and serum creatinine of 1.7 mg / dl on blood test led us to start an fluid therapy . Glycopyrrolate 0.2 mg i m was premedicated at 30 minutes pre - operation . The patient's blood pressure (bp) was 130/50 mmhg, ventricular response 90 - 100 times / min, and arterial oxygen saturation 97% when she arrived at the operation room . The induction of anesthesia was initiated with injecting 2 ml of 2% lidocaine to reduce injection pain and propofol (diprivan astrazeneca, uk) and remifentanil (ultiva glaxosmithkline, uk) were injected using a target - controlled infuser (orchestra fresenius vial, france). After confirming the patient's being unconscious, rocuronium 40 mg was injected and then endotracheal intubation was performed with close monitoring of arterial blood pressure . Ventilation with 100% o2 was given while central venous catheterization was placed in right jugular vein . After the induction of anesthesia, the patient's vital sign showed no hemodynamic disorder with systolic bp 130 - 150 mmhg, diastolic bp 40 - 60 mmhg, ventricular response approximately 100 - 110 times / min, and central venous pressure (cvp) 8 - 9 mmhg . The effect site concentration was injected as 2.5 - 3.0 g / ml of propofol and and 2.0 ng / ml of remifentanil . After the lower abdomen laparotomy at obstetrics and gynecology for the right ovary cystectomy, a small bowel infarction from jejunum to ileum was detected . The authors suspected a mesenteric arterial embolism based on the patient's previous medical history . A transesophageal echocardiology (sonosite micromaxx, bothell, usa) probe was immediately inserted and a spontaneous echo contrast (sec or " smoke ") in the left atrium (la) and a 13 18 mm size thrombus in the laa was detected (fig . The findings in tee made a mesenteric arterial embolism highly suspicious for the cause of the small bowel infarction . No hemodynamic instability was observed through whole procedure of operation and the patient was transferred to the intensive care unit with endotracheal tube inserted . After confirming her awareness and the absence of any neurologic disorder, extubation was performed in intensive care unit . To minimize the risk of hemorrhage of the anastomosis site it was started with low molecular weight heparin 3 days after the operation and oral warfarin was added 5 days after the operation . Mesenteric infarction is induced by the embolism in the left atrium, left ventricle, and heart valves; the superior mesenteric artery is anatomically the most vulnerable to infarction due to its large diameter and the acute angle takeoff from the aorta, but the inferior mesenteric artery is rarely vulnerable due to its small diameter . For preventing injury from ischemia, the main mesenteric blood vessels have extensive collateral circulation; however, when the mesenteric arterial embolism occurs, the mid - segment of the jejunum, which is the farthest from the collateral circulation of the celiac artery and inferior mesenteric artery, becomes most vulnerable to ischemia . An acute hypoperfusion state induced by mesenteric arterial vascular disorder forms 60 - 70% of the total mesenteric ischemia with mortality rate exceeding 60% . Symptoms can vary from acute severe, nonremitting abdominal pain to vague abdominal pain . And associated symptoms may include nausea and vomiting, transient diarrhea, and bloody stools . However the absence of any specific clinical symptom and no indicator shown in blood test or plain x - ray make early diagnosis challenging which may end up in delayed treatment . It is highly suspicious in aged patients with af and a previous history of cardiac and vascular disease like recent myocardial infarction, rheumatic heart disease, and recent cardiac catheterization . Mesenteric angiography is the most reliable diagnostic tool and also helps locating exact site of blockage . An early angiography on a patient suspected of arterial ischemia is widely accepted to decrease the mortality rate of mesenteric ischemia patients . There was no comment about the thrombus in the la and the laa on the pre - operative tte examination; it is difficult to observe a laa by tte, so the sensitivity of the tte of the la or laa is known as 39 - 63% . In contrast, by tee, observing the posterior structures of the heart, such as the la and laa is much easier, the sensitivity and the specificity of thrombus in the la was reported as 93 - 100% and 99 - 100%, respectively . Tee of patients with af shows a sec in the la or laa, which is known to increase the risk of thromboembolism . Estimation of blood flow velocity in the left or right atrium and left and right atrial appendage permits a more quantifiable measure of stasis . The risk of stroke increases sharply with marked reductions in blood flow velocity (<15 cm / sec), particularly in the laa or posterior left atrium . Sec refers to the presence of dynamic, smoke - like echoes seen during echocardiogram in the la or laa; it is known that sec is formed by a red blood cell rouleaux formation on stagnant flow condition . Difficulty to quantificate sec by identifyimg as a fluid whirlwind of smoke is due to differences between the frequency and the gain of an echocardiogram probe . Sec may be missed in the case where the gain is too low or the test room is too bright . Sec should be observed under high gain with a high - frequency probe over 5 mhz which a uniform noise is seen in the la . The laa peak outflow velocity can be estimated by tee and sec semiquantitatively graded as marked or dense if present throughout the entire cardiac cycle, or faint when intermittent . The goal of treating af include maintaining sinus rhythm, controlling heart rate, and preventing thromboembolism by using anticoagulant therapy; the risk stratification of thromboembolism is most important for anticoagulant therapy . The high risk factors of thromboembolism include a previous history of thromboembolism, mitral valve stenosis, and a mechanical valve; moderate risk factors are ages over 75, hypertension, heart failure, left ventricular ejection fraction less than 35%, and diabetes . Females, ages from 65 to 74, coronary arterial diseases, and thyrotoxicosis correspond to low risk factors . During anticoagulation therapy, aspirin 81 - 325 mg / day should be taken in the case where there are no risk factors or only low risk factors; aspirin or warfarin (inr 2 - 3) should be taken in case of 1 moderate risk factor . Warfarin should be taken in case of 2 or more moderate risk factors or the existence of a high risk factor in case of a mechanical valve, inr should be maintained more than 2.5 at minimum . Chads2 (cardiac failure, hypertension, age, diabetes, stroke [doubled]), another stroke risk stratification, scores 1 for heart failure, hypertension, ages over 75, and diabetes and scores 2 for a previous history of a stroke or transient ischemic attack and adds the scores, and then classifies score 0 as a low risk group, scores 1 as a moderate risk group and scores over 2 as a high risk group . Aspirin is used for the low risk group, aspirin or warfarin is selectively used for the moderate risk group, and the high risk group with scores over 2 should be performed with anticoagulant therapy . In this case, the patient should have been initiated an anticoagulant therapy considering her high blood pressure, congestive heart failure, and chronic cerebral infarction detected by the brain ct . In addition, aged patients with af showing mitral annular calcification on echocardiography, there is a high potency of thromboembolism, even without mitral stenosis, mitral regurgitation, or prolapsed leaflet . The authors suggest that risk stratification before operation should be performed on af patients with thromboebolism risk factors; hemodynamic and neurologic observation are needed on the induction, maintenance, and emergence period of anesthesia; tee during operation can be helpful for these patients.
Since the introduction of the concept or sublethally injured or viable but nonculturable (vbnc) cells by byrd and colwell in the 1980's, there is increasing evidence for the existence of such a state in microbes, particularly in the aquatic environment that elicits a myriad of interrelated sub - lethal microbial stresses such as nutrient starvation and osmotic stress [2, 3] (table 1). This is a cause for concern because of evidence that microbial pathogens in such a state may still retain their capacity to cause infections after ingestion by fish, animals, or by humans, despite their inability to grow under conditions employed in laboratory - based procedures for determining their presence in water . Albeit currently unknown in terms of its severity or scope, it is now generally appreciated that heavily stressed pathogenic microbial species existing in a vbnc (or not immediately culturable state) may potentially pose as yet an undefined risk to public health, which is attested by the fact that there is increasing evidence to support the viewpoint that stressed cells in this quiescent state may actually be more virulent than well - fed laboratory - tamed microorganisms due to augmented virulence factor expression . Were the first to bring experimental evidence of the existence of vbnc state in pathogenic bacteria, where they showed that e. coli and v. cholera cells that were suspended in artificial seawater quickly lost their ability to grow on the culture media normally used for their detection . According to oliver, a bacterium in the vbnc state is defined as a cell which is metabolically active, which being incapable of undergoing the cellular division required for growth in or on a medium normally supporting grown of that cell . Besnard et al . Suggest that the transition to the vbnc state in l. monocytogenes represents a survival strategy that bacteria can adopt under adverse conditions (starvation, salt stress, etc . ). Vbnc microorganisms are considered to represent a subpopulation of cells that are unable to grow in the usual culture media and cannot resuscitate by traditional resuscitation techniques, but yet remain physically active for several functions such as cellular elongation, respiratory chain activity [6, 7, 17], or incorporation of radio - labelled substrates . For example, cappelier and coworkers recently reported that avirulent vbnc cells of l. monocytogenes incubated in filtered sterilized distilled water need the presence of an embryo to be recovered in egg yolk and regain virulence after recovery . The vbnc state was observed after a 25 to 47 days incubation period (concentration of culturable cells less than 1 colony forming unit per ml). As microorganisms are extremely diverse and dynamic, it is not surprising that the many different types of microbial species present in the water environment exist in a number of physiological states that possess different requirements for survival and to sustain growth . Indeed, the number of waterborne bacteria in which the vbnc state has been reported has greatly increased, particularly in recent times that reflect technological advances . For instance, campylobacter jejuni has been reported to exist in two different cellular morphotypes, where the atypical coccus - form (currently associated with nongrowing vbnc state) occurs in water under extended nutrient depletion conditions . A number of different research groups have reported that these atypical culture forms are still capable of infection mice and poultry . Moreover, rowan and coworkers recently reported on that different culture morphotypes of l. monocytogenes generated in water after exposure to novel pulsed - plasma gas - discharge treatment can survive internalization by human polymorphonuclear leukocytes . While lindbck et al . Reported that the ability to enter into an avirulent vbnc form is widespread among l. monocytogenes isolated from salmon, patients and the environment . L. monocytogenes were tested for virulence in a cell plaque assay and by intraperitoneally inoculation in immunodeficient rag1 mice . Moreover, moreno et al . Described successions in cellular alterations in helicobacter pylori nctc 11637 after inoculation into chlorinated drinking water . They concluded that h. pylori could survive disinfection practices normally used in drinking water treatment in the vbnc form, which would allow them to reach final consumption points and, at the same time, enable them to be undetectable by culture methods . Whereas kastberg et al . Recently reported that l. monocytogenes cells, whether planktonic or attached, were homogenous with respect to sensitivity to acidic disinfectants at the single - cell level . Directvisualization of actively respiring bacteria is gaining in popularity amongst research groups investigating this vbnc state [3, 6, 7]. Researchers have exploited use of metabolic staining to reveal an underestimation in the level of microbial survival compared to similar samples cultured on traditional agar plates . Recent research in our laboratory has also shown that subpopulations of waterborne pathogens such as e. coli and pseudomonas spp . Treated with novel pulsed - power disinfection technologies (such as use of pulsed - plasma gas - discharge technology that will be expanded on later in this paper) were capable of reducing the redox dye 5-cyano-2, 3-ditolyl tetrazolium chloride (ctc) that is an indicator of electron acceptor function, yet similarly treated samples were unable to form colonies on a variety of laboratory - based culture media [6, 7]. This corroborates more recent research undertaken by sawaya and coworkers who used a combined 4'6-diamidion-2-phenylindole (dapi) and ctc stains to highlight the occurrence of physiologically active bacteria in river and wastewater treatment plants that were much higher than those obtained by plate counting . These researchers also reported that microscopic viable bacteria were more chlorine resistant than culturable bacteria . That said, a demonstration of active respiration does not necessarily infer that these stressed bacteria are capable of future growth . As numerous researchers continue to report on the use of redox stains for highlighting differences in agar - plate counts, it is important that we holistically explore and identify specific microbial cues at the cellular level which govern the transition to the vbnc state along with exploiting commensurate advances in media formulations that are tailored for optimal resuscitation of these sublethally stressed cells (cited in [10, 24]). The latter authors showed that the addition of a commercially available antioxidant oxyrase and a heat - stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine, resuscitated e. coli, and salmonella enteric serovar typhimurium that were stressed by prolonged incubation in water microcosms . Advancing the earlier pioneering work of dodd et al . And aldsworth et al ., who previously postulated that self - destruction or cell suicide may be attributed to sublethally stressed or damage microbes being incapable of coping with oxidative burst when rapidly growing on nutrient rich media, it is important that we also exploit advances in toxicology (such as methods exploring cellular apoptosis and necrosis, cell membrane lipid peroxidation assays, and nuclear chromatin / comet assays) that will provide valuable insights into the possible role of intracellular free radicals in combination with the direct physical action of the applied environmental stress on the generation and persistence of vbnc organisms in water . Indeed, servais and coworkers recently reported that certain environmental factors such as nutrient scarcity and solar irradiation lead to a high proportion of vbnc e. coli in freshwater . The latter fecal microorganisms are brought into freshwater environments mainly through wastewater release, surface runoff, and soil leaching . Interestingly, modified guidelines for bathing water states that enumeration of e. coli will replace total coliforms and fecal (also called thermotolerant) coliforms as bacterial indicators of water quality . The latter authors articulated that the number of e. coli in freshwater is systematically underestimated by traditional culture - based methods such as multiple tube fermentation and or membrane filtration techniques), which is cause for concern from a public health perspective . On a related theme, numerous researchers have also recently reported on the occurrence of bacterial autophagy (i.e., microbial adaptation to autophagic microbicidal host immune cell defences), which is an important cell survival process initiated during nitrogen starvation conditions . Among the minority of bacteria that have been discovered it is estimated that more than 90% are as yet nonculturable as attested by the fact that international committee on systematic bacteriology has recognised a new category for nonculturable bacteria that it named candidatus for which phylogenetic relatedness has been determined by amplification and sequence analysis of prokaryotic rna genes with universal prokaryotic primers and authenticity has been verified by use of in situ probes . Such nonculturable organisms may only be detected by use of such molecular techniques based on probes such as 16s and 23s rrnas or on determination of mrna, either by quantitative real - time pcr and/or by fluorescent techniques such as in situ hybridization (fish), microradiography, epifluorescence microscopy, and flow cytometry [2, 11, 12]. While fiksdal and tryland advocated use of rapid enzyme assays for monitoring of water quality, which may also detect organisms in the injured or vbnc state . Garcia - armisen and servais showed that the ratio of direct viable- (dvc-) fish count and the culturable count increased with decreasing abundance of culturable e. coli in river water, and therefore the slope of the linear - log - log correlation of dvc - fish versus colony forming unit numbers was less than one . The authors hypothesized that the more stressful conditions, such as nutrient deprivation and increase solar stress at low turbidities met in low contaminated environments, were responsible for the larger fraction of vbnc e.coli . As mentioned previously by many research groups [13, 2933], all field trials plotting log -d - galactosidase (galase) activity or log -d - glucuronidase (gluase) activity versus log culturable target bacteria in fresh or marine water have shown regression straight lines with a slope less than one, suggesting that enzyme activity calculated per culturable indicator bacteria increases when their numbers decrease (e.g., when sewage effluent becomes diluted in receiving waters). Pure culture studies of e. coli have also shown that after exposure to other types of extrinsic stress such as chlorination, the galase activity is less reduced than the direct viable count . While zimmerman and coworkers demonstrated that e. coli can be present in higher numbers in recreational water samples using fluorescent antibody direct viable counting that what are detected with standard culture methods . The latter advances the early landmark study of bjergbaek and roslev who reported on the occurrence and persistence of vbnc e. coli in nondisinfected drinking water using different cultivation dependent methods, fluorescence in situ hybridization (fish) using specific olignucleotide probes, direct viable counts (dvc), and by enumeration of gfp - tagged e. coli (green fluorescent protein, gfp). These studies specifically reemphasises the need for a rapid, accurate, and precise method for detecting health risks to humans from contaminated water . Other likely candidate methods for efficient and rapid - detection of vbnc bacteria in the aquatic and soil environments include rna - based genotypic approaches . Recently reported on the rapid and accurate quantification of vbnc pathogens in biosolids via monitoring and quantifying stress - related genes in salmonella spp . Using cdna microarrays combined with quantitative reverse transcription polymerase chain reaction (qrt - pcr). Okabe and shimazu also describe detection of host - specific bacteroides - prevotella 16s rrna genetic markers (total, human, cow- and pig - specific) as promising alternative indicators for identifying the sources of fecal pollution in environmental water because of their abundance in the feces of warm - blooded animals . The authors clearly state that detection of aforementioned genetic markers mainly reflected the presence of vbnc bacteroides - prevotella cells in water, suggesting that seasonal and geographical variations in persistence of these host - specific bacteroiides - prevotella 16s rrna genetic markers must be considered if used as alternative fecal indicators in environmental waters . This corroborates emerging studies that suggest that increases in coliform concentration after stp and dewatering processes may be attributed to cells going into vbnc state implying traditional coliform enumeration methods are not sufficient to determine number of viable cells . This also reinforces common viewpoint shared by many scientists that a variety of problematic bacteria can enter vbnc states as a survival response when exposed to deleterious environmental stresses such as nutrient starvation, osmotic stress, and so forth . However, in order to gain a greater appreciation of environmental and public health risks associated with persistence of microbial pathogens in different culturable states, it is imperative that we acquire an understanding of the interrelated molecular responses governing tolerance to these applied sub - lethal stressors . Such as detailed investigations reported by brackman et al . Who recently found that autoinducer- (ai-) 2 quorum - sensing inhibitors affected starvation response and reduce virulence in several vibrio species, most likely by interfering the signal transduction pathway at the level of luxpq . The aforementioned detection methods are not as yet commonly used for routine measurement as they either are not simple enough and/or the equipment is too expensive . Therefore, in terms of enabling effective risk - based assessment and environmental management to address the occurrence and persistence of vbnc pathogens in water, critical data must be acquired to convert vbnc - phase potential pathogens from unknown to known and defined hazards . In terms of satisfying these information gaps, accurate enumeration of total microbial load and real time identification to the various types present in water are pivotal to hazard identification and characterization, which will contribute significantly to assuring water safety . Regarding the latter, smeets et al . Recently described improved methods for modelling drinking water safety using a robust quantitative microbial risk assessment (qmra), where a case study monitored campylobacter data for rapid sand filtration and ozonation processes . This study showed that currently applied methods do not predict monitored data used for validation . Therefore, underestimation in the levels of problematic microbes in water, and failure to identify the presence of pathogenic organisms in representative water samples also pose significant threats to public health . Greater information is also required on elucidating the existence of commonly shared cellular mechanisms (and associated gene expression regulators and gene markers) that govern cellular conversion to this vbnc state . Moreover, there is a dearth of knowledge regarding specific underlying molecular and associated cellular mechanisms governing transition and persistence of waterborne microorganisms in this vbnc state, in addition to obviously establishing what specific environmental conditions or triggers cause these changes in culturable state . Greater studies are also required to investigate the presence and role of this vbnc state in microbial pathogens that cause disease in the aquatic natural setting such as in fish.taking for example, flavobacterium psychrophilum, the causative agent of rainbow trout fly syndrome and cold water disease in salmonids, where subpopulations were still culturable after starvation for 300 days in sterilised fresh water . The virulence of starved f. psychrophilum was maintained for at least 7 days after the transfer of the bacterial cells to fresh water . The vnbc state was earlier reported for this fish pathogen by madetoja and wiklund who revealed differences between enumeration using advanced immunoflourescence and genetic probes (i.e., nested pcr) compared to that of using traditional agar plate cultivation . While the long - term survival cellular responses to salinity, temperature and starvation have been studies in the eel pathogen vibrio vulnificus for over a decade . To combat such severe microbial infections affecting fish, many researchers have advocated water recirculation and good management as potential methods to avoid disease outbreaks particularly with f. psychrophilum . Thus, highlighting the importance of augmenting water quality through improvements in wastewater treatment, which should be augmented in efficiency and capacity to cater for under - appreciated and for emerging microbial threats . To comprehensively address such issues, detailed analysis of the proteome that arise during this morphological transition will enable identification and characterization of key proteins that are responsible for this vbnc state . In addition, tandem use of microarray gene analysis along with real - time quantitative pcr will help unravel specific gene functions and will pin - point over - arching regulatory framework(s). The aforementioned studies should also be carried out in parallel with developing improved protocols for resuscitating vbnc organisms, particularly availing of advances made in fermentation technology such as exploiting chemostat - based bioreactor approaches that can simulate and monitor multiple environmental stresses (either applied simultaneously or sequentially) over extended time periods . Indeed, kooi and coworkers recently exploited use of a chemostat to report on the dynamic behaviour of simple aquatic ecosystems with emphasis on nutrient recycling in order to monitor toxicants . Thus, use of the latter provides a useful vehicle for investigating the effects of deleterious microbial stressors on the structure and functioning of ecosystems . Other related studies that merit attention include the development of more rapid, user - friendly, field - based technologies that will reliably and repeatably detect and quantify various waterborne pathogens that may persist in different culture states . Less than four hours was recommended as rapid by noble and weisberg in a recent review of rapid detection methods for bacteria in recreational waters . Due to plethora of different detection methods currently being developed combined with obvious interlaboratory variability in terms of associated operating protocols, it is imperative that we develop and agree upon a standardized battery of reliable tools for detecting and quantifying culturable and nonculturable bacteria so as to establish future unified, quantitative - risk management protocols for monitoring our aquatic environments . The latter will greatly facilitate our endeavours to collectively comply with emerging environmental policies such as eu's water framework directive . Cryptosporidium species have emerged over the past decades as major waterborne pathogens causing gastroenteritis in humans . The occurrence of the environmentally resistant thick - walled oocyst stage of this organism has become a worldwide concern due to its resistance to disinfection with chlorine at concentrations typically applied in drinking water treatment plants (2 to 6 thus, the control of cryptosporidium oocysts remains a major challenge for drinking water utilities due to fact that the common chemical disinfectants - free and combined chlorine, when used singly, are practically ineffective for inactivating this protozoan under conditions encountered in most treatment facilities . This protozoan is transmitted via the faecal - oral route, where consumption of contaminated drinking water and use of recreational water are major sources of infection . Cryptosporidium oocysts are resistant to conventional disinfectants at concentrations and exposure times commonly used, and their infectious doses in humans have been estimated to be as low as 30 oocysts . Indeed, more than 160 waterborne outbreaks of cryptosporidiosis have been reported globally, with the greatest documentation occurring in the us and the uk [46, 48]. This situation has become a major concern for water authorities and consequently, significant modifications to drinking water regulations have been proposed for the detection and surveillance of this protozoan in the us and in other developed countries . The new european drinking water directive advocates that all the state members should provide drinking water supplies with absence of pathogenic organisms . However problems associated with the determination of oocysts viability / infectivity make the establishment of maximum acceptable concentrations very difficult, and concentrations of 330 oocysts/100 litres in treated water have been proposed as action levels . Recent contamination of drinking water supplies in the west of ireland have led to a significant number of confirmed cases of cryptosporidiosis, intimating that the use of conventional decontamination methods failed to eliminate this enteroparasite in treated water (cited in garvey et al . ). This situation has become a major concern for water authorities and consequently, significant modifications to drinking water regulations have been proposed for the detection and surveillance of this protozoan in the us and in other developed countries . Development of alternative methods of cryptosporidium disinfection for water applications (such as ozone and/or uv) has been hindered by the uncertainty surrounding efficacy of using in vitro surrogate viability assays due to their overestimation of oocysts survivors posttreatments and the lack of critical data on the preferred use of in vitro cell culture and/or in vivo animal - based infectivity assays to determine interrelated factors governing repeatable disinfection of oocysts suspended in water . Although recent studies that utilised at least 20 different cell lines have advocated the preferential use of the human ileocecal adenocarcinoma hct-8 cell line as an equivalent in vitro method to that of using the gold standard mouse assay for measuring infectivity of cryptosporidium, there has been limited evidence to date on the combined use of these approaches for assessing critical operational parameters governing pulsed uv light (puv) as a means of disinfecting water contaminated with this enteroparasite . This study demonstrated that there is good agreement between use of in vitro culture - qpcr and the scid - mouse - infectivity assays for evaluating the disinfection efficacy of pulsed uv for inactivating c. parvum suspended in saline, thus reducing the requirement to unnecessarily use animals for these particular research studies . However, the authors recommended that pressing investigations are needed to comprehensively demonstrate efficacy of using this novel disinfection uv approach for treating lower concentrations of infectious oocysts under dynamic conditions found in wwtps and in the aquatic environment . Development of a reliable and repeatable method of measuring fluence values from uv - delivered pulses for water disinfection applications is also required, and should take on board critical interrelated factors identified previously by bolton and linden for continuous low- (lp) and medium - pressure (mp) uv units . Development of puv has recently received attention as a potentially novel strategy for decontaminating water as it offers many benefits including rapid microbial reductions and efficiency of energy usage due to underpinning high peak - power dissipation during treatments [55, 56]. Indeed, use of ultraviolet (uv) light have become widely accepted as alternative methods to chlorination for wastewater disinfection [57, 58]. There are also over 2,000 wastewater treatment plants worldwide using either lp or mp ultra - violet technologies . Recent studies investigating continuous - use uv lamp technology has demonstrated the effectiveness of uv in inactivating pathogens in wastewater . Research has also shown that, to ensure permanent inactivation and prevent the recovery of microorganisms following exposure to uv, a broad polychromatic spectrum of uv wavelengths is necessary such as doses delivered by mp and puv systems . These wavelengths inflict irreparable damage not only on cellular dna, but on other molecules such as enzymes as well . Moreover, numerous studies have also highlighted limitations of decontamination techniques such as conventional low pressure mercury lamps designed to produce energy at 254 nm (called monochromatic or germicidal light) that include microbial repair and the necessity for lengthy durations of exposure to obtain suitable levels of decontamination . More recently, medium - pressure mercury uv lamps have been used because of their much higher germicidal uv power per unit length and because of their ability to emit polychromatic light comprising germicidal wavelengths from 200 to 300 nm). This approach kills microorganisms by using ultrashort duration pulses of an intense broadband emission spectrum that is rich in uv - c germicidal light (200 to 280 nm band). Puv is produced using techniques that multiply power manifold by storing electricity in a capacitor over relatively long times (fractions of a second) and releasing it in a short time (millionths or thousandths of a second) using sophisticated pulse compression techniques [56, 60, 61]. The emitted flash has a high peak power and usually consists of wavelengths from 200 to 1100 nm broad spectrum light enriched with shorter germicidal wavelengths [62, 63]. This technology has received several names in the scientific literature: pulsed uv light [6466], high intensity broad - spectrum pulsed light, pulsed light, intense pulsed light, and pulsed white light . Despite the fact that uv light appears effective for inactivating waterborne enteropathogens, including cryptosporidium spp . It is recognised that many organisms have mechanisms for repairing light - induced dna damage . However, rochelle et al . Recently reported that cryptosporidium spp . Oocysts could neither repair uv light - induced damage nor regain infectivity under standard conditions used for storage and distribution of treated drinking water . This is despite the fact that both c. parvum and c. hominis were shown to harbour genes encoding uv repair proteins for mechanisms such as nucleotide excision repair and photolyase enzymes . Indeed, irradiated oocysts were unable to regain preirradiation levels of infectivity, following exposure to broad array of potential repair conditions, such as prolonged incubation, preinfection excystation triggers, and post - uv holding periods . . Reported that adaptive microbial survival (tailing phenomenon) occurs when samples are treated in high turbidity solutions using continuous uv sources whereas tailing did not occur when similar samples were treated with pulsed xenon lamp . In terms of potential future alleviation strategies for combating c. parvum in water, it is likely the combined use mp and puv technologies would offer considerable advantages as the next - generation decontamination bolt - on approach including rapid processing and efficiency of oocyst destruction, and should accommodate dynamic operational requirements at wwtp level . Use of ozone is gaining in popularity as an alternative or complementary approach for disinfection in drinking water facilities worldwide . Some landmark studies have recently reported on the possible efficacy of using ozone for destroying cryptosporidium oocysts . However, there are very limited published findings to date that holistically investigate critical factors governing the effective and repeatable destruction of this recalcitrant enteroparasite in drinking water supplies using ozone or other oxidative agents . Of those researchers that have reported on this complex oxidation process, it would appear that using ozone singly or combined with (or without) free chlorine or monochloramine (in addition to other synergistic factors such as disinfection concentration, contact time, dissolved organic content concentration, ph, and temperature) are of critical importance for the inactivation of treated c. parvum [72, 73]. Despite limited understanding of the dynamic complexities underpinning advanced oxidation processes, ozonation is often preferred to chlorination because former leads to smaller concentrations of potentially harmful halogenated disinfection by - products . Yargeau and leclair also recently reported that use of ozone appears to be a promising technique for degradation of antibiotics, even in wastewater . After 4.5 min of ozone treatment, the concentration of sulphamethoxazole was below the hplc detection limit of 0.6 mgl indicating a degradation efficiency higher than 99.24% . Intense research has been recently focused on the development of novel pulsed plasma gas discharge technology as a complementary means of treating water containing unwanted microbial pathogens and chemicals . Pulsed plasma gas - discharge (ppgd) technology involves applying high voltage pulses to gas - injected test liquids resulting in the formation of a plasma that causes free radicals such as dissolved ozone and hydrogen peroxide, free electrons, ultraviolet light (uv), acoustic shock waves, and electric fields at levels between 1050 kv / cm to be generated in the test liquids (figure 1). Rowan and coworkers has been shown that application of ppgd successfully reduces unwanted campylobacter and salmonella spp . In poultry wash water . Ppgd (akin to puv) is an enabling technology that requires transient generation of high voltage and high current that in turn results in the generation of large peak powers ranging from megawatts to terrawatts . Depending upon the application (ppgd or puv), a pulse generator will deliver a large energy level on a single shot basis or alternatively will deliver a modest amount of energy (110 j) at a repetition rate from 10 to 10,000 pulses per second . Thus ppgd offers a radical new approach to energy delivery that involves the use of repetitive switching techniques to deliver stored energy in intense ultrashort bursts (85100 nanoseconds). During each pulse, very high levels of peak power are generated (1020 mw), and treatment is achieved using the required number of pulses, which is favourable in terms of requirements for new energy efficient technologies for the fast - approaching post peak oil era . Other research groups have also reported on the successful use of pulsed high voltage for decontaminating microbial populations and phenol in liquid solutions . Interestingly, research in our laboratory has shown that the myriad of biocidal properties generated by use of ppgd technology has also demonstrated promising results for the removal of prions on surgical material intimating potentially other healthcare applications for this decontamination approach . However, a nonoptimized pulsed plasma - gas discharge process may also lead to the formation of undesirable by - products such as brominated organic compounds and halogenates in treated water . Brominated organic compounds are also considered potentially harmful and remain the subject of international study in order to elucidate mechanistic and kinetic information regarding their formation during ozonation so as to identify effective strategies for their reduction or elimination . Considerable attention should be given to reducing or eliminating the occurrence of harmful by - products of ozone during this project via adjustments in sparged - gas composition, decreasing ph and complementary use of pulsed uv technology . Surprisingly, despite increased interest in the development of nonthermal advanced oxidative processes (such as corona plasma discharges, ozone combined with h2o2, low / medium pressure uv combined with h2o2, etc . ), this author has been unable to source any published reports on possible toxicological issues associated with use of these new technologies for disinfection applications in healthcare, agri - food or the environment . On a related point, our research group has recently reported on the occurrence of vbnc state for food and water - borne microorganisms generated in liquid suspensions after separate exposure to ppgd and pulse electric field technologies [6, 7]. Although antibiotics have been used for decades, only recently has an increasing number of studies highlighted the lack of understanding and knowledge about persistence of antibiotics in the aquatic environment and the potential environmental risks that this may pose . Particularly as antibiotics are often poorly degraded or removed in conventional wastewater treatment plants (wwtps) which causes formation of toxic degradation products that may impact negatively on the aquatic environment and public health . Antibiotics in the broader sense are chemotherapeutic agents that inhibit or abolish the growth of microorganisms, and have been used extensively in human and veterinary medicine as well as aquaculture for the purpose of preventing or treating microbial infections . Wise estimated antibiotic consumption worldwide to lie between 100,000 and 200,000 ton per annum . The reader is also directed to the recently published landmark reviews of kmmerer [83, 84] for further detailed information on the possible input, occurrence, fate, and effects of antibiotics in the aquatic environment . The concentrations of antibiotics in municipal sewage and in sewage treatment plants are typically lower by a factor of 100 compared to hospital effluent . While bacterial resistance to antibiotics have also been found in soil . Some studies revealed that many different types of antibiotics are biodegradable under aerobic or anaerobic conditions, while other related research has reported that certain aquatic microbes can actually use these free active compounds as a sole carbon source . However, research has also shown that the concentrations and activity spectrum of compounds found in the environment does not correlate with the presence of resistant bacteria isolated from the same environments . Concern over the development of secondary resistance to commonly - used antibiotics (either through vertical and/or horizontal gene transfer among related and unrelated bacteria) in the environment with potential for adversely affecting aquatic and terrestrial organisms along with the potential for a cyclic unwanted return to humans again via consumption of drinking water has stimulated numerous research groups to investigate these important issues . Kmmerer clearly articulated that the issue of acquired resistance was nearly always addressed in publications by describing the presence of antibiotics in the environment, with often mere speculation as to the possible relationship between low concentrations of antibiotics in water and emergence of resistance . In truth, however, the trend of accumulating and accelerating resistance to antibiotics is of concern as this is also juxtaposed by the marked reduction in mankinds' current arsenal of effective tools for combating this phenomenon . From a review of best published literature it would appear that possibly the most significant undefined risk in terms of antibiotics are microorganisms harbouring multiple antibiotic resistance genes such as vancomycin - resistant enterococci (vre), methicillin - resistant staphylococcus aureus (mrsa), and multiresistant pseudomonads that are living in close proximity to each other (such as in biofilms sewage sludge flocs) as opposed to the free active compounds present at low concentrations in the aquatic environment . Indeed, davison provided evidence that antibiotic resistance is already present in natural environments and that it can be exchanged between bacteria for at least a decade . Therefore, under intense discussion is the possibility that nutrient - poor, oxygen - limited and cold aquatic environments exemplified by sewage sludge may be selecting for and acting as a reservoir for slowing growing resistant organisms . This environment provides for a higher biodiversity of microbial types and numbers affording a greater probability for accelerated exchange of antimicrobial drug resistance and virulence determinants in the presence of other neighbouring microbes deficient in such deleterious properties . Indeed, ohlsen et al . Revealed that antibiotics even at sub - inhibitory concentrations can have an impact on cell function and can stimulate expression of quiescent virulence factors or potentiate transfer to an antibiotic resistant state . The latter is strongly aligned with findings from unrelated environmental stress studies which previously showed that many food and waterborne microbial species can sense and adapt to related and unrelated sub - lethal environmental stresses (such as osmotic stress or starvation) through complex quorum sensing and respond through an orchestrated controlled subsequence of preferential gene expression . Thus, not alone may the latter confer microbial tolerance to the applied stress through a process of elevating activities in important genes that regulate key house - keeping functions such as osmotic stress response and protection vital proteins (chaperone), but such environmental tempering may also act as stimuli for up - regulating microbial virulence factor expression . The classic example being l. monocytogenes, where its' transcriptional activator prfa up - regulates virulence factor expression in the presence of a known stress (e.g., body temperature), yet down - regulates transcription of pathogenic determinants in the presence of soil - related carbohydrates . Interestingly, zupan and raspor recently reported on the development of an invasion - agar assay for determining in vitro invasiveness of saccharomyces cerevisiae, and showed that this yeast augmented virulence when exposed to temperatures typical of human fever (37 to 39c) yet exhibited strong repression effect on invasion in the presence of salts, anoxia and some preservatives . The aforementioned also highlights the extreme versatility of microbial species present in complex communities (such as in biofilms in wwtps) to rapidly change and adapt when confronted with a sustained external selective pressure . Kmmerer stated that there is a dearth of information on the interrelated factors governing the fate and effects of antibiotics in the environment (i.e., microbial ecology), highlighting that the majority of published studies to date are limited to single compound approaches . He also recently postulated that the effects of antibiotics in wwtps or in the aquatic environment may be under - estimated, particularly as there is evidence that microbial exposure to antibiotics from the same group or from different groups may result in additive effects . For -lactams it has been shown that their potency is much higher in the presence of 5-fluorouracil, a cytotoxic compound also present in sewage in concentrations at the mg or gl range . Use of biocides such as triclosan and quaternary ammonium compounds used in hospitals and homes may also select for antibiotic resistance in microbial pathogens . Additionally, there is no published information as yet on the potential impact of low levels of other pharmaceutically active compounds (such as endocrine disrupting chemicals) on development of antibiotic resistance as the former also tolerate sewage treatment . There is also limited information on how long bacteria maintain antibiotic resistance in the absence of continued selective pressure for that resistance . That said, recent findings also suggest that bacteria which have already have become resistant through the application of antibiotics will not necessarily have a selective advantage in sewage treatment [92, 93]. Interestingly kmmerer reported that resistance was found to be high in hospital effluents and in sewage treatment plants, yet hospital effluents contribute to less than 1% of the total amount of municipal waste suggesting that hospitals are not the main source of resistant bacteria in municipal sewage . The latter would also suggest that antibiotic usage in the community accounts for the main input of resistant bacteria into municipal sewage . The question should also be posed as to whether or not multi - drug resistant bacteria that transcend to the quiescent vbnc state after periods of extended exposure to a nutrient depleted stressful environment (such as in water or in soil) are still capable of gene transfer and whether or not these important molecular determinants remain unaltered and stable . Kmmerer also reported that the concentration of antibiotics may be much higher if the active compounds are persistent and accumulate, for example, by sorption to solid surfaces such sewage sludge, sediments, or soil . While most antibiotics tested to date have not been biodegradable under aerobic conditions(kmmerer [83, 94, 95]),biodegradability has been poor for most of the compounds investigated in laboratory tests, including some to the -lactams . Out of 16 antibiotics tested, only benzyl penicillin (penicillin g) was completely mineralized in a combination test . No evidence of biodegradation for tetracycline was observed during a biodegradability test, and sorption was found to be the principal removal mechanisms for tetracycline in activated sludge . Substances extensively applied in fish farming had long half - lives in soil and sediment as reported in several investigations . However, some substances were at least partly degradable . Maki et al . Found that ampicillin, deoxycycline, oxytetracycline, and thiamphenicol were significantly degraded, while josamycin remained at initial levels . Despite the aforegoing, it has yet to be established that permanent exposure of antibiotics in sewage treatment systems promotes the development of antibiotic resistance and selective effects on bacterial communities . Studies have also revealed that bacteria that are resistant to antibiotics are present in surface water . Furthermore, antimicrobial resistance has also been found in marine bacteria and bacteria living in estuaries or coastal waters polluted with sewage water . Other researchers have reported that antibiotic resistance genes (args) can be found in a region where no selection pressure exists . Whereas pruden et al . Noted that tetracycline args tet(w) and tet(o) were present in treated drinking water and recycled wastewater, suggesting that these are potential pathways for the spread of args to and from humans . High loads of antibiotics in sediments at concentrations potent enough to inhibit the growth of bacteria have been reported for aquaculture . While antibiotic - resistant bacteria have been detected in drinking water supplies as far back as the 1980s . In fish farming sector such as aquaculture and mariculture, the widespread use of antibiotics for treating bacterial diseases has been associated with the development of resistance in a range of bacterial pathogens including aeromonas hydrophilia, aeromonas salmonicida, edwardsiella tarda, edwardsiella icttaluri, vibrio anguillarum, vibrio salmonicida, pasteurella piscida and yersinia ruckeri . There are also considerable gaps in current knowledge concerning the possible transfer of chemical contaminants and microbial pathogens (including those harbouring args) into the food chain through land spreading of some treated organic municipal and industrial material on agricultural land used for food production . The reader is directed to the comprehensive report produced recently by the food safety authority of ireland that addresses critical issues underpinning these putative concerns . Other pharmaceutically active chemicals . A marked observation from this paper is the pressing need for greater risk - based assessment and for new and/or improved alleviation strategies for the optimal decontamination of wastewater at treatment plant level so as to reduce or eliminate the microbial (and their associated metabolite) risks to public health . In the context of improving wastewater treatment and planning for safe drinking supplies, one must also acknowledge growing international concerns about the release of certain chemicals into the aquatic environment that may result in alterations in the reproductive health of humans and wildlife [108110]. The environmental presence of such man - made and naturally occurring compounds, properly referred to as endocrine disrupting chemicals (edcs), can mimic or interfere with the binding and action of natural hormones, thus disrupting normal physiological processes [111, 112]. Consumption of water contaminated with edcs may also cause reproductive disorders in humans as such chemicals have the ability to mimic the function of natural estrogens as well as disrupting the synthesis and metabolism of hormones by binding to hormonal receptors (cited in [108, 110]). Thyroid system - disrupting activity in water from municipal domestic sewage treatment plants was also detected recently .the list of known edcs is extensive and includes natural and synthetic steroid hormones (such as 17 -estradiol(e2),estrone (e1), and 17 -ethinylestradiol (ee2)), phytoextrogens, pesticides, pharmaceuticals, and surfactants, all of which have been detected worldwide in processed water from domestic treatment plants (wwtps) at the ng / l level that may cause abnormalities to aquatic organisms [113, 114]. Edcs were recently detected in effluents from wwtps and from receiving waters at levels exceeding ng / l in ireland . Recent studies conducted by fogarty and mcgee have demonstrated that fish habituating downstream of waste - water treatment plants (wwtps) in the midlands region in ireland exhibited delayed spermatogenesis compared with fish upstream and intersex (feminization) was discovered in roach [115117]. The scientific community has particularly focused on estrogenic edcs (i.e., compounds interacting with the human estrogen receptor), which enter the environment from a variety of sources including effluent discharge pipes, agricultural runoff and landfills . In particular, such edcs are considered to be dominant contributors to estrogenic activity in treated water from wwtps and have been found in treated effluent at the ng / l level [113, 118]. Whilst intensive conventional - based treatment approaches have investigated many interrelated factors in the design and operating conditions of wwtps for reduction or elimination of edcs in treated wastewater, none have reported on their effective removal . Moreover, due to the common practice of residual chlorine in drinking water distribution, halongated by - products such as haloacetic acids and trihalomethanes form that exhibit a range of potential carcinogenic potentials (cited in). The european commission (com 706) acknowledged that there is an urgent need for further scientific research to denature deconjugated edcs in the environment . Therefore, it is essential that such compounds be efficiently and effectively removed from processed water discharged from wwtps . On a similar theme, yamamoto and coworkers recently reported the persistence of 8 pharmaceuticals with relatively high ecological risk and high consumption (namely acetaminophen, atenolol, carbamazepine, ibuprofen, ifenprodil, indomethacin, metenamicacid, and propranolol) using river water . Previous researchers have recently shown that use of similar non - thermal corona discharge processes such as the ppgd technology may be potentially effective at destroying structurally - related organic compounds such as dyes, phenol and aniline in aqueous solutions [121125]. Other nonplasma related studies have shown that the use of uva can also facilitate removal of edcs including e1, e2, and ee2 from water by photolysis [123, 126]. Given that the majority of known edcs are phenolic in chemical structure, the benzene rings underpinning these edcs should be rapidly photodegraded by hydroxyl radical attack during ppgd treatments . It is worth noting that the use of ozone become widely accepted as alternative methods to chlorination for wastewater disinfection [57, 58], and ozone has traditionally been applied in drinking water treatment plants for disinfection . While other researchers have demonstrated the production of short - lived, high - oxidative, plasmochemical elements in pulsed - plasma - treated test liquids such as water and effluent for bacterial decontamination, no study to date has reported on the use of corona discharges singly or combined with pulsed uv light as a novel approach for the destruction of established and emerging microbial threats to public health in water or effluents . These dynamic environmental fate studies must be carried out in tandem with experiments that ascertain the likelihood of by - product toxicant formation and unwanted persistence in water, and address these associated undefined risks by either adjusting existing and/or implementing new complementary alleviation strategies . There is a pressing need to generate critical data in gaps highlighted above via provision of funding to establish consortia of meaningful stakeholders comprising scientists, engineers, and end - users in order to facilitate policy makers and implementers (suchaswater managers) strive towards meeting ambitious objectives set for the eu's water framework directive that requires member states to attain a good ecological status for all water bodies by 2015 . While a global response to emerging environmental problems has been positive with the provision of substantial research funding been made available to the broad and diverse researcher communities (such as eu fp7 initiatives), one obvious real challenge that still remains is how does mankind harness and channel vast amounts of relevant sophisticated data that is produced and disseminated from a multiplicity of research groups worldwide into meaningful streamlined forums that can be shared and understood by all in a timely fashion . Exploiting advances in information technology will assist greatly with this challenge and will drive effective risk - based assessment, evaluation, and management of present and emerging problems for the aquatic environment . Currently, there is insufficient information available to reach definitive conclusion on the significance and impact of the vbnc state organisms and antibiotic resistant bacteria in the environment which would allow for the assessment of the potential risk related, for instance, to human health and ecosystem functions . Indeed impact of antibiotics present in the aquatic environment on the frequency of resistance transfer is questionable, with greater concern placed on the input of resistant bacteria into the environment from different sources . This suggests that greater emphasis should be placed on reducing the likelihood of antibiotic resistance occurrence in the first instance at the point of source by prudent management and careful rotation of antibiotic usage in both the hospital and community settings and by systematic continued monitoring of resistance, which will collectively impact positively on public health and wellbeing . The lack of understanding and knowledge surrounding a broad range of established and emerging risks to the aquatic environment is apparent . For example, mena and gerba recently reviewed best published data for risk assessing the opportunist pathogen p. aeruginosa in water and concluded that process of estimating risk is currently significantly constrained because of the absence of specific (quantitative) occurrence data for pseudomonads . In order to gain a greater appreciation and understanding of the critical environmental fate and associated impact of such undefined and variable microbial risks, more controlled laboratory investigations are needed to be undertaken in combination with conducting field studies . There is also pressing need to obtain definitive data on proposed risk to public health and to the aquatic environment combined with developing appropriate short- and long - term mathematical and computer models with capacity for monitoring and predicting ecotoxicological effects of these microbial stressors, particularly under dynamic naturalistic settings akin to those recently described by bontje and coworkers and gevaert et al . It is quite apparent that proper judgement of the impact of microbial pathogens, their metabolites, and pharmaceutically active compounds require a thorough evaluation of their risk and hazard . Regarding the latter, where risk is normally expressed as the ratio between the predicted environmental concentration of the active ingredient (ai) and its predicted no - effect concentration . Hazard is expressed in terms of ai's persistence, potential for bioaccumulation, and ecotoxicity . The combined use of monitoring data aligned with development and application of dynamic pollutant (contaminant) fate models is recommended . Pharmaceutical producers should also highlight environmental precaution when designing new more environmental friendly ais, and that the environmental data should be transparent to the general public . In terms of holistic monitoring and prediction, we must also factor in seasonal environmental changes such as atmospheric and oceanic processes that occur in response to increasing greenhouse gases to map disease potentiation dynamics as this will aid development of appropriate strategies for controlling microbial risks across a range of human and natural systems . Indeed, sedas recently showed that climate also influences the abundance and ecology of pathogens, and the links between pathogens and changing ocean conditions, including human disease such as cholera . It is recognised that environmental risk - based management is typically uncertain due to different perceptions of the risk problem and our limited knowledge about the inter - play of biological, physical and chemical processes underlying these risks . While a plethora of real - time quantitative microbial detection tools combined with more rapid and efficient decontamination approaches are on the horizon, we must be equally prudent about exhaustively confirming their efficacy such as agreed standards for sensitivity and reliability for detection, and eco - friendliness for decontamination . It is clear and apparent that the emergence of such complex problems has heralded a new dawn in research and innovation (i.e., a marked departure from the solitary phd student environment), where collection, analysis and timely dissemination of such vast and meaningful information can only be effectively addressed using a well - managed consortia of networked researchers from various complementary disciplines by way of using a plethora of appropriate frontier funding initiatives such those offered by the eu (http://cordis.europa.eu/fp7/home.html). While it is recognised that undertaking such far - reaching cross - boundary initiatives will enable the potential impact, implications and future proofing of established and emerging risks to be managed and catered for properly . Is must be equally recognised that effectively managing and harnessing the potential of such diverse consortia comprising academics, industrial partners (smes to multinationals), policy makers and so forth pose significant logistic and complex challenges, for example, attempting to holistically cater for all stakeholders in a united global society on a single theme who have different needs and goals are real challenges . Therefore, such important issues must also be managed with a strong over - arching foresight, particularly in the context of embracing and exploiting advances in the communication and information technology landscape so that we can accommodate and provide for the real - time flow of knowledge to all stakeholders in order to identify potential synergies, emerging trends (problematic, beneficial or otherwise), and/or opportunities.
All animal procedures were performed in a facility accredited by the association for assessment and accreditation of laboratory animal care international in accordance with protocols approved by ut austin (#2012 - 00084, #13080701) and the university of oklahoma health sciences center (#14 - 072-i) animal care and use committee and the principles outlined in the guide for the care and use of laboratory animals . A total of 15 baboons between six and nine months old were infected intra - nasally and intra - tracheally with 1010 cfu b. pertussis strain d420 on day 0 . Of these, eight animals were treated with human anti - ptx antibodies on day 2/3 (denoted t1-t8), while the remaining seven were untreated controls (denoted c1-c7). Serial dilutions of nasopharyngeal washes were plated on selective regan - lowe agar plates supplemented with cephalexin (40 g / ml). The specific details of procedures performed at the oklahoma baboon research resource at the university of oklahoma health sciences center have been described previously . Microbial identification was performed by idexx (westbrook, me, u.s.a .) Using maldi - tof for a preliminary identification based largely on 16s ribosomal rna followed by a panel of standard microbiological tests, including gram stain, lactose fermentation on macconkey agar, urease and oxidase activity and the triple sugar iron test . Elisa was used to detect and quantify antibodies binding fha, los and ptx antigens from b. pertussis (list labs, campbell, ca, u.s.a .) As described . Curves were fit using a 4-parameter logistic model and compared to a reference monoclonal human antibody or high titer baboon serum run on each plate . The reference was standardized by comparison to the who reference reagent pertussis antiserum 06/142 (nibsc, potters bar, hertfordshire, u.k . ), and serum antibody concentrations converted to iu / ml . Day 0 serum anti - fha titers were log - transformed, and the values for baboons in the typical and atypical groups were found to be statistically different with a p - value of <0.001 in a t - test . For los elisas, plates were coated with 5 g / ml los, with results reported as raw absorbance values in the dose - response regime at a serum dilution of 1:625 . All elisas were performed in duplicate with standard deviations of the measurements reported as error . All animal procedures were performed in a facility accredited by the association for assessment and accreditation of laboratory animal care international in accordance with protocols approved by ut austin (#2012 - 00084, #13080701) and the university of oklahoma health sciences center (#14 - 072-i) animal care and use committee and the principles outlined in the guide for the care and use of laboratory animals . A total of 15 baboons between six and nine months old were infected intra - nasally and intra - tracheally with 1010 cfu b. pertussis strain d420 on day 0 . Of these, eight animals were treated with human anti - ptx antibodies on day 2/3 (denoted t1-t8), while the remaining seven were untreated controls (denoted c1-c7). Serial dilutions of nasopharyngeal washes were plated on selective regan - lowe agar plates supplemented with cephalexin (40 g / ml). The specific details of procedures performed at the oklahoma baboon research resource at the university of oklahoma health sciences center have been described previously . Microbial identification was performed by idexx (westbrook, me, u.s.a .) Using maldi - tof for a preliminary identification based largely on 16s ribosomal rna followed by a panel of standard microbiological tests, including gram stain, lactose fermentation on macconkey agar, urease and oxidase activity and the triple sugar iron test . Elisa was used to detect and quantify antibodies binding fha, los and ptx antigens from b. pertussis (list labs, campbell, ca, u.s.a .) As described . Curves were fit using a 4-parameter logistic model and compared to a reference monoclonal human antibody or high titer baboon serum run on each plate . The reference was standardized by comparison to the who reference reagent pertussis antiserum 06/142 (nibsc, potters bar, hertfordshire, u.k . ), and serum antibody concentrations converted to iu / ml . Day 0 serum anti - fha titers were log - transformed, and the values for baboons in the typical and atypical groups were found to be statistically different with a p - value of <0.001 in a t - test . For los elisas, plates were coated with 5 g / ml los, with results reported as raw absorbance values in the dose - response regime at a serum dilution of 1:625 . All elisas were performed in duplicate with standard deviations of the measurements reported as error . During a series of studies designed to assess the potential for passive immunization to treat pertussis, several baboons had unexpectedly variable wbc and bacterial colonization levels 2-to-3 days after inoculation with b. pertussis (fig . 1afig . 1.serum antibody levels indicate susceptibility to pertussis infection . A: wbc and b. pertussis colonization levels were measured on day 2 or 3 after experimental infection . Levels typical of the baboon model on day 2/3 are shown for wbc (> 13,000/l; solid line) and colonization (> 10/ml, dashed line). Baboons exhibiting expected symptoms fell into the upper right quadrant, while baboons that appeared to be protected from disease fell into the lower left quadrant . B: the day 9/10 los elisa absorbance values correlated with the log - transformed day 9/10 fha antibody responses with a pearson correlation coefficient of 0.90 . Animals exhibiting low values in both assays were classified as nave, while animals exhibiting high values in both assays were classified as previously exposed . C: anti - fha antibody sera concentrations collected on day 0 correlated with typical and atypical model responses; * * * p<0.001 in a t - test of the log - transformed concentration values . Typical responses include high levels of colonization, a marked wbc rise and lack of a secondary antibody response . Shown are responses for animals without b. bronchiseptica co - infection; bars indicate geometric means . ). Six of 15 animals exhibited typical responses for this model: namely, a rapidly rising wbc and high bacterial colonization level on day 2/3 . However, six animals had low wbc and colonization, one was heavily colonized but had a low wbc count, and two were not heavily colonized but had a two - to - four - fold wbc rise . Additionally, nasopharyngeal wash samples from four baboons (c2, c3, c4 and t5) yielded rapidly growing colonies apparent one day after plating on regan - lowe media selective for bordetella species and overgrew bordetella pertussis colonies appearing one to three days later . Serum antibody levels indicate susceptibility to pertussis infection . A: wbc and b. pertussis colonization levels were measured on day 2 or 3 after experimental infection . Levels typical of the baboon model on day 2/3 are shown for wbc (> 13,000/l; solid line) and colonization (> 10/ml, dashed line). Baboons exhibiting expected symptoms fell into the upper right quadrant, while baboons that appeared to be protected from disease fell into the lower left quadrant . B: the day 9/10 los elisa absorbance values correlated with the log - transformed day 9/10 fha antibody responses with a pearson correlation coefficient of 0.90 . Animals exhibiting low values in both assays were classified as nave, while animals exhibiting high values in both assays were classified as previously exposed . C: anti - fha antibody sera concentrations collected on day 0 correlated with typical and atypical model responses; * * * p<0.001 in a t - test of the log - transformed concentration values . Typical responses include high levels of colonization, a marked wbc rise and lack of a secondary antibody response . Shown are responses for animals without b. bronchiseptica co - infection; bars indicate geometric means . Cultures of the rapidly growing bacteria from animal c4 were positively identified as b. bronchiseptica . Maldi - tof was used to make a preliminary species identification, followed by a panel of biochemical tests to confirm the bordetella assignment and discriminate between b. bronchiseptica and b. pertussis . The results showed that the cultured organism was gram negative, did not ferment lactose on macconkey agar, did not ferment lactose, sucrose or dextrose but catabolized peptone in the triple sugar iron test (k / k result) and was positive for oxidase and urease . Notably, b. bronchiseptica but not b. pertussis is urease - positive, and only b. bronchiseptica has flagella and is motile . While not definitively confirmed, the other three animals were strongly suspected of b. bronchiseptica coinfection by virtue of similar culture characteristics . In all, nine of 15 baboons behaved aberrantly in this model (table 1table 1.baboon responses to experimental pertussis infectionbaboonactive b. br infection anti - fha titer>100 iu / mlanti - los absorbance> 0.1 wbc <13,000/lcfu <10/mlresponse to pertussis infection c1 - ----typicalc2yes----excludedc3yes--yes - excludedc4yesyesyesyesyesexcludedc5-yesyesyesyesatypicalc6 - ----typicalc7 - ----typicalt1-yesyesyesyesatypicalt2-yesyesyesyesatypicalt3-yesyesyesyesatypicalt4-yesyesyesyesatypicalt5yesyesyes - yesexcludedt6-yesyes - yesatypicalt7 - ----typicalt8 - ----typicala) b. br = b. bronchiseptica; -animal did not exhibit this behavior; = b . B) anti - fha and anti - los titers were measured in serum samples collected on day 9/10 after experimental pertussis infection . D) typical baboon responses to experimental pertussis infection are defined by wbc> 13,000 /l, cfu> 106/ml on day 2/3 and low fha and los serum titers; animals with known or suspected b. bronchiseptica infection were excluded from this classification ., supplementary table 1). A) b. br = b. bronchiseptica; -animal did not exhibit this behavior; * = b . B) anti - fha and anti - los titers were measured in serum samples collected on day 9/10 after experimental pertussis infection . D) typical baboon responses to experimental pertussis infection are defined by wbc> 13,000 /l, cfu> 106/ml on day 2/3 and low fha and los serum titers; animals with known or suspected b. bronchiseptica infection were excluded from this classification . With compelling evidence of an active b. bronchiseptica infection in several animals, we sought to determine whether any other baboons in this study had been previously exposed to the organism . This question was addressed by examining antibody titers to fha and los prior to infection and the kinetics by which those titers rose after infection . Specifically, high titers before pertussis infection and a rapid rise after infection indicate a secondary immune response and strongly suggest prior b. bronchiseptica exposure . Prior experiments indicated that nave baboons do not exhibit primary responses to fha until after day 12, while secondary responses are detectable on day five [9, 13]. Thus, a high antibody titer on day 9/10 to fha indicates prior exposure to any bordetella species, while a similar response against los indicates prior exposure to either b. bronchiseptica or b. pertussis . Detection of antibodies against ptx at day 9/10 was used to identify prior exposure to b. pertussis as opposed to b. bronchiseptica, which does not express this antigen due to mutations in the promoter . Accordingly, serum titers against fha and los on day 9/10 were determined for each baboon serum sample . Comparison of the resulting anti - fha and anti - los serum titers revealed two distinct groups: seven baboons with low anti - fha and low anti - los titers and eight baboons with high titers for both bordetella antigens (pearson correlation 0.90; fig . Next, anti - fha titers were determined for all time points, as these responses were stronger than those recognizing los . Animals with high anti - fha titers on day 9/10 also exhibited high titers early in the experiment, and these remained high for the ~21 day experiment (supplementary fig . Six of the seven baboons with no apparent secondary response had rising anti - fha titers starting around day 16 or later, indicative of a primary response to the experimental infection . One baboon, c2, had no response at day 9/10, but a very strong response starting at day 13 . Rapidly growing bordetella colonies were recovered from this animal on day 9, suggestive of b. bronchiseptica co - infection . Only the seven untreated baboons could be analyzed for the presence of anti - ptx igg on day 9/10 due to interference from humanized anti - ptx antibodies administered to the treated baboons . One baboon, c5, was positive for anti - ptx antibodies as well as anti - fha and anti - bp los antibodies on day 9/10 (supplementary table 1, supplementary fig . C5 had a colony count of 5 cfu / ml on day 2/3 and no wbc rise, indicating near complete protection from b. pertussis colonization . While we cannot exclude the possibility that this baboon was exposed to b. bronchiseptica as well, this result indicates that c5 had likely been previously exposed to b. pertussis . We next aimed to develop a quantitative description of typical versus atypical baboon responses to pertussis infection . Published data [14, 16] indicate typical responses on day 2/3 include bacterial colonization> 10 cfu / ml, an increase in wbc count to> 13,000/l and the absence of secondary immune responses to bordetella antigens . Atypical responses do not meet one of more of these metrics (table 1). Baboons with confirmed or suspected active b. bronchiseptica infection (c2, c3, c4 and t5) were excluded from this analysis to avoid potential confounding factors, such as competition with b. pertussis, unpredictable wbc rise and unpredictable timing of the secondary antibody response . For future studies, a predictive test to exclude baboons likely to exhibit atypical responses to b. pertussis infection thus, the presence of serum anti - fha antibodies on day 0 was evaluated as a potential screening tool . When baboons with active b. bronchiseptica infections were removed from analysis, anti - fha antibody presence on day 0 was significantly predictive of the baboon s subsequent responses (p<0.001; fig . The baboon model of pertussis brings substantial improvements over previous models and has generated considerable enthusiasm due to its similarity to human disease . Unfortunately, the majority of baboons (nine of 15) in this study did not develop symptoms as expected (fig . 1a). In an effort to improve the utility of this model, we aimed to understand why some baboons varied in their response to b. pertussis infection . Three baboons (c3, c4 and t5) were likely infected with b. bronchiseptica when the study began and were partially protected against b. pertussis infection . Prior studies in mice have shown that exposure to an engineered b. pertussis strain can protect against subsequent b. bronchiseptica infection; conversely, an engineered b. bronchiseptica strain can protect against b. pertussis infection . There are numerous overlapping antigens between these subspecies which are likely responsible for cross - protection . Specifically, anti - los and anti - pertactin antibodies are often bactericidal and importantly, they would be expected to have the substantial impact on subsequent b. pertussis infection levels and symptoms caused by infection observed in this study . Additionally, baboon c2 was cohoused with b. bronchiseptica infected baboon c3 and appears to have been infected during the study, as this organism was not observed on plates until day 9 . An additional six animals were suspected of prior b. bronchiseptica or b. pertussis exposure, based on antibody profiles indicative of secondary responses after experimental b. pertussis infection (fig . These animals all exhibited lower than expected colonization levels either with or without a suppressed wbc rise . While informative retrospectively, these data cannot be used to screen baboons prior to initiating an experiment . To this end, day 0 anti - fha antibody concentrations were predictive of the baboons subsequent response to infection, although these exhibited greater variability than day 9/10 titers (fig . Because fha is a shared antigen present in b. pertussis, b. parapertussis and b. bronchiseptica, it is particularly useful as a simple screen to rule out confounding prior exposures for studies involving b. pertussis infection . While prior bordetella exposure complicates scientific studies, it further supports the baboon model s relevance to human disease . In humans, whooping cough in humans can be difficult to diagnose, because of widely varying infection severity and clinical manifestations which depend on the patient s prior exposures to b. pertussis and other microbes with overlapping antigens and timing of that exposure, exact age and level of general health . These factors may play a similar role in the diversity of the baboon responses seen here and must be carefully controlled . In comparison to other model animals used for pertussis, baboons are typically housed with access to the outdoors where small rodents, such as rabbits or mice, for whom b. bronchiseptica is endemic could pass disease to the research animals . In our hands, commercial baboon serum (sigma) had a relatively high anti - fha antibody concentration of ~90 iu / ml (supplementary table 1), indicating that bordetella infections are likely common in baboon colonies . The well - documented, highly communicable nature of both b. pertussis infection in baboons and b. bronchiseptica infection in other mammals requires extremely careful handling, containment and observation of baboons involved in pertussis studies . The university of oklahoma health science center is home to the only specific pathogen free indoor baboon colony that was the source of baboons c7, t7 and t8 . These three specific pathogen free baboons responded to infection with b. pertussis as expected and were typical of the model . In our ongoing work, neonatal baboons from the specific pathogen free colony are pre - screened for anti - fha titer and consistently fall below 5 iu / ml at the time of infection and exhibit high colonization as expected (data not shown). To maximize the impact of pertussis experiments in baboons, we recommend the following baboon exclusion criteria: (1) clinical evidence of illness, (2) b. bronchiseptica - culture positive nasopharyngeal wash and (3) anti - fha titers> 5 iu / ml indicating prior exposure to bordetella sp . With close attention to these factors, the baboon model of pertussis is poised to be an important new tool in development of next - generation pertussis vaccines and therapeutics . Funding . This work was supported by national institutes of health [ai066239 and ai122753 to j.a.m . And p40od010431 and p40od010988 to r.f.w .] And synthetic biologics [j.a.m . ]. Conflicts of interest . This work was supported in part by funding from synthetic biologics.
To develop methods for mapping a qtl to a phylogenetic tree, we begin with several simplifying assumptions: the taxa are represented by inbred lines, the tree relating the taxa is known without error, the quantitative trait of interest is affected by a single diallelic qtl, and there are no background effects (i.e., the effect of the qtl is the same in the different crosses in which it is segregating). We consider the case of intercrosses among pairs of taxa, consider only autosomal loci, and assume a common genetic map . The basic idea, illustrated in figure 1, is that each possible location for the origin of a diallelic qtl on the tree corresponds to a different partition of the taxa into two groups, with the two groups corresponding to the two qtl alleles . For different partitions, the qtl will segregate in different sets of crosses . In the case of very large crosses, with each having high power to detect the qtl, if present, we could simply consider the crosses individually and use the pattern of presence / absence of qtl to identify the correct partition of the taxa . Note that one does not need data on all possible crosses . For the case illustrated in figure 1, with four taxa, it would be sufficient to consider the crosses a b, a c, and b d, as with just these three crosses, the five possible partitions have distinct patterns of presence / absence of the qtl . In the following, we focus on partitions of the taxa into two groups, in place of locations of the qtl on the tree . Illustration of the basic concepts behind the mapping of a qtl to a phylogenetic tree . On the left the locations of possible origins of a diallelic qtl are indicated by the numbers 15 . In the table on the right, we indicate the presence or absence of a qtl in each of the six possible crosses among pairs of taxa, according to the location of the qtl on the tree . Each possible qtl location on the tree corresponds to a partition of the taxa into two groups . Given limited resources and crosses of limited size, there will be incomplete power to detect the qtl in a given cross, and so the naive approach based on the presence or absence of the qtl in the different crosses will likely be misleading . A more formal approach, in which the likelihoods for the different possible partitions are evaluated and compared, will provide a clear assessment of the evidence for the different locations for the qtl on the tree . Consider a particular location in the genome as the site of a putative qtl, and consider a particular partition of the taxa into two qtl alleles . We assume a linear model with normally distributed errorsyij=i+aij+dij+ij, where yij is the phenotype for individual j in cross i, i the average phenotype in cross i, and are the additive and dominance effects of the qtl, respectively, and the ij are independent and identically distributed normal (0,). The aij and dij denote encodings of the qtl genotypes, with aij = dij = 0 if the qtl is not segregating in cross i. for convenience, we call the two qtl alleles defined by the partition as the high allele (h) and the low allele (l), although we wo nt actually constrain the high allele to increase the phenotype . If the qtl is segregating in cross i, then we take aij = 1, 0, or + 1, if individual j has qtl genotype gij = ll, hl, or hh, respectively, and dij = 1 if individual j has qtl genotype hl and dij = 0 otherwise . For most putative qtl locations, the qtl genotypes are not be observed, but we may calculate (e.g., by a hidden markov model) the conditional probabilities of the qtl genotypes given the available multipoint marker genotype data, pijk = pr(gij = k\|mij). It is critical that we have a common map for the set of crosses, so that a putative qtl location is clearly defined in all crosses . It is not necessary, however, that the same markers be used in all crosses or that they be informative in all crosses . We may then use standard interval mapping (lander and botstein 1989) or an approximation such as haley knott regression (haley and knott 1992) to fit the model, estimate the parameters i,,, and, and calculate a lod score, lod(), where denotes the partition of the taxa and denotes the location of the putative qtl . The lod score is the log10 likelihood comparing the hypothesis of a single qtl at that location to the null hypothesis of no qtl but with the multiple crosses allowed to have separate phenotypic means, that is, yij normal (i,). (2005), in that one recodes the genotypes in the crosses in which the qtl is segregating, stacks them on top of one another, as if they were a single intercross, and performs interval mapping with cross indicators as additive covariates . The only difference is that we are considering all possible partitions of the taxa, while li et al . There is one technicality: the crosses in which the qtl does not segregate also need to be included in the likelihood, and they contribute to the estimate of the residual variance . We thus consider each possible partition,, one at a time, and scan the genome to obtain a set of lod curves, lod(). We summarize these at the chromosome level, calculating the maximum lod score for partition on chromosome i, mi = maxi lod(). The maximum on chromosome i, maxmi, indicates the evidence for a qtl on chromosome i. to evaluate the relative support of the different partitions, we use an approximate bayes procedure . Assuming the presence of a single diallelic qtl on chromosome i, we assign equal prior probabilities to the different possible partitions,, treat the profile log likelihoods mi (in which we have maximized over all nuisance parameters, including the location of the qtl on the chromosome) as if they were true log likelihoods, and obtain posterior probabilities by taking 10mi and rescaling so that they sum to 1 . That is, we further use these approximate posterior probabilities to form a 95% bayesian credible set of partitions . One could assign unequal prior probabilities to the partitions, for example, based on the branch lengths in the assumed phylogenetic tree, giving more weight to longer branches . One might also use a prior on partitions that assigns greater weight to partitions induced by the tree and lesser (but nonzero) weight to the other (possibly more numerous) partitions . The 95% credible set of partitions is relevant only if there is sufficient evidence for a qtl on that chromosome . To evaluate the evidence for a qtl, we consider the maximum of the mi on chromosome i and derive a significance threshold, adjusting for the genome scan, by a stratified permutation test (churchill and doerge 1994). The permutation test is stratified in that we permute the phenotype data, relative to the genotype data, separately in each cross . For each permutation replicate, we calculate the lod curve for each possible partition and then take the maximum lod score across the genome and across partitions . The 95th percentile of these permutation results may be used as a significance threshold, or we may calculate a p - value that accounts for the search across partitions and across the genome . One may restrict the analyses to the set of partitions induced by the assumed phylogenetic tree, or one may consider all possible partitions of the taxa into two groups . For example, for the four - taxon tree in figure 1, one may consider only the five partitions that correspond to qtl locations on the tree, as in the accompanying table, or one may also consider the two additional partitions, ac\|bd and ad\|bc . The consideration of all possible partitions will be accompanied by some loss of power, particularly if there is a large number of taxa . However, the correct phylogenetic tree will seldom be known with certainty and will likely vary along the genome, particularly if the taxa are closely related . Moreover, if there is strong support for one of the partitions that is not associated with a qtl location on the assumed phylogenetic tree, one would certainly want to know this . Thus, we are inclined to always consider all possible partitions and not focus on those induced by an assumed phylogenetic tree . In this section, we address a theoretical question of considerable interest: which subsets of crosses are sufficient to identify the location of a qtl on the phylogenetic tree? With very large crosses, we can exactly determine which crosses are segregating a qtl and which are not . As discussed in the introduction for example, for the case in figure 1, if one performs only the crosses a b, a c, and a d, the ideal results perfectly discriminate among the possible locations of the qtl on the tree . However, if one performs only the crosses a b, a c, and b c, several of the possible partitions of strains exhibit the same pattern of presence / absence of qtl and so are confounded . It is useful, in considering this problem, to represent a set of crosses by a graph, with nodes corresponding to taxa and edges indicating a cross between two taxa . Three possible choices of a subset of five crosses among the six taxa are displayed in figure 2, b d . A phylogenetic tree with six taxa (a) and three possible choices of five crosses among the six taxa, with nodes denoting taxa and edges denoting crosses (b d). A sufficient condition for identifying the true partition of the strains is the use of a set of crosses that connect all of the taxa, as in figure 2b . Choose an arbitrary taxon (e.g., a) and assign it an arbitrary qtl allele . With sufficient numbers of individuals in each cross, we may determine whether the qtl is segregating in a cross, which indicates that the two taxa have different qtl alleles, or is not segregating, which indicates that the two strains have the same qtl allele . Thus, one may move between taxa connected by a cross and assign qtl alleles, and so if the set of crosses connect all of the taxa, one can assign qtl alleles to all taxa and so identify the correct partition of taxa . If the set of crosses are not connected (as in figure 2, c and d), then some partitions of taxa will be confounded . For example, for the crosses in figure 2c, the partition abc\|def will give the same set of qtl results as under the null hypothesis of no qtl . Other pairs of partitions are confounded in this example, such as ab\|cdef and abdef\|c . If one is considering all possible partitions of the taxa (and not just those induced by the tree), then graph connectivity is also a necessary condition for identifying the true partition: if the crosses do not connect all taxa there will always be some partitions that are confounded . However, if one focuses solely on those partitions induced by the tree (that is, partitions that result from a split on an edge in the tree), then it is not necessary that the crosses connect all taxa . For the pairs of partitions that are confounded with this choice of crosses, no more than one of each pair corresponds to a split on the tree in figure 2a; each possible partition induced by the tree gives a distinct set of qtl results for these crosses . Moreover, in this case one may omit any one of the three crosses, b c, b e, c e: only four crosses are necessary to distinguish among the nine partitions induced by the tree in figure 2a . That the crosses connect all taxa is a necessary and sufficient criterion to distinguish among all possible partitions, but it is not a necessary condition to distinguish among the partitions induced by the tree . Note that a cross between two taxa corresponds to a path along the tree from one leaf to another . Further, the qtl will be segregating in crosses whose paths go through the edge with the qtl, but it will not be segregating in crosses whose paths do not go through that edge . A necessary and sufficient criterion for a set of crosses to distinguish the partitions induced by the tree (i.e., to distinguish the possible locations of the qtl on the tree) is that each edge is covered by at least one cross and that no two edges appear only together . If an edge was not covered by a cross, then a qtl on that edge could not be distinguished from the null model, of no qtl . If two edges only appear together in crosses, then those two qtl locations cannot be distinguished . Note that a cross in which the qtl is segregating will limit the possible qtl locations to the edges on the corresponding path through the tree . As every pair of edges along such a path will appear separately in different crosses, we see that the specific edge containing the qtl may be identified . For n taxa (with n3), the minimal number of crosses to distinguish among all possible partitions is n1 . To distinguish among the partitions induced by the tree, the minimal number of crosses is 2n/3 (the smallest integer that is greater than 2n/3; a proof appears in the appendix). For n5, these are the same; for n6, fewer crosses are needed to distinguish among the tree partitions . As discussed in the previous section, we recommend that one not restrict oneself to the partitions induced by the tree but rather always consider all possible partitions, possibly with different prior weights . As a result, we recommend that one use, at a minimum, a set of crosses that connect all taxa . However, this is based on the assumption of a small number of taxa . If the number of taxa, n, is large, the total number of non - null partitions (2 1) will vastly exceed the number of partitions induced by the tree (2n 3), and so there is great potential advantage in focusing on the tree partitions . Of course, in practice crosses are of finite size and so one cannot identify the true partition of the taxa without some degree of uncertainty . In the next section we explore, via computer simulation, the relative performance of the proposed method with different possible choices of crosses . We begin by comparing our proposed method to the naive approach of considering the crosses individually and comparing the pattern of presence / absence of a qtl in the crosses to what is expected for different possible partitions . We then compare the performance of our approach with all possible crosses to different choices of a minimal set of crosses . We consider the case of four taxa and use of all six possible intercrosses among pairs of taxa, with 75 individuals per cross (a total sample size of 450). We consider a single autosome of length 127 cm, with markers at an approximately 10-cm spacing, and with a single diallelic qtl placed in the center of an interval between two markers, near the middle of the chromosome . The qtl alleles were assumed to act additively (that is, no dominance), and the percentage phenotypic variance explained by the qtl, in the crosses in which it was segregating, was 10% . We assumed either the partition a\|bcd or ab\|cd; other possible partitions are equivalent to one of these . To reduce computation time, we used haley knott regression (haley and knott 1992) for all simulation studies, with lod score calculations performed on a 1-cm grid . For the naive approach, we applied a given significance threshold and inferred the presence or absence of a qtl in a cross if the maximum lod score on the chromosome was above or below the threshold, respectively . If the presence / absence pattern matched that for a possible partition, that partition was inferred . For the proposed approach, we applied a given significance threshold on maxm and then formed a 95% bayesian credible set of partitions, using equal prior probabilities on all seven possible partitions . If maxm was greater than the threshold but the 95% credible set did not contain the truth, the result was considered a false positive . The results, based on 10,000 simulations, are displayed in figure 3 as receiver operating characteristic (roc) curves: the power (the rate of true positives) vs. the false positive rate, for varying significance thresholds . We display two sets of curves for the proposed method: for the dashed curves, the power indicates that maxm exceeded the threshold and the true partition was contained within the 95% credible set; the dotted curves are more stringent and require that the credible set contained only the true partition . Points are plotted at the results with a nominal 5% significance threshold, adjusting for an autosomal genome scan, with the genome modeled after the mouse and the thresholds estimated by 10,000 simulations under the null hypothesis of no qtl . (the estimated thresholds are displayed in supporting information, table s1 and table s2 .) Estimated receiver operating characteristic (roc) curves for the naive method (solid curves), the proposed method, with power indicating that the true partition is contained within the 95% credible set (dashed curves), and the proposed method, with power indicating that the 95% credible set contains only the true partition (dotted curves), in the case of four taxa, with each of the six possible intercrosses having a sample size of 75, and a qtl responsible for 10% of the phenotypic variance in the crosses in which it is segregating . The red and blue curves correspond to the case that the true partition is a\|bcd and ab\|cd, respectively . The roc curves for the naive method form interesting shapes, with the lower part of each corresponding to low thresholds and the upper part corresponding to high thresholds, and indicate terrible performance: the false positive rate is well controlled, but power is low . The problem is that, with only moderate power to detect the qtl in a given cross, one has low power to detect the qtl in all of the crosses in which it is segregating, which is necessary to identify the correct partition of the taxa . Lowering the significance threshold below the 5% level helps somewhat, but the power to detect the true partition is no higher than 21% . The naive approach might actually perform better if one considered a smaller set of crosses, but we have not explored this further . The proposed method performs reasonably well, and the false positive rate is well controlled at the nominal 5% significance threshold (the points in figure 3). Lowering the threshold could give some improvement in power while maintaining the false - positive rate below the target level, at least in the simulated situations . In the previous section, we noted that it is not necessary to use all possible crosses among taxa . To distinguish among all possible partitions, one need sets of crosses that connect all taxa and are of minimal size (i.e., n1 crosses for n taxa) are called minimal sets . We now turn to the question of whether it is better to use all crosses, with a smaller number of individuals per cross, or a minimal set of crosses, with a larger number of individuals per cross . We use the same general settings as for the simulations comparing the proposed method to the naive approach, with four taxa and the true partition being either a\|bcd or ab\|cd, but here we vary the total sample size among 300, 450, and 600 individuals, and we vary the percentage phenotype variance explained by the qtl from 2.5 to 15% . We consider either all six crosses or a minimal set of three crosses, and we consider all 16 choices of three crosses that include all four taxa . We also compared the consideration of all seven possible partitions, or just the five partitions induced by the tree in figure 1 . We estimated 5% genome - wide significance thresholds by simulations under the null hypothesis of no qtl (see table s2). Figure 4 displays the simulation results, as a function of the effect of the qtl, for the case that the total sample size was 450 (i.e., 75 individuals per cross when considering all crosses and 150 individuals per cross when considering a minimal set of three crosses) and when all possible partitions were considered . The results with other sample sizes and with analysis restricted to the five partitions induced by the tree in figure 1 are shown in figure s1, figure s2, figure s3, figure s4, figure s5, and figure s6 . The top of each figure indicates the power (the chance that maxm exceeded its threshold and the true partition was contained in the 95% credible set); the middle indicates the exact power (the chance that maxm exceeded its threshold and that the credible set contained only the true partition); the bottom indicates the false positive rate . The left and right correspond to the true partition being a\|bcd or ab\|cd, respectively . The black dashed curves correspond to the use of all six possible crosses; the solid curves correspond to the different choices of a minimal set of three crosses, with blue, red, and green corresponding to cases in which 3, 2, or 1 of the crosses are segregating a qtl . Estimated power (top), exact power (middle), and false - positive rates (bottom) in the case of four taxa with a total sample size of 450, as a function of the percentage phenotypic variance explained by the qtl . The other curves are for the various choices of a minimal set of three crosses, with the curves in blue, red, and green corresponding to cases in which three, two, and one of the crosses are segregating the qtl, respectively . The results are based on 10,000 simulation replicates, with analyses considering all possible partitions of the taxa . In choosing among the possible minimal sets of crosses, power is highest when a larger number of crosses are segregating the qtl . For a fixed total sample size, the use of all possible crosses (with fewer individuals per cross) has better performance than the worst of the possible minimal sets of crosses, but is not as good as the best of the possible minimal sets of crosses . The use of all possible crosses has greater power when the true partition is ab\|cd (in which case four of the six crosses are segregating the qtl) than when the true partition is a\|bcd (in which case three of the six crosses are segregating the qtl). The false - positive rate (figure 4, bottom) is well controlled throughout . The use of a total sample size of 300 or 600 gives qualitatively similar results (see figure s1, figure s2, figure s3, figure s4, figure s5, and figure s6; figure s7 and figure s8 contain the false negative rates), although we note that while a larger sample size results in a great improvement in power, it gives only a slight improvement in the chance that the credible set includes only the true partition . Restricting the analysis to the five partitions induced by the tree has little effect on power (compare figure s1 and figure s2), but improves the chance that the credible set includes only the true partition (compare figure s3 and figure s4), and results in a somewhat lower false - positive rate (compare figure s5 and figure s6). The performance of the proposed method with different possible choices of minimal crosses is largely predicted by the number of crosses that are segregating a qtl: the solid curves of a given color (which indicates the number of crosses segregating a qtl) are largely coincident, but there are some differences (red curves in figure 4, middle right). To explore this further, the results for the individual choices of crosses, when the percentage phenotypic variance explained by the qtl is 10% and the total sample size is 450, are displayed in figure 5 . (for other sample sizes and for the analyses restricted to the partitions induced by the tree in figure 1, see figure s9, figure s10, figure s11, figure s12, figure s13, figure s14, figure s15, and figure s16 .) Detailed results on the estimated power (top), exact power (middle), and false positive rates (bottom), for individual choices of crosses, in the case of four taxa with a total sample size of 450, and with the qtl being responsible for 10% of the phenotypic variance in crosses in which it is segregating . Blue, red, and green correspond to cases in which three, two, and one of the crosses are segregating the qtl, respectively . The results are based on 10,000 simulation replicates, with analyses considering all possible partitions of the taxa . The black vertical line segments indicate 95% confidence intervals . In the case that the true partition is ab\|cd, there are some differences among the choices of three crosses when two of the three are segregating the qtl, in terms of the chance that the 95% credible set contains only the true partition (figure 5, middle). For example, the use of the crosses a b, a c, and b d gives exact power of 50%, while the use of a b, a c, and a d gives, we need to consider the sign of the qtl effect in different crosses for the true partition and the best alternative partition; these are shown in table 1 . If the true partition is ab\|cd, with c and d having an allele that results in an increase in the phenotype, the a b cross does not segregate a qtl, while each of a c, b d, and a d have a segregating qtl with the latter taxon in each cross increasing the phenotype . With the crosses a b, a c, and a d, the best alternative partition after ab\|cd would be a\|bcd, in which a c and a d are also segregating the qtl, but a b should also be segregating the qtl, and note that for both partitions ab\|cd and a\|bcd, the qtl has effect in the same direction in the a c and a d crosses . On the other hand, with the crosses a b, a c, and b d (which was seen to have better performance), in the only alternative partition with two crosses segregating a qtl, ad\|bc, the two crosses should have qtl effects in opposite directions (the a and d alleles both result in a decrease in phenotype), and so this should be easy to distinguish from the ab\|cd partition . For this choice of three crosses, all other partitions have a qtl segregating in just one of a c or b d but not both . As a result, the chance that the credible set contains only the true partition is slightly higher . For each partition, the taxa to the right of the vertical bar have the high allele . For each cross, the sign of the effect is for the right vs. the left taxon . While no such differences among the choices of minimal crosses are seen when the true partition is a\|bcd and all possible partitions are considered in the analysis, these sorts of differences do arise when the analysis is restricted to the five partitions induced by the tree in figure 1 . We consider the case of four taxa and use of all six possible intercrosses among pairs of taxa, with 75 individuals per cross (a total sample size of 450). We consider a single autosome of length 127 cm, with markers at an approximately 10-cm spacing, and with a single diallelic qtl placed in the center of an interval between two markers, near the middle of the chromosome . The qtl alleles were assumed to act additively (that is, no dominance), and the percentage phenotypic variance explained by the qtl, in the crosses in which it was segregating, was 10% . We assumed either the partition a\|bcd or ab\|cd; other possible partitions are equivalent to one of these . To reduce computation time, we used haley knott regression (haley and knott 1992) for all simulation studies, with lod score calculations performed on a 1-cm grid . Recombination was simulated assuming no crossover interference . For the naive approach, we applied a given significance threshold and inferred the presence or absence of a qtl in a cross if the maximum lod score on the chromosome was above or below the threshold, respectively . If the presence / absence pattern matched that for a possible partition, that partition was inferred . For the proposed approach, we applied a given significance threshold on maxm and then formed a 95% bayesian credible set of partitions, using equal prior probabilities on all seven possible partitions . If maxm was greater than the threshold but the 95% credible set did not contain the truth, the result was considered a false positive . The results, based on 10,000 simulations, are displayed in figure 3 as receiver operating characteristic (roc) curves: the power (the rate of true positives) vs. the false positive rate, for varying significance thresholds . We display two sets of curves for the proposed method: for the dashed curves, the power indicates that maxm exceeded the threshold and the true partition was contained within the 95% credible set; the dotted curves are more stringent and require that the credible set contained only the true partition . Points are plotted at the results with a nominal 5% significance threshold, adjusting for an autosomal genome scan, with the genome modeled after the mouse and the thresholds estimated by 10,000 simulations under the null hypothesis of no qtl . (the estimated thresholds are displayed in supporting information, table s1 and table s2 .) Estimated receiver operating characteristic (roc) curves for the naive method (solid curves), the proposed method, with power indicating that the true partition is contained within the 95% credible set (dashed curves), and the proposed method, with power indicating that the 95% credible set contains only the true partition (dotted curves), in the case of four taxa, with each of the six possible intercrosses having a sample size of 75, and a qtl responsible for 10% of the phenotypic variance in the crosses in which it is segregating . The red and blue curves correspond to the case that the true partition is a\|bcd and ab\|cd, respectively . The roc curves for the naive method form interesting shapes, with the lower part of each corresponding to low thresholds and the upper part corresponding to high thresholds, and indicate terrible performance: the false positive rate is well controlled, but power is low . The problem is that, with only moderate power to detect the qtl in a given cross, one has low power to detect the qtl in all of the crosses in which it is segregating, which is necessary to identify the correct partition of the taxa . Lowering the significance threshold below the 5% level helps somewhat, but the power to detect the true partition is no higher than 21% . The naive approach might actually perform better if one considered a smaller set of crosses, but we have not explored this further . The proposed method performs reasonably well, and the false positive rate is well controlled at the nominal 5% significance threshold (the points in figure 3). Lowering the threshold could give some improvement in power while maintaining the false - positive rate below the target level, at least in the simulated situations . In the previous section, we noted that it is not necessary to use all possible crosses among taxa . To distinguish among all possible partitions, one need sets of crosses that connect all taxa and are of minimal size (i.e., n1 crosses for n taxa) are called minimal sets . We now turn to the question of whether it is better to use all crosses, with a smaller number of individuals per cross, or a minimal set of crosses, with a larger number of individuals per cross . We use the same general settings as for the simulations comparing the proposed method to the naive approach, with four taxa and the true partition being either a\|bcd or ab\|cd, but here we vary the total sample size among 300, 450, and 600 individuals, and we vary the percentage phenotype variance explained by the qtl from 2.5 to 15% . We consider either all six crosses or a minimal set of three crosses, and we consider all 16 choices of three crosses that include all four taxa . We also compared the consideration of all seven possible partitions, or just the five partitions induced by the tree in figure 1 . We estimated 5% genome - wide significance thresholds by simulations under the null hypothesis of no qtl (see table s2). Figure 4 displays the simulation results, as a function of the effect of the qtl, for the case that the total sample size was 450 (i.e., 75 individuals per cross when considering all crosses and 150 individuals per cross when considering a minimal set of three crosses) and when all possible partitions were considered . The results with other sample sizes and with analysis restricted to the five partitions induced by the tree in figure 1 are shown in figure s1, figure s2, figure s3, figure s4, figure s5, and figure s6 . The top of each figure indicates the power (the chance that maxm exceeded its threshold and the true partition was contained in the 95% credible set); the middle indicates the exact power (the chance that maxm exceeded its threshold and that the credible set contained only the true partition); the bottom indicates the false positive rate . The left and right correspond to the true partition being a\|bcd or ab\|cd, respectively . The black dashed curves correspond to the use of all six possible crosses; the solid curves correspond to the different choices of a minimal set of three crosses, with blue, red, and green corresponding to cases in which 3, 2, or 1 of the crosses are segregating a qtl . Estimated power (top), exact power (middle), and false - positive rates (bottom) in the case of four taxa with a total sample size of 450, as a function of the percentage phenotypic variance explained by the qtl . The other curves are for the various choices of a minimal set of three crosses, with the curves in blue, red, and green corresponding to cases in which three, two, and one of the crosses are segregating the qtl, respectively . The results are based on 10,000 simulation replicates, with analyses considering all possible partitions of the taxa . In choosing among the possible minimal sets of crosses, power is highest when a larger number of crosses are segregating the qtl . For a fixed total sample size, the use of all possible crosses (with fewer individuals per cross) has better performance than the worst of the possible minimal sets of crosses, but is not as good as the best of the possible minimal sets of crosses . The use of all possible crosses has greater power when the true partition is ab\|cd (in which case four of the six crosses are segregating the qtl) than when the true partition is a\|bcd (in which case three of the six crosses are segregating the qtl). The false - positive rate (figure 4, bottom) is well controlled throughout . The use of a total sample size of 300 or 600 gives qualitatively similar results (see figure s1, figure s2, figure s3, figure s4, figure s5, and figure s6; figure s7 and figure s8 contain the false negative rates), although we note that while a larger sample size results in a great improvement in power, it gives only a slight improvement in the chance that the credible set includes only the true partition . Restricting the analysis to the five partitions induced by the tree has little effect on power (compare figure s1 and figure s2), but improves the chance that the credible set includes only the true partition (compare figure s3 and figure s4), and results in a somewhat lower false - positive rate (compare figure s5 and figure s6). The performance of the proposed method with different possible choices of minimal crosses is largely predicted by the number of crosses that are segregating a qtl: the solid curves of a given color (which indicates the number of crosses segregating a qtl) are largely coincident, but there are some differences (red curves in figure 4, middle right). To explore this further, the results for the individual choices of crosses, when the percentage phenotypic variance explained by the qtl is 10% and the total sample size is 450, are displayed in figure 5 . (for other sample sizes and for the analyses restricted to the partitions induced by the tree in figure 1, see figure s9, figure s10, figure s11, figure s12, figure s13, figure s14, figure s15, and figure s16 .) Detailed results on the estimated power (top), exact power (middle), and false positive rates (bottom), for individual choices of crosses, in the case of four taxa with a total sample size of 450, and with the qtl being responsible for 10% of the phenotypic variance in crosses in which it is segregating . Blue, red, and green correspond to cases in which three, two, and one of the crosses are segregating the qtl, respectively . The results are based on 10,000 simulation replicates, with analyses considering all possible partitions of the taxa . The black vertical line segments indicate 95% confidence intervals . In the case that the true partition is ab\|cd, there are some differences among the choices of three crosses when two of the three are segregating the qtl, in terms of the chance that the 95% credible set contains only the true partition (figure 5, middle). For example, the use of the crosses a b, a c, and b d gives exact power of 50%, while the use of a b, a c, and a d gives, we need to consider the sign of the qtl effect in different crosses for the true partition and the best alternative partition; these are shown in table 1 . If the true partition is ab\|cd, with c and d having an allele that results in an increase in the phenotype, the a b cross does not segregate a qtl, while each of a c, b d, and a d have a segregating qtl with the latter taxon in each cross increasing the phenotype . With the crosses a b, a c, and a d, the best alternative partition after ab\|cd would be a\|bcd, in which a c and a d are also segregating the qtl, but a b should also be segregating the qtl, and note that for both partitions ab\|cd and a\|bcd, the qtl has effect in the same direction in the a c and a d crosses . On the other hand, with the crosses a b, a c, and b d (which was seen to have better performance), in the only alternative partition with two crosses segregating a qtl, ad\|bc, the two crosses should have qtl effects in opposite directions (the a and d alleles both result in a decrease in phenotype), and so this should be easy to distinguish from the ab\|cd partition . For this choice of three crosses, all other partitions have a qtl segregating in just one of a c or b d but not both . As a result, the chance that the credible set contains only the true partition is slightly higher . For each partition, the taxa to the right of the vertical bar have the high allele . For each cross, the sign of the effect is for the right vs. the left taxon . While no such differences among the choices of minimal crosses are seen when the true partition is a\|bcd and all possible partitions are considered in the analysis, these sorts of differences do arise when the analysis is restricted to the five partitions induced by the tree in figure 1 . These data concern four intercrosses among five inbred mouse strains, cast / ei (c), dba/2 (d), i / lnj (i), pera / ei (p), and 129s1/svimj (s). The four intercrosses performed were c d, c s, d p, and i p. the c d and c s crosses were all males and had 277 and 275 mice, respectively . The d p and i p crosses had approximately equal numbers of males and females and had a total of 282 and 322 mice, respectively . As in li et al . (2005), we focus on a single phenotype, the square root of plasma hdl cholesterol . Note that the four intercrosses form a daisy chain, s c d p i, and so satisfy the connectedness condition necessary for inference of the correct partition of the strains at a diallelic qtl . (2009), with marker locations obtained using the mouse map converter at the jackson laboratory (http://cgd.jax.org/mousemapconverter). We used standard interval mapping (lander and botstein 1989) and considered all 15 possible partitions of the five strains, without attempting to infer a phylogenetic tree relating the strains . To handle the two sexes, we included sex as an additive covariate (that is, we allowed for a shift in the average phenotype between the sexes and assumed no qtl sex interaction). We used permutation tests with 10,000 replicates to obtain 5% significance thresholds for the individual crosses and for maxmi . The estimated significance thresholds for the individual crosses were approximately 3.44 for all four crosses; the estimated threshold on maxmi was 5.39 . (2005), we focused on chromosomes 1, 2, 4, 5, 6, and 11 . The lod curves for the top five partitions on each chromosome are in figure 6, middle . The posterior probabilities of the different partitions, assuming the presence of a single diallelic qtl, are on the right . In all cases, (2005): lod curves for individual crosses (left), lod curves for the top five partitions (middle), and approximate posterior probabilities for each partition (right). The partitions corresponding to the five lod curves in the middle are indicated on the right . The labeled points on the right indicate the partitions included in the 95% bayesian credible sets . On the left and in the middle, dashed horizontal lines are plotted at the 5% significance thresholds . For chromosome 1, significant evidence for a qtl is seen in the crosses c s and d p but not in c d or i p. by the naive approach, we would infer the partition cd\|ips, and this is the partition that li et al . Our proposed method does give this partition the highest posterior probability (57%), but also gives reasonable weight to the alternative ps\|cdi (posterior probability 39%), in which case the qtl would also be segregating in the i p cross . For chromosome 2, we see a qtl just in cross c d. by the naive approach (given the set of crosses performed), we would infer the partition cs\|dip, which is the partition that li et al . However, by the proposed method, cs\|dip has a posterior probability of only 20%, while the partition c\|sdip (in which the qtl would also be present in the c s cross) has a posterior probability of 80% . For chromosome 4, we have evidence for a qtl in all four crosses (although in the cross i p, the maximum lod score was 3.42, just missing the threshold of 3.44). If we assume that there is no qtl segregating in i p, we would infer the partition ds\|cip, while if we take the evidence for a qtl in i p as sufficient, we would infer the partition cp\|dis, and this is the partition that li et al . The latter is the partition with the highest posterior probability (78%), while the former has posterior probability 7%, and a third partition, c\|dips, in which case the qtl is segregating in neither i p nor d p, has posterior probability 16% . For chromosome 5, we see a qtl only in cross i p, and so by the naive approach we would infer the partition i\|cdps; this partition does have the highest posterior probability (83%) and was the partition that li et al . But the maximum lod score for this partition was 3.98, which does nt meet the 5% significance threshold . (the genome - scan - adjusted p - value was 0.37 .) Thus, by our proposed approach, we would not infer the presence of a qtl . But if we do allow that there is a qtl, two other partitions are contained within the 95% credible set: dp\|cis, with posterior probability 9%, in which case the qtl is also segregating in the cross c d, and is\|cdp, with posterior probability 6%, in which case the qtl is also segregating in the cross c s. for chromosome 6, we have significant evidence for a qtl only in cross c d (the other three crosses have maximum lod scores of 1.51.9 on chromosome 6), and so the naive method would give the partition cs\|dip, which has posterior probability <0.01% and is not contained in the 95% credible set . The partitions with highest posterior are c\|dips (47%), with the qtl also segregating in c s, and ci\|dps (45%), with the qtl also segregating in c s and i p. the 95% credible set also contains a third partition, ds\|cip, with posterior probability 7% . (2005) had assumed the partition c\|dips, which is the partition with highest posterior probability . For chromosome 11, there was significant evidence for a qtl only in the cross i p, although the cross d p has a maximum lod score of 3.16 (corresponding to a genome - scan - adjusted p - value of 0.093). The naive approach would give the partition i\|cdps, which has posterior probability 0.9% and is not contained in the 95% credible set . If we consider the evidence for a qtl in d p to be sufficient, we would infer the partition p\|cdis, which has posterior 16% and was the one that li et al . The partition with highest posterior probability is di\|cps (posterior probability 60%), in which case the qtl is also segregating in c d. the 95% credible set also contains the partition ps\|cdi (posterior probability 21%), in which case the qtl is segregating in c s but not c d. as with chromosome 5, the maximum lod score across partitions (4.70) does not meet our 5% significance threshold, and so by our proposed method we would not infer the presence of a qtl . We have described a formal approach for the joint analysis of multiple crosses to map the origin of qtl alleles to a position on a phylogenetic tree . Our approach unites qtl mapping with phylogenetic comparative methods to provide a view of the genetic mechanism underlying phenotypic evolution . Further, our approach partitions taxa according to their qtl allele, facilitating haplotype analyses for the fine mapping of qtl . In addition, as part of this work, we have begun to evaluate a variety of experimental design issues for such research, which provides some guidance to researchers seeking to take advantage of this approach . The goal of the work in li et al . The key difficulty in applying this idea is that one must define a unique partition of the strains into the two qtl alleles, a priori . In the presence of multiple qtl, the phenotypes of the strains cannot be trusted for inferring the qtl alleles, and in the current application, the six qtl partition the five strains in diverse ways . (2005) used the pattern of qtl in the different crosses to infer the appropriate partition, which we have (perhaps overly harshly) characterized as the naive approach . We have proposed a formal method for comparing the different possible partitions . For two of the six loci, we find that the partition with strongest support is different from that assumed by li et al . (2005), and for all six loci there are multiple partitions with reasonable support . As seen in figure 6, middle, the different partitions can have quite different lod curves and so provide different information on the likely location of the qtl . Thus, our formal approach to identifying the well - supported partitions can improve localization of a qtl . Moreover, one could combine the information from the multiple partitions to better define the location of the qtl, taking account of the uncertainty in the partition . Furthermore, while the application of these ideas to evolutionary studies remains our primary interest, the more straightforward application is in biomedical or agricultural research, as in li et al . (2005), for the combined use of multiple crosses to more precisely map a qtl and, subsequently, with an inferred partition (or partitions) of strains in hand, to inform the analysis of the haplotypes of the strains (see, for example, burgess - herbert et al . The results are also valuable for the design of future experiments, if additional crosses are to be performed . Our approach has some similarities to the use of local phylogenetic trees to define possible partitions of multiple alleles (pan et al . 2009; zhang et al . 2012) and to coalescent - based approaches (zllner and pritchard 2005) for genome - wide association studies . The key distinction of our method is that we seek not just to establish association but also to identify the appropriate partition and so define the origin of the mutant qtl allele on the local phylogenetic tree . In our approach, the qtl location on the tree is not a nuisance parameter but rather is the target of inference . In our simulation studies, we compared the use, for a fixed total sample size, of all possible crosses to different choices of a minimal set of crosses . Depending on the underlying true partition of taxa at a qtl, one can choose a minimal set of crosses with considerably higher power . However, given the prior uncertainty in the true partition, and the possibility of multiple qtl that each partition the taxa differently, it is prudent to consider all or at least a larger number of possible crosses . An even more important experimental design question, which we have not considered here, is how to choose which taxa, out of a large number of related taxa, to consider, in the effort to characterize the genetic architecture of a quantitative trait . The approach could be adapted for the analysis of a set of backcrosses, although these would likely need to be of a special form, with the f1 hybrids all crossed to a common parent . There are a number of additional ways in which our analytical framework could be extended . Most quantitative traits are affected by multiple qtl, rather than single qtl as assumed here . The restriction that a qtl has a common effect in all crosses in which it segregates might be relaxed, particularly for traits that are heavily shaped by epistasis, such as hybrid sterility and hybrid inviability (coyne and orr 2004). Prior distributions of qtl partitions could incorporate phylogenetic branch lengths (taxa separated by shorter evolutionary distances are more likely to share qtl alleles) as well as topologies . Finally, future developments might account for variation in the tree . This variation includes both statistical uncertainty associated with phylogenetic inference and real phylogenetic discordance across the genome, which results from incomplete lineage sorting and introgression in recently diverged taxa (pamilo and nei 1988; maddison 1997; pollard et al . 2006; white et al . 2009). The power of reconstructing qtl evolution as well as the increasing capacity for genetic mapping of complex traits and phylogenetic reconstruction should provide motivation for these extensions in the evolutionary, biomedical, and agricultural communities . Software incorporating the proposed methods are available as part of r / qtl (broman et al . 2003, http://www.rqtl.org), an add - on package to the general statistical software r (r development core team 2010).
A 69-year - old man was referred due to newly developed hemoptysis and was diagnosed as operable adenocarcinoma of the right upper lobe of the lung . Because the tumor was invading the right main bronchus and azygos vein, however, from the 6th postoperative day, he developed tachypnea, desaturation (sao2 85% to 90% under room air), and atrial fibrillation with rapid ventricle response around 150 beat per minute . Despite increased oxygen supply, desaturation gradually progressed, and the patient was intubated for mechanical ventilation . After initiating positive mechanical ventilation, severe subcutaneous emphysema developed . Bronchoscopic examination revealed a bronchopleural fistula (bpf) on the right main bronchus stump (fig . Exudates in the right pleural cavity were evacuated by closed thoracostomy, and microrganisms such as streptococcus pneumonia and pseudomonas aeruginosa were isolated from the pleural fluid . After 5 days of conservative treatment with antibiotics and pleural cavity drainage, surgery was decided to obliterate bpf because the onset of empyema was relatively acute and stump fistula was too large to expect spontaneous closure . After redo thoracotomy through previous incision, primary closure of bpf was performed by multiple interrupted sutures along with transposition of omental flap to the bronchial stump (fig . After thorough debridement and irrigation of pleural cavity, the right pleural cavity was packed with sterilized gauze and pericostal sutures were applied to approximate the intercostal space . Vacuum - assisted closure (vac) sponge (curavac; daewoong, seoul, korea) was applied just above the rib leaving the pleural cavity communicated through intercostal space . A 12 french chest tube was placed into the vac sponge to apply negative pressure . The skin above the vac sponge was widely covered using adhesive drape and negative pressure of 120 mmhg was applied for exudates evacuation . Three additional vac dressing changes were performed under general anesthesia on every 3 days after bpf closure . After confirming that no more organisms from the right pleural cavity were isolated by follow - up microbial culture, clagett operation was performed on 9th postoperative days from bpf closure and thoracotomy wound could be closed without additional vac apply . The patient remains asymptomatic without any evidence of infection or wound problem for 7 months follow - up . Postpneumonectomy empyema (ppe) is a life - threatening complication, and is often related with a bpf . Treatment of ppe should be targeted on closure of bpf, effective drainage of pleura cavity, and obliteration of the dead space . One of the most popular approaches to treat ppe with bpf is the closure of bronchial stump followed by irrigation of the pleural space through open window thoracotomy . Delayed closure is performed with dead space filled by antibiotics solution (so - called clagett operation). Although this approach has been shown to be highly successful, it forces the patient long hospitalization during the sterilization and stressful daily irrigation with dressing change ., novel technique using vac has been widely used for various wound infections, such as deep sternal wound infection and open orthopedic fractures . Vac device promotes wound healing by generating negative pressure which leads to granulation tissue formation, removal of exudates, and reduction of edema with increased tissue perfusion by arteriolar dilatation . We borrowed this advantage of vac for the sterilization of pneumonectomy space instead of daily irrigation with dressing change through open thoracotomy . In this patient, peribronchial tissue was fresh enough for early surgical repair because the detection of ppe with bpf was relatively earlier . Therefore we thought early sterilization with vac was possible, if we could obliterate the bpf with omental flap transposition . Before we decided to conduct this procedure, one of our main concerns was complication induced by negative suction pressure, such as bleeding, disruption of repaired fistula, or mediastinal shifting . In this case, we packed sterilized gauze into pleural cavity to avoid direct contact of negative pressure to mediastinum (fig . Exudates were evacuated by the vac system through communication along the intercostal space . By these methods stated above, we could control the ppe effectively for relatively short period, less cost and trouble for patient . Although vac dressing in ppe with bpf is not widely used method, it could be an effective alternative method for the open window sterilization with shorter treatment period in selected patients.
Osteoarthritis (oa) is a chronic musculoskeletal disease characterized by loss of articular cartilage and changes in the subchondral bone . Several risk factors (e.g., age, female gender, hypothyroidism, race, genetic susceptibility, and obesity) are associated with oa1 . Thyroid hormones might be of key importance in the maintenance of articular cartilage and play a role in the pathogenesis of oa3 . Radiographic oa is characterized by osteophytes, sclerosis, and joint space narrowing, which is a result of cartilage erosion and subchondral sclerosis4 . Ultrasonography is also used to evaluate the condition of the articular cartilage, and pre - oa evaluation of the knee by ultrasound may have prognostic value5 . The aim of the present study was to investigate the effects of hypothyroidism on femoral cartilage thickness by using ultrasound, which is a valid and reliable method in this regard and has previously been found to be useful in the early diagnosis of knee oa6, 7 . Forty patients diagnosed with hypothyroidism and 30 age-, gender-, body mass index (bmi)-, smoking status-, and physical activity - matched healthy subjects were enrolled . Subjects with a history of knee trauma or previous knee surgery; additional (other than hypothyroidism) systemic and/or chronic diseases including diabetes mellitus and rheumatoid arthritis; and any abnormal laboratory results regarding renal, hepatic, thyroid, or parathyroid function were excluded . All subjects were informed about the study procedure, and they provided consent to participate.this study was approved by the local ethics committee of recep tayyip erdogan university medical school . The thickness of the femoral articular cartilage was measured using a 7- to 12-mhz linear probe (eizo nanao corporation, esaote, italia) while the subjects comfortably sat on the examination table with their knees in maximum flexion, and the probe was placed in the axial plane on the outer edge6, 8 . Three mid - point measurements were taken from each knee, from the right lateral condyle (rlc), right intercondylar area (ria), right medial condyle (rmc), left medial condyle (lmc), left intercondylar area (lia), and left lateral condyle (llc) (fig . (suprapatellaraxial view) demonstrating femoral cartilage measurementsrlc: right lateral condyle; ria: right intercondylar area; rmc: right medialcondyle; llc: left lateral condyle; lia: left intercondylar area; lmc: left medial condyle). The cartilage thickness was interpreted as the distance between the thin hyperechoic line at the synovial space / cartilage interface and the sharp hyperechoic line at the cartilage - bone interface7 . Ultrasonogram (suprapatellaraxial view) demonstrating femoral cartilage measurements rlc: right lateral condyle; ria: right intercondylar area; rmc: right medialcondyle; llc: left lateral condyle; lia: left intercondylar area; lmc: left medial condyle all statistical analyses were performed using spss version 16.0 . The normal distribution of continuous variables was tested using the kolmogorov - smirnov test . Student s t - test was used to compare normally distributed data, and the mann - whitney u test was used to compare non - normally distributed data . Eighty knees of the 40 patients with hypothyroidism (29 females and 11 males) and 60 knees of the 30 healthy subjects (22 females and 8 males) were examined in this study . The demographic characteristics of the participants are shown in table 1table 1.characteristics of study population (mean standard deviation) patients (n=40)control (n=30)age (years)25.50 3.3424.46 3.15gender, n (%) female29 (72.5)22 (73.3)male11 (27.5)8 (26.7)bmi (kg / m)26.38 1.3825.45 3.06disease duration (months)30.3 15.4physical activity, n (%) physical activity34 (85.0)25 (83.3)no physical activity6 (15.0)5 (16.7)smoking status, n (%) smokers7 (17.5)6 (20.0)non - smokers33 (82.5)24 (80.0)significant at p<0.05 . The mean age of the patients with hypothyroidism was 25.50 3.34 years, while that of the healthy subjects was 24.46 3.15 years (p>0.05). The mean bmi of the former was 26.38 1.38 kg / m, while that of the latter was 25.45 3.06 kg / m (p>0.05). The mean disease duration in the patients with hypothyroidism was 30.32 15.41 months . Gender, smoking status, and physical activity were similar between the groups (p>0.05 for all). significant at p<0.05 table 2table 2.comparison of femoral cartilage thickness (mm) between patients with hypothyroidism and healthy controls (mean standard deviation)patients (n=40) control (n=30)rlc (mm)1.83 0.141.88 0.08ria (mm)1.89 0.111.92 0.09rmc (mm)1.79 0.111.83 0.08llc (mm)1.86 0.121.91 0.08lia (mm)1.88 0.141.93 0.10lmc (mm)1.77 0.12 * 1.83 0.08rlc: right lateral condyle; ria: right intercondylar area; rmc: right medial condyle; llc: left lateral condyle; lia: left intercondylar area; lmc: left medial condyle . Patients with hypothyroidism had thinner femoral cartilage than the healthy controls at all measurement sites, but the differences were not statistically significant except in the case of the lmc (p<0.05). Rlc: right lateral condyle; ria: right intercondylar area; rmc: right medial condyle; llc: left lateral condyle; lia: left intercondylar area; lmc: left medial condyle . Hypothyroidism is known to be associated with oa and inflammatory forms of arthritis and connective tissue diseases, which can cause arthritis9 . The clinical findings of hypothyroidism include epiphyseal disgenesis, aseptic necrosis, and viscous non - inflammatory arthropathy particularly affecting the knees, wrist joints, and hands10 . The thyroid hormones are essential for endochondral ossification and stimulating the expression of the gene that controls chondrocyte maturation and matrix synthesis3 . Mature chondrocytes in the cartilage synthesize type ii collagen and proteoglycans, which begin to degradation in early oa . As this degradation progresses oa involves the entire joint, including the subchondral bone and articular cartilage, and the prevalence of periarticular soft tissue lesions is high14, 15 . In addition to degradation and loss of the articular hyaline cartilage, progressive thickening, remodeling, and sclerosis of the subchondral bone; formation of osteophytes; and chronic inflammation of the synovial membrane are other features of oa17 . Ultrasound is an inexpensive noninvasive method for imaging the musculoskeletal system that is readily accepted by patients . It can be provide information about the joint cartilage, synovitis, periarticular soft tissues, and bony cortical abnormalities in peripheral joints with oa . It seems to be a promising and accurate method for monitoring cartilage response to drug therapy in oa18 . Cartilage defects may predict cartilage loss in asymptomatic knee oa and can indicate early oa, which is demonstrated by a loss in the sharpness of the margin19 . Lee et al . Reported a significant correlation between ultrasonographic measurements and histologic grading of oa femoral condylar cartilage6 . A previous study reported that low hemoglobin levels had a negative effect on femoral cartilage thickness as detected by ultrasonography in healthy subjects20 . Ultrasound is also useful to monitor the effect of biologic therapy in rheumatoid arthritis and can be used to evaluate both inflammatory and destructive changes21 . Tsai et al . Suggested that ultrasound could be used to monitor changes in the cartilage of patients with rheumatoid arthritis22, and tunc et al . Revealed that the femoral cartilage is thinner on the hemiparetic side of stroke patients23 . The results of the present study show the negative effects of hypothyroidism on the thickness of the distal femoral cartilage . To the best of the authors knowledge, this is the first study exhibiting thinning of the distal femoral cartilage in correlation with hypothyroidism . The sample size was relatively small, and only the thickness and not the volume of the cartilage was measured, although it has been shown that differences in cartilage volume result primarily because of differences in joint surface areas (epiphyseal bone size) rather than cartilage thickness24 . Overall, on the basis of the study findings, patients with hypothyroidism seem to have thinner femoral cartilage . Thus, the presence and severity of femoral cartilage thinning may be useful for early diagnosis of knee oa . Ultrasonography is a noninvasive, cost - effective method to measure the thickness of the articular cartilage, and screening of joint cartilage by ultrasonography may enable early diagnosis of knee oa in patients with hypothyroidism.
Alcohol, autoimmune diseases, injury, and drugs are identified causes for recurrent pancreatitis . Phpt is associated with high parathyroid hormone (pth) levels, which usually leads to hypercalcemia and hypophosphatemia . Patients may present with recurrent nephrolithiasis, or be asymptomatic, detected on routine biochemical screening . Phpt patients are also associated with altered carbohydrate metabolism characterized by insulin resistance, hyperinsulinemia, and glucose intolerance and even frank diabetes . However, the corelation between the two remains controversial and has been explained only based on few case reports . We present a patient with recurrent episodes of pancreatitis primarily due to hypercalcemia of phpt and eventually leading to diabetes mellitus . A 21-year - old woman, presented with acute onset of abdominal pain over epigastric region radiating towards back with vomiting . The patient was overweight with a body mass index (bmi) of 24.8 kg / m . General physical examination was noncontributory apart from a nodule in the lower pole of right lobe of thyroid gland . The patient's past history shows repeated episodes of abdominal pain over last 3 years, which required hospitalization . There was recurrent history of pain in the flanks and on two occasions she had passed stones while passing urine . Straight x - ray of kidney, ureter, and bladder region showed the presence of renal stones in both the kidneys . The patient was then diagnosed with recurrent pancreatitis and nephrolithiasis . During the course of pancreatitis she never had any hypocalcemia . On repeated occasion she was detected to have diabetes mellitus with hba1c 6.9% and was put on insulin . Lipid profile, liver function, renal function, and thyroid function tests were normal . Prothrombin time was 11.6 s (control 11.5 s) with international normalized ratio (inr) 1.01 . Further investigation of laboratory parameters, such as serum calcium, albumin, and pth was abnormal [table 2]. Thyroid ultrasonography showed small, well defined, oval shaped hypoechoic structure just inferior to the lower pole of right lobe of thyroid gland suggestive of parathyroid adenoma or small lymph node [figure 1]. Usg showing hypoechoic mass suggestive of parathyroid adenoma abdominal contrast - enhanced computed tomography (cect) showed features suggestive of mild acute pancreatitis with left renal calculus . Magnetic resonance cholangiopancreatography showed chronic pancreatitis with no evidence of biliary obstruction or choledocholithiasis [figure 2]. Magnetic resonance cholangiopancreatography showing chronic pancreatitis patient was diagnosed with phpt causing hypercalcemia which resulted in nephrolithiasis and recurrent pancreatitis further leading to pancreatic diabetes . The patient was overweight with a body mass index (bmi) of 24.8 kg / m . General physical examination was noncontributory apart from a nodule in the lower pole of right lobe of thyroid gland . The patient's past history shows repeated episodes of abdominal pain over last 3 years, which required hospitalization . There was recurrent history of pain in the flanks and on two occasions she had passed stones while passing urine . Straight x - ray of kidney, ureter, and bladder region showed the presence of renal stones in both the kidneys . She was detected to have diabetes mellitus with hba1c 6.9% and was put on insulin . Lipid profile, liver function, renal function, and thyroid function tests were normal . Prothrombin time was 11.6 s (control 11.5 s) with international normalized ratio (inr) 1.01 . Further investigation of laboratory parameters, such as serum calcium, albumin, and pth was abnormal [table 2]. Thyroid ultrasonography showed small, well defined, oval shaped hypoechoic structure just inferior to the lower pole of right lobe of thyroid gland suggestive of parathyroid adenoma or small lymph node [figure 1]. Usg showing hypoechoic mass suggestive of parathyroid adenoma abdominal contrast - enhanced computed tomography (cect) showed features suggestive of mild acute pancreatitis with left renal calculus . Magnetic resonance cholangiopancreatography showed chronic pancreatitis with no evidence of biliary obstruction or choledocholithiasis [figure 2]. Patient was diagnosed with phpt causing hypercalcemia which resulted in nephrolithiasis and recurrent pancreatitis further leading to pancreatic diabetes . Hypercalcemia due to hpt is not a very common cause for acute pancreatitis, usually seen between 1.5 and 7% in the different series . Presentation of pancreatic disease in phpt can be classified in four classes: phpt presenting as acute pancreatitisphpt presenting as acute recurrent pancreatitis with no evidence of chronic pancreatitisphpt presenting as chronic pancreatitis with or without pancreatic calcificationphpt complicated by acute pancreatitis in the postoperative period . Phpt presenting as acute pancreatitis phpt presenting as acute recurrent pancreatitis with no evidence of chronic pancreatitis phpt presenting as chronic pancreatitis with or without pancreatic calcification phpt complicated by acute pancreatitis in the postoperative period . Literature review on case series suggested, phpt patients presenting with acute pancreatitis are more common (44%) than those with chronic pancreatitis . But, no evidence has been reported for patients who developed acute pancreatitis in the postoperative state . Few researchers have reported that patients with hypercalcemia secondary to hpt have low pancreatic secretion but normal enzyme activity . In such cases, pancreatitis may result due to damage of parenchymal cell and pancreatic duct caused by activation of intrapancreatic trypsinogen to trypsin due to high calcium concentration in pancreatic juice . Few theories also suggest the mutations of genes such as serine protease inhibitor kazal type 1 (spink1) or cystic fibrosis transmembrane conductance regulator (cftr) may also be involved . Hypercalcemia during pancreatitis can be an indication for hpt, whereas hypocalcemia can occur during an attack of pancreatitis . Patients with chronic pancreatitis due to phpt also have significant incidence of renal colic, nephrolithiasis, and nephrocalcinosis . In our case so any patient with pancreatitis should be investigated for hypercalcemia to avoid missing the diagnosis of hpt.
Differential quantification of complex mixtures using high - performance liquid chromatography coupled to electrospray ionization and tandem mass spectrometry (hplc esi ms / ms) can help researchers study biological variation at the molecular level and gain insights into the molecular machinery of cellular activity and disease progression . Researchers reveal biological variation most often by comparing two or more populations, typically collecting data for thousands of distinct molecules in each population, for example, genes, peptides, or metabolites, and then statistically analyzing the differences among populations . Here a population comprises biological or technical replicates having a biological state in common, for example, healthy or diseased . Ms / ms workflows aimed at discovering biological variation fall into two categories: labeled and label - free quantification strategies . Labeled quantification strategies are popular because they allow researchers to analyze multiple peptide mixtures (often derived from protein samples) in a single hplc (while we use peptide as a representative analyte, the set of analytes is not limited to peptides .) Researchers compute relative abundance (fold changes) of resulting ion intensities between the concurrently analyzed samples and determine which peptides (or inferred proteins) are differentially abundant . Thus, labeling strategies do no scale to the level required for large - scale comparative studies, for example, a clinical study with tens, hundreds, or even thousands of biological samples . Because labeled strategies do not scale, researchers are increasingly turning to label - free quantification strategies, either intensity - based (ms1) or spectral counting (ms2). Spectral counting is straightforward but is biased toward peptides derived from high abundance proteins because spectral counting requires multiple ms2 scans matched to each protein to obtain statistically valid results . Intensity - based quantification is less straightforward than spectral counting, requiring the area under the curve computation using ms1 scans . However, intensity - based quantification is well - suited to study lower abundance peptides, which are often more interesting than higher abundance peptides . Intensity - based strategies require repeatability and reproducibility, which are inherently problematic in hplc ms / ms workflows and lead to excessive false - positive and false - negative results . Researchers will eventually discard the false - positive peptides via hypothesis - driven experiments, such as selected reaction monitoring (srm), but false - negative results are detrimental because these candidates are never pursued . As a result, poor repeatability and reproducibility cause researchers to miss possible insights and draw incorrect inferences . Ms / ms chromatographic data stem from extraneous variability, which includes systematic bias (sample variability and instrument variability) and complex variability . Sources of sample variability include incomplete enzymatic digestion and pipetting errors made during sample preparation . Instrument variability can stem from physical changes in the liquid chromatography, mass spectrometry (ms) hardware, or environment, including hplc performance degradation, ms calibration drift, and volatiles in the lab air that affect ionization efficiency . Instrument variability is global in nature because each change similarly affects each ion s intensity in a run . It stems from signal distortion due to transient stochastic events that occur during an hplc esi ms / ms run . For example, variability in esi performance due to the mobile phase composition or flow rate fluctuations distorts the measured ion signal . It is complex because each event affects only a narrow temporal window within an hplc esi ms / ms run, where window widths vary and can overlap among runs when analyzing separate samples . Normalization strategies in hplc ms / ms workflows attempt to remove systematic biases from the data before statistical inference . Traditionally, normalization strategies use a combination of a global scaling function and a peptide selection method . Global scaling functions include median scale, mean scale, quantile, ranking, and least - squares fitting using linear or polynomial regression . Unfortunately, these global scaling functions often require a complete matrix on which to compute, specifically, no missing data . While it is possible to impute missing values, it is recommended to do so after normalization . Thus, the selection of peptides for inclusion in the global scaling function is critical . Peptide selection methods include: (1) common within sample (cws); (2) top l order statistics (los); (3) percentage of peptides present (ppp); and (4) peptides with rank invariant peptide (rip). Webb - robertson et al . Developed a useful application named statistical procedure for the analyses of lc ms proteomics normalization strategies (spans), which recommends the best global scaling function and peptide selection method combinations based on rigorous statistical tests . Unfortunately, spans only includes global scaling functions and, regardless of the peptide selection method, global scaling functions cannot capture and mitigate complex variability . Although largely unexplored, complex variability during an hplc esi ms / ms run seems inevitable, even when researchers follow strict protocols . The national cancer institute s clinical proteomic tumor analysis consortium (cptac) studies provide an example . Cptac established standard operating procedures to enable interlaboratory comparisons of proteomic studies, particularly in the context of cancer biomarker discovery . In their sixth study, they used their standard operating procedures to produce publicly available, community reference data sets generated from a yeast proteome digest with 48 spiked - in proteins (ups1 standard from sigma aldrich). Rudnick et al . Found irregularities attributed to electrospray instability in one of the technical replicates (sample c, replicate 2) from this data set generated by the instrument aliased ltq - xl - orbitrapp@65 . The chromatogram s distinctive sawtooth pattern (supplemental figure 1 in the supporting information) is a textbook example of complex variability . While rudnick et al . Reported modestly diminished peptide identification performance for this replicate analysis, we suspected that the complex variability also diminished intensity - based peptide quantification performance . Further investigating this examining the extracted chromatograms plots (xcs), each representing all peptide signals in a run (experimental procedures), reveals a trough in replicate 2 (rep 2) (figure 1a) corresponding to the same time frame as the observed electrospray instability in the tic generated from the raw data (supplemental figure 1 in the supporting information). Unfortunately, global scaling functions fail to mitigate complex variability (figure 1b). Furthermore, global normalization can have unintended consequences and can adversely affect regions where no complex variability exists . Figure 1b shows that two regions of the xc for rep 2 now have more extraneous variability than before normalization, potentially disguising true biological variation . Complex variability in technical replicates . Extracted chromatograms (experimental procedures) from three hplc esi ms / ms technical replicates (cptac study 6, ltq - xl - orbitrapp@86) show that even well - controlled hplc (a) extracted chromatogram for sample c s replicate 2 s un - normalized data (exp c rep 2, long - dashed red line) contains a distinctive trough during the same time frame as the electrospray instability in its corresponding tic (supplemental figure 1 in the supporting information). (b) same data normalized by median scale result in extracted chromatograms where the distinctive trough remains . Extracted chromatograms for replicates 1 and 3 (exp c rep 1, solid blue line, and exp c rep 3, short - dashed orange line) only slightly diverge . However, replicate 2 s (exp c rep 2, long - dashed red line) extracted chromatograms show that the complex variability is now exaggerated, showing the adverse effects of median scale normalization . (c) same data normalized by pin result in similar extracted chromatograms for each of the three replicates . Pin removes the trough in replicate 2 (exp c rep 2, long - dashed red line). This demonstrates pin s ability to mitigate complex variability . In response to the shortcomings of current normalization strategies exemplified in this data from cptac, we developed proximity - based intensity normalization (pin), which mitigates complex variability and systematic bias in ms1 chromatographic data . Here we describe the underpinnings of this strategy and demonstrate that pin improves repeatability and reproducibility, allowing researchers to better detect biological variation from biological ms data . We collected and processed salivary endogenous peptides as previously described . In brief, we collected all saliva samples according to protocols approved by the university of minnesota institutional review board . Donors declared that they were healthy nonsmokers and were free of confounding conditions . Clarified saliva was prepared from fresh whole saliva samples by centrifuging at 3000 g at 4 c for 15 min, followed by 16 100 g at 4 c for 1 min . The supernatant was mixed in a 10:1 ratio with denaturing buffer consisting of 4% sds, 100 mol / l dithiothreitol, and 100 mmol / l tris, ph 7.4 . The samples were boiled for 5 min, cooled to room temperature, then added to a centrifugal filter (amicon ultra, 0.5 ml, 10 kda, millipore). Two hundred microliters of buffered urea (8 mol / l urea with 100 mmol / l tris ph 8.5) was added to the sample, and the mixture was centrifuged at 14 000 g at room temperature for 40 min . An additional 200 l of buffered urea was added, and the sample was centrifuged at 14 000 g at room temperature for 40 min . The filters were discarded, and the collected peptides were alkylated, by the addition of iodoacetamide in buffered urea to 50 mmol / l in the dark for 20 min . Mcx (oasis 3 cc, 60 mg, waters) cleanup was performed by diluting the samples to 3 ml with 2% formic acid and h2o to ph 3 . The mcx columns were equilibrated with 2 ml of 1:1 methanol: water followed by the addition of the entire sample, then washed with 3 ml of 0.1% formic acid and 2 ml of methanol; peptides were eluted with 1 ml of 95% methanol/5% ammonium hydroxide . The eluted peptides were dried in a speed - vacuum, redissolved in water, and quantified by a modified bca assay (thermo scientific, waltham, ma) using trypsin - digested saliva as a standard . Three micrograms of peptides were further purified and concentrated using the stage - tip protocol . Fresh saliva was collected from a single donor and processed for isolation of endogenous peptides . Sufficient sample quantity was loaded in a single autosampler vial to run three replicate injections in succession . Fresh saliva was collected from a single donor and divided into three portions . Each aliquot was processed for endogenous peptide isolation with the identical protocol, placed into individual autosampler vials and analyzed in succession . Fresh saliva was collected from a single donor and processed for isolation of endogenous peptides and aliquoted with increasing amounts (0.5, 1.0, 1.5, 2.0, 2.5, and 3 g) into individual vials . Ms / ms on an ltq - orbitrap xl mass spectrometer (thermo scientific, waltham, ma) equipped with an eksigent (eksigent technologies, redwood city, ca) 1dlc nanoflow system and a microas autosampler . An in - house, pulled tip capillary column with a 100 m inner diameter was packed to 13 cm with magic c18aq 5 m, 200 pore particles (michrom bioresources). Peptide mixtures were dissolved in an aqueous solution containing 2% acn with 0.1% formic acid and then separated by a 240% acn gradient in 0.1% formic acid over 60 min at 250 nl / min . Full - scan mass spectra were acquired in the orbitrap at 60 000 resolution at m / z 400, followed by tandem mass spectrometry (ms / ms) in the ltq of the five most intense ions from the full scan . National cancer institute s cptac network s study 6 data set ltq - xl - orbitrapp@65, which was downloaded from tranche, is now available at https://cptac-data-portal.georgetown.edu/cptac/public . Because data acquisition for this study was performed in profile mode, we converted the .raw files to mzxml files using msconvert version 3.0.3364 specifying centroid = true . Peptide signals were extracted using an in - house software application . In brief, the software takes in a list of mzxml files and processes the file sequentially . Second, after all scans were processed, extracted ion chromatograms (xics) were constructed from the deisotoped peaks . The peptide signals m / z values and retention times were adopted from the xic s apex peak . In lieu of computing an xic s area under the curve, a peptide signal s intensity was computed by summing its xic peak intensities . We arbitrarily selected 2 min from the xics apex peak .) Finally, corresponding peptide signals were grouped across multiple analyses based on m / z and retention time tolerances . As a quality measure, we required at least two ms2 scans (among all sequentially analyzed files) corresponding to each peptide signal . This software and thermo .raw files used for analyses are available from the authors upon request . All ms / ms data were analyzed using sequest version 27, rev 12 (thermo scientific). First, we downloaded the yeast uniprot fasta database (ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/proteomes/yeast.fasta.gz, february 3, 2012) and the crap contaminant fasta database (downloaded from ftp://ftp.thegpm.org/fasta/crap, february 28, 2012). The crap ups entries were replaced with entries from an updated ups fasta database (http://www.sigmaaldrich.com/life-science/proteomics/mass-spectrometry/ups1-and-ups2-proteomic.html, february 22, 2012). The yeast and crap fasta databases were concatenated and designated as the forward database . Each protein sequence was then reversed with a perl script (matrix science, boston, ma), designated as the decoy database, and concatenated to the forward database . For the c versus e experiment, sequest parameters included a fragment ion mass tolerance of 1 da, oxidation of methionine as a variable modification, and 2 da mass tolerance . Scaffold version 3.6.1 (proteome software, portland, or) was used to validate ms / ms - based peptide and protein identifications . Peptide identifications were accepted if they met minimum criteria 7 ppm precursor mass tolerance, one tryptic terminus minimum, six amino acid minimum length, and 90.0% probability as specified by the peptide prophet algorithm . Protein identifications were accepted if they could be established at> 80.0% probability (protein prophet) and contained at least one identified peptide . Proteins with similar peptides that could not be differentiated based on ms / ms analysis alone were grouped to satisfy the principles of parsimony . The resulting false discovery rate (fdr) within scaffold was 1.4% at the protein level and 0.1% at the peptide level (supplemental files 1 and 2 in the supporting information). Unless otherwise specified, all statistical analyses were performed using the r statistical package, version 2.14 - 0 2011 - 10 - 31, r.app 1.41 or 3.02 2013 - 09 - 25 . Two sample t tests were conducted using the r function t.test and the default confidence level was 0.95 . Prior to multivariate analyses, for example, pooled estimate of variance, data were first log - transformed to obtain a normal distribution prior to computing variance . We generated xcs by first determining each peptide s xic and then summing their recorded intensities within each scan . The resulting summed intensities (y axis) were plotted over time (x axis) using r s lowess function (locally weighted polynomial smoother); the smoothing span parameter set to 0.07 for figure 4 and 0.05 for figure 6 . Linear regression normalization was performed by applying least - squares regression on minus versus average (ma) scatter plots using a pairwise iterative algorithm . First, the algorithm selected peptide signals using the cws method and then ma - transformed peptide signal intensities for each pair of runs using eqs 1 and 2 in supplementary note 2 in the supporting information . Fitted data were generated in r with the function lm, for example, lm(ma), and subtracted from observed ratios with eq 3 . The data then were deconvoluted using eq 4 in supplementary note 2 in the supporting information . We performed the iteration process twice because the difference between the mean of all intensity ratios from the previous iteration was <0.005, as previously described . Author: loess normalization was performed in r using the normalizecyclicloess function found in the limma package . The algorithm first selected peptide signals using the cws method; next, the algorithm selected log2 transformed intensities (as required by normalizecyclicloess) prior to submitting them to normalizecyclicloess using default parameters . Quantile normalization was performed in r using the normalizequantile function found in the limma package . The algorithm selected peptide signals using the cws method prior to normalizedquantile analysis with default parameters . The algorithm first selected signals using the cws method prior to computing the median of peptide signal intensity ratios, which was used as a normalization factor . Median scale was performed by scaling peptide signal intensities values within each run by the median of peptide signals selected using the cws method . While we mathematically define pin s neighborhood construction (see results and discussion), in practice we define a peptide signal s neighborhood boundary using a retention time window around the peptide signal s retention time (figure 2) a static retention time window is centered at the peptide signal s retention time, plus and minus a specified time period, for example, 2 min . A dynamic retention time window is based on the width of a peptide signal s xic . Once the neighborhood boundaries are established, the neighborhood is then populated with all peptide signal xic peaks within the retention time window . A peptide signal s intensity is then normalized by computing the proportion of signal within the neighborhood . The proportion is computed by dividing the peptide signal s intensity by the sum of neighborhood xic peak intensities . For all analyses discussed herein, neighborhood boundaries were determined by a dynamic retention time window corresponding to the peptide signal s xic width . Pin s neighborhood construction . Xics for three peptide signals (a c) are shown in three dimensions . Here peptide signal b s neighborhood boundaries (rt window depicted by the horizontal red lines) are determined by its xic width . Peptide signal b s neighborhood construction includes all xic peaks within its retention time window (peaks in red and blue). In this case, the neighborhood includes a portion of peptide signals a s xic and a portion of peptide signal b s xic . In pin, we take a nontraditional approach . We observe that complex variability, such as esi instability, affects bounded temporal regions within chromatographic data, as opposed to systematic bias which affects the entire hplc therefore, to mitigate complex variability, pin normalizes each peptide signal s intensity by computing its proportion of intensity relative to its neighboring peptide signals (figure 2). Mathematically, we define a peptide signal s neighborhood aswhere n is the set of neighboring peptide signals, n is a peptide signal, rtmin is the index of the peptide signal corresponding to the neighborhood s lower retention time boundary, and rtmax is the index of the peptide signal corresponding to the neighborhood s upper retention time boundary . With the neighborhood defined, a peptide signal s normalized intensity, that is, its proportion of neighborhood intensity, iswhere nj is the intensity of peptide signal j and ni is the intensity of peptide signal i falling within the neighborhood retention time boundaries . The premise for this new approach is that we view biological samples and hplc esi mathematically, a composition is defined as...x of d parts is a d x 1 vector (x1, x2,..., xd) of positive components whose sum is 1. (20) because the components quantities must sum to 1, compositional data are an example of sum - constrained data . While the concept of sum - constrained data may seem foreign to most, sum - constrained data are actually quite prevalent . For example, simple percentages and parts per million (ppm) are sum - constrained measurements . Percentages are sum - constrained because the total is constrained to 100, and ppm measurements are sum - constrained because the total is constrained to one million . In analyzing compositional data, statistical analyses must be done with care because nave analysis of compositional data can lead to incorrect inferences . The only abundance information that remains for a single component is relative to the other components making up the whole . In other words, prior to statistical analyses, compositional data must meet two conditions: (1) the components of interest must be relatively small parts of the whole; (2) components within the whole must remain relatively constant in size and composition . When these conditions are met, univariate statistical tests, such as a student s t test, on compositional data should not lead to incorrect inferences . The rationale for our treatment of biological samples as compositions is two - fold . First, biological systems, whether at the molecular, cellular, or organ level, are dynamic and interactive . For example, within a proteome, the presence, absence, or change in abundance of one or more proteins can affect the presence, absence, or change in abundance of one or more other proteins in the system . Therefore, the abundance of a particular protein, relative to other proteins is important . As a result, researchers reveal biological variation by finding differences in a biological sample s composition . Second, sample collection and preparation impose constraints on the number of proteins available for measurement . For example, aliquoting a portion of a sample, perhaps based on a bradford assay, puts a cap on the amount of protein used for comparison . As a result the rationale for our treatment of chromatographic data as compositional stems from the fact that constraints are imposed within hplc esi for example, during esi, coeluting peptides compete for a finite number of charges and a limited space on droplets . The finite number of charges and the limited space are constraints . As a result, mass spectral intensities of ionized peptides depend on their coeluting peptides, and thus we deem the resulting chromatographic data are compositional . Therefore, when we treat chromatographic data resulting from the analysis of proteomic samples as compositional, we can detect statistically significant differences in proportions across populations . This compositional data meets the two prerequisite conditions for its statistical analysis: (1) the amount of a single component (peptide) is small relative to the whole, which remains true in its corresponding chromatographic data, and (2) in the vast majority of biological systems, the core proteome and its digested peptides, accounts for more than 90% of the measured protein mass in a sample; that is, only 10% is compositionally different between similar biological samples . Thus, when we view complex biological samples and chromatographic data as compositional, we can use simple univariate statistics (such as student s t test) to reveal biological variation . This reasoning stems from the fact that when using the same (or very similar) lc column, peptides elute in approximately the same order . As a result, retention time windows within resulting chromatograms will contain approximately the same set of peptide signals when analyzing different samples with overall similar composition . Thus, this region becomes a subcomposition, which, for mathematical purposes, is just another composition . We can do so even when complex variability is present because complex variability similarly affects peptide signal intensities within close temporal proximity . Finally, when we view regions (also called neighborhoods) of chromatographic data as compositional, we can detect statistically significant differences in peptide signal proportions across populations . This is true because the data again meet the two prerequisite conditions for comparisons of compositional data: (1) provided that neighborhood boundaries are properly set, a peptide signal s neighborhood population is large enough so that the intensity of a peptide signal remains small relative to the sum of its neighbors intensities and (2) provided that similar lc (column type and gradient duration) is used, neighborhoods will contain sufficiently overlapping populations . With underlying assumptions as a basis, we assessed our pin strategy s ability to mitigate systematic bias and complex variability and reveal statistically significant biological variation . As an initial evaluation, we applied pin to the cptac data described in the introduction data that alluded to the capabilities of global scaling functions and motivated our development of a new method . Use of pin on this data produced the ideal result expected from normalization (figure 1c): nearly identical xcs between replicates, even for the run containing the complex variability due to electrospray instability . Encouraged by these results, we further evaluated pin vis - - vis median scale and other current normalization strategies . We applied several different peptide selection strategies and global scaling functions to archived data sets from four experiments: instrument variability, sample variability, serial dilution (as a proxy for loading amount differences), and cptac study 6 . The results of these experiments, when taken together, demonstrate pin s superior mitigation of systematic bias and complex variability while retaining biological variability . To assess reduction in instrument variability, we generated three replicates by analyzing a single aliquot of salivary endogenous peptides using an autosampler and hplc esi (see experimental procedures and supplemental tables 3 and 4 in the supporting information .) First, we assessed reduction in variability between un - normalized data, data normalized by five global scaling functions, and data normalized by pin . We used two commonly employed metrics: pooled estimate of variance (pev) and median standard deviation coefficient of variance (cv). Although we evaluated numerous scaling functions, we only report pin and the five best performing normalization strategies determined by pev and cv reduction . For the global scaling functions, we employed the cws peptide selection strategy prior to normalization . For pin variability experiment, pin outperformed the five best current normalization strategies by reducing pev by 73% and cv by 46%, compared with an average of 13 and 19% respectively for the global scaling functions . Four experiments show pin s superior performance in reducing variance using two commonly used measurements: pooled estimate of variance (pev) and coefficient of variation (cv). (a, c) pin (right - most column in each figure) versus five common normalization strategies in reducing pev for each of the four experiments . (b, d) pin (again, right - most column in each figure) versus the same five common normalization strategies in reducing cv for each of the four experiments . To assess reduction in the variability resulting from sample handling, we followed the same protocol as the instrument variability experiment, except we prepared three aliquots of salivary endogenous peptides in parallel and analyzed each aliquot using an autosampler and hplc esi (see the experimental procedures and supplemental tables 5 and 6 in the supporting information .) Again, we employed cv and pev and compared pin s results to the top five performing normalization strategies . Pin continued to outperform these normalization strategies by reducing pev by 71% and cv by 41% compared with an average of 11 and 8%, respectively, for global scaling functions . To assess reduction in variability resulting from loading amount differences, we conducted serial dilution experiments using a complex mixture of salivary endogenous peptides and bradykinin as a spiked - in standard . (see the experimental procedures and supplemental tables 7 and 8 in the supporting information .) Traditionally, researchers have conducted serial dilution experiments to determine peptide abundances in a concentrated sample or produce calibration curves . We used serial dilution experiments unconventionally, as a proxy for loading amount differences we prepared six aliquots of this mixture by combining increasing amounts (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g) of salivary endogenous peptides with an equal amount of bradykinin (50 fmol). We again employ pev and cv and compared pin to the top five performing normalization strategies . Pin reduced pev by 75% and cv by 55% compared with an average of 34 and 23%, respectively, for global scaling functions . To assess reduction in variability in the face of biological variation, we used data from the cptac study 6 data set for instrument aliased ltq - xl - orbitrapp@65 . In brief, cptac study 6 evaluated mixtures of yeast with sigma ups1 spiked in at five different levels (a e), each level three times greater than the previous level . Each sample was then analyzed three times by hplc esi ms / ms . We selected samples c and e because sample c contained complex variability and sample e contained nine times the amount of ups1 (experimental procedures, supplemental tables 9 and 10 in the supporting information). Using the c versus e data set, pin again outperformed global scaling function pin reduced pev by 61% and cv by 19% while global scaling functions, on average, pev by 9% and surprisingly increased cv by 14% (figure 3d, fourth row). In this case, global scaling functions had a negative effect rather than a positive effect in normalizing intensities . To assess pin s ability to mitigate systematic bias again, we used the serial dilution data set as a proxy for loading amount differences . We chose to visualize loading amount differences because it was a classic example of systematic bias (figure 4 and supplementary figure 2 in the supporting information). When we plotted the un - normalized peptide signal intensities, we observed divergent regression lines, indicative of systematic bias due to loading amount differences (figure 4a). As described in supplemental note 1 in the supporting information, data with no systematic bias would result in regression lines lying on the horizontal line positioned at 0 on the y axis . We then plotted the same data after normalization using median scale as the global scaling function (figure 4b). We observed that the regression lines have slightly converged and were repositioned below and above y = 0, indicating improvement in systematic bias . Finally, we plotted the data after normalization using pin (figure 4c). We observed that the regression lines on the right end of the plot were then positioned on or very near y = 0 . However, we also noted that the lines remained below the horizontal flat line on the left end of the plot . Unlike median scale, the regression lines converged, making the regression lines nearly indistinguishable . From these observations, we concluded that using pin performed well to mitigate the systematic bias, although a small amount remains . Furthermore, we concluded that pin outperformed median scale normalization in making systematic bias consistent between runs . Serial dilution - pin versus median scale minus versus average (ma) pots . (a) ma plot visually demonstrates that pin outperforms median - scale normalization in mitigating systematic bias . (a) ma plot of un - normalized data for six different loading amounts reveals systematic bias (regression lines diverge from y = 0). (b) ma plot of median scale data for six different loading amounts demonstrates a slight improvement in systematic bias (regression lines begin to converge around y = 0). (c) ma plot for pin normalized data for six different loading amounts demonstrates a substantial improvement in systematic bias (regression lines converge around y = 0). Typically, in a serial dilution experiment, the data are normalized using an internal or spiked in peptide with the goal of determining absolute abundance and relative abundance, rather than composition . The standard metric for absolute abundance is the coefficient of correlation (r), with the goal of perfect correlation (r = 1.0). The standard metric for relative abundance is fold change, with the goal of monotonically increasing fold changes corresponding perfectly to the original amounts loaded . Here we expected fold changes of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 corresponding to the saliva peptide load amounts of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g, respectively . We first plotted the un - normalized intensity for a single salivary peptide (gpgifpppppqp), indicative of other salivary peptides found in each of the six serial dilution runs and computed the r and fold changes (figure 5a, e). We noted that r = 0.81, and fold changes were compressed and not monotonically increasing . We then plotted the spike - in normalized intensity (with 500 fmol bradykinin peptide) of the same peptide and computed the r and fold changes (figure 5b, e). The measured correlation improved to r = 0.98, and pin versus spiked - in standard normalization for a single peptide (gpgifpppppqp) intensity with six different sample loading amounts . (a) un - normalized intensity plot shows mediocre correlation (r = 0.82). (b) spiked - in (bradykinin) normalized intensity plot shows improved correlation (r = 0.98) but does not remove systematic bias stemming from loading amount differences (slope is very high). (c) pin normalized intensity plot shows decreased correlation (r = 0.81) but removes systematic bias stemming from loading amount differences (slope = 0.01). (d) pin normalized data followed by original loading amount scaling shows improved correlation (r = 0.99), which is better than spiked - in standard normalization . (e) table showing fold changes for each intensity plot (a d) using the 1.0 g sample loading amount as the common denominator, while pin compresses the fold changes . However, when we use the serial dilution experiment as a proxy for loading amount differences, then, our goal should not be to achieve perfect correlation to absolute amounts; rather, our goal is to find no compositional differences (slope = 0.0). This stems from the fact that even though overall loading amounts varied between these samples, within each sample the proportion of any given peptide to the whole did not change because overall composition did not change . Furthermore, relative fold changes should all be equal to 1, indicating no changes in the composition . We plotted the pin - normalized intensities for the same peptide and computed the slope and fold changes (figure 5c, e). We observed that normalizing with pin achieves a slope = 0.01, and fold changes are near 1, indicating little change in biological variation . Because we knew the loading amounts, we estimated the absolute abundance of peptides initially loaded onto the hplc column by scaling pin normalized intensities by the run s loading amount . Scaling pin normalized data by the loading amount showed r = 0.99 (figure 5d) and monotonically increasing fold changes (figure 5e). Scaled pin outperformed spike - in normalization for estimating absolute peptide abundance and in estimating fold changes . We next turned our attention from a single peptide fold change to the analysis of composite xcs representing all peptide signals in a run . When we plotted the un - normalized xcs, we observed a clear separation of monotonically increasing xcs, indicating the presence of systematic bias (figure 6a). However, upon closer inspection, we found that the xcs for the 2.0 g and 3.0 g samples were not in the expected order . We then plotted the spike - in normalized xc and observed that the 3.0 g xc shifted its position but still lay below the 2.0 g xc (figure 6b). Next we plotted the xcs for the pin normalized data and observed a convergence of the xcs (figure 6c). Furthermore, we observed an undulation in the xcs, consistent across the different runs . Finally, we plotted the pin normalized data scaled by loading amounts and observed that the xcs became monotonically increasing and were in the correct order for nearly all time points (figure 6d), with the exception of the lowest loading amounts . Peptide signal extracted chromatograms pin versus spiked - in standard normalization for six different loading amounts . (the 2.0 and 3.0 g extracted chromatograms are not in the correct order .) (b) spiked - in (bradykinin) normalized extracted chromatogram plots show that the 2.0 g extracted chromatogram is now above the 1.0 g extracted chromatogram but is still below the 2.0 g extracted chromatogram . (c) pin normalized extracted chromatogram shows convergence, indicating the removal of systematic bias . (d) scaling pin normalized data by the original loading amounts plot shows extracted chromatograms monotonically increasing and in the correct order . Finally, we assessed pin vis - - vis common normalization strategies in the context of detecting of biological variation . Again, we used the cptac study 6 and selected samples c and e. here we used a student s t test to determine the number of differentially abundant (or proportional) ups1 and yeast proteins and peptides found . We used spans to recommend and perform the optimal peptide select strategy and global scaling function combinations . Spans performed correctly, finding no global scaling functions were appropriate for the cptac c versus e data set . These results confirmed our previous findings that global scaling functions cannot capture and mitigate complex variability . Nonetheless, spans was run using three normalization strategies: (1) los (5%) peptide selection strategy with mean global scaling; (2) rip peptide selection strategy with mean global scaling; and (3) rip peptide selection strategy with median global scaling (supplemental table 11 in the supporting information). The up designation indicates proteins present lower abundance (or proportion for pin) in sample e compared with sample c. the down designation indicates lower abundance (or proportion in pin) in sample e compared with sample c. (a) ups1 proteins statistically different between samples c and e. (b) yeast proteins that are statistically different between samples c and e. (c) number of true positives, false - positives, and false - negatives for ups1 and yeast proteins . Pin outperformed median and mean global scaling functions in finding statistically significant ups1 proteins (figure 7a). With pin, we found 36 statistically different ups1 proteins going up (true positives) and only 1 ups1 protein going down in e versus c (false - positive). Global scaling functions performed abysmally, finding fewer ups1 proteins as statistically different compared with the un - normalized data . Pin found 20% more (6) ups1 proteins as statistically different that were false negatives in the un - normalized data (figure 7c). One important point is that when we treat chromatographic data as compositional data, we also measure statistically significant decreases in background yeast proteins in sample c versus e. we assert these are true positives, reflecting physical realities of these samples . Our reasoning is that when viewing the proteome as compositional, then it follows that as the proportion of ups1 proteins goes up, the proportion of yeast proteins must go down . It is also well known that increasingly abundant peptides can suppress ionization of coeluting peptides, leading to decreased proportional intensities of some yeast peptides in response to increasing load of ups1 peptides . This may be even more prevalent an affect within adding relatively large amounts of the ups1 standard into the yeast background . Therefore, with pin, we expect to find a larger number of yeast peptides with reduced proportion to the whole when compared with other scaling functions (figure 7b, c). To our knowledge, we are the first to demonstrate that biological variation can be revealed in proteomic chromatographic data by viewing it as compositional . Here we also introduce pin, a new local method that normalizes a peptide signal s intensity by computing its proportional intensity relative to its neighborhood . We show that pin dominates competing normalization strategies when measuring reduction in variability and finding biological variation, even when complex variability is present . The only related work is very recent work by lyutvinskiy et al ., which describes a new instrumental response correction method, a local normalization method, for significant variations in the eluent and analyte composition for improving accuracy of predictive models . We compared and contrasted pin with their method and found some similarities and some striking differences . Both methods employ temporally bounded neighborhoods to normalize each peptide signal . However, the composition of the neighborhoods is decidedly different in the two methods . While pin takes an unbiased approach to populating its neighborhood by including all peptide signal xic peaks within a retention time window, their method takes a biased approach by populating their neighborhoods using only peptides confidently identified from ms2 spectra . Furthermore, the method for correction is not clearly defined, making it difficult to evaluate . In one instance, they describe an abundance alignment method using unchanged peptides as internal standards . In another instance, they report using the square root of the median of all peptides within the retention time window . Unfortunately, their report did not evaluate performance vis - - vis common normalization strategies, making it difficult to compare to our pin strategy . They did report that their normalization strategy improved their predictive model, but it is unclear to us whether the improvement stemmed from the local nature of their strategy or if other common normalization strategies would have produced similar results . Regardless of the differences, we both agree on the central need to account for complex variability in intensity - based quantification . A key benefit of pin over common normalization strategies is its simultaneous mitigation of multiple types of measurement error, including systematic bias and complex variability . This benefit stems from treating chromatographic data as compositions and temporal regions within the data as subcompositions . Thus, any bias affecting the composition as a whole will also affect subcompositions . As a result, when pin mitigates complex variability within the subcomposition, it inherently also mitigates systematic bias . A second benefit of pin over common normalization strategies is that pin requires no a priori knowledge of the type or source of measurement error . For example, a pipetting error doubling the amount of the sample does not necessarily correspond to a linear response in corresponding ion intensities in the resulting data . Therefore, simply normalizing by a single global scaling factor, such as the median of measured ion intensities, is not accurate . Computing the appropriate global scaling factor to mitigate systematic bias involves a priori knowledge of both absolute loading amounts and absolute ion counts . However, because pin treats chromatographic data as compositional, the only information about peptide abundance and ion counts is relative information; thus, pin requires no knowledge of absolute loading amounts or absolute ion counts . A third benefit of pin over common normalization strategies utilizing global scaling functions is that pin does require a complete matrix on which to compute and thus implicitly handles the missing value problem . Then, to compare a single peptide signal s pin normalized intensities (proportions) across runs, its neighborhoods must be compositionally similar . If needed, missing values can still be imputed after normalization but prior to statistical inference, as is recommended . Of course, as with any method, pin s performance depends on some factors . First, pin relies on high - resolution instrumentation and accurate mass measurements (<10 ppm error) to extract and quantify relevant chromatographic information . Second, as with other methods, the peptide elution order for analyzed mixtures must be similar; pin requires similar order to form similar neighborhoods . This means mixtures analyzed using different types of chromatographic systems may not be easily compared . Third, although pin tends to compress the dynamic range of fold changes, the end results are unaffected because it is fold change statistical significance (not numerical value) that counts for determining biological variation . However, if the original loading amount is known, for example, in a serial dilution experiment, that information can be used to scale results and accurately compute fold changes . Fourth, with any normalization method, overfitting and underfitting data is a concern . With pin, we first construct neighborhoods using a temporal window, and the width of this window primarily controls data fitting quality . We conducted several experiments (results not shown) varying the window width and found that varying the window width between 2 to 5 min as well setting window size to the temporal width of a true peptide signal s xic made little difference in the results . However, setting the window <2 min and> 5 min tended to overfit and underfit the data, respectively . We expect that optimal window width will correlate somewhat with chromatographic characteristics, primarily gradient duration . Therefore, we utilize the width of the peptide signal s xic as input to a function dynamically computing each peptide signal s temporal boundaries . Despite pin s improvements in reducing variability and the number of false - negatives reported, ms - based results must still be confirmed via hypothesis - driven experiments, such as a western blot or targeted ms . Targeted ms, for example, srm, is a powerful tool gaining popularity over traditional biochemical analyses due to the ever - increasing scale of today s high - throughput experiments . Such experiments require as input a transition list or an inclusion list containing feature values (m / z retention time pairs, which are simply peptide signals). Because pin operates on a list of peptide signals, each with an associated ms / ms spectrum containing potential srm transitions, the software implementing pin thus, pin is well - suited to drive targeted ms experiments . Given that common normalization strategies cannot capture and correct systematic bias and complex variability inherently present in hplc ms / ms workflows, we expect pin to dramatically improve intensity - based quantification from hplc, although we studied complex protein digests and endogenous peptide mixtures, we believe pin will be widely applicable to many omics fields using hplc ms / ms to analyze complex mixtures, for example, lipidomics, glycomics, and metabolomics . The upshot will, we expect, be reproducibility- and repeatability - improved, and otherwise falsely reported or missed statistically significant biological variation will be discovered.
Pulmonary sling is a vascular abnormality wherein the left pulmonary artery arises from the right pulmonary artery and then traverses between the esophagus and the trachea toward the hilum of the left lung . It was first described by glaevecke and doehle in 1897 . Since then, about 100 cases have been reported in the world . The average age at the onset of symptoms, such as respiratory distress and cyanosis, is 2 months and about half have symptoms from birth . So, adult cases are very rare, are most often asymptomatic and usually found incidentally . When combined with complete cartilaginous tracheal rings in the absence of posterior membranous portion, it is called ring - sling complex . In that case, we here report an adult case who was diagnosed as pulmonary sling with complete tracheal ring by ct and bronchoscopy along with a literature review . She had had symptoms of airway obstruction, such as wheezing, hoarseness and mild dyspnea since a young infant . Blood pressure was 140/90 mmhg, pulse rate was 80/min, body temperature was 36.5c and respiration rate was 16/min . On physical examination, she was alert, but had a slight dyspneic appearance . There were no cyanosis, chest deformities and cardiac murmurs . Fev1 was 1.52l (66.2% of predicted value) and fvc was 1.90l (54.5% of predicted value) on pulmonary function test . Complete cartilaginous ring without posterior membranous portion was found on the entire trachea (fig . 1). The plain chest x - ray showed a right mediastinal mass - like density and a long - segment tracheal narrowing (fig . 2). The chest ct revealed the anomalous left pulmonary artery originating from the right pulmonary artery and its posterior course between the trachea and the esophagus (fig . She was discharged without specific treatment because of mild symptoms which are mainly caused by complete tracheal ring and not by tracheal compression due to vascular anomaly . The term vascular ring was introduced to describe the mediastinal vascular anomalies causing tracheobronchial compression by dr . The pulmonary sling is a kind of vascular ring and the term was first used by contro et al . In 1958 to distinguish it from other vascular anomalies . Carl et al . Reported that when vascular rings were classified into four groups, as follows, 1) complete vascular rings with double aortic arch or right aortic arch 2) pulmonary sling 3) innominate artery compression and 4) miscellaneous, pulmonary sling was about 4.5% . Embryologically, it has been suggested that it is caused by a maldevelpment of the left ventral pulmonary artery bud due to growth failure or reabsorption . The left lung may capture its arterial supply by connection of the left postbronchial plexus with the right sixth arch through capillaries caudal to the lung bud . Then, as the lung bud grows caudally, the course of the left pulmonary artery is behind the developing trachea - bronchial tree . About half have cardiac or tracheo - bronchial anomalies which determine the prognosis . According to christoph dohleman s review, the associated cardiac anomalies were 49% and among those, patent ductus arteriosus, ventricular septal defect and atrial septal defect were most common . Tracheal stenosis with complete tracheal rings was the most common anomaly, followed by stenosis of main stem bronchus, tracheal bronchus and tracheo - esophageal fistula . Wells et al . Proposed a classification system assigning pulmonary sling without complete tracheal rings to type 1 and pulmonary sling with complete tracheal ring to type 2 . Ring - sling complex can be classified into a lethal form and a form in which there is enough pars membranacea left to allow sufficient tracheal growth . The two forms can be distinguished only by their clinical course, i.e., either severe respiratory symptoms with progression or mild symptoms without progression . Bronchoscopy and echocardiography should be performed to evaluate the combined cardiac or tracheo - bronchial anomalies . The plain radiographic features include unequal aeration due to compression of the right main bronchus by the anomalous vessel, low carina, horizontal equal - length right and left mainstem bronchi and long - segment tracheal stenosis . Diagnostic considerations such as dilated azygous vein, lymphadenopathy, foregut cyst or esophageal neoplasm should be excluded . Most cases can be diagnosed by ct or mri, but misinterpretation can occur because of a bifurcation - like structure of the pulmonary artery . The left pulmonary artery can be missed because of its different level of crossing the carina or main bronchus . Was the first to propose the now most common corrective surgical procedure: division and reimplantation of the anomalous left pulmonary artery into the pulmonary trunk anterior to the trachea with division of the ligamentum arteriosum . Because of high operation mortality and loss of function of the left lung due to obstruction of the reconstructed vessel, a conservative approach with symptomatic management was recommended initially . But recent development of operation technique has resulted in low operation mortality and good patencty of the reconstructed vessel . So, in view of the seriousness of this condition and the fact that reports of surgical management have been encouraging, current policy is to offer an operation as soon as the diagnosis is made.
The identity of a human being should be preserved even after death, as there are consequences, often financial, that lead to the court of law, which demands that the identity of the deceased be established before a verdict is passed . Forensic science based evidence is accepted in a judicial setting by the court and plays a major role in the identification of individuals who cannot be identified visually or by other simple means . However, it is difficult to perform after marked post - mortem changes such as decomposition and skeletonization have taken place due to environmental factors such as humidity, temperature, and exposure to microorganisms . Nevertheless, a post - mortem is obligatory in terms of the law and social norms . The identification process involves anthropological analysis for sexing skeletal material, which provides relatively fast and accurate data that help the police investigator narrow down his field of search to a limited geographic area or within a gender . Gender has been determined from pelvis, skull, and long bones, with assessment of epiphysis and metaphysis in unknown skeletons . The distance between the basion and the prosthion, the circumference of the head, the length of the supraorbital ridge, mastoid process, and mandibular ramus, the shape and length of palate, the circumference of occipital condyle, the sizes of teeth, the length and height of head, the distance between the basion and nasion, the height of the mandibular symphysis, the foramen magnum, the sphenoidal sinus, the sella turcica, and the frontal sinuses have also been used recently for gender determination in unknown remains . It has been reported that maxillary sinuses remain intact despite the skull and other bones getting badly disfigured in victims who are incinerated . Jovinic stated that maxillary sinuses reach their mature sizes at about the age of 20 years . During adulthood, their shapes and sizes the size of the maxillary sinus can be affected due to environmental factors, genetic diseases, or post infections . As the image represents a contiguous series of cross - sections and three - dimensional information and the machine is available in most of the hospitals, ct has been applied in the study of fossil skulls . Ct scan can be used for determination of gender by measurement of maxillary sinus when other methods are inconclusive, though this method is not error - free . The aim of the study was to determine the size [mediolateral (ml), superoinferior (si), and anteroposterior (ap) linear dimensions] and the volume of the maxillary sinuses using ct scanning, and investigate whether these parameters can be used to determine the gender of an individual for forensic identification . The study was designed to measure the ml, si, and ap dimensions along with the volumes (v) of both the right and the left maxillary sinuses of an individual using ct . The study was carried out on a small regional population of patients presenting to the imaging center of institute of dental studies and technologies, modinagar, western uttar pradesh, india, between december 2009 and august 2011 . Patients were selected on the basis of strict inclusion and exclusion criteria that favored intact maxillary sinuses without any pathological, physiological, or surgical deformity . Thirty patients (15 male and 15 female) were included in the study, who were between 20 and 50 years of age, had retained all their permanent teeth, and were referred to the imaging center to have a ct scan of the paranasal sinuses (ct pns) for various reasons, in which no pathological findings were detected on ct images . Patients who were found to have maxillary sinus pathology on ct scan, facial deformities involving the maxilla, clinical facial asymmetries, or had previously undergone a surgery of the maxillary sinus were excluded from the study . Non - contrast coronal ct scan was performed on all patients to visualize the maxillary sinuses using ge ct / e dual slice ct scanner (ge healthcare technologies, waukesha, wi, usa). Prior to the scan, the patient was instructed to remove all metallic objects including hair pins, jewelry, and so on from the head and neck region and positioned on the ct table in prone position . The patient's neck was hyper - extended with the chin resting on a pad . For stabilization, sections of 3 mm thickness were planned on the preliminary scout view extending from the anterior margin of the frontal sinus to the posterior margin of the sphenoid sinus, with a reconstruction matrix size of 512 512 at 120 kv, 100 ma . Coronal ct was performed after instructing the patient to remain steady during the entire procedure . For the purpose of standardization, all scans were performed by the same operator on the same machine using similar exposure parameters . The ct image stack thus acquired was transferred to the advantage workstation 5 (ge healthcare technologies) for post processing . The ml and si measurements were made where the maxillary sinus was in its widest position, with the help of the on - screen linear measurement tool on the ct workstation . The linear distance between the two points was annotated on the screen [figure 1]. To measure the ap dimension of the maxillary sinus, the first and the last appearance of the sinus was noted in the sequential coronal ct sections, and the number of sections between them was determined . Finally, the number of sections obtained was multiplied by 3 (thickness of a single section) to find the ap dimension of the sinus . Linear measurement of mediolateral and superoinferior dimensions of maxillary sinus maxillary sinus volume (v) was calculated using the paint on slices tool on the workstation . To define a volume, the outline of the sinus was traced manually on each slice of the image stack using the on - screen mouse pointer in the coronal plane [figure 2]. Once the tracing was complete, the workstation automatically segmented the entire volume of the sinus from the surrounding structures and the segmented portion could be visualized and manipulated in 3d [figure 3]. Tool at this point, switching to the histogram view on the workstation [figure 4] automatically reflected the volume of the sinus in cubic centimeters (cc). The entire procedure was repeated for the right and the left maxillary sinuses separately for every patient . Workstation showing the sinus volume the t - test for independent samples was used to compare these values in two groups . Discriminative analysis was used to detect the gender by using the data obtained from ct scans . The patients were distributed into three age groups starting from 20 to 50 years with a class interval of 10 years . The mean, along with the standard deviation was calculated for all the dimensions of the right and the left maxillary sinuses, namely ml, si, and ap, for both males and females [table 1]. Distribution of maxillary sinus dimensions measured on ct and their standard deviation for the right maxillary sinus, the mean value of the ml dimension was found to be 27.53 4.26 mm in males and 25.12 6.75 mm in females . The mean of si dimension was 38.21 5.77 mm in males and 33.34 6.57 mm in females . Also, the mean of ap dimension was 42.60 3.79 mm in males and 36.00 4.09 mm in females . For the left maxillary sinus, the mean value of the ml dimension was found to be 27.01 5.04 mm in males and 23.22 6.21 mm in females . The mean value of the si dimension was 36.99 4.45 mm in males and 33.11 6.71 mm in females . Also, the mean value of ap was 40.80 2.73 mm in males and 37.20 2.96 mm in females . A statistically significant (p <0.05) difference was found in the right si dimension, left si dimension, and the left ap dimension of the left maxillary sinuses between males and females . A significant (p <0.01) difference was found in the right ap dimension of the maxillary sinus between males and females . The other maxillary sinus dimensions showed a pattern of being larger in males than in females; however, the difference was statistically insignificant . A comparison was made between the dimensions of the right and left maxillary sinus within males and females separately [table 2]. On non - statistical comparison in males, the right maxillary sinus gave an impression of being slightly larger than the left maxillary sinus in its overall dimensions . Similarly, in females, the right maxillary sinus was marginally larger in dimensions than the left maxillary sinus, with the exception of the ap dimension . Aberrantly, the ap dimension of right maxillary sinus was found to be less than that of the left maxillary sinus in females . However, these intra - gender findings were statistically insignificant (p> 0.05). Distribution of right and left maxillary sinus dimensions in males (n=15) and females (n=15) the mean with standard deviation was calculated for the volume of the right (vr) and the left (vl) maxillary sinuses for both males and females [table 3]. For the right maxillary sinus, the mean volume was found to be 16.63 4.54 cc in males and 11.61 5.15 cc in females . For the left maxillary sinus, the mean volume was found to be 15.19 3.94 cc in males and 10.95 4.98 cc in females . Distribution of maxillary sinus volume measured on ct and its standard deviation the volume of the maxillary sinuses in males was overall larger than in females . A significant (p <0.01) statistical difference was found in the right maxillary sinus volume between males and females . A significant (p <0.05) difference was found in the left maxillary sinus volume between males and females . A comparison was made between the volume of the right and left maxillary sinus within males and females separately [table 4]. Overall, both in males and females, the right maxillary sinus volume was found to be larger than the left maxillary sinus volume . However, this intra - gender volume difference was statistically insignificant (p> 0.05). Distribution of right and left maxillary sinus volume in males and females a discriminative analysis was performed using the spss software to determine whether these linear measurements and volume could be used for gender determination . The following formula could be used for gender determination from measurements of the right maxillary sinus: gender = 5.116 - 0.159mlr - 0.014 sir + 0.171 apr + 0.218 vr where mlr is the ml dimension of the right maxillary sinus, sir is the si dimension of the right maxillary sinus, and apr is the ap dimension of the right maxillary sinus . The accuracy of gender predicted using the linear measurements and volume of the right maxillary sinus was found to be 80.0% in both males and females . The following formula can be used for gender determination from measurements of the left maxillary sinus: gender = 0.720 - 0.258 mll - 0.146 sil + 0.120 apl + 0.586 vl where mll is the ml dimension of the left maxillary sinus, sil is the si dimension of the left maxillary sinus, and apl is the ap dimension of the left maxillary sinus . The accuracy of gender predicted from the linear measurements and volume of the left maxillary sinus was found to be 66.7% in males and 80.0% in females, with an overall gender prediction accuracy of 73.3% . The following formula can be used for gender determination from measurements of the right and the left maxillary sinuses together: gender = 4.033 - 0.101 mlr - 0.21 sir + 0.397 apr + 0.118 vr - 0.23 mll - 0.014 sil - 0.417 apl + 0.358 vl the accuracy of gender predicted from the linear measurements and volume of the right and the left maxillary sinuses together was found to be 80.0% in males and 86.7% in females . Gender determination from measurements of right and left maxillary sinuses when the variables in the formulae derived above are substituted with the desired values, a numeric value is obtained for the gender . When this value is positive, the gender is predicted to be male . A negative value is predicted to be female . It was observed that the accuracy of gender prediction from the measurements of the right maxillary sinus (80.0%) was more than that of the left maxillary sinus (73.3%). When the measurements of the right and the left maxillary sinuses were accounted together for predicting the gender, the accuracy rate increased to 83.3% . The mean, along with the standard deviation was calculated for all the dimensions of the right and the left maxillary sinuses, namely ml, si, and ap, for both males and females [table 1]. Distribution of maxillary sinus dimensions measured on ct and their standard deviation for the right maxillary sinus, the mean value of the ml dimension was found to be 27.53 4.26 mm in males and 25.12 6.75 mm in females . The mean of si dimension was 38.21 5.77 mm in males and 33.34 6.57 mm in females . Also, the mean of ap dimension was 42.60 3.79 mm in males and 36.00 4.09 mm in females . For the left maxillary sinus, the mean value of the ml dimension was found to be 27.01 5.04 mm in males and 23.22 6.21 mm in females . The mean value of the si dimension was 36.99 4.45 mm in males and 33.11 6.71 mm in females . Also, the mean value of ap was 40.80 2.73 mm in males and 37.20 2.96 mm in females . A statistically significant (p <0.05) difference was found in the right si dimension, left si dimension, and the left ap dimension of the left maxillary sinuses between males and females . A significant (p <0.01) difference was found in the right ap dimension of the maxillary sinus between males and females . The other maxillary sinus dimensions showed a pattern of being larger in males than in females; however, the difference was statistically insignificant . A comparison was made between the dimensions of the right and left maxillary sinus within males and females separately [table 2]. On non - statistical comparison in males, the right maxillary sinus gave an impression of being slightly larger than the left maxillary sinus in its overall dimensions . Similarly, in females, the right maxillary sinus was marginally larger in dimensions than the left maxillary sinus, with the exception of the ap dimension . Aberrantly, the ap dimension of right maxillary sinus was found to be less than that of the left maxillary sinus in females . However, these intra - gender findings were statistically insignificant (p> 0.05). Distribution of right and left maxillary sinus dimensions in males (n=15) and females (n=15) the mean with standard deviation was calculated for the volume of the right (vr) and the left (vl) maxillary sinuses for both males and females [table 3]. For the right maxillary sinus, the mean volume was found to be 16.63 4.54 cc in males and 11.61 5.15 cc in females . For the left maxillary sinus, the mean volume was found to be 15.19 3.94 cc in males and 10.95 4.98 cc in females . Distribution of maxillary sinus volume measured on ct and its standard deviation the volume of the maxillary sinuses in males was overall larger than in females . A significant (p <0.01) statistical difference was found in the right maxillary sinus volume between males and females . A significant (p <0.05) difference was found in the left maxillary sinus volume between males and females . A comparison was made between the volume of the right and left maxillary sinus within males and females separately [table 4]. Overall, both in males and females, the right maxillary sinus volume was found to be larger than the left maxillary sinus volume . However, this intra - gender volume difference was statistically insignificant (p> 0.05). A discriminative analysis was performed using the spss software to determine whether these linear measurements and volume could be used for gender determination . The following formula could be used for gender determination from measurements of the right maxillary sinus: gender = 5.116 - 0.159mlr - 0.014 sir + 0.171 apr + 0.218 vr where mlr is the ml dimension of the right maxillary sinus, sir is the si dimension of the right maxillary sinus, and apr is the ap dimension of the right maxillary sinus . The accuracy of gender predicted using the linear measurements and volume of the right maxillary sinus was found to be 80.0% in both males and females . . The following formula can be used for gender determination from measurements of the left maxillary sinus: gender = 0.720 - 0.258 mll - 0.146 sil + 0.120 apl + 0.586 vl where mll is the ml dimension of the left maxillary sinus, sil is the si dimension of the left maxillary sinus, and apl is the ap dimension of the left maxillary sinus . The accuracy of gender predicted from the linear measurements and volume of the left maxillary sinus was found to be 66.7% in males and 80.0% in females, with an overall gender prediction accuracy of 73.3% . The following formula can be used for gender determination from measurements of the right and the left maxillary sinuses together: gender = 4.033 - 0.101 mlr - 0.21 sir + 0.397 apr + 0.118 vr - 0.23 mll - 0.014 sil - 0.417 apl + 0.358 vl the accuracy of gender predicted from the linear measurements and volume of the right and the left maxillary sinuses together was found to be 80.0% in males and 86.7% in females . Gender determination from measurements of right and left maxillary sinuses when the variables in the formulae derived above are substituted with the desired values, a numeric value is obtained for the gender . A negative value is predicted to be female . The accuracy of gender prediction, however, was variable . It was observed that the accuracy of gender prediction from the measurements of the right maxillary sinus (80.0%) was more than that of the left maxillary sinus (73.3%). When the measurements of the right and the left maxillary sinuses were accounted together for predicting the gender, determination of gender is an important aspect of forensic investigation as it narrows down the investigator's field of search . Various methods which have been discussed below have been used to identify the gender of the human remains from a crime scene or a site of mass disaster . It has been reported that 100% accuracy can be achieved in gender determination from skeleton, 98.0% from both pelvis and skull, 95.0% from pelvis only or the pelvis and long bones, 90.095.0% from both the skull and the long bones, and 80.090.0% from the long bones only . Morphometric parameters such as mesio - distal and bucco - lingual dimensions of the right permanent teeth were shown to predict the gender with an accuracy of 87.0% . Other parameters such as odontometric differences, root length, and crown diameter have been used to determine the gender with varying degrees of accuracy . The circumference and area of the foramen magnum were used to differentiate the gender with an accuracy of 67.0% and 69.3%, respectively . In their study of the linear measurement of palatal bone and skull base showed significant sexual dimorphism, with reliability rates of 63.0% and 65.0%, respectively . A study based on radius and ulna for gender determination showed an accuracy for forearm ranging between 76.0% and 86.0%, whereas osman showed a sex determination accuracy as high as 96.0% . Eshak in his study of gender determination showed that metacarpals, proximal phalanges, and distal phalanges are sexually dimorphic with an accuracy of 80.0%, 76.6%, and 80.0%, respectively . Three - dimensional volume - rendering reconstructed image of metacarpal gave more accurate result (92.0%). Measurements of hand length and phalanges of the fingers and dimensions of the palm have been used for gender determination with varying degrees of accuracy . Various methods involving the soft tissues, such as cheiloscopy (lip prints), radiographic methods for gender determination such as distance between the crest of alveolar ridge and the superior margin of mental foramina on orthopantomogram of edentulous individuals, and radiograph of the calcaneus, have been used . Torwalt and robert were able to predict the gender using width of the fourth rib and sternal area on chest radiographs with an accuracy of 95.8% for males and 90.3% for females . Sex could be determined with an accuracy of 95.6% by making cephalometric plots on lateral teleradiography with an orthodontic software . Hsiao et al ., attempted to develop a method to determine sex from lateral cephalometric and discriminant functional analysis and determined sex with 100% accuracy in a random sample of 100 taiwanese adults . The studies discussed above for gender determination used plain radiographs, which included lateral cephalograms, and panoramic view for mental foramen, gonial angle, and mandibular ramus, along with ankle and hand it seems that plain radiographs have a higher average degree of accuracy in predicting the gender when compared to ct used in our study . However, plain radiographs, along with other conventional methods discussed above can only be used when the bones, skull, or the skeleton are found intact . When human skeleton is found in fragmented or incomplete state, it is necessary to look for denser bones, such as the maxillary sinus, which has a higher possibility of remaining intact by resisting decomposition, fracture, and incineration . Ct has been reported to be a robust method in the estimation of different dimensions of the maxillary sinus . Previous studies have shown that the dimensions of maxillary sinuses from measurements of human skulls were similar to those obtained by ct scans and the consistency of measurements of the paranasal sinuses using ct images have been evaluated in the last decade . It may, therefore, be reasonably assumed that ct is a reliable method for measuring the dimensions of the maxillary sinuses . In the literature, the mean values of maxillary sinus measurements were 32, 25, and 35 mm in length, width, and height, respectively . Our study measured the ml, si, and ap dimensions of the maxillary sinus on ct in 30 patients including both males (n = 15) and females (n = 15). Maxillary sinus showed an average size of 27.53 38.21 42.60 mm in males and 25.12 33.34 36 mm in females . The left maxillary sinus showed an average size of 27.01 36.99 40.80 mm in males and 23.22 33.11 37.20 mm in females . We found that the overall size of the maxillary sinus was larger in males than in females . These findings were in agreement with those of authors like fernandes, teke et al ., and sahlstrand - johnason et al ., who have separately reported that the overall size of the maxillary sinus is larger in males than in females . Also, the right maxillary sinus gave an impression of being larger than the left maxillary sinus in overall dimensions in both males and females, but the difference was found to be statistically insignificant . The volume of the maxillary sinus has been previously measured using ct . Sahlstrand - johnson et al ., reported that the volume of the maxillary sinus can also be accurately estimated by using a simple formula: (ml dimension si dimension ap dimension) divided by 2 . Such an estimation of volume is debatable and was not considered for use in this study; however, this tool might be beneficial in clinical practice for approximate estimation of the maxillary sinus volume, where volume measurement applications are not available or precision is not critical . In a study about gender determination, it was found that the volume of maxillary sinuses was larger and wider in males than in females in europe but narrower in males than in females in zululand . Our study used the ct workstation to measure the volume of the right and left maxillary sinuses in males and females by tracing the outline of the sinus manually on each slice of the ct image stack using the on - screen pointer in the coronal plane . The software module on the workstation then automatically calculated the maxillary sinus volume in cubic centimeters . We found that for the right maxillary sinus, the average volume was 16.63 cc in males and 11.61 cc in females . For the left maxillary sinus, the volume was calculated to be 15.19 cc in males and 10.95 cc in females . Therefore, it could be concluded that males have significantly larger maxillary sinus volumes than females . Also, the volume of the right maxillary sinus is slightly larger than the left maxillary sinus in both males and females . Sahlstrand - johnson et al ., measured the dimensions of 120 maxillary sinuses from head cts and found the volume to be larger in males than in females with a mean value of 15.7 5.3 cm . These authors reported differences in the volume of the maxillary sinus between males and females, and this finding is in accordance with the findings in our study . But they did not find any significant difference between the volumes of the right and the left maxillary sinuses, which is in contrast to our study, as we found that the volume of the right maxillary sinus was larger than that of the left maxillary sinus, though the difference was statistically insignificant . Teke et al ., employed ct to measure the length, width, and height of the maxillary sinus and predicted the gender with an accuracy of 69.4% in females and 69.2% in males and an overall mean accuracy of 69.3% . Uthman et al ., studied the accuracy and reliability of maxillary sinus dimensions measurement in gender classification through the use of reconstructed helical ct images and reported an accuracy of correctly sexing 74.4% of male and 73.3% of female sinuses . Amin and hassan investigated the possibility of estimation of sex from some radiologic measurements of the maxillary sinus using multi - detector ct among a known cross - section of egyptian population, which gave a predictive accuracy of 70.8% in males and 62.5% in females . Improvising on the ct - based studies, which did not include the volume of the maxillary sinuses, our study measured the volume of the maxillary sinus along with its three other linear dimensions (ml, si, ap) to predict the gender of an individual . It was observed that if the volume is also included as a variable in the discriminative analysis along with the other three dimensions, the accuracy of gender prediction increases to 80.0% in males and 86.7% in females, with an overall accuracy rate of 83.3% . The maxillary sinuses remain intact although the skull and other bones may be badly disfigured in victims who are incinerated, and therefore, maxillary sinuses can be used for identification . Ct measurements of maxillary sinuses may be useful to support gender determination in forensic medicine . This study, building upon similar previous studies, demonstrates that the length, width, and height of the maxillary sinus, along with its volume can be used to predict the gender of an individual with a fair degree of accuracy . The accuracy rate in this study is comparable, if not higher than many other methods that have been used to predict the gender of an individual from skeletal remains . However, the result of the study does not prove this method to be infallible . Further studies with larger sample size are required to make this procedure conclusive and achieve standardization.
Thyroid gland disease in children are in second place by frequency among all endocrine disorders (1). When interpreting the results of the assessment of thyroid function should be taken into account the significant differences in the concentrations of tsh, thyroid hormones, thyroid a binding protein and calcitonin between children of different ages . Also, different content of iodine in the diet has an impact on the results of hormone tests and the results of radio - nucleotide tests of the thyroid gland . Measurement of serum tsh is the most sensitive test for the detection of primary hypothyroidism . Significantly better indicator of thyroid function are the concentrations of free t4 and t3 in relation to their total concentration in the blood . In cases of suspected thyrotoxicosis, measuring serum t3 is the most sensitive test for hypersecretion (or exogenous input) of thyroid hormone . Confirmation of primary hyperthyroidism today is based on the determination of very low (previously undetectable) concentration of tsh levels (<0.1 mj is used the j fixation . Within the false hyperthyroidism, caused by exogenous intake of thyroid hormones, congenital hypothyroidism (ch) includes all conditions in which exist insufficient function of the thyroid gland, regardless of etiology, and is clinically or laboratory detected at birth . The incidence of ch of 1:1,500 is comparable to reports in other jurisdictions (1,2). Hypothyroidism in the newborn period is almost always overlooked and delayed diagnosis leads to the most severe outcome, mental retardation (emphasizing the importance of neonatal screening) (3). The development of the central nervous system during the first trimester of pregnancy up to age of 18 - 24 months depends on the presence of normal amounts of thyroid hormones . Given the fact that the early introduction of substitution therapy is essential for the achievement of normal psycho motor development, it needs to start immediately after confirming the diagnosis, or finding high levels of tsh in a repeated sample of blood . Thyroid hormone levels change markedly during childhood, and that adult reference intervals are not universally applicable to children (4). The treatment of choice is oral administration of l - thyroxine . For infant s dose is 10 - 15 g / kg or at least 37.5 g / day, but the recommended dose is 50 g / day . The levels of t4 and tsh levels should be regularly monitored and maintained in the upper half of the normal range for the chronological age of the patient . The only known side effects of sodium - l - thyroxine are determined by an overdose of the drug . Monitoring the rate of growth and bone maturation in older children is an excellent indicator of the therapy adequacy . To present the age and sex structure of the patients diagnosed with hypothyroidism, evaluate diagnostic methods for making diagnosis, evaluation of etiology of hypothyroidism, with special review of the therapeutic modality . The study has a retrospective character, and includes all patients who have the diagnosis of hypothyroidism treated at the endocrinology counseling center of the pediatric clinic, university medical centre in sarajevo, and at the moment of data collection were aged 0 - 18 years . Patients were in relation to age divided into three groups (0 - 1 months, 1 month to 10 years, and over 10 years of age). The diagnosis of patients was based on physical findings, hormonal findings of the thyroid gland (ria method), with ultrasound findings (linear probe of 7.5 mhz). Distribution of patients on the basis of gender, revealed more significant representation of female patients (65.93%) (figure 1). There were no significant differences in the appearance of the disease in relation to patient s age (p>0.05). Gender and age distribution of patients physical examination of the struma was not detected in the majority of cases (74; 81.32%, p<0.05) among patient suffering from hypothyroidism (figure 2). Thireomegaly confirmation by physical examination average value of hormones in time when is diagnosis confirmed the maximum deviation from the average baseline hormone levels shows tsh level, while t3 is within the reference values, and t4 values are slightly lower . The average tsh value at the time of diagnosis was significantly higher compared to the reference value for all age groups except that the largest deviation was noticed in the age group of 0 - 1 months . Ultrasound examination was used in 44 (48.35%) cases, and among patients examined with ultrasound in 22 (50%) cases the struma was confirmed . Ultrasound findings in most cases 14 (31.81%) demonstrated diffuse struma and hashimoto thyroiditis together, followed by normal results in 11 (25%) cases, hypoplastic thyroid gland in 7 (15.90%) cases, the very diffuse struma in 6 (13.63%) of cases, while the nob - like and cystic glands and the hashimoto thyroiditis is registered in 2 (4.54%) cases, the parenchyma was not visualized in 2 (4.54%) cases, of which one ectopia glands and the second state after total thyroidectomy due to ca of the gland . In relation to the etiology of hypothyroidism most patients belong to a group where hypothyroidism is associated with other diseases and conditions (27; 29.67%), and the least with the congenital hypothyroidism with 18 (19.78%) of cases . Most patients with congenital hypothyroidism was female (11; 61.11%), while the overall average age at diagnosis was 12 days . Most patients with hashimoto thyroiditis are female (19; 90.47%), while the overall average age at diagnosis was 11.33 years . Most patients with ordinary hypothyroidism was female (16; 64.00%), while the overall average age at diagnosis was 7.32 years . Distribution of hypothyroidism in relation to sex is approximately equal what is to be expected given that this is a hypothyroidism related with other diseases and conditions, and the average age at diagnosis was 7.21 years . The average dose of l - thyroxine in the age of 0 - 1 months was 50 g, 1 month10 years and 37.5 g, and the group of patients over 10 years, 65 g . Epidemiological studies suggest a higher incidence of thyroid disease and hypothyroidism especially in female population up to six times . Our data correlate with the general conclusion on the higher incidence of hypothyroidism in girls . Congenital hypothyroidism (ch) occurs in approximately 1:2,000 to 1:4,000 newborns (6). Prior to the onset of newborn screening programs, the incidence was in the range of 1;7,000 to 1:10,000 (6,7). Before the diagnostic process is necessary to take high - quality medical history of the patient (from perinatal age, including gestational and prenatal history). At the time of diagnosis nearly 40% of patients were aged up to 10 years and the same number at age over 10 years . The incidence of hypothyroidism in our community is twice as high in the pre - teen and children aged over 10 years compared to congenital hypothyroidism . The assessment of the existence of struma was performed by physical examination which reliably according to the literature is around 60% even for an experienced doctor examiners . Data on the incidence of struma testifies to the fact that hypothyroidism in most of the patients did not last long and the struma has not developed . The existence of struma without hormone deficiency may be the expression of the status of iodine deficiency in the region . Tsh mostly deviates from the upper reference value and if we take into account the literature data on desirable variability during therapy of tsh from 0.1 to 5.5 miu / l, then it is six times more than the upper limit of the reference value . Tsh is a long - term indicator of thyroid function or is slow to respond to the change of the target hormone and is of interest to observe the dynamics in the period of 4 - 6 months . The value of t3 hormone is within the reference value, despite extremely high tsh and t4 just below the lower limit value of euthyroid state . It is obvious that young body puts great effort to synthesize in this time the maximum desirable amount of t3 and t4, and markedly elevated tsh indicates hypothyroidism durability . Miu / l, which undoubtedly confirm this diagnosis and suggest an urgent initiation of therapy . This finding of tsh is three times higher than tsh 20 representing by itself evidence of congenital hypothyroidism, and four times higher than the level of tsh 15 in which the newborn is immediately call for retesting after the screening on congenital hypothyroidism . Ultrasound examination of the thyroid gland is used in the process of diagnosis in about half of patients . This is a useful diagnostic element to display the volume and morphology of the thyroid gland but because of limited opportunities due to prompt admission and review as part of the initial diagnostic is not always performed . A special benefit for the monitoring of these patients would have a portable (hand held) ultrasound device and machine that would produce imaging and continuously monitor the evolution of morphology in relation to the functional status of the thyroid gland . The frequency of the ultrasound struma in examined patients of about 50% indicates failure of clinical palpation as thyroid volume estimation method, because of that it is an indication for the administration ultrasound in case of suspected struma or the existence of the node in the thyroid gland . Most patients were with diffuse struma and morphological substrate that on ultrasound looks like hashimoto thyroiditis, which is consistent with literature data . Normal ultrasound and both volumetric and morphological finding is found in as many as 25% of the examined patients . Often children with gracile neck or with protruding thyroid cartilage larynx appears magnified as ultrasound refute findings although clinical and laboratory there is verified hypothyroidism . Finding hypoplastic thyroid gland is not rare and corresponds to a later stage of hashimoto thyroiditis or congenital hypoplasia of the thyroid . It is interesting that in two children ultrasound obtained findings did not visualize the thyroid gland and in the case of congenital hypothyroidism and thyroidectomy in children with thyroid cancer . The incidence of nodes and cyst formation in ultrasound examination of the thyroid gland is not large and it is only 4.54% (2 patients), which indirectly speaks about fortunately a small number of potential thyroid cancer, which generally always evolve from a cold thyroid nodule . A quarter of our patients have the so - called ordinary hypothyroidism without elevated antibodies . This form of thyroid insufficiency begins on average in prepubertal age (the average age of diagnosis 7.21 years) and is more common in girls . As a separate entity, we pointed out the occurrence of hypothyroidism with other diseases and conditions existing at the time of diagnosis . It occurs with type 1 diabetes mellitus, slowed growth, down syndrome, vitamin d - resistant rickets, panhypopituitrity and other chronic diseases and conditions . Patients with congenital hypothyroidism are usually introduced with higher initial dose of the drug in order to minimize the already possibly resulting consequences of reduced thyroid function . Our therapy is completely in accordance with these rules and is in the first month was 50 g per dose . As child grows the dose of the drug decreases, and an average of 4 mg / kg of body weight, a further increment of body weight dose is relatively smaller calculated on kg of bw . The american academy of pediatrics recommends: at two and four weeks after the initiation of l - thyroxine treatment; every 1 - 2 months during the first 6 months of life; every 3 - 4 months between 6 months and three years of age; every 6 - 12 months thereafter until growth is complete; four weeks after any change in dose (more frequently if results are abnormal or non - compliance is suspected) (9). Hypothyroidism is the second most common endocrine disease in children s age (after type 1 dm), with a predominance of females . Congenital hypothyroidism has about one - fifth of patients . Physical examination revealed in about one fifth of patients the struma of the thyroid gland; tsh levels is critical for the diagnosis and correction of therapy in pediatric patients with hypothyroidism . Congenital hypothyroidism is diagnosed on average at the age of 12 days, which is optimal period for therapeutic response; substitution treatment is carried out by l - thyroxine which have relatively reduced doses from neonatal age onwards; future research should be directed to the assessment of iodine deficiency in the region in which our patients lives, as an important etiological factor for the development of struma and thyroid gland pathology.
Vitiligo is an acquired, idiopathic disorder characterised by circumscribed depigmented macules and patches and can have a major impact on personality clinically, with any treatment for vitiligo, the repigmentation usually begins at the orifice of hair follicles, which enlarges and coalesces to cover whole vitiliginous areas; meanwhile, on the palms and soles, mucosal surfaces, where hair follicles are absent, vitiliginous macules are very difficult to treat . A vitiliginous area with leukotrichia is considered to be a sign of poor prognosis . The concept of follicular unit transplantation is based on the fact that hair, in general, does not grow singly, but with the exception of hairline, emerges from the scalp in groups called follicular units, histologically these units are comprised of 1 - 4 terminal and 1 - 2 vellus hairs that form a distinct group bounded by a circumferential band of adventitial collagen, the perifolliculum . The existence of undifferentiated stem cells in the hair follicle in these units, which can serve as a source of melanocytes for repigmentation is the rationale behind follicular unit transplantation . A 12-year - old female patient presented with depigmented macule on the right eyebrow of 8 years duration [figure 1a]. No new lesions were seen elsewhere in the body and size of the lesions remained stable for the past 2 years . On examination, she had single depigmented macule of the size 2 cm 1.5 cm . A diagnosis of focal type of vitiligo was made based on the clinical findings . Since the patient did not respond to the medical management, surgical correction with follicular unit transplant using the follicular unit extraction (fue) technique was suggested . Donor hairs were harvested from the post auricular region using 1 mm skin biopsy punches . The follicular units thus obtained were transplanted using an 18-g needle in the depigmented macules with 3 mm gap between the follicles . Paraffin gauze dressing was done for the recipient area and the dressing was removed after 5 days . Repigmentation of the vitiligo patch was seen at the end of 6 weeks and complete pigmentation was seen at 12 weeks, with resolution of leukotrichia at the end of 6 months [figure 1b]. In the late 1950s, staricco showed amelanotic melanocytes in the external root sheath of hair follicles that suggested immature pigment cells . Further a melanocyte reservoir was proposed in the hair follicle by ortonne et al ., in 1980 who described the presence of dopa negative, non - dendritic pigment cells along the external root sheath that migrated towards the basal cell layer to become functionally active in patients treated with psoralen and uva therapy for vitiligo it has been found that only active (dopa positive) melanocytes existed in the epidermis of normal skin . There are some inactive melanocytes in the outer root sheath (ors) of hair follicles, which form the melanocyte reservoir in the human skin . Suggested that melanocytes from the implanted lower third of the hair follicle can act as a reservoir and are able to migrate and repigment achromic areas in vitiligo . Studied the different stages of repigmentation of vitiligo and confirmed the existence of a melanocyte reservoir in the ors of hair follicles . Vitiligo is a process in which only active (melanin - producing) melanocytes are destroyed and the inactive melanocytes in the ors are preserved and serve as the only source for repigmentation . Recovery of vitiligo is initiated by the proliferation of these inactive melanocytes, followed by the upward migration to the nearby epidermis to form perifollicular pigment islands and the downward migration to the hair matrices to produce melanin . A retrograde movement of melanocyte to the depigmented hair follicles in vitiligo lesions has also been proposed to explain the repigmentation of leukotrichia seen after surgical treatment of vitiligo . After surgical repigmentation of vitiligo, the epidermis has a high concentration of melanotic melanocytes . Migration of these melanocytes from the area of high concentration in the epidermis to the hair follicle in the dermis, where the pigmented melanocytes are deficient, can explain the phenomenon of pigmentation of leukotrichia . The existence of undifferentiated stem cells in the hair follicle in these units, which can serve as a source of melanocytes for repigmentation is the rationale behind follicular unit transplantation . This procedure for vitiligo has been reported earlier, in one study by na et al ., perifollicular repigmentation around the grafted hair was observed in (71% patients) within 2 - 8 weeks . In cases of localised / segmental vitiligo, perifollicular pigmentation was seen in 82% patients and the study concluded follicular unit grafting to be an effective method for treating localised / segmental vitiligo, especially on hairy parts of the skin, including the eyelids and eyebrows and for small areas of vitiligo . Malakar and dhar in 1999 conducted a study of three patients with five vitiliginous patches . Single follicular unit transplant conducted by kumaresan 2011 showed excellent repigmentation after 4 - 8 weeks with no recurrences in a case of vitiligo . All these studies have used the traditional follicular unit transplantation technique where in a strip was taken from post auricular region of the scalp, follicles dissected and transplanted . In our case, we have used fue technique; the follicles were not dissected and transplanted as a whole unit in hopes of maintaining their natural milieu and promoting better proliferation of the cells, thus helping to improve the results . Apart from this, fue technique is much simpler, suture less with less patient downtime and less complications (e.g., scarring). Thus, fue appears to be an effective method for treating localised / segmental vitiligo, especially on hairy parts of the skin, including the eyelids and eyebrows and for small areas of vitiligo . Though labour intensive, it was found to be associated with quicker patient recovery time, less morbidity and good colour match.
Stainless steel crowns (sscs) have been the most preferred material for restoration of pulpotomized primary molars and their success has been extensively established to date . Current improvements in the bond strength, wear resistance and the increasing demand of parents to provide esthetic restorations for children have made resin - based composites popular for the restoration of primary posterior teeth . It has been agreed that in primary molars with pulp therapy treatments the main problem may be the cavity depth, as the floor of the pulp chamber effectively constitutes the cavity floor, resulting in long unsupported cusps . Bonded restorations splint the cusps together and decrease cusp flexure, preventing their subsequent separation by fracture . In addition, placement of a considerable amount of adhesive restorative material in the pulp chamber may provide additional reinforcement by altering the fulcrum of cuspal flexing . In primary teeth adhesive restorations have many advantages over sscs, some of those are preservation of sound tooth structure and normal contact area and increased resistance to microleakage . In pulpotomized primary teeth a base of zinc oxide eugenol (zoe), either plain or reinforced is placed over the amputation site to cover the pulpal floor following the coronal pulp amputation . According to many investigations, a resin - based composite material should not be used over zoe because it increases microleakage and produces poor bond strength to dentin because eugenol suppresses the polymerization of composite resin . Therefore, it is advisable to cover zoe with a suitable material in order to prevent the harmful effect of zoe on composite restorations . On the other hand, using different bases with different compositions under composite resin may influence its properties [1315] and subsequently jeopardize the final success of the restoration . The presence of gaps in the marginal area and between various layers of restoration is one of the major causes of microleakage which is considered one of the main factors responsible for treatment failure . The present study evaluated microleakage and gap formation between different bases and composite restoration in pulpotomized primary molars . Seventy - eight extracted human primary second molars which had at least three intact surfaces, consisting buccal, lingual and one proximal surface were selected and stored in 0.5% chloramine solution for 24 hours . Proximo - occlusal cavities were prepared involving two surfaces only using a high - speed bur under water coolant and the cervical margins were placed in the enamel . All the pulpotomy procedures were carried out using a conventional technique in which caries was completely removed and upon removal of the roof of the pulp chamber the pulp tissue was removed and irrigation was performed with normal saline solution . Reinforced zoe paste (zonalin, kemdent, purton, swindon, wiltshire, uk) was mixed according to the manufacturer s recommendation by 5:1, powder: liquid ratio with it was placed on the pulp chamber floor in 2-mm thickness (determined by a periodontal probe) and an approximately 2-minute interval was necessary for the setting of zoe . The teeth were divided into 6 groups (n=13) using the simple randomization method with the flip of a coin . In order to eliminate the anatomic variations of the teeth as confounding factors only the second primary molars were used and the number of maxillary and mandibular second primary molars was the same in the groups under study (flipping of a coin has been done separately for maxillary and mandibular molars). A metal t band matrix was prepared for each tooth and in all cases, cavity preparations were filled with composites using incremental light cure technique . 35% phosphoric acid (ultraetch, ultradent products, south jordan, usa) was used for acid etching for 20 seconds followed by a 30-second water rinse and the excess water was removed from the surfaces with cotton pellets . Two coats of single bond adhesive (3m / espe, st paul, mn, usa) were applied onto the cavity walls in sequence for 15 seconds with gentle agitation and light - cured for 10 seconds with a halogen light source (arialux, apadanatak, tehran, iran). Filtek z-250 composite resin (3m / espe, st paul, mn, usa) was placed on the zoe layer in 2-mm - thick oblique increments and light - cured for 40 seconds . However, in this group, the 2-mm - thick layer of resin - modified glass - ionomer (rmgi) (gc fuji ii lc, tokyo, japan) which was mixed according to the manufacturer s instructions by using one level scoop of powder to two drops of liquid covered the zoe base by the closed sandwich technique and light - cured for 20 seconds . However, the 2-mm - thick layer of self - cured glass - ionomer (gc fuji i, tokyo, japan) which was mixed according to the manufacturer s instructions by using one level scoop of powder to two drops of liquid covered the zoe layer . The restorative procedures were the same as described above and the 2-mm - thick layer of light - cured calcium hydroxide (lime lite, pulpdent, watertown, ma, usa) was placed on zoe layer and light - cured for 20 seconds . However, the 2-mm - thick layer of self - cured calcium hydroxide (dycal ivory, dentsply, milford, de, usa) which was mixed according to the manufacturer s instruction in equal volumes of base and catalyst and was homogeneous and streak free was placed over the zoe layer . A 2-mm - thick layer of high silver, non gamma 2, spherical amalgam (lojic plus, sdi, bays - water, australia) after 8 seconds trituration was condensed in the cervical region and allowed to set for 5 minutes . Consequently, the etching, bonding and restorative procedures were done similar to those described above . The teeth were thermocycled using 500 cycles at 5c/55c with a dwell time of 30 seconds . The entire tooth surface was covered with two layers of nail varnish, except for the restorations and 1 mm around their margins . The teeth were embedded in a self - curing acrylic base by using metallic molds to allow ease of handling . The specimens were immersed in 0.5% basic fuchsine solution for 24 hours, followed by washing under tap water . Then each tooth was invested in a clear self - curing acrylic resin and sectioned mesiodistally through the restoration by using a diamond blade (isomet, germany). The specimens were examined under a stereomicroscope (nikon, tokyo, japan) at 20 magnification for evidence of dye penetration using the following criteria (figure 1) 0=no leakage; 1=leakage originated at the occlusal surface only; 2=leakage originated at the cervical surface only; 3=leakage originated from the occlusal and cervical margins; 4=leakage is present at both cervical and occlusal aspects and is continuous . They were gold sputter - coated with gold palladium and observed under a scanning electron microscope (xl30, philips international inc, potomac, md, usa) (figures 3 and 4). The space between the zoe layer base and the base - composite layers were measured quantitatively by manual microstructure distance measurement software (nahamin pardazan asia co, iran). 35% phosphoric acid (ultraetch, ultradent products, south jordan, usa) was used for acid etching for 20 seconds followed by a 30-second water rinse and the excess water was removed from the surfaces with cotton pellets . Two coats of single bond adhesive (3m / espe, st paul, mn, usa) were applied onto the cavity walls in sequence for 15 seconds with gentle agitation and light - cured for 10 seconds with a halogen light source (arialux, apadanatak, tehran, iran). Filtek z-250 composite resin (3m / espe, st paul, mn, usa) was placed on the zoe layer in 2-mm - thick oblique increments and light - cured for 40 seconds . . However, in this group, the 2-mm - thick layer of resin - modified glass - ionomer (rmgi) (gc fuji ii lc, tokyo, japan) which was mixed according to the manufacturer s instructions by using one level scoop of powder to two drops of liquid covered the zoe base by the closed sandwich technique and light - cured for 20 seconds . The adhesive procedures were the same as those in group 1 . However, the 2-mm - thick layer of self - cured glass - ionomer (gc fuji i, tokyo, japan) which was mixed according to the manufacturer s instructions by using one level scoop of powder to two drops of liquid covered the zoe layer . The restorative procedures were the same as described above and the 2-mm - thick layer of light - cured calcium hydroxide (lime lite, pulpdent, watertown, ma, usa) was placed on zoe layer and light - cured for 20 seconds . However, the 2-mm - thick layer of self - cured calcium hydroxide (dycal ivory, dentsply, milford, de, usa) which was mixed according to the manufacturer s instruction in equal volumes of base and catalyst and was homogeneous and streak free was placed over the zoe layer . A 2-mm - thick layer of high silver, non gamma 2, spherical amalgam (lojic plus, sdi, bays - water, australia) after 8 seconds trituration was condensed in the cervical region and allowed to set for 5 minutes . Consequently, the etching, bonding and restorative procedures were done similar to those described above . The teeth were thermocycled using 500 cycles at 5c/55c with a dwell time of 30 seconds . The entire tooth surface was covered with two layers of nail varnish, except for the restorations and 1 mm around their margins . The teeth were embedded in a self - curing acrylic base by using metallic molds to allow ease of handling . The specimens were immersed in 0.5% basic fuchsine solution for 24 hours, followed by washing under tap water . Then each tooth was invested in a clear self - curing acrylic resin and sectioned mesiodistally through the restoration by using a diamond blade (isomet, germany). The specimens were examined under a stereomicroscope (nikon, tokyo, japan) at 20 magnification for evidence of dye penetration using the following criteria (figure 1) 0=no leakage; 1=leakage originated at the occlusal surface only; 2=leakage originated at the cervical surface only; 3=leakage originated from the occlusal and cervical margins; 4=leakage is present at both cervical and occlusal aspects and is continuous . They were gold sputter - coated with gold palladium and observed under a scanning electron microscope (xl30, philips international inc, potomac, md, usa) (figures 3 and 4). The space between the zoe layer base and the base - composite layers were measured quantitatively by manual microstructure distance measurement software (nahamin pardazan asia co, iran). There was less leakage in the amalgam group compared with other groups by using dunn test (p=0.004). The mean (sd) amount of distance between zoe base layer (l1) and these amounts for composite base layer (l2) are shown in table 2 . The differences between the groups were not statistically significant in l1 (p=0.94) or in l2 (p=0.47); however, the amalgam and self - cured calcium hydroxide groups had the lowest space in l1 and l2 zones . Zinc oxide eugenol is widely used in pulp therapy to obturate the root canals or to cover the pulpal floor in pulpotomized teeth . Use of zoe for the latter reason is inevitable since replacing zoe with calcium hydroxide was not successful . Although use of resin - based composite material in direct contact with zoe is contraindicated traditionally [17, 18, 19], in some studies evaluating the effect of zoe on composites no adverse effect have been shown [20, 21]. In addition, it has been reported that the detrimental effects of zoe on composite resin are only seen at a distance of less than 100 m from the zoe base . Zoe un der composite restoration showed the greatest microleakage; therefore, it seems necessary to cover zoe with other materials . It has been established that various cavity bases have an influence on composite restorations as marshall et al indicated that polycarboxylate and glass - ionomer bases caused reduction in the hardness of composite restorations . In addition, the findings of berrong et al confirm the effect of glass - ionomer base on composite properties . Liners may be used to counterbalance the cusp deformation as a consequence of polymerization shrinkage of composite resin . The liner must not allow polymerization shrinkage forces to create a debonding force or to form gaps between itself and the tooth or composite interface . In this study, microleakag increased respectively in the following order: amalgam <calcium hydroxide <glass ionomer <light - cured calcium hydroxide <zincoxide eugenolresin - modified glass - ionomer . Although it has been confirmed that resin - based bases have a better bond with composite materials and it is believed that light - cured calcium hydroxide has better physical properties compared with conventional calcium hydroxide, the findings of this study are contradictory . According to papadakou et al, light - cured calcium hydroxide base under composite restoration is pulled away from the dentin floor of the cavity as a result of an apparent adhesion to composite resin during polymerization shrinkage . The justification may be relevant for resin - modified glass - ionomer too; therefore, the greater amount of microleakage in these groups might be attributed to pulling away from the zoe layer because of better bonding to the composite . Although in determining the gap, there was no significant difference between the groups, it should be emphasized that gap is a three - dimensional phenomenon and sem evaluation is a two - dimensional tool . It might be possible to obtain more clear results by enhancing the samples in future studies . In this study, the amalgam group exhibited the least microleakage and the lowest gap formation in l1 layer, which could be related to insolubility of amalgam and its condensability because amalgam does not create pulling forces from the cavity and its condensation force may be considered the most important factor in its marginal adaptation . These findings are contradictory with those reported by junior et al, who indicated that placement of amalgam under composite restorations (amalcap technique) resulted in considerable microleakage . It should be pointed out that they used a single bottle etch - and - rinse adhesive system and cured it before insertion of the amalgam, which may have caused the leakage in that study . In addition, they placed amalgam only at the gingival margins of the restoration and in the present study amalgam was placed on the zoe layer . In the present study, the resin - modified glass - ionomer group revealed great amounts of microleakage due to the fragile nature of the powder / liquid glass ionomer cement . Addition of the resinous content did not improve the strength of the material sufficiently to withstand the shrinkage forces during composite polymerization . Considering the lower amount of microleakage in the glass ionomer group in comparison with resin modified glass ionomer, possibly apparent adhesion of the latter to the composite resin and pulling away from the cavity floor is the reason for this difference between the two materials . Despite various advantages attributed to calcium hydroxide, its role in microleakage of composite restorations has not been fully elucidated and it is believed that it may have a softening effect on composite resins . On the other hand, in a study carried out by lingard et al, dycal had little interaction with composite resin . In the present study, self - cured calcium hydroxide had better microleakage inhibitory results compared to the light - cured one, which might be explained by findings of papadakouet al, who indicated that prisma vlc dycal base was found to be pulled away from the dentin floor of the cavity as a result of an apparent adhesion to the composite resin during polymerization shrinkage . It should be emphasized that microleakage grade 2 was evident in all the groups and as a result, none of the groups had leakage only at the occlusal surface . It is obvious that in the gingival margins the enamel is thinner and it is difficult to achieve good adhesion with dentin . The situation may highlight the importance of base materials under composite resins in preventing microleakage . Finally, the null hypothesis was refuted since some differences were observed between different techniques . In composite restorations of primary pulpotomized molars: covering of zoe layer with amalgam exhibited the lowest amount of dye penetration . Microleakage with other bases in an ascending order was as follows: calcium hydroxide, glass - ionomer, light - cured calcium hydroxide, zoe and resin - modified glass - ionomer.
Duodenal diverticulum was first reported by a french pathologist chomel in 1710 and was diagnosed radiologically by case in 1913 . Duodenum is the second most common site of diverticula in alimentary tract after colon, followed by jejunum, ileum, and stomach [3, 4]. Although associated with complications like diverticulitis, perforation, obstruction, or haemorrhage, the majority of duodenal diverticula are asymptomatic [5, 6], more often coming as a surprise on gastrointestinal series . Duodenal diverticula are found in 0.16 to 6% of upper gastrointestinal barium series and up to 23% of endoscopic retrograde cholangiopancreaticographies [6, 7]. However, reported incidence from cadaveric studies could be as high as 31.8% . In recent review of the literature, there were a few cadaveric studies on the incidence of duodenal diverticulum throughout the world, and none from south india . To the best of our knowledge, the present study should be the first to highlight the prevalence and anatomical location of duodenal diverticula in south indians . One hundred and twenty specimens of duodenum from 108 male and 12 female subjects, in the age group of 25 to 63 years, procured from the department of anatomy, thanjavur medical college, thanjavur, and department of anatomy, aarupadai veedu medical college, puducherry, were utilized for this study, between the years 2005 and 2010 . The specimens of duodenum and pancreas en block were removed, serially numbered, and preserved in 10% formalin . Specimens with duodenal diverticula were identified and carefully dissected, and its anatomical location on the duodenal wall was studied . Of the 120 specimens, diverticula were found in five specimens, the prevalence being 4.2%, and were located on the medial wall, which is morphologically the mesenteric border of the duodenum . They were solitary, globular - shaped, and extraluminal with the fundus of the diverticula posterior to the duodenum, partially buried in the pancreas . Of the five specimens with diverticula, three (60%) were present in the second part and two (40%) in the third part of the duodenum . Diverticula in the second part of duodenum were located in the periampullary region, above the major duodenal papilla . Sizes of the diverticula in the second part (figures 1 and 2) were 2.5, 0.7 and 0.5 cm and in the third part (figure 3) 3 cm and 0.5 cm . Duodenal diverticula can be classified into intraluminal duodenal diverticulum (idd) and extraluminal duodenal diverticulum (edd). Idd are congenital, resulting from defective recanalisation of duodenal lumen during fetal development with coexistent congenital anomalies . Edd are acquired or false diverticula, resulting from mucosal herniation at the point where blood vessels penetrate the intestinal wall, which also explains their typical location at the medial or pancreatic border, in 88% of cases . Only 4% of them occur on the lateral wall of the duodenum . In the present study also, the diverticula were extraluminal and found on the medial wall, which is morphologically the mesenteric border of the duodenum . Even though some studies state that there is no gender predisposition [4, 5, 13], grant boileau came across two diverticula in 11 female subjects as compared to 13 from 122 male subjects . In case's series of 85 cases of duodenal diverticula, 60% occurred in females . Mackenzie et al . Have also reported a female preponderance in the ratio from 1.6 to 1 . However, in the present study, diverticula were found only in the male subjects, and the absence of the diverticula in female subjects could be due to less study population, which was also one of the limitations of our study . Review of the literature demonstrates that the incidence of duodenal diverticulum is highly variable according to the diagnostic procedure used . The incidence in upper barium series is 0.16 to 6% and in ercp studies 0923% [57]. A study in mexico by acua et al . Reported an incidence up to 11.6% by endoscopic cholangiography . In autopsy series by akhrass et al ., an incidence of 0622% has been reported . In a study done in 105 specimens, 14 cases (13%) of duodenal diverticula with 13 solitary and one multiple of the 133 cadavers examined by grant boileau, diverticula were found in 15 subjects (11.2%), with 11 solitary and four multiple . Among them, 12 were globular in shape, three were conical, and five were tubular . Ackermann, in an anatomical study of 50 cadavers, reported an incidence of 22%, with eight solitary and three multiple diverticula . The highest on record to the present date is 31.8%, in a cadaveric study by minoru and atsuyoshi . Thus, incidence of duodenal diverticula from various cadaveric studies is certainly higher than the incidence from any other diagnostic procedure, in view of the fact that the search for diverticula is more accurate in cadavers than visualizing them in any other methods of investigations . However, the present study revealed a lower prevalence of 4.2% when compared to that in the literature (table 1). Most of the duodenal diverticula occur in the periampullary region of the duodenum, within 2.0 cm of the ampulla of vater [17, 20, 21]. Several studies confirmed that the second part is the most common site followed by the third and fourth part of the duodenum . In a study by lapin et al ., 62% of duodenal diverticula occurred in the second part, followed by the third (30%) and fourth part (08%)., in their study, showed that 82% of duodenal diverticula occurred in second part, 10% in the third, and 08% in the fourth part . In the present study as well, three diverticula (60%) were found in the second part, in the periampullary region, above the major duodenal papilla, and two (40%) in the third part of the duodenum, of the total five diverticula . Distribution of duodenal diverticula in various parts in duodenum is shown in table 2 . Despite the fact that duodenal diverticula are asymptomatic in 90% of cases, the higher frequency in the second and third part is associated with increased incidence of biliary stones, pancreatitis, and biliary and pancreatic anomalies . Concurrently, size of the diverticula is also of clinical importance since jaundice, cholangitis, and obstruction of pancreatic duct or bile duct have been reported due to the pressure effects . In case's radiological study of 85 cases, the average size of the diverticulum was 2.8 cm . In the present study, the size of the diverticula was 0.5 to 3 cm (mean: 1.4 cm), as compared to the mean value of 1.7 cm (range: 0.4 cm to 4.5 cm) by wiesner et al . However, the dimensions of the diverticula in cadavers may be smaller than in life because of shrinkage during the embalming process, which is a disadvantage in our study . Since less than 10% of patients develop nonspecific clinical symptoms like abdominal pain or discomfort, diagnosis of duodenal diverticula is incidental, found only during other diagnostic or therapeutic procedures . However, 6.5% of patients may develop complications . In the most common complications, being hemorrhage and pancreaticobiliary diseases, the clinical presentation may mimic acute cholecystitis, acute pancreatitis, or peptic ulcer disease [3, 7, 28]. Even though complications of diverticula are recognized with the advent of ct, misdiagnosis is still problematic, as it is not commonly considered in differential diagnosis and due to the fact that its asymptomatic presentation masks the true incidence of duodenal diverticula . However, in the present study we found that the prevalence of duodenal diverticula is lower in south indians, when compared to other studies . A vegetarian diet and high intake of fiber could be significantly associated with lower risk of diverticular diseases for they were correlated with rapid bowel transit times [31, 32], thus reducing the intraluminal pressure . Small bowel diverticula, in fact can be found in patients older than 50 years with peristaltic disorders, such as progressive systemic sclerosis, visceral myopathy, and visceral neuropathies leading to an increase in intraluminal pressure . Thus, a low fiber diet and advancing age might contribute to the risk of developing acquired duodenal diverticula . The low prevalence rate of duodenal diverticula in south indians could be attributed to the indian diet, which consists of rice, wheat, ragi, lentils, vegetables, yoghurt, and less animal protein . Duodenal diverticula being asymptomatic in 90% of cases makes it difficult to ascertain the incidence and constitutes a diagnostic and therapeutic challenge due to their non - specific presentation . Thus, awareness of the frequency and location of duodenal diverticula is of great importance in the diagnosis and management of pancreaticobiliary diseases . In conclusion, our report serves to highlight the prevalence and anatomical location of duodenal diverticula in south indians . The present study revealed a lower prevalence (4.2%) of duodenal diverticula comparable to that in the literature, and whether this could be attributed to the indian diet pattern needs further study.
Mycotoxins are toxic secondary metabolites produced by organisms of the fungi kingdom, commonly known as molds . Aflatoxins (afs) are mycotoxins derived by aspergillus flavus (a. flavus) and aspergillus parasiticus (a. parasiticus) and are listed as group i carcinogens by the international agency for research on cancer (iarc), a body of the world health organization (who). There are four major natural aflatoxins (i.e., af b1, af b2, af g1, and af g2). Af b1 and af b2 are produced by a. flavus, whereas af g1 and af g2 are produced by both a. flavus and a. parasiticus . The hierarchy of toxicity, carcinogenicity, and mutagenicity of different aflatoxins is in the order af b1> af g1> af b2> af g2 . Hence, the extent of contamination will vary with geographic location, agricultural and agronomic practices, and the susceptibility of commodities to fungal invasion during preharvest, storage, and/or processing periods . On a worldwide scale, aflatoxins are found in stored food commodities and oil seeds such as corn, peanuts, and so forth . To achieve quantitative analysis of aflatoxins, analytical methods based on various instrumental techniques high - performance liquid chromatography mass spectrometric (hplc ms) methods have drawn the most interest due to their excelling selectivity . However, hplc ms analysis is prone to matrix effects, which are dependent on analytes, sample matrices, lc ms conditions, and sample preparation . To some degree, use of internal standards, especially stable isotope dilution, alleviates the problems associated with matrix effects . A major disadvantage of hplc - ms stable isotope dilution analysis is the need for expensive isotope - labeled analogues of the analytes . In addition, when analyte concentrations in a sample solution are below the limit of quantitation (loq) of the method, enrichment of the analytes must be carried out prior to analysis . Immunoaffinity columns have been used for long to extract aflatoxins from various sample matrices . To reduce the cost of analysis, chemical mini - columns, particularly florisil - packed columns were proposed and proven effective for sample cleanup in aflatoxin analysis . Major drawbacks of these column - based procedures include a time - consuming elution of the retained analytes and use of significant quantities of toxic organic solvents . Solid - phase extraction (spe) of aflatoxins with c18 cartridges has been successfully developed . Magnetic solid - phase extraction (mspe) is a new version of spe . The adsorbent needs not to be packed into a cartridge as in traditional spe . Instead, a suspension of the nanometer - sized adsorbent is added and mixed well with the sample solution to extract analytes . After extraction, the adsorbent can be easily separated from the solution and collected through magnetic decantation by means of an external magnet . Therefore, mspe is quick, easy to perform, and importantly, well - suited to handle liter volumes of samples, which means a large enrichment factor can be conveniently obtained . Use of mspe in quantitative analysis of estrogens and isoflavones in milk samples has been reported . The aim of the present research was to develop a rapid, cost - effective, and efficient mspe procedure for hplc this procedure serves not only for sample cleanup, eliminating any potential matrix effects on the subsequent hplc ms / ms analysis, but also for af enrichment, allowing the quantification at very low levels . To achieve high extraction efficiency, superparamagnetic fe3o4 nanoparticles with different surface modifications were synthesized, characterized by using tem and ftir and evaluated as the mspe adsorbent . Mspe parameters affecting the extraction efficiency, including extraction time, eluting solvent, and so forth were investigated . Finally, rapid analysis of low levels of aflatoxins in red wine samples by using the proposed mspe hplc ms / ms method was demonstrated . Aflatoxin standards, dopamine (structures are shown in figure 1), 1,6-hexamethylenediamine, anhydrous sodium acetate, iron(iii) chloride hexahydrate (fecl36h2o), ethylene glycol, hplc grade acetonitrile, methanol, and formic acid were purchased from sigma - aldrich chemicals (st . Milli - q water (millipore, bedford, ma) was used throughout the work . Chemical structures of the compounds involved in this work: af b1, af b2, af g1, af g2, and dopamine . Amine - terminated fe3o4 magnetic nanoparticles (amnps) were prepared by a one - pot hydrothermal procedure previously reported (illustrated in figure 2). Briefly, 4.33 g of 1,6-hexanediamine, 1.33 g of anhydrous sodium acetate, and 0.66 g of fecl36h2o were dissolved in 25 ml of ethylene glycol by vigorously stirring at 50 c to obtain a clear solution . It was then transferred to a teflon - lined autoclave for 6 h at 198 c to obtain amnps . After each rinse step, the amnps were separated from the supernatant by using an external magnet . Amnps were dried at 50 c under n2 overnight . To prepare polydopamine coated mnps (pd - mnps), an in situ oxidative self - polymerization procedure was used . A suspension of amnps was prepared by dispersing 220 mg of amnps in 10 ml of 10 mm tris - hcl buffer solution (ph 8.5) through sonication for 15 min . Dopamine hcl (2.5 mg / ml) was added to the amnps suspension with vigorous stirring, and the ph of the mixture was adjusted to 8.5 by addition of 10 mm sodium hydroxide . The solution was placed on a shaker for 12 h after which pd - mnps were collected by magnetic decantation, washed three times with water, and finally redispersed in 5.0 ml water by sonication for 15 min . Illustration of pd - mnps preparation involving two steps: (1) one - pot synthesis of amine - terminated magnetic nanoparticles (amnps) from fecl3 and hexanediamine and (2) coating amnps with polydopamine via in situ oxidative self - polymerization of dopamine . Transmission electron microscopic (tem) images were acquired on a jeol, jem-1011 with a resolution of 0.2 nm lattice . Aflatoxin standard mixture containing 1000 ng / ml af b1, af g1 and 300 ng / ml af b2, af g2 (sigma - aldrich, st . Louis, mo) was allotted at 100 ul per centrifuge tube and stored at 20 c until use . The work standard solutions were prepared daily by appropriate dilution with methanol / water (50:50, v / v). To an erlenmeyer flask containing 50 ml of sample, 100 l of the pd - mnps suspension prepared above was added . The flask was placed on a magnet for 30 s to let pd - mnps settle down . After washing twice with 500 l of water, aflatoxins were eluted from the pd - mnps with 250 l of warm acetonitrile / methanol (1:1) at about 60 c for 3 min . After magnetic separation, 100 l of supernatant was transferred to a centrifuge vial and mixed with 100 l of water . After being filtered through a 0.22 m filtration membrane, portions (5 l) illustration of the proposed mspe procedure for facile extraction of aflatoxins followed by hplc the system consisted of two pumps (lc-10advp, shimadzu, toyoto, japan), an online degasser (dgu-12a, shimadzu), and a triple quadrupole mass spectrometer equipped with a heated esi source (tsq quantum, thermo scientific, san jose, ca). Both the lc and mass spectrometer were controlled by xcalibur software (thermo finnigan). A c18 reversed - phase column (ascentis, 3 m particle size, 10 cm 2.1 mm, sigma - aldrich, st . Meoh / water mixture (60/40, v / v) containing 5 mm ammonium acetate was used as the mobile phase at a flow rate of 0.150 ml / min . Sample injection volume was 5 l . Data were acquired in full scan and srm mode . The ms detector was operated in the positive ion mode with the following settings: spray voltage of 3 kv, vaporization temperature of 270 c, capillary temperature of 300 c, sheath gas pressure of 35 (arb), auxiliary gas pressure of 10 (arb), tube lens voltage of 150 v, and capillary voltage of 35 v. srm parameters for ms detection of aflatoxins are summarized in table 1 . Mspe is gaining attention because it is quick, easy to perform, inexpensive, and suitable for working with large volume samples . The success of a mspe procedure depends largely on the surface chemistry of the superparamagnetic nanometer - sized adsorbent particles . Polydopamine - coated nanoparticles have been widely used in biological and pharmaceutical technology arenas . In this study, it was found that a highly stable polydopamine coating could be formed on the surface of amine - terminated fe3o4 superparamagnetic nanoparticles via oxidative self - polymerization of dopamine . Because the polydopamine coating thus formed is composed of dihydroxyindole, indoledione, and dopamine units (as illustrated by figure 2), pd - mnps are expected to have a high affinity for aflatoxins through a combination of charge transfer, -stacking, and hydrogen - bonding interactions, and therefore, they are expected to serve as a highly effective mspe adsorbent for extracting these compounds from solutions . One - pot synthesis of amine - terminated mnps (amnps) was achieved by following a procedure previously reported using fecl36h2o as the precursor of magnetic particles and hexamethylenediamine as an amino group source . From the tem results, amnps prepared were 30 nm in diameter in average and had a circular shape . The ft - ir spectrum (figure 4a) shows strong absorptions at 575 cm arising from the vibration of the fe o bond and at 3133.5 and 3420.9 cm from n h bond in the amine group . Pd - mnps were easily prepared by incubating amnps with dopamine in a tris buffer at ph 8.5 for 3 h. tem analysis showed that pd - mnps obtained had an average diameter of 40 nm . In the ft - ir spectrum of pd - mnps (figure 4b), a strong absorption band at 1400 cm and a moderate band at 1600 cm are observed, clearly indicating the presence of polymerized aromatic structures . In addition, an absorption band at 31003450 cm is much stronger than that in amnps spectrum, which is due to the overlapping of hydroxyls and amines in polydopamine . It was also noted that the suspendability of pd - mnps was much improved from that of the precursor amnps or bare fe3o4 nanoparticles . A diluted suspension of pd - mnps was stable for several days in terms of its appearance . Vis monitoring of the suspension for 1 week did not detect any leaching of polydopamine or its components from the nanoparticles . Ft - ir spectra of amnps (a) and pd - amnps (b). In the mspe study, pd - mnps were added to 50 ml of a standard solution in each extraction test . Aflatoxins extracted from the solution were subsequently retrieved from the pd - mnps and quantified by hplc ms / ms . Ms methods have been reported for aflatoxin quantification . In the present study, several mobile phases, including meoh / h2o (60:40) containing 5 mm ammonium acetate, meoh / h2o (60:40) containing 0.1% formic acid, acn / h2o (60:40) containing 5 mm ammonium acetate, and meoh / acn / h2o (60:20:20), were tested for the separation on a c18 column . It was found that isocratic elution with meoh / h2o (60:40) containing 5 mm ammonium acetate resulted in the best analytical results in terms of separation efficiency and detection sensitivity . Intriguingly, introducing ammonium acetate to the mobile phase significantly improved the detection sensitivity . However, no similar effects were observed with formic acid (0.1% v / v), a commonly used additive to enhance ionization efficiency in hplc ms analysis . Effects of pd - mnps amount on extraction efficiency were investigated . In a set of extraction tests, 10, 25, 50, 75, 100, 150, 200, and 500 l of pd - mnps suspension were added to different 50.0 ml portions of a standard solution containing 0.100 ng / ml af b1, respectively . Ms / ms analysis of the eluent from each test, the extraction efficiency of af b1 was assessed . It was found that extraction efficiency increased [from 33.1 7.2% to 89.2 2.6% (n = 3)] with the increase in pd - mnps amount and remained nearly constant when> 75 l of pd - mnps suspension was used . For further studies, 100 l of pd - mnps suspension was added to 50 ml of sample to extract aflatoxins . The extraction efficiency of af b1 obtained were 86.7 2.2%, 89.7 1.7%, and 89.5 0.3% desorption of aflatoxins retained on pd - mnps is also critical to the success of the mspe development . Elution conditions, including eluting solvent, elution time (1, 2, 3, 5, and 10 min), and elution temperature (30, 45, 60, 80 c) were investigated to achieve a high retrieval efficiency . Methanol / acetonitrile (1:1) was found most effective to retrieve aflatoxins from pd - mnps . On the basis of these studies, methanol / acetonitrile (1:1) as the eluting solvent and an elution time of 3 min at 60 c were selected for desorption of aflatoxin from pd - mnps . An equal amount of water was added to the elution solution prior to hplc ms / ms analysis in order to minimize the solvent effects on the separation . Interestingly, when using amnps or bare fe3o4 mnps for mspe of aflatoxin, the extraction efficiency was found to be significantly lower . This may be due to either a low affinity of aflatoxins to these adsorbents or poor desorption of aflatoxins from them under the selected elution conditions . Pd - mnps exhibit a very high affinity for aflatoxins because of combined interactions arising from charge transfer, -stacking, and hydrogen bonding, most of which do not exist in the case of amnps or bare fe3o4 mnps . Comparing the effectiveness of retrieving af b1 retained on pd - mnps with different eluting solvents . Under the selected mspe conditions, two standard solutions of aflatoxins at concentrations ranging from 0.01000.300 ng / ml were extracted to determine the extraction recovery for all the four aflatoxins tested . Aflatoxins extracted were quantified by using a calibration curve prepared from standard water solutions of aflatoxins . The recovery for the four aflatoxins ranged from 58.6 to 91.0% (table 2). The difference in recovery is likely because of the fact that af b1 and af b2 are more hydrophobic than af g1 and af g2 . These recovery values are significantly higher than many of those obtained from spe procedures . It should be pointed out that the proposed mspe offers an enrichment factor of 100, which is attractive for analysis of samples containing aflatoxins at very low levels . Is highly important . In this work, quick, selective, and sensitive quantification of trace aflatoxins in red wines by coupling the proposed mspe with hplc ms / ms analysis is demonstrated . To obtain the analytical figures of merit for the mspe ms / ms method, simultaneous quantification of authentic af b1, af b2, af g1, and af g2 solutions was performed . The behaviors of chromatographic retention and product ion spectra of these four aflatoxins are shown in figure 6 . Ms analytical conditions, the four aflatoxins were well - separated from each other . Ion transitions (i.e., m / z 313 285 for af b1, m / z 315 259 for af b2, m / z 329 243 for af g1, and m / z 331 245 for af g2) were monitored using the srm detection mode . Five - point calibration curves were prepared with authentic aflatoxin solutions at concentrations ranging from 0.00600 to 3.00 ng / ml in water . These solutions were submitted to the proposed mspe procedure and injected into the hplc regression analysis of the results yielded linear calibration equations for all four aflatoxins tested with r values> 0.995 . Interday (5 days) precisions of the slope and intercept of the calibration curves were found to be in the range between 3.5% and 5.6% (rsd, n = 5). From the calibration curves, the limits of detection were estimated to be in the range from 0.0012 ng / ml for af b1, af b2, and af g1 to 0.0031 ng / ml for af g2 (signal / noise = 3). These results indicate that the present method is very sensitive for the analysis of aflatoxins . It should be pointed out that these lods are so low because the mspe extraction provides a 100-fold enrichment factor (i.e., the sample is preconcentrated by a factor of 100 through the extraction), which is a significant gain of using the proposed mspe procedure . Ms / ms determination of aflatoxins in water and red wine: chromatograms and ms spectra for each aflatoxin involved . Sample a: water spiked with 0.0300 ng / ml of af b1 and g1 and 0.0100 ng / ml of af b2 and g2 . Sample b: water spiked with 0.150 ng / ml of af b1 and g1 and 0.0500 ng / ml of af b2 and g2 . Red wine: sample spiked with 0.0600 ng / ml of af b1 and g1 and 0.0200 ng / ml of af b2 and g2 . The proposed mspe ms / ms method was applied to quantification of aflatoxins in liquid foodstuff samples, taking red wine as a model system . Two red wine samples were purchased from a local store and analyzed to determine the four aflatoxins . To verify the analytical results these samples were spiked with authentic aflatoxins and analyzed again . No unknown peaks appeared in the chromatogram, indicating that the method was specific for the determination of aflatoxins . The analytical results are summarized in table 3 . As can be seen, both the accuracy and the repeatability of the present mspe ms method are good, because all recovery values are> 90% and rsds are <8% . These results suggest that the proposed method is useful for rapid quantification of aflatoxins in red wines . As shown in table 3, aflatoxins were not detected in the wine samples tested . These results are in agreement with those reported previously . Unlike some other mycotoxins such as ochratoxin a red wine samples are analyzed in this work as a model to demonstrate the usefulness of the proposed mspe procedure . It is expected that the proposed method can be applied to analysis of other liquid foodstuff samples such as beer, vegetable fluids, among others . It is worth noting that because af contents in these samples are normally below the lods of hplc ms methods, enrichment of the analytes is needed prior to the analysis . The present mspe procedure offers an enrichment factor of 100 for aflatoxins, which is attractive in analysis of these liquid foodstuff samples . In conclusion, shell superparamagnetic nanoparticles (pd - mnps) can be easily prepared from amine - terminated fe3o4 mnps and dopamine via an in situ oxidative self - polymerization approach . More importantly, they exhibit a high affinity to aflatoxins, thus enabling a facile and effective magnetic solid phase extraction (mspe) of these toxins from large volume liquid samples . Using pd - mnps as the adsorbent ms / ms analysis and offers a considerable enrichment factor that is highly desired for quantification of trace aflatoxins . To the best of our knowledge, this is the first report on mspe of this important group of toxins from foodstuff samples . As demonstrated in this work, the proposed mspe ms / ms method is fast, very sensitive, and applicable to fast quantification of aflatoxins in water, red wine, and likely other liquid foodstuff samples.
In rheumatoid arthritis (ra), a circadian rhythm of disease activity has been well documented . Morning stiffness, joint pain, and swelling (one of the ra classification criteria) are worse in morning, which could be explained by diurnal variations in the metabolism or secretion of endogenous cortisol and cytokines, especially il6 . Corticotropin - releasing hormone (crh) release leads to the pituitary production of corticotrophin (acth), followed by glucocorticoid secretion by the adrenal cortex . These components constitute the hypothalamic - pituitary - adrenocortical (hpa) axis, which has a circadian rhythm . The acth level varies during the day due to its pulsatile secretion . As a result, in physiologic conditions, plasma acth and serum cortisol concentrations are highest at about the time of waking in the morning (at 8 am), decrease irregularly during the day, are low in the evening, and reach their nadir after beginning sleep (at 2 am). In ra patients, impaired cortisol secretion to acth has been described, supporting the concept of a relative adrenal glucocorticoid insufficiency . Moreover, il6 is also considered to be responsible for stimulating the production of acute phase of proteins, and increased levels of it have been reported in serum and synovial fluid in ra . Increased levels of il6 were found from 2 am to 7 am in ra patients, which has been associated with a flare in disease activity in early morning [6, 7]. Therefore, diurnal variations in the clinical signs and symptoms of ra may be partly due to changes in the cortisol and proinflammatory cytokine levels . Glucocorticoids (gcs) are among the most frequently used drugs in treating ra, and most commonly are used to suppress inflammation and pain . But hpa axis support is a crucial rule of low - dose gcs in stable ra . When a potentially harmful drug such as a gc is prescribed, a balance between side effects and therapeutic benefits is attempted, and it is prudent to administer the minimal dose for the shortest duration to avoid adverse effects . With assumption of minimizing interference with the normal diurnal hormonal pattern, in usual practice, in single - dose schedule, prednisolone is administered in the morning . It seems that administration of prednisolone at bedtime, which precedes the circadian flare of inflammatory activity, can potentially better control disease activity in somewhat lower doses . In order to reduce the chance of hpa suppression, we should use the lowest dose of short half - life gcs, preferably in alternate fashion . Accordingly, using the drug at the optimal time may permit the smallest possible dose to be used . Prescribing gcs at night may in part lead to the suppression of an explosive surge of cytokines between 2 am and 7 am by better hpa axis support, so it is assumed that when a low dose gc is used for relief of morning stiffness, night administration could be more effective than an equivalent dose given in the morning . In this study, we decided to test the hypothesis that administration of low - dose prednisolone at bedtime could be more effective in control of disease activity and morning symptoms . Sixty patients who fulfilled the criteria of the 2010 american college of rheumatology for ra were included in this study from april to september 2008 . All patients were referred to the yazd rheumatology center and were studied after obtaining informed consents and approval of the local ethics committee according to the declaration of helsinki . At the start of the study, all of the patients had been on long treatment with a single daily dose of prednisolone (7.5 mg / day) and one or more disease modifying antirheumatic drugs (dmards). Those who were taking drugs other than nonsteroidal anti - inflammatory drugs (nsaids) and dmards and those with any comorbid conditions such as diabetes, hypertension, and other chronic diseases were excluded . 5 mg prednisolone was reinstituted for the patients in the morning (8 am) for the first three months, and, for the next three months, the patients received 5 mg prednisolone at 10 pm (before - after method). Patients took their drug at 10 pm to prevent interference with their sleep . A questionnaire including age, gender, the number of involved and tender joints, pain score (none = 0, mild = 1, moderate = 2, severe = 3), fatigue score (none = 0, mild = 13, moderate = 46, severe = 710), morning stiffness score (0 = none, 1 30 min, 2 = 3060 min, 3 = 1 - 2 h, 4 = 24 h, 5 4 h and 6 = all day), erythrocyte sedimentation rates (esr), c reactive protein (crp), and supposed adverse effects profile of tachycardia, nocturia, sleep disturbance, infection, and blood pressure disturbance was designed by the authors on the basis of similar studies . Esr and crp were measured at the beginning of the study and end of the first and second trimester . The clinical data were collected at the end of each month during the study . We used the clinical disease activity index (cdai), a numerical index that is calculated by summing the number of tender and swollen joints using the 28-joint count and the patient and physician global assessments on a 10 cm visual analogue scale . The cut points for remission and various degrees of disease activity are as follows: low disease activity 10, moderate disease activity 22, high disease activity> 22 . Means of each variable within the first and second trimester and at the end of each trimester were compared . Statistical significance was calculated by nonparametric wilcoxon's signed ranks test using spss version 11.5 (spss inc ., chicago, il, usa) for windows . Sixty patients, nine male (15%) and fifty - one female (85%), with a mean age of 46 11.09, were included in the study . Disease duration among our patients was between 12 months to 40 years with mean of 7.1 7.5 years as mentioned in table 1 . But they were on dmards from beginning of disease in some and months after initiation of disease in the others . Minimum duration of dmard used in our patient when they enrolled in the study was around 6 months . Thirty - seven patients (53.3%) were treated with two dmards, and ten (16.7%) patients were treated with three dmards (table 2). The mean of the morning stiffness was decreased and had significant statistical differences (p value = 0.000). The mean of the cdai was reduced at the end of the second three months, when the patients were treated with prednisolone at night (p value = 0.000). The mean of the fatigue score and crp level were decreased when prednisolone was administrated at 10 pm, and differences were significant (p value <0.05). The mean of the pain score was decreased when the patients took prednisolone at night, but there was no significant statistical difference (p value = 0.146). Also, the mean of tender joint was decreased when the patient took prednisolone at nigh; however, there was no significant statistical difference (p value = 0.129). The mean of the esr was decreased, but the difference was not significant (p value = 0.283). Means of each variable at the end of the first and the second trimester are compared in table 4 . Nocturia was seen in 3 patients (5%) when prednisolone was administrated at night, and sleep disturbance was seen in 1 patient in this group (table 5). In figure 1, comparison of variable means at baseline and end of before and after phase was shown . Better control of inflammation and improving patient symptoms related to inflammatory state with minimal drug adverse effect is the best management in chronic inflammatory conditions like ra . In the current study, administration of prednisolone at night induced a significant beneficial effect on the morning stiffness (p <0.001), which was similar to the results of the arvison and buttgereit report . It should be stated that in the arvidson study, prednisolone was taken at 2 am, which could potentially have a negative effect on the sleep pattern of patients . De silva et al . Showed a significantly shorter duration of morning stiffness in patients when prednisolone was given at night . Similar to our study, the patients were on oral prednisolone at 10 pm deandrade et al . Also noted that a small evening dose was more effective than the same dose given in the morning . Klinefelter and his coworkers evaluated the safety of single low - dose prednisone therapy . In their study, a number of patients required nocturnal doses to control their morning stiffness and did not show any more suppression of the hpa axis suppression than the patients receiving morning doses . Fatigue is frequently identified as a significant symptom by ra patients . The prevalence of clinically relevant fatigue is commonly seen between 40% and 80% of patients with ra . We showed that the mean of fatigue scores also decreased when the prednisolone was administrated at night (p <0.000). However, this variable was not taken into consideration in the previous studies, although it is a main complaint of patients . The theoretical optimal timing of prednisolone administration could be at 2 am but due to interfering with pre - disturbed sleep in ra patients, modified release prednisolone was introduced to overcome this problem . We, in our study, showed that shifting single oral prednisolone from morning to bedtime schedule also could have similar beneficial effect on morning symptom which is universally available . In our patients, there was a tendency for a decrease in the mean of pain scores when the patients took prednisolone at night, although employing wilcoxon's test showed no statistical significant difference . Result of means of variables was similar within and at the end of each trimester . However, in the study conducted by arvidson and buttgereit, pain scores decreased statistically significant after bedtime administration of gcs . In another study, clinical outcome after administration of prednisolone at 8 am, 1 pm and 11 pm was compared . Four weeks later, there was no difference in pain or stiffness scores between these times of prednisolone administration . Also, karatay and his colleagues reported that there were no differences between pain and morning stiffness scores when gcs were administered at different times of the day . The differences between our and their findings could be attributed to the total daily doses and/or ethnic differences in the patients . A comparison of objective measurements of joint inflammation revealed no significant statistical differences, although there was a decline in the number of tender joints . Nevertheless, it should be mentioned that in most of our patients, joint tenderness was not a predominant symptom . In our study, cdai was also used for clinical assessment and there was a significant statistical difference between the mean of two groups . In spite of a reduction in the mean of esr, there was no significant statistical difference (p = 0.283). As in the studies done by arvidson et al . And karatay et al ., the mean of crp levels decreased significantly following nocturnal use of prednisolone [8, 20]. We emphasized that adverse profile of shifting prednisolone from morning to bedtime was negligible in our survey and none of them was related to hpa axis derangement . Addressing to our limitations in the study, we should mention that, due to clinical nature of our study we did not determine the cytokines such as il6 and hormones involving in hpa axis notably serum and urine cortisol that it could be designed as a separate supplementary study . Also, we think that due to potential beneficial of bedtime dosing of prednisolone, we could test smaller doses of prednisolone at bedtime along with this study and compare the mean of prednisolone doses in two phases (groups) of study . In conclusion, our study showed that similar doses of prednisolone could be safely administrated at bedtime with better clinical responses in most (but not all) of ra patients.
Raised intracranial pressure (icp) after traumatic brain injury (tbi) is common and associated with increased risk for death and disability . Despite decades of animal and human research, successful prevention and treatment of this deadly complication largely eludes the medical community . Because of the considerable heterogeneity and severity of the disease, well designed, prospective, randomized studies in neurotrauma are rare . All academic attempts to generate reliable trial data are noteworthy . In this context, we enthusiastically applaud prez - brcena and colleagues for their thoughtful and ambitious research . The study evaluates the use of two barbiturates, pentobarbital and thiopental, in the treatment of refractory intracranial hypertension after tbi . Forty - four patients were randomly assigned to receive pentobarbital or thiopental after first - level measures had failed to control icp . In the pentobarbital and thiopental groups, icp was controlled in 18% and 50% of patients, respectively, without any statistically significant difference between groups in the rate of infectious complications or hemodynamic compromise . Glasgow outcome scale (gos) scores at 6 months revealed a poor neurologic outcome (death, vegetative state, and severe disability) in 17 and 12 patients in the pentobarbital and thiopental groups, respectively . No statistical analysis of gos scores was reported, but the study was not powered to detect a difference in outcome . Specifically, cranial computed tomography revealed bilateral brain swelling in significantly more patients in the pentobarbital group, and more patients in the thiopental group had evacuated lesions or no swelling at all . In addition, the doses of barbiturate were not equivalent between groups . To compensate for this, the authors attempted to ensure equivalent potency by titrating the dose to electrographic burst suppression (ebs) or flat pattern . However, the number of patients in each group that reached ebs was not reported; therefore, it is unclear whether bioequivalent doses were actually achieved between groups . Previous research has shown that the serum concentration at which ebs is reached varies between patients, and serum and cerebrospinal fluid levels correlate poorly with ebs . Therefore, ebs is not necessarily an accurate surrogate for barbiturate dose . Given that thiopental is more lipophilic than pentobarbital and was infused at a higher initial maintenance rate, it is possible that the thiopental group maintained a higher cerebral concentration of barbiturate . Despite these potential confounders, the relevance of this trial cannot be overstated . In the current age of multimodality monitoring, individualized treatment paradigms, and combination therapy, these data have important implications and bring to the forefront a number of questions . What is the appropriate goal of barbiturate therapy icp control, ebs, neuroprotection, or a combination of these? Experimental models of ischemia demonstrate that anesthetic doses of barbiturates provide no additional attenuation of brain free fatty acid release than subanesthetic doses . In models of focal infarction, animals that were anesthetized with pentobarbital dosed to preserve an active electroencephalogram had equivalent reductions in infarct volume as those anesthetized to ebs . Barbiturates lessen the release of s-100b, excitatory neurotoxins, and amino acid markers of energy failure, but it has not been proven that ebs is a requirement for these neurochemical changes . These data cast doubt on the argument that patients benefit maximally from barbiturate protocols that include ebs as a therapeutic target, especially given the frequent adverse effects of hypotension and immune dysfunction . Nevertheless, several studies convincingly demonstrate that barbiturates can treat elevated icp, particularly in patients who are refractory to other management strategies . If barbiturates can lower icp and provide neuroprotection even without ebs, then why has an association with good outcomes not been realized in clinical trials? Perhaps by the time barbiturates are employed for refractory icp often several days after the trauma alternatively, it is plausible that only specific pathophysiologic and cerebral hemodynamic profiles will respond to barbiturate treatment . Prez - brcena and coworkers reported that the association of focal lesions with icp control was 3.6 times higher than that for diffuse lesions . Improved pressure reactivity indices and cerebral tissue oxygen tension in response to barbiturates, even in the presence of continued elevations in icp, these findings raise the possibility that in select patients barbiturates have the ability to prevent localized ischemia, reverse dysautoregulation, and improve cerebral oxygenation . Finally, barbiturates may indeed provide an outcome benefit, but were not superior to other drugs including opiates, mannitol, hypertonic saline, and benzodiazepines that were used in controls . Despite insufficient evidence that either pentobarbital or thiopental improves outcomes after tbi, or that one drug is better than the other early combination therapy of barbiturates with hypothermia or progesterone, in concert with multimodality invasive neuromonitoring, holds promise in the treatment of this devastating condition [11 - 14]. Ebs: electrographic burst suppression; gos: glasgow outcome scale; icp: intracranial pressure; tbi: traumatic brain injury.
Cyclosporine is a lipophilic cyclic peptide of 11 amino acids, while tacrolimus is a macrolide antibiotic . Both drugs have been isolated from fungi and possess similar suppressive effects on cell mediated and humoral immune responses . Patients treated with the calcineurin inhibitors are at high risk of developing renal injury1). Although renal insufficiency induced by calcineurin inhibitors has received the most attention, tubular dysfunctions are also clinically important and will be briefly reviewed in this article . Calcineurin inhibitor - associated tubular dysfunction is manifested by metabolic acidosis, hyperkalemia, calcium, phosphate wasting, and magnesium loss . The first case report with post - transplant renal tubular acidosis was described by massry et al . Renal tubular acidosis (rta) is non - anion gap metabolic acidosis and is generally mild and a symptomatic in kidney recipients3). The reported prevalence of calcineurin inhibitor - associated rta is 13 - 17% intransplanted patients4 - 6). The former form of acidosis is characterized by bicarbonate wasting due to the toxic effects of calcineurin inhibitors . In contrast, distal or type iv rta is characterized by the inability to excrete hydrogen ions6). Use of calcineurin inhibitor cyclosporine can frequently cause type 4 rta, a mild hyperchloremic acidosis, sometimes with elevated potassium . This may be reflecting decreased aldosterone activity and suppression of ammonium excretion by hyperkalemia7). There are some reports now that provide some insight as to how that might occur . Collecting ducts have 2 types of intercalated cells - the acid or hydrogen ion - secreting alpha - intercalated cells and the bicarbonate - secreting beta - intercalated cells (fig . 1). The preponderance of which cell dominates rests on the type of diet . An acid diet leads to more alpha - intercalated cells, whereas an alkaline diet leads to more expression of beta - intercalated cells . It has been reported that protein hensin is actually important in mediating transformation between beta - and alpha - intercalated cells . Deposition of hensin leads to the conversion of bicarbonate - secreting beta - intercalated cells into the acid - secreting alpha - intercalated cell . Fk506 and consequently acid - secreting cells will be less abundant and risk for amild normal anion gap metabolic acidosis will increase8, 9)(fig . Treatment of calcineurin inhibitor - associated acidosis is mainly with oral supplement of bicarbonate4, 12). Synthetic mineralo corticoid is apotential treatment option, but has more frequent side effects10). An elevation in plasma potassium concentration due to reduced efficiency of urinary potassium excretion is common in calcineurin inhibitor - treated patients . It may be severe and potentially life - threatening with concurrent administration of an angiotensin converting enzyme inhibitor or angiotensin receptor blocker . Cyclosporine may reduce potassium excretion by altering the function of several transporters, decreasing the activity of the renin - angiotensin - aldosterone system, and impairing tubular responsiveness to aldosterone15, 16). Renal potassium excretion is primarily derived from potassium secretion in the distal cortical nephron via potassium channels in the luminal membrane . This process is stimulated by sodium reabsorption, aldosterone, and the basolateral na, k - atpase pump . In vitro studies suggest that cyclosporine may directly impair the function of the potassium secreting cells in the cortical collecting tubule by affecting each of these steps: reduced activity of the na, k - atpase pump17 - 19), inhibition of the luminal potassium channel20), and increased chloride reabsorption16). Cyclosporine increases paracellular or transcellular chloride reabsorption, which prevents generation of lumen - negative potential that drives potassium secretion . Calcineurin inhibitors inhibit luminal potassium channels and increase chloride reabsorption via alteration of wnk kinases21)(fig . Cyclosporine may also have a secondary effect on potassium homeostasis in patients concurrently treated with a beta - blocker . In this setting, there is often a modest and transient elevation in the plasma potassium concentration due to movement of potassium out of cells into the extracellular fluid22). Treatment is similar to that of hyperkalemia in chronic kidney disease: reduction in potassium intake, adjustment in medications, and so forth . One exception occurs in the early posttransplant period, in which renal insufficiency also contributes to the impairment in potassium excretion . Administration of a cation exchange resin increases the risk of intestinal necrosis at this time23). Cyclosporine inhibits the type iia sodium phosphate cotransporter and probably the type iib sodium phosphate cotransporter of the intestine24). Post - transplant hypophophatemia is usually asymptomatic, but can rarely be severe enough to cause severe muscle weakness, including weakness of respiratory muscle . Several case reports suggest that hypomagnesemia may contribute to calcineurin inhibitor - induced encephalopathy25 - 27). Hypomagnesemia has also been implicated as a contributor to the nephrotoxicity associated with cyclosporine28). Animal studies suggest that hypercalciuria induced by cyclosporine administration is associated with an inhibition of calbindin d28k expression, resulting in calcium transport defect in the distal tubule29). In conclusion, attention must also be paid to the calcineurin inhibitor associated renal tubular dysfunction such as renal magnesium wasting, calcium and phosphate wasting, distal tubular acidosis, and impaired renal potassium excretion, in addition to the well known side effects, such as tubulointerstitial fibrosis and glomerular or vascular damage.
Cancer is a major public health problem worldwide; it is the primary and secondary causes of death in economically developed and developing countries, respectively . The global concern on cancer continues to intensify as a result of the aging and expanding world population and the increasing adoption of cancer - causing habits . The mechanism of carcinogenesis remains largely unknown although genetic susceptibility is a known possible explanation for the interindividual variation in cancer risk . Nuclear factor-b (nf-b) was initially identified in 1986 as a transcription factor which binds to a 10 bp dna element in kappa immunoglobulin light - chain enhancer in b cells . The nf-b family consists of p50 (nf-b1), p52 (nf-b2), p65 (rela), c - rel (rel), and relb . The major form of nf-b is a heterodimer of the p50 and p65/rela subunits which are encoded by the nfkb1 and nfkb2 genes, respectively . The human nfkb1 gene is mapped to chromosome 4q24 and encodes a 50 kda dna - binding protein (p50) that can act as a master regulator of inflammation and cancer development [57]. A common insertion / deletion polymorphism (94ins / del attg, rs28362491) in the promoter region of the nfkb1 gene elicits a regulatory effect on the nfkb1 gene . A previous meta - analysis concluded that the deletion allele serves as a risk or protective allele for cancer susceptibility in caucasian or asian populations, respectively; however, it revealed no association between the polymorphism and cancer risk . An increasing number of studies have assessed the association between the nfkb1 promoter 94ins / del attg polymorphism and cancer risk [1012]. However, these studies obtained conflicting results . Therefore, we collected all available data to perform an updated meta - analysis that generates a precise estimation to comprehensively and objectively investigate the association between the nfkb1 promoter 94ins / del attg polymorphism and cancer risk . A comprehensive literature search for relevant articles published (last search updated in september 15, 2013) in pubmed (http://www.ncbi.nlm.nih.gov/pubmed/) was performed with the following key words: (genetic polymorphism, polymorphism, snp, single nucleotide polymorphism, gene mutation, or genetic variant), (neoplasm, cancer, tumor, carcinoma, or carcinogenesis), and (nfkb1, nf-b1, nuclear factor kappa b1, nf kappa b1, or the reviews and references of eligible studies were hand - searched for additional relevant publications . The most recent or complete study was selected when more than one publications contain overlapping data . Case - control studies that evaluated the association of the nfkb1 promoter 94ins / del attg polymorphism with cancer risk and described in detail the genotype distributions of the polymorphism in cases and controls were included in this meta - analysis . Studies that were not for cancer research, were only case population, and were duplication of previous publication were excluded in this meta - analysis . Information was carefully extracted from eligible studies independently by two investigators (xiao yang and pengchao li) according to the inclusion criteria listed above, and the result was reviewed by a third investigator (jun tao). The following data were collected from each study: surname of first author, year of publication, ethnicity, genotyping method, source of controls, frequencies of the genotypes in cases and controls, cancer type, and hardy - weinberg equilibrium (hwe) of genotype distribution among controls . Studies that investigated more than one type of cancer were regarded as individual datasets only in subgroup analyses according to cancer type . Articles that reported different ethnic groups and countries or locations were considered different study samples for each category cited above . The strength of association between the nfkb1 promoter 94ins / del attg polymorphism and cancer risk was estimated through pooled odds ratio (or) with its corresponding 95% ci . Pooled ors were calculated for insertion allele versus deletion allele, ins / ins versus del / del, ins / del versus del / del, ins / ins + ins / del versus del / del, and ins / ins versus ins / del + del / del . Subgroup stratification analyses by ethnicity and cancer type were conducted to identify the association of the 94ins / del attg polymorphism with cancer susceptibility . The between - study heterogeneity of the studies included in this meta - analysis was evaluated using the q and i statistic tests, where i> 50% indicated heterogeneity . The random - effects model was selected when i was significant (> 50%); otherwise, the fixed - effects model was selected . The allele frequencies of the nfkb1 promoter 94ins / del attg polymorphism from the respective study were determined by allele counting . In addition, a chi - square test was used to determine whether or not the observed frequencies of genotypes conform to hwe . Pooled or in the current meta - analysis in addition to the comparison among all subjects, we performed stratification analyses by cancer type (if one cancer type contained only one studies, it was combined into the other cancers group) and ethnicity . Begg's funnel plot and egger's test were adopted to evaluate the publication bias in our meta - analysis [14, 15]. All statistical analyses were performed by stata 10.0 software (statacorp, college station, tx, usa). A total of 21 case - control studies involving 6127 cases and 9239 controls were analysed . The allele and genotype frequencies of the nfkb1 promoter 94ins / del attg polymorphism were extracted from all eligible studies . In total, this meta - analysis included 3 bladder cancer studies, 4 colorectal cancer studies, 2 ovarian cancer studies, 2 oral cancer studies, 2 prostate cancer studies, and 8 studies with the other cancers . Of the 21 studies, 14 were conducted among asians and 7 were conducted among caucasians . The results of hwe test for the genotype distribution in the control population are shown in table 1 . Six of the eligible studies were not in hwe [10, 11, 18, 21, 31]. The pooled ors of the included case - control studies revealed a statistically significant association between the nfkb1 promoter 94ins / del attg polymorphism and cancer risk across the four genetic models ins / ins versus del / del, or = 1.47, 95%, ci = 1.111.93; dominant model, or = 1.26, 95% ci = 1.031.53; recessive model, or = 1.26, 95% ci = 1.051.51; and ins allele versus del allele, or = 1.19, 95%, ci = 1.051.35 (table 2, figure 2). Stratified analyses also revealed a significant association between the polymorphism and ovarian, oral, and prostate cancers in the various models . Ethnic subgroup analyses revealed significant increases in cancer risk in the four models among asians but not among caucasians . The results became prominent when the six studies that deviated from hwe were excluded (see supplementary table 1 and supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/612972). Publication bias was evaluated by begg's funnel plot and egger's test, and the visual asymmetry was determined in the funnel plot analysis (figure 3). The results of egger's tests for all genetic models are shown in supplementary table 2 (ins allele versus del allele, p = 0.004). Nf-b serves important functions in pathogenetic regulation and influences cancer development and aggressiveness by enhancing tumour angiogenesis, antiapoptosis, and proliferation and by repressing immune response [7, 33, 34]. Several investigators reported the constitutive activation of nf-b in various malignancies [35, 36], including nonsmall cell lung carcinoma and colon, prostate, breast, bone, and brain cancers . P50 overexpression is frequently observed in various tumour tissues; hence, p50 is potentially involved in tumourigenesis . A polymorphism in the promoter region of nfkb1 encoding the p50 subunit of nf-b modulates gene activity . A meta - analysis of all eligible studies in 2010 suggested that the deletion allele serves as a protective or risk allele for cancer susceptibility among asians or caucasians, respectively . After the reported study, numerous studies further assessed the relationship between the nfkb1 promoter 94ins / del attg polymorphism and cancer among asians and caucasians [10, 12, 32]. However, the association remains inconclusive because of the inconsistent results from the published studies . Li et al . Found an association between del / del genotype and bladder cancer risk but none between the polymorphism and hepatocellular carcinoma susceptibility . In this study, we analysed 21 eligible case - control studies with 6127 cases and 9239 controls . The results of this meta - analysis revealed a significant association between insertion allele careers and enhanced cancer risk . The probable mechanism behind the observed association may be linked to the enhanced expression and activity of p50 (nf-b1). The insertion allele is reportedly associated with the increased promoter activity and enhanced nfkb1 mrna expression [8, 12, 17]. The major effect of p50 (nf-b1) is mediated by its function as a component of the transcription factor nf-b, which is among the major signalling pathways involved in the cellular response to environmental stress . P50 serves an important function in inhibiting cell apoptosis by modulating the expression levels of several survival genes, such as bcl-2 homologue a1, pai-2, and iap gene family . Certain antiapoptosis proteins, such as bcl - xl and fas - associated death domain - like il-1-converting enzyme inhibitor protein, are upregulated through the nf-b signalling pathway [4042]. In addition, accumulated evidence illustrated that the p50 (nf-b1) signalling pathways participate in cellular proliferation by increasing il-5, promoting mapk phosphorylation [7, 44], and modulating cyclin d1 expression . Therefore, the observed association between the 94ins / del attg polymorphism and cancer risk can be accounted for by the insertion allele that can inhibit apoptosis and promote cellular proliferation by upregulating the expression of p50 (nfkb1) [8, 12, 17], which was implicated in the abovementioned mechanism . In the stratified analyses, the increased cancer risk remained in subgroups of asians but not in those of caucasians . The ethnic differences in the allele frequencies may be caused by natural selection or balance to other related genetic variants . The increased cancer risk also remained in the subgroups of ovarian, oral, and prostate cancers . This result suggested that the nfkb1 gene might function as a prominent factor in these cancers . Therefore, further investigations are warranted to validate ethnic difference and cancer specificity in the effect of this functional polymorphism on cancer susceptibility . First, significant between - study heterogeneity was detected in some comparisons and may be distorting the meta - analysis . However, the association between the insertion allele and cancer risk in the overall population and in the asian population became pronounced when the six studies that deviated from hwe were excluded . Third, the studies included in the analysis used different genotyping methods with different quality control issues that may have also influenced the results . Fourth, publication bias was observed in our study, which may affect the validity of conclusion . In the stratified analysis, we found that the publication bias was significant among the asian groups and other cancer groups but not significant among the caucasian, bladder, and colorectal cancer groups . The sample sizes of the included studies were diverse, and most of them were insufficiently large . Finally, only three controls were population based; thus, they may not represent the general population . Therefore, the results of this study should be interpreted with caution . In conclusion,
The occurrence of hypothyroidism in patients with diabetes mellitus has attracted attention since joslin et al . Revealed this association . Several studies have reported a higher prevalence of thyroid dysfunction in diabetes [24]. This association may be related to autoimmunity as well as other factors associated with either type 1 or type 2 diabetes and suggests that persons with diabetes be screened for hypothyroidism [2, 57]. The executive summary of standards of medical care in diabetes (2014) published by the american diabetes association (ada) as well as the clinical practice guidelines for hypothyroidism in adults (2012) copublished by the american association of clinical endocrinologists (aace) and the american thyroid association (ata) suggest screening patients with type 1 diabetes for thyroid diseases . Age may be a factor associated with the higher prevalence of hypothyroidism among patients with type 2 diabetes [4, 10]. In elderly patients, however, it may be difficult to determine if an abnormal thyroid stimulating hormone (tsh) pattern is due to actual thyroid disease or an unrelated cause or possibly is secondary to drug interference . Although some studies investigated this, it was not always taken into consideration that the upper reference range for tsh in elderly individuals may be higher than in the general population [1012]. It is now clear that screening for thyroid dysfunction in elderly subjects with diabetes should take into account that an increase in serum tsh levels is expected . Previously, an increase in tsh levels in elderly persons was attributed to elevated levels of circulating thyroid antibodies in this specific population . However several recent studies demonstrated that, even after excluding those with known thyroid diseases and circulating antithyroid antibodies, the tsh distribution curve remained shifted to the right; the 97.5th percentile tsh values rose as the population aged [9, 1317]. When considering screening for thyroid diseases in patients with diabetes, the diverse types of drugs used by a significant number of these patients ought to be taken into account, as many reportedly interfere with tsh levels, especially metformin and sulfonylureas [1822]. The major aim of this study was to evaluate the prevalence of previously undiagnosed cases of hypothyroidism in the elderly with diabetes by applying specific ri for serum tsh . A cross - sectional study of 1160 subjects over 60 years of age was performed, of whom 751 had diabetes . All participants underwent routine thyroid function tests, especially as thyroid function or autoimmunity tests had not been performed for all patients previously . The mean time since diabetes diagnosis was 10.2 years (range: 2.614.1 years). Only patients with diabetes who had been diagnosed at least 2 years priorly were included; diagnosis was according to the ada criteria . The blood counts as well as renal and liver functions of all patients were normal . Subjects normally resided in the metropolitan area of rio de janeiro and self - identified as belonging to middle and upper social classes . As for race, 68% were white, 20% were mulatto, and 12% were black . Urinary iodine assessment was not performed, since salt iodization in brazil is determined by federal law and a previous study showed sufficient iodine intake in the rio de janeiro population . A national health surveillance agency review of salt from samples used in the city a year before the beginning of our study confirmed appropriate iodine levels . Exclusion criteria were patients under treatment for thyroid diseases or with a history of thyroid disease, use of medications known to interfere with measurement of tsh or free thyroxine (ft4) in the previous three months (except for diabetes treatments), use of contrast media and other medications containing iodine in the last six months, illness, accident, or surgery in the last six months, and chronic disease except for hypertension and dyslipidaemia . There were 409 patients without diabetes who attended a clinical laboratory to collect routine tests for which function and/or autoimmunity thyroid evaluation had not been requested and that had normal blood count, kidney, and liver functions . All of them fulfilled the same exclusion criteria of patients with diabetes and were not under antidiabetes drugs for any other reason . This study was approved by the institutional review board of hospital clementino fraga filho, universidade federal do rio de janeiro . Patients with diabetes were divided into subgroups according to the medications they used as follows: subgroup mtf: metformin (5003000 mg / day); subgroup su: sulfonylureas (glibenclamide 515 mg / day or glimepiride 14 mg / day); subgroup dpp4: dipeptidyl peptidase 4 (dpp4) inhibitors (vildagliptin or sitagliptin, 50100 mg / day); subgroup tzd: thiazolidinedione (pioglitazone, 1545 mg / day); subgroup ins: insulin analogues glargine or detemir 1342 ui, plus analogues lispro or aspart 614 ui / day; subgroup mtf+: metformin in combination with one or more other medications (sulfonylurea, dpp4 inhibitor, thiazolidinedione, or insulin analogues); the mtf+ group used lower doses of some medications compared to the doses when they were used as the sole medication: mtf: 5001500; su (glibenclamide: 510 mg / day, glimepiride: 1 - 2 mg / day); tzd: 1530 mg / day; dpp4 and ins doses did not differ from the previous subgroups . All patients with diabetes had been on the same medication for at least one year . Other antidiabetes drugs were not evaluated in the study, as there was an insufficient number of patients to allow statistical analysis . Body mass index (bmi) was obtained by dividing the body weight (kg) over the square of the height (m). Tsh, ft4, antithyroperoxidase antibodies (tpoab), fasting glucose (fg), and glycated hemoglobin (hba1c) were measured in all patients . Serum tsh, ft4, and tpoab were measured by electrochemiluminescence immunoassays using the roche modular analytics e170 instrument (roche diagnostics australia pty ltd ., serum tsh concentrations were measured by an immunometric method, with an intra - assay percent coefficient of variation (% cv) of 3.0% at concentrations of 0.040 0.001 the tsh reference interval (ri) established in our previous study of the population in the same region was 0.4 to 5.8 mu / l for subjects aged between 60 and 79 years and 0.4 to 6.7 mu / l for those aged 80 years and older . The% cv was 1.4% at concentrations of 9.0 0.1 pmol / l (0.7 0.01 ng / dl), 1.8% at 16.7 0.3 pmol / l (1.3 0.02 ng / dl), and 2.0% at 34.7 1.3 pmol / l (2.7 0.1 ng / dl). The ri for age groups, 60 years and older, was in the range of 9.021.9 pmol / l (0.71.7 ng / dl). The intra - assay% cv for tpoab was 6.3% at concentrations of 21.3 1.34 iu / ml, 5.1% at 51.2 2.6 iu / ml, and 2.7% at 473 12.7 iu / ml; a score lower than 34 iu / ml indicated the absence of thyroid disease according to the assay's manufacturer . Fg was measured by an enzymatic reference method with hexokinase using roche / hitachi cobas c, gluc3 (roche diagnostics gmbh, mannheim, germany). The lower limit of assay detection is 0.1 mmol / l (2.0 mg / dl), with an intra - assay% cv of 0.8% at concentrations of 5.2 0.04 mmol / l (93.2 0.7 mg / dl), 0.7% at 13.4 0.11 mmol / l (241.0 2.0 mg / dl), and 0.7% at 36.1 0.28 mmol / l (651.0 5.0 mg / dl). According to the ada, the normal value is less than 5.6 mmol / l (100.0 mg / dl). Hba1c was measured by high - performance liquid chromatography (hplc) on the bio - rad variant ii hemoglobin a1c system (bio - rad laboratories, inc . The hba1c reportable range is between 3.1 and 18.5 percent (%) in national glycohemoglobin standardization program (ngsp) units and between 10 and 179 mmol hba1c / mol hb (mmol / mol) in international federation of clinical chemistry and laboratory medicine (ifcc) units . The master equation for designated comparison method is as follows: ifcc = (10.93 ngsp) 23.50 (http://www.ngsp.org/convert1.asp). According to the manufacturer, the intra - assay cv was 0.9% for concentrations in healthy subjects and 0.59% for measurements obtained from individuals with diabetes . A patient with diabetes with hba1c levels below 7.0% (53 mmol / mol ifcc) mu / l in patients aged 6079 years and above 6.7 mu / l in patients aged 80 years or older, with ft4 levels ranging between 9.0 and 21.9 pmol / l (0.7 and 1.7 ng / dl); overt hypothyroidism was diagnosed with the same values of tsh and with serum ft4 below 9.0 pmol / l (0.7 ng / dl). Autoimmunity was considered present when tpoab titres were greater than 34 iu / ml . General data were analyzed using the graphpad prism software, version 6.0 (graphpad software, inc ., kolmogorov - smirnov tests were performed to assess the normal distribution . Log 10 transformations of nonnormal variables were performed for analysis . To test if patients with diabetes and persons without diabetes ages were matched, the unpaired t - test with welch's correction was used, as age had normal distribution . Descriptive analyses of serum tsh, fg, and hba1c were reported as medians and 25 and 75 percentiles, because they were not normally distributed . Descriptive analysis of serum ft4 was reported as mean with standard deviation, as it was normally distributed . Two tailed mann - whitney and kruskal - wallis tests were used to compare the nonparametric tsh distributions in different subpopulations . Comparison between two tsh subgroups was performed by an independent test of dunn multiple comparisons . For all parameters, p <0.05 was considered statistically significant . To describe the relationship between tsh (mu / l) and mtf dose (mg / day), a statistical model was used assessing the possible significance of mtf effect in the tsh variability and providing estimated values for tsh according to different doses . The class of generalized linear models (glm) likelihood - ratio tests were used to assess the significance of parameters estimates, and half - normal plots with simulation envelopes were used to assess goodness - of - fit of the models . The analysis was performed using r software (r core team, 2014), and the r package hnp was used to the half - normal plots . In addition, predicted and average curves were compared and residuals were analyzed . Data of parameters not normally distributed are shown as follows: median, 2575 percentiles (95% ci). Those normally distributed are shown as mean sd . There were no statistically significant differences in the mean of age between patients with diabetes and persons without diabetes, so that both groups were matching for study according to the age . The baseline demographic, clinical, and general laboratory data are presented in table 1 . Data of tsh, ft4, and autoimmunity in euthyroid patients are in table 2, as well as the occurrence of hypothyroidism and autoimmunity in hypothyroid patients . A lower rate of circulating antibody among hypothyroid patients was observed in the subgroup of pioglitazone [25% (1/4)]; however, the small number in the group did not justify a statistical analysis . In relation to hormonal data between antidiabetic drugs mtf+ subgroup is shown separately, since we had interest in checking whether this subgroup behaved as the subgroup only on mtf or as the other subgroups, since the mtf doses used were lower . Table 3 shows the results of euthyroid patients and table 4 those of hypothyroid patients . When we compared clinical and subclinical hypothyroidism between patients with diabetes as a whole group and persons without diabetes, the results in patients with clinical hypothyroidism were as follows: tsh values: 17.5, 13.017.3 (95% ci 12.216.4) mu / l in patients with diabetes and 15.6, 13.416.8 (95% ci 11.916.1) mu / l in persons without diabetes (p = ns); ft4 values: 7.1 0.3 (95% ci 6.67.6) pmol / l [0.55 0.02 (95% ci 0.510.59) ng / dl] and 7.7 0.1 (95% ci 6.87.9) pmol / l [0.6 0.01 (95% ci 0.530.61) ng / dl], respectively (p = ns). For patients with subclinical hypothyroidism tsh values were 12.3, 10.414.0 (95% ci 11.214.8) mu / l in patients with diabetes and 10.9, 9.413.8, (95% ci 10.713.7) mu / l in persons without diabetes, with a marginal significance (p = 0.049); and ft4 values were 14.2 0.39 (95% ci 11.512.0) pmol / l [1.1 0.03 (95% ci 0.890.93) ng / dl] and 14.2 0.3 (95% ci 11.814.2) pmol / l [1.1 0.02 (95% ci 0.921.1) ng / dl], respectively, in addition to the data cited, it is noteworthy that tsh in mtf users was also lower than in persons without diabetes (p = 0.032). Ins compared to all other subgroups together had highest levels of fg, hba1c, and bmi . These parameters were as follows (mtf others, resp . ): fg: 8.4, 6.610.6 (95% ci, 7.310.4) 7.3, 5.69.3 (95% ci 6.58.8), p = 0.004; hba1c: 7.6, 6.49.0, (95% ci 5.810.7) 7.1, 6.28.9 (95% ci 6.89.2), p = 0.0381; and bmi: 30.9 6.3 28.4 5.2, p = 0.0061 . With regard to the relationship between tsh levels and mtf dose the selected model assumes an inverse distribution for the square root of tsh in the random component of the glm, an identity link function, and a systematic component (linear predictor) with intercept and terms until third order to the dose of metformin . Symbolically, we have the estimated tsh with mtf dose varying from 500 to 3000 mg / day . Parameters estimates and standard errors, according to likelihood - ratio tests, were all significant . The half - normal plot related to this model indicates a good fit, since less than 5% of the points fell out of the simulation envelope . Even more, the residuals have mean, first, and third quartiles close to zero: 0.0017, 0.2540, and 0.1398, respectively . A graphical representation of the model is presented in figure 1, showing that the two curves are close . The overall conclusion is that there was a significant reduction of tsh as the mtf dosage increased . It was also possible to obtain a confidence interval for the estimated tsh, based on the asymptotic properties of the maximum likelihood estimators . Figure 2 shows the 95% confidence interval . Regarding a possible influence of mtf+ subgroup on the tsh, this was not observed in the same model, and therefore, the curves were not performed . In order to assess whether an age - specific tsh ri to older patients had a clinical impact with respect to the diagnosis of hypothyroidism in patients with diabetes, we compared the amount of hypothyroidism diagnosis in these patients when using tsh adjusted for age with the amount that would be diagnosed if using the ri defined by the kit manufacturer . In the first case 6.4% (45/751) this diagnosis was increased to 14.6% (110/751), that is, more than twice, if the ri were adopted without discrimination by age . In this study, we aimed to evaluate the prevalence of undiagnosed hypothyroidism in patients with diabetes over 60 years of age by adopting age - specific ri . Using this method, we observed that the diagnosis of hypothyroidism was slightly but significantly higher in patients with diabetes than in persons without diabetes, albeit less frequent than reported in some previous studies . A pubmed search revealed that most studies on the prevalence of hypothyroidism in patients with diabetes were conducted before recent revelations in the field about tsh values in the elderly . This rate is quite variable, ranging from 5.7% to 27.7% in previous studies [2, 3, 5, 10, 12]. In the patients with diabetes population we studied, the difference among the various studies could be attributed to which ri were used for the diagnosis of hypothyroidism, to the heterogeneity of patients' ages, including in the same group those younger than 60 years and those older than 60 years, and to the characteristics of each population . There were higher rates of positive tpoab in patients with diabetes (13.8%), especially in hypothyroidism patients (64.4%). Of note, the percentage of patients with diabetes with hypothyroidism and tpoab positivity was higher than that observed in other studies, which ranged from 14.6% to 43% [6, 7, 10]. Autoimmunity directed against the thyroid can vary depending on factors such as patients' ages, environmental factors, and the laboratory methods used . As this study focused on a select population of elderly subjects, the involvement of environmental factors including differences in iodine diet content, stress, and drugs could be other factors to be considered . Although tsh levels were equivalent when comparing patients with diabetes to persons without diabetes overall, patients with diabetes who were given mtf had slightly but significantly lower tsh compared to persons without diabetes as well as to patients with diabetes treated with other antidiabetes agents . Mtf is the most widely used drug to treat diabetes and is the first choice medication recommended for the treatment of type 2 diabetes by ada . Some reports suggest that all subjects treated with mtf likely have lower tsh levels [30, 31], while others report no significant lowering of tsh by the drug, except in those subjects with subclinical hypothyroidism or who are on levothyroxine treatment [19, 32]. The reason mtf may reduce tsh may be due to its reported capacity to cross the blood brain barrier . It directly acts in several regions of the brain and can concentrate in the pituitaries and hypothalamuses of rats and can also suppress amp - activated protein kinase activity (ampk) [33, 34]. As hypothalamic triiodothyronine administration also decreases this enzyme, it is postulated that mtf could decrease tsh in the same manner as thyroid hormone, but to a lesser degree . This idea is further supported by a study that suggests that mtf may have an impact on thyrotrope function in hypothyroid patients, lowering tsh in polycystic ovary syndrome, which was attributed to a probable effect increasing thyroid hormone action in the pituitary . We observed an inverse correlation between doses of mtf and tsh . When evaluating patients being given a combination of mtf and one of the other drugs, lower tsh levels were not significant . A possible explanation is that the mtf doses in mtf+ subgroup were smaller than those on mtf as a sole medication . This could be the reason tsh have not been affected by mtf in combination with other antidiabetic drugs . We question whether the relation between tsh and mtf doses could explain the different results observed regarding the effect of this medication on tsh levels when comparing different series . At this point, these results cannot be considered definitive but are preliminary data that offer new perspectives regarding the relationship between mtf and tsh, requiring confirmation with a larger number of cases . It has been suggested that the effect of mtf on tsh could be secondary to weight reduction . In the model adopted, we did not compare the impacts of each medication on patients' weights, but the bmi of each treatment subgroup was compared with the others . At the time of data collection, all subjects except those on ins treatment had similar bmi . Therefore, weight did not appear to be a significant factor in the effect of mtf on tsh levels compared to other medications . Investigators also pointed to a goitrogenic effect as well as a decrease in iodine uptake by the thyroid gland with second - generation su gliclazide . In our study, these effects were also not observed in another study of second - generation su glyburide . Regarding dpp4, no changes were observed in tsh between this subgroup and either persons without diabetes or subjects in the other diabetes treatment groups . A search of the pubmed database using the terms dpp4 inhibitors, thyroid, tsh, hypothyroidism, and hyperthyroidism did not yield any studies related to tsh changes with dpp4 inhibitors . The tsh levels of patients who used pioglitazone (tzd) did not differ from other subgroups . Pioglitazone is a tzd, a class of drugs that act as an exogenous peroxisome proliferator - activated receptor- (ppar) agonist . Besides controlling glucose metabolism and fatty acid storage, ppar pioglitazone is also capable of lowering the expression and release of certain cytokines in thyroid cells . We observed that tpoab levels were lower in tzd subgroup of patients with diabetes; however we could not make conclusions regarding the effect of pioglitazone on autoimmunity because of the small number of patients . Future studies with ppar agonists in autoimmune thyroid diseases are necessary to determine any such effect . We postulate that this may be due to selection bias because this group also had the highest bmi and hba1c levels . Previous studies showed a positive correlation between tsh, even within its normal range, and body weight / bmi and hba1c [12, 4042]. One study linked increased weight with the susceptibility to harbor thyroid autoimmunity . We did not consider this aspect, as tpoab values in patients using ins (the subgroup with higher bmis) were not different when compared to the other subgroups . Considering the most appropriate evaluation of the prevalence of undiagnosed hypothyroidism among patients with diabetes, especially the elderly, our results possible bias in this study is that of other drug interference, since polymedicine is prevalent in elderly patients; besides that, thyroid function was only measured once and thus cases of subclinical thyroid dysfunction could be due to nondetected nonthyroidal illness during the selection . We attempted to minimize some of these possible biases, by excluding those subjects who used medications that are known to interfere with tsh levels . The authors suggest that an annual tsh measurement in dm patients over 60 years would be appropriate for hypothyroidism screening . If tsh levels do begin to increase at any time in relation to the specific ri for age, ft4 and tpoab levels should be measured . In conclusion, in patients with diabetes aged 60 years or older, hypothyroidism was more prevalent than in persons without diabetes of the same age, justifying the suggestion of annual screening in these patients . Use of tsh ri appropriate for the elderly can avoid the misdiagnosis of hypothyroidism . In this study we found an interesting association between the mtf dose when used as a single medication and tsh levels, encouraging that further studies are conducted on this issue.
Infections are frequently encountered in day to day practice of oral and maxillofacial surgery which has the potential to spread through the fascial planes to the head and neck region to compromise the vital structures . These infections often respond to surgical and antimicrobial management if diagnosed and treated appropriately . To know whether the inflammatory process is in the stage of cellulitis that can be treated by antibiotics alone or abscess formation which requires primary evacuation of pus and administration of antibiotics is inevitable in the case of acute odontogenic infections . Clinically it is often difficult to diagnose the stage of infection and to define its exact location . The anatomical location of abscess in patients with odontogenic infection is commonly determined by physical examination, but abscess of deep subcutaneous layer can be difficult to diagnose [2 - 4]. The relatively blind surgical incision and drainage of an abscess based on diagnosis by physical examination alone usually results in excessive harm, unnecessary extensive incisions, excess time, and failure to locate and evacuate the abscess cavity . There are various diagnostic tools available which have minimized this therapeutic dilemma for surgeons to precisely diagnose and delineate the extent, and location of the lesion . Computer tomography (ct) scans and magnetic resonance imaging (mri) are effective in diagnosing inflammatory conditions but are expensive and, suffer from some disadvantages . In light of these factors, the aim of the study was to evaluate the use of ultrasonography as a diagnostic tool for fascial spaces infections in maxillofacial region . This clinical study was approved by the institutional review board and the ethical commission and written informed consent was obtained from patients . Twenty five patients reporting to kle hospital and medical research centre, belgaum, india during the period 2007 to 2008 were included in the study . Patients presenting with signs and symptoms of maxillofacial space infections, 12 male and 13 female with an average age of 40 (sd 7.4) years were studied . A detailed history and clinical and radiographic examination of the swelling was carried out by a single examiner (p.k.p .) In a systematic manner . Swellings with history of short duration, diffuse margins, lack of fluctuancy and inflamed overlying skin were considered clinically as cellulitis (figure 1). Well localized swellings with history of longer duration and presence of fluctuancy were diagnosed as abscess . After obtaining the informed consent complete blood investigations were done and patients were subjected to ultrasonographic (usg) examination and reports obtained (figures 2 and 3). A 7.5 mhz linear or a convex transducer (l&t medical selectra lx, biometric cables, chennai, india) was applied over the skin with ultrasound equipment (acuson x300, siemens ag, erlangen, germany) covering the suspected area in transverse and axial sections to determine the presence or absence of fluid collection and its location . Usg guided needle aspiration was then carried out to conform the fluid collection and pus sent for culture and sensitivity . The difference between two groups was considered as statistically significant, when p <0.05 . Of the 25 patients, 4 presented with buccal swelling, 6 with submandibular swelling, 10 with buccal and submandibular swelling, 3 had buccal and infraorbital swelling and 2 presented with buccal, submandibular and infraorbital swelling (table 1). Fluid collection identified by ultrasonography (usg) all these 25 patients were evaluated clinically and radiographically . In total 64 spaces were diagnosed clinically in 25 patients, out of which 34 spaces had abscess formation and the rest 30 spaces were in the stage of cellulitis . Twenty eight spaces were reported to have fluid collection, suggestive of abscess formation and rest 36 spaces were diagnosed to be in the stage of cellulitis (table 2). Considering the number of spaces involved, clinical findings were correlated with usg findings (figure 4) and there was statistically significant agreement between these two methods of assessment (kappa index = 0.814, p <0.001). It was found that usg is sensitive in 65% of the cases, having specificity of 80% . Patients diagnosed with abscess formation were primarily subjected to surgical incision and drainage under antibiotic coverage . Patients diagnosed as having cellulitis were admitted to the wards and parenteral antibiotics were administered along with supportive therapy . Although spatially confined, purulent material may spread deeply into contiguous fascial spaces such as the submandibular, sublingual and pterygomandibular spaces . Severe complications such as mediastinitis, intra - cranial abscess and parapharyngeal spread with airway obstruction can result if the infection is not recognized and treated promptly . In case of rapidly spreading infections, securing the airway and broad spectrum intravenous antibiotic therapy a finding of fluctuance is often difficult on clinical examination, especially in spaces such as the submasseteric, where purulent material is deep within the soft tissues and muscle . Despite of various diagnostic modalities which are available as an adjuvant to clinical examination, usg seems to play an important role in reducing the therapeutic dilemma to a great extent . Radiographs and other imaging studies can be used to diagnose the spreading infections in the head and neck area but plain radiographs do not often provide a good definition of soft tissues . Ct and mri are effective in diagnosing inflammatory conditions and choice between these two techniques usually depends on the area involved . However, both techniques are expensive . Ct exposes the patient to large doses of radiation especially if repeated follow - up examinations are to be performed . The major disadvantage of mri is the prolonged time for image acquisition, and also the image may suffer from the effects of the patient motion . The high static magnetic field also poses a danger to those individuals with cardiac pacemakers, neurostimulator units and intraocular therapeutic devices . For many years, usg has played a major role as a diagnostic tool in various medical conditions . In maxillofacial surgery, it is relatively a new diagnostic aid . The usg examination has been used to evaluate various masses in the neck and cysts, tumours, swellings, and similar processes in soft tissues of the cranio - facial region . It offers potential advantage because it can be performed noninvasively, repeatedly, and easily, even at the bed side . With the aid of high - resolution transducer usg is an effective diagnostic tool to confirm abscess formation in the superficial fascial spaces and is highly predictable in detecting the stage of infection (figures 1, 2 and 3). It has the ability to pinpoint the relation of the abscess to the overlying skin, accurately measure the dimensions of the abscess cavity, and its precise depth below the skin surface . The principle of usg is based on the fact that, there are large differences in the impedance for ultrasound waves between soft tissue and air, and between soft tissue and bone . Bone and air are absolute barriers to an ultrasound beam, this means that no image within or behind bony or air containing structure can be produced by ultrasound . Therefore some regions of maxillofacial field cannot be evaluated by ultrasound, such as the retropharyngeal region and paranasal sinuses . No echoes are returned by fluids and thus usg is very sensitive in detecting fluid collections as in case of maxillofacial infections . Though usg cannot differentiate an abscess from surrounding blood vessels, but combination of colour doppler ultrasonography with grey scale has solved this problem . The target of colour doppler imaging is the moving blood cells within the blood vessel . The vessels of the inflammatory tissue which has a higher blood volume due to increased permeability of the vessel wall are depicted as a colour flow signal . Blood flowing towards the usg transducer is displayed as red and that moving away from transducer as blue . In contrast the retained pus which does not contain flowing blood cells is delineated as no colour flow signal . This property of doppler ultrasonography allows it to differentiate blood vessels from static regions of images . As a diagnostic tool ultrasonography imaging stands as a non - invasive, cost effective, readily available and repeatable technique . The machine is portable and can be used in real time during surgery . Except with few short - comings usg surpasses all the other diagnostic modalities especially in cases of maxillofacial space infections . In our study ultrasonography has been useful in detecting abscess formation in maxillofacial spaces and highly predictable in determining the stage of infection.
The cluster of pathologies known as metabolic syndrome, including obesity, insulin resistance (ir),type 2 diabetes, and cardiovascular disease (cvd), has become one of the most serious threats to human health . The dramatic increase in the incidence of obesity in most parts of the world has contributed to the emergence of this disease cluster, particularly insulin resistance and type 2 diabetes . Ir is associated with a number of diseases including obesity, metabolic syndrome, type 2 diabetes, lipodystrophies, polycystic ovary syndrome, and chronic infection . The main characteristics of ir are disinhibited lipolysis in adipose tissue, impaired uptake of glucose by muscle, and disinhibited gluconeogenesis . Ir most often precedes the onset of overt type 2 diabetes and is compensated initially by hyperinsulinemia . But this chronic secretion of large amounts of insulin to overcome tissue insensitivity can itself finally lead to beta cell failure and occurrence of hyperglycemia . In ir, visceral adipose tissue is resistant to the antilipolytic effect of insulin and consequently releases excessive amounts of ffa . A major contributor to the development of ir is an overabundance of circulating fatty acids . Insulin - resistant people with obesity and/or type 2 diabetes have been identified a defect in mitochondrial oxidative phosphorylation that relates to the accumulation of triglycerides and related lipid molecules in muscle . In type 2 diabetes, elevated blood glucose levels are clearly an important secondary cause of dyslipidemia in susceptible patients, and poor control of glycaemia can sometimes result in profound dyslipidaemia, including hypertriglyceridaemia and low high density lipoprotein (hdl) blood levels . Sustained hyperglycemia in type 2 diabetes induces macrovascular and microvascular complications . In a recent report of world health organization and international diabetes federation it was found that about 80% diabetic morbidity and mortality is caused by diabetic cardiomyopathy which is closely related with diabetic dyslipidemia . The low density lipoprotein (ldl) blood level in a patient with diabetes may be somewhat misleading; a patient with diabetes may have an increased proportion of small dense ldl particles and an increase in atherogenic risk, compared with a nondiabetic patient with similar ldl blood level . Moreover, patients with small, dense ldl will also typically have lower hdl and elevated triglyceride blood levels, which may further increase atherosclerosis risk . Plant derivatives with purported hypoglycemic properties have been used in folk medicine and traditional healing systems around the world . Many modern pharmaceuticals used in conventional medicine today also have natural plant origins . Among them, metformin was derived from the flowering plant galega officinalis (goat's rue or french lilac), which was a common traditional remedy for diabetes . Oleander (nerium oleander, no) of the dogbane (apocynaceae) family grows along the whole mediterranean coast starting in southern portugal in the west, in syria, and in streambeds of the western and southern anatolia . Central nervous system depressant activity and dose - dependent cardiotonic effect of no were exhibited in studies . Ishikawa et al . Were reported that postprandial rise in blood glucose when maltose and sucrose were loaded in nondiabetic healthy rats was reduced by hot water extract of nerium indicum leaves . Recently ligands for the peroxisome proliferator - activated receptors (ppars) which play a key role in glucose and lipid metabolism are defined as new insulin sensitizing drugs and hypolipidemic fibrates . In the present study, we investigated the effects of no distillate on hyperglycemia and dyslipidemia and its activities on liver and adipose tissue ppars in type 2 diabetic rats . September period from mediterranean region of turkey, identified, and authenticated at the department of biology . Firstly collected plant was washed, fresh shoots were chopped, and adequate distilled water was added . After the liquid started to evaporate, container lid was covered, and vapor was separated to other clean glass containers by causing it to come in contact with a surface cooled with cold water . No distillate was lyophilized in small glass bottle (20 ml) by using lyophilizator . Lyophilized no distillates were dissolved at concentrations of 7.5, 75, and 750 g / ml in distilled water . Eighty male sprague dawley rats (1012 weeks) were allocated to metabolic cages individually in an automatic ambient humidity (50 5%), temperature (22 2c), and light - dark (12: 12) controlled room . Animals were obtained from the experimental research center of akdeniz university, faculty of medicine, antalya, turkey and divided into eight groups . Commercially available rat normal pellet diet and water were given ad libitum to all animals prior to dietary manipulation . The experimental protocol was approved by the ethics committee in animal experimentation of selcuk university, turkey . After 2 weeks feeding of high fat diet, diabetes was induced in fasted animals by a single intra - peritoneal injection of streptozotocin (stz) (35 mg / kg bw) dissolved in citrate buffer (ph 4.5) when only buffer was received by control groups . In one week after stz injection, rats with 16.65 mmol / l (300 mg / dl) nonfasting blood glucose level were considered to be type 2 diabetic . Healthy control (c) and healthy control administered the highest level of no (cno-10) group had normal pellet diet although high fat group (hf) and other all type 2 diabetic groups had high fat diet which 58% of the metabolic energy is provided from animal fat . Nutritional substances of normal pellet diet were dry matter 89%, crude protein 21%, metabolic energy 2850 kcal / kg, crude fiber 5%, methionine and cystein 0.75%, calcium 1.02.0%, phosphor 0.51.0%, and sodium 0.5% (optima feeds, turkey). After induction of diabetes, animals were randomly allocated to five groups in which one of them did not had any treatment (d); other groups had active substances once a day by gavage for 12 weeks of the treatment period . The experimental groups according to diets and administrations applied to animals were represented in table 2 . Although fasting blood samples were taken from tail vein of all rats at 15 day intervals through the experiment, the results in tables 3 and 4 represent only the last sampling . About 0.5 ml whole blood from each animal serum samples were analyzed immediately for fasting blood glucose (fbg), total cholesterol (total - c), hdl, ldl, triglyceride, alkaline phosphatase (alp), aspartate transaminase (ast), and alanine transaminase (alt) by using commercially available colorimetric diagnostic kits (il test, instrumentation laboratory, milano, italy) by autoanalyzer (ilab, 300, milano, italy). After sampling the animals in all test groups was euthanized under general anesthesia with thiopental sodium (50 mg / kg bw) subsequent to the final sampling time . Liver and white adipose tissue samples from subcutan adipose tissue were taken and kept under required conditions . Data for homa - ir (homa - ir: fasting insulin (u / ml) fasting glucose (mmol / l)/22.5), beta cell functions as homa- (homa-(%): (20 fasting insulin (u / ml)/{fasting glucose (mmol / l) 3.5}), atherogenic index (ai: ([total - c] [hdl])/[hdl]), hdl% in total - c, and triglyceride to hdl ratio of the all studied groups were calculated . Serum insulin (drg, millipore, ma, usa) and leptin (r&d, mn, usa) levels were analyzed by elisa according to kit procedures . Total rna is extracted, and oligo - dt primed first - strand cdna is synthesized . A reverse transcription polymerase chain reaction (rt - pcr) is performed using a thermal cycler system, specific primers for ppar-, ppar-, and -actin (roche diagnostics, rotkreuz, switzerland) are used . Were performed one - way anova followed by a multiple comparison test (postdoc duncan's test) using spss 17.0 (spss, chicago, usa). Differences were considered significant at p less than 0.05 . Also kruskall wallis and mann whitney - u test (use these when the data is not normally distributed) were used to determine statistically differences of in vitro data between groups . Table 3 shows values of body weight, fbg, hba1c, total - c, hdl, ldl, and triglyceride, ai, hdl% in total - c, triglyceride to hdl ratio of the all studied groups . Data for insulin, leptin, homa - ir, homa-, alp, ast, and alt were presented in table 4 . No gastrointestinal disorders were observed during or after no treatment . Although being diabetic, the considerable elevation in body weight of no treated groups was estimated compared to d and g (p <0.0001). Fbg levels were significantly decreased by using no when compared to d and g (p <0.0001). In parallel with the improvement of fbg as shown in table 3, all other diabetic groups were hyperglycemic and had significantly higher hba1c than healthy groups and diabetic no groups (p <0.0001). Although having high fat diet, no-1 and no-10 displayed similar total - c concentrations compared to c (p> 0.05, table 3). Total - c was numerically lower in cno-10 and higher in hf than c. the increased values of hdl were found in no-0.1 and no-10 compared to c (p <0.0001, table 3). The reducing effect of all no regimens and g on ldl concentration was noticeable and the values were similar to healthy groups (p> 0.05). Ldl levels in type 2 diabetic no-1 and no-10 groups were significantly lower than d (p <0.05). Hdl percentage in total - c of no-0.1 and no-1 groups was similar to c (p> 0.05) and the highest hdl percentage was estimated in no-10 among healthy and diabetic groups except cno-10 . The similarity in terms of ai of no groups to healthy groups was noticeable (p> 0.05, table 3). The reducing effect of no-10 on ai was significant when compared to d, g, no-0.1, and no-1 (p <0.001). There were significant reductions in triglyceride - hdl ratio of all no regimens compared to d (p <0.0001, table 3). These reducing effects of no on the ratio were noticeable and the results were similar to c (p> 0.05). Triglyceride - hdl ratio was numerically lower in cno-10 and was higher in hf than c. similar results in g and d were noted in terms of triglyceride - hdl ratio . When we assessed insulin levels, the antihyperglycemic effect of no was seen on data that no significantly decreased insulin concentration compared to d (p <0.0001, table 4). Although insulin levels in all no groups were numerically higher than other healthy control groups, the results were statistically similar with those groups (p> 0.05).insulin level was decreased by g and data were not different with all no groups and healthy groups (p> 0.05). There was 26.72% fall in insulin level of cno-10 compared to c. leptin levels of all no groups were not significantly different compared to healthy control groups (p> 0.05). Almost twofold increases were estimated in other diabetic groups when compared to all no treated groups (p <0.01, table 4) calculated homa - ir in no groups and in g was significantly lower than d (p <0.0001, table 4). Insulin sensitivity has been improved by no treatment . Also numerically the lowest insulin resistance was found in cno-10 . Homa- impaired dramatically in groups d and g. improved beta cell function was observed in all no groups and was similar to healthy group's values (p> 0.05, table 4). The highest alp activity was found in d (p <0.0001, table 4). All no treatment dosages decreased alp activity when compared to d (p <0.0001). The alp activity of g was similar to all no regimens (p> 0.05). The elevated alt activities, compared with c, were observed in d and g (p <0.05, table 4). The primer sequences, pcr protocol, and product sizes are presented in table 5 . Agarose gel electrophoresis of pcr products are shown in figures 1(a) and 2(a). The densities of each band evaluated by diana v1.6 and aida 2.4.3 analysis programs (raytest imaging system, germany) and the ratio of liver and adipose tissues ppar-, -, - mrna expressions to -actin are given in figures 1(b), 1(c), 1(d),2(b), 2(c), and 2(d), respectively . In liver, ppar- mrna expression decreased in d compared to c (p <0.05), whereas its expression increased in no-10 treated group (p <0.01). Ppar- mrna expression increased in no-0.1 (p <0.001) treated group compared to d. ppar- mrna expression increased in no treated groups as a dose dependent manner but the increase in no-10 group is not significant compared to c (p> 0.05). In adipose tissue, ppar- and - mrna expression increased in no-10 group compared to c and diabetic rats (p <0.01). The increase in ppar- mrna expression is more prominent in no-10 treated group compared to c and diabetic group (p <0.001). During the experiment, administration of combined drug and insulin was not in question . Studies, most notably the dcct, have defined quantitatively the relationship between glycated hb and average glycemia . The increase in vascular disease in patients with diabetes is thought to be due to the deleterious effects of metabolic abnormalities, such as hyperglycemia, insulin resistance, dyslipidemia, and advanced glycation end products [18, 19]. In diabetes, hba1c levels predict the risk of microvascular complications and glycemic control to a hba1c of less than 7% will reduce microvascular complications and could decrease risk for macrovascular disease as well . The most important result in terms of the fbg is that no administered groups exhibited a level which was close to the data from c and that this condition was confirmed with the hba1c levels obtained from the above - mentioned groups . No treatment significantly reduced hba1c levels that were similar to healthy groups when glibenclamide had no significant effect over that . No treatment reduced hba1c 15.97, 15.94, and 19.54% in no-0.1, no-1, and no-10, respectively, when compared to d. all no regimens also decreased fbg compared to other diabetic groups at the end of trial . Therefore, no may be beneficial to reduce microvascular and macrovascular risk of type 2 diabetes . Homa - ir is an independent predictor of cvd in type 2 diabetes and the improvement of ir might have beneficial effects not only on glucose control but also on cvd in patients with type 2 diabetes mellitus . No administration resulted in significant lowering of both insulin level and homa - ir compared to d as observed in g. improved beta cell function (homa-) in all no dosages was established but the improvement was noticeable in no-0.1 . Homa- was numerically higher in cno-10 than c. the evaluated data suggest that no ameliorates glycemic control by insulin secretagogue and sensitizing effects . Dyslipidemia, as associated with diabetic metabolism and the metabolic syndrome, is characterized by a so - called proatherogenic blood lipid profile, comprising low levels of hdls, increased ldls, and serum triglycerides associated with vldls . In fact, hypertriglyceridemia is considered to represent an important risk factor for atherosclerosis and subsequent cardiovascular complications in type 2 diabetic patients . The atherogenic index has recently been proposed as a marker of plasma atherogenicity because it is increased in people at higher risk for coronary heart disease and is inversely correlated with ldl particle size . The increased risk of coronary heart disease in patients with the metabolic syndrome suggests that the insulin - resistant state is atherogenic without concomitant elevations in plasma glucose and glycosylated hemoglobin [26, 27]. The present data showed that all no administration numerically decreased ai but the highest no administration level significantly lowered atherogenicity in blood compared to other diabetic groups . A positive correlation between mean homa - ir and ai (p <0.05, r = 0.71) and also between mean hba1c and ai (p <0.01, r = 0.86) suggests that improving blood glucose level and insulin receptor sensitivity may support to decline atherogenicity and cardiovascular complications in type 2 diabetes by using no distillate . Moreover, the highly significant positive correlation between homa - ir and triglyceride levels (p <0.0001, r = 0.74) and inclination of the reduction of triglyceride by no administration indicates no may have a good beneficial effect on glucose and lipid metabolism as inferred from homa - ir results . The atherogenic link between high triglycerides and hdl is due to the higher plasma concentration of triglyceride - rich, vldl that generates small, dense ldl during lipid exchange and lipolysis . These ldl particles accumulate in the circulation and form small, dense hdl particles, which undergo accelerated catabolism, thus closing the atherogenic circle [28, 29]. The significant similarity of c levels especially in total - c of no-1 and no-10 administration and ldl concentrations in all no regimens of diabetics support lipid lowering effects of no . Diabetic dyslipidemia includes an overall increase in atherogenic particles identifiable, by measuring apolipoprotein b, and a predominance of small dense ldl particles (phenotype b). A high ratio of triglycerides to hdl - cholesterol correlates with ldl phenotype b, small hdl particles, and ir [3032] and found to be a powerful independent indicator of extensive coronary disease . According to the above explanations, the ratio of triglycerides to hdl results in this paper indicates that no treatment may prevent the extensive coronary disease due to decreasing atherogenic particles since the ratio found to be statistically very similar to the ratio of c. also, in healthy rats, no made reduction in triglycerides to hdl ratio . High hdl levels in blood are considered to be cardioprotective, since the apo - a - containing hdl particles that help transport cholesterol to the liver from peripheral tissues, as well as away from macrophages associated with cholesterol deposits within the vascular wall . However, this cardioprotective effect may not be solely due to cholesterol transport . For example, hdl may have direct antioxidant and anti - inflammatory effects on the vessel wall . The reduction in triglyceride levels together with the higher concentrations of hdl especially in no-0.1 and no-10 groups compared to c were remarkable effects of no over dyslipidemia of type 2 diabetic rats . The ratio of serum hdl cholesterol to total - c used a marker of cardiovascular risk . All no treatment levels were associated with increase in hdl percentage in total - c but a highly significant arise was found in no-10 compared to other diabetics although the lower percentages of hdl in total - c of diabetic groups were estimated especially in g and d. numerically increase in hdl percent in total - c of cno-10 compared to c also indicates the beneficial effect of no over the lipid metabolism of healthy individuals.all these positive results in respect to lipid lowering effects of no may indicate reducing risk factor for atherosclerosis and subsequent cardiovascular complications in type 2 diabetes . Leptin is a hormone secreted predominantly by adipose tissue and is a signal of sufficiency of energy . Leptin effects are mediated by its action on the hypothalamus and directly on target tissues (muscle, gonads, beta - cells, and liver). In normal conditions of weight maintenance, leptin concentrations are positively correlated with total body fat mass . In short - term food deprivation, serum leptin levels decrease and soluble leptin receptors are thought to be important for transport to or over the blood brain barrier (bbb), and it is the saturation of this transport or impairment of leptin receptor signal transduction that may be the cause of leptin resistance . So that, the significant elevated levels of leptin in d and g, except no treated ones, reflect leptin resistance and/or insulin resistant state . The effect of leptin on inducing insulin resistance was exhibited by positive correlation between mean homa - ir and leptin (p <0.05, r = 0.76) in this research . No positively affected leptin levels and was found to be similar to healthy control level . This remarkable result indicates that no may help to compensate the energy metabolism by improving leptin and insulin levels, and homa - ir, homa- in type 2 diabetes . Triglycerides are an important cause of leptin resistance as mediated by impaired transport across the bbb . In this research, positive correlation was found between mean triglyceride and leptin levels (p <0.05, r = 0.70). This data may indicate that high triglyceride levels resulted in high blood leptin levels in d and g groups . A number of studies have reported that alt, ast, and/or ggt levels independently predict incidents of type 2 diabetes, metabolic syndrome, and cvd . In addition, these markers have been shown to be associated with indirect measures of insulin resistance including fasting insulin levels and homa - ir [37, 38] since alt was associated with insulin resistance independently and an inexpensive way to improve the identification of subjects with insulin resistance . The reduction of ast activity in no-10 was noticeable when compared to d. alt activity was significantly higher in d than c group but was similar in no groups compared with c (p> 0.05). Also, mean alt activity was positively correlated with homa - ir (p <0.05, r = 0.80). Although alp activities were not different between type 2 diabetic and nondiabetic patients, the increased alp activity has been reported in ketotic and nonketotic diabetic rats primarily due to an increase in intestinal and bone / liver alp isoenzyme . All no regimens exhibited the beneficial effect on reducing alp activity in type 2 diabetic rats . The results of liver enzymes by using no dosages show that no had no detrimental effect on the liver function . In rodents, adipose tissue ppar- mrna and protein levels are reduced after an overnight fast [42, 43] in stz - induced diabetes, which is consistent with the stimulatory effect of insulin on ppar- expression . In addition, chronic feeding with high fat diets was shown to increase ppar- expression in adipose tissue, whereas fasting stimulates especially liver ppar- expression in rodents [42, 43]. The overexpressions of ppar- and - in adipose tissue may indicate that the effect of no-10 group dosage on reducing insulin resistance and indirectly reducing the risk of atherosclerosis is being through the storage of fatty acids to adipocytes and regulating adipocyte differentiation . Significantly increased ppar- expression in liver and adipose tissue of no-10 group suggests that no-10 dosage may have important effects on regulating fatty acid oxidation system, lipoprotein synthesis, and inflammatory responses . Single agents that promote both ppar- and ppar- agonism could theoretically offer significant benefits in improving dyslipidaemia and reducing hyperglycaemia and thus reduce these cardiovascular risk factors associated with type 2 diabetes and metabolic syndrome . In addition, such a therapy could reduce the underlying insulin resistance and help to break the cycle of altered glucose and lipid metabolism that promotes type 2 diabetes . It has been considered that increasing fatty acid oxidation in liver by overexpression of liver ppar- led not to use too much fat from adipose tissues for energy and also storage of fatty acids in adipocytes by overexpression of adipose tissue ppar- of no treated animals improved insulin action . This progress prevented body weight loss of no treated animals due to an antilipolitic effect of insulin when compared to uncontrolled diabetic ones . Homa - ir and homa- results also support this improvement . Also the pulling down effect of no-10 dosage on body weight might be associated with overexpressions of ppar- and - in adipose tissue of healthy rats since overexpression of ppar- specifically in adipose tissue decreases fatty acid levels and protects against obesity . Moreover ppar- and ppar- are both active participants in energy burning; ppar- plays a critical role in fatty acid oxidation and is thus responsible for energy expenditure; ppar- also enhances fatty acid catabolism and energy uncoupling in skeletal muscle and adipose tissue as well, as recently shown in the liver . The considerable beneficial effects of especially no-10 group treatment (no distillate administration at a dose of 375 g/0.51 ml of distilled water / d) on glucose metabolism, insulin resistance, insulinotropic activity, leptin, dyslipidemia, liver enzymes, and ppars may point out the insulin - like effect of no and offer new approaches to treatment strategies that target both fat and glucose metabolism ultimately leadind to a reduction in both the chronic microvascular complications of type 2 diabetes and the risk of macrovascular events such as cvd . In addition, this concentration of no may prevent and control elevated glucose and blood lipid levels of the remainder of the population.
Guinea pig anti - insulin, guinea pig anti - glucagon, guinea pig anti - pancreatic polypeptide, and rabbit anti - somatostatin were obtained from dako, carpinteria, ca . Secondary antibodies used were fluorescein isothiocyanate conjugated donkey anti - rabbit igg; fluorescein isothiocyanate conjugated donkey anti - guinea pig igg; and cy3-conjugated donkey anti - mouse igg (jackson immunoresearch, west grove, pa). Goat anti - mouse igg conjugated to alexa fluor 488 was from invitrogen, carlsbad, ca . 2): p1, 5-cactgagcaggacatcctccga-3; p2, 5-catcctcaaagcagtggatcca-3; p3, 5-cttcctcaacaagaaagacctct-3; p4, 5-ggtgagcggtttttgctttcaaa-3; p5, 5-caagtggttcacagacacatcta-3; p6, 5-ccttggatgtgagccacagct-3. Oligos used for insulin expression assays are as follows: 5-cagcaagcaggtcattgttt-3 and 5-gggaccacaaagatgctgtt-3. For -cell mass assay, pancreata were weighed and cut as 20-m frozen sections . One - tenth of the sections confocal images (covering 1/5 of all stained pancreatic areas) were randomly captured using a 5 objective lens and analyzed with bioquant software (24) to calculate the area ratio between -cells and the whole pancreas to calculate islet mass . Intraperitoneal ptx injection (at 1 g/100 g body wt), intraperitoneal glucose tolerance test (ipgtt), insulin sensitivity assays, and serum insulin assays followed published procedures (25,26). Islet isolation and perifusion followed an established protocol (27). For camp assays, islets were incubated in ringer's solution (with 2 mmol / l glucose) with 0.1 mmol / l isobutylmethylxanthine (ibmx) for 2 h and then used for assaying camp levels with the camp biotrak enzyme immunoassay system (ge healthcare). For perifusion, islets of similar size and shape were used . Hand - picked islet cells were isolated and placed in a 1-ml perifusion chamber, equilibrated in 5.6 mmol / l glucose for 30 min and then challenged with 16.7 mmol / l glucose (16.7 mmol / l glucose + 100 mol / l ibmx), 300 mol / l tolbutamide, and 20 mmol / l kcl . The perifusion fractions were collected in 3-min intervals at 1 ml / min flow rate and assayed for insulin by radioimmunoassay ., islets were first isolated from six animals and fixed (0.25% gluteraldehyde in pbs) for sectioning and imaging . For quantification of vesicles docked on the cell membrane, images were captured using tem at magnification 10,00015,000 . The number of docked vesicles was counted before genotype identification, with vesicles whose outer surface was within 10 nm of the plasma membrane as docked granules . At least 50 randomly captured microscopic fields (from different -cells) of each genotype were analyzed before identifying their genotype . For calcium imaging, a series of images were acquired from isolated islets under glucose levels of 2, 4, 6, 8, 10, and 15 mmol / l . Images were background subtracted, and the mean fluo-4/fura - red intensity ratio was calculated across the whole islet (28). This ratio was then normalized to the ratio calculated at 4 mmol / l glucose stimulation . Total internal reflection fluorescence microscopy (tirfm)-based live cell imaging followed published procedures (29,30). Briefly, islets were isolated and dispersed in calcium - free krebs - ringer buffer (krb) containing 1 mmol / l egta and cultured on high refractive index cover glass (olympus) in rpmi medium . -cells were then infected with recombinant adenovirus adex1ca insulin - green fluorescent protein (gfp) (29) for observation in krb containing 2.2 mmol / l glucose (37c). The olympus tirfm system was used with a high - aperture objective lens to observe the fluorescence of gfp with a charge - coupled device camera at 300-ms intervals using metamorph version 7.1 (universal imaging). Diiodomethane sulfur immersion oil (n = 1.81, cargille laboratories) was used to make contact between the objective lens and the high refractive index cover glass . Light propagates through the cover glass at an angle of 65 and undergoes total internal reflection at the glass cell interface . The refractive indexes for the glass (n = 1.8 at 488 nm) and cells (n = 1.37) predict an evanescent field decline of threefold within 44 nm from the interface and of 10-fold within 100 nm . Most analyses, including tracking (single projection of different images) and area calculations were performed using metamorph software, added with manual event selection . In this evanescent field setting, a granule would have a vertical distance of 9.6 nm from the plasma membrane and qualify as a morphologically docked granule (granule distance from plasma membrane <10 nm in electron microscopic studies) (31). We immunostained endogenous insulin granules in fixed pancreatic -cells . Then, we manually counted bright spots as the docked granules . We examined go protein expression in both embryonic and adult pancreata using a monoclonal antibody that recognized both go1 and go2 . Robust go production is detected in all hormone - expressing cells in all stages examined, including e11.5, e17.5, and 3-month - old adults (fig . 1). We do not detect go in exocrine acinar or pancreatic duct cells (fig . 1 and data not shown). Further rt - pcr analyses showed that both go1 and go2 mrna could be detected in adult islet cells, suggesting that both isoforms might be involved in islet cell function (fig . Expression patterns at three mouse stages, e11.5 (a), e17.5 (b e), and 3-month - old adult (f i), are shown . Three panels: go, hormone (green), and a merged image . Note that all hormone - expressing cells express go . (a high - quality digital representation of this figure is available in the online issue .) Go is not required for islet cell differentiation . A: a diagram showing the go (f) allele . Only some exons are shown (from 5 to 8). . Arrows p1p6 indicate the oligonucleotides used for detecting go mrnas in b, which shows rt - pcr detection of go mrna in 4-month - old adult islet cells . Rt reactions with insulin - specific oligos were used as controls (with or without reverse transcription). (p3 + p4) detects go2 mrna (cre refers to go; pdx1). C: body weights of f / f and f / f;cre animals at 12 weeks of age . D: total insulin content in 4- and 8-week - old mice (p28 and p56). (a high - quality color representation of this figure is available in the online issue .) We used a go conditional allele, in which two loxp sites flanked the fifth and sixth exons of go, common to go1 and go2 (go, fig . Deletion of the flanked exons produces a truncated mrna that only codes for the nh2-terminal 156 amino acids, which lacks all motifs that bind to adenylyl cyclase, phospholipase cs (plcs), and the subunits . We expect that this above manipulation results in a null go allele (go). Indeed, go animals display identical phenotypes as previously reported for null mutants (data not shown), whereas go mice showed a similar phenotype as wild - type littermates . Furthermore, the truncated protein did not perturb insulin secretion in a cultured -cell line (supplementary fig . 1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1719/dc1). Go;pdx1 (f / f;cre) adult animals were derived from standard genetic crosses . Pdx1 animals express cre in all undifferentiated pancreatic progenitors and inactivate go in all pancreatic progenitor cells of f / f;cre mice . In addition, no cre toxicity in pdx1 animals has been observed (22). Rt - pcr assays showed that the mrna sequence corresponding to the fifth and sixth exons of go was no longer detectable in islets of 4-month - old f / f;cre animals (fig . The f / f;cre animals were no different in body weight from their control littermates (go or f / f) at all ages examined: 6, 9, 12, and 20 weeks (fig . 2c). Additionally, no structural or behavioral (aggression, feeding, moving, and mating) defects were obvious in these animals . At postnatal day 1 (p1), the insulin contents in f / f;cre and f / f pancreata were not significantly different (supplementary fig . 2a, available in an online appendix), suggesting that go is not required for -cell differentiation . By p28, the insulin content in f / f;cre animals was reduced by 20% over that of control littermates (fig . 2d). At p56 (8 weeks), the insulin content of f / f;cre animals consistent with this finding, the -cell mass was reduced in f / f;cre animals at p56 as well (fig . We analyzed islet morphology and expression of several genes that are required for endocrine islet cell differentiation and function, including mafa, mafb, myt1, nkx6.1, and pdx1 (25) by immunofluorescence . These data suggest that go is not required for islet neogenesis, even though it is expressed in early ngn3-expressing endocrine progenitor cells (32). Gi and go inactivation by ptx uncouples the inhibitory effects of some neural hormones, such as adrenaline, on insulin secretion (12). Because both gi and go are expressed in islet cells and they both can be adp - ribosylated by ptx (14,33,34), it is not clear which g - protein is mediating the ptx effect on insulin secretion . The fasting blood glucose levels in f / f;cre and f / f animals were similar (fig . However, ipgtt showed that f / f;cre animals have significantly improved glucose clearance over control littermates (fig . Consistent with this observation, the fasting serum insulin levels are similar between f / f and f / f;cre animals . Fifteen minutes after glucose challenge, the serum insulin levels in f / f control animals increased by 2-fold but increased up to 10-fold in f / f;cre mice (fig . 3b). Because the insulin sensitivity in f / f;cre and control animals was similar (fig . 3c), the above findings demonstrate that losing go potentiates insulin secretion from -cells . We next tested whether gi proteins function to repress insulin secretion in the absence of go . If they do, we expect that ptx treatment of f / f;cre animals would further potentiate insulin secretion . Ptx injection into f / f animals resulted in a significant increase in glucose tolerance . 3d), suggesting that although gi proteins are expressed in islet cells and may be adp - ribosylated by ptx, go is the major mediator of ptx's effect on insulin secretion . Pancreas - specific go inactivation enhances glucose - clearing capability in adult animals . A: ipgtt results in 6- and 12-week - old animals . Go;pdx1 (f / f;cre) and go (f / f) animals were used . B: serum insulin levels 0 and 15 min after intraperitoneal glucose injection in 12-week - old f / f;cre and f / f males . Note the fold increases in both control (gray bars) and f / f;cre (black bars) animals after glucose stimulation . C: insulin sensitivity of 12-week - old f / f;cre and f / f animals . The y - axis is presented as the ratio of glucose levels of the assay point over that of the 0-min value . Islet perifusion assays were used to directly test how go inactivation affects insulin secretion in vitro . Islets from 2-month - old animals were assayed for insulin secretion in response to glucose, ibmx, tolbutamide, and kcl stimulation . Glucose induces insulin secretion through metabolism to alter the atp / adp ratio and other metabolites . Ibmx inhibits camp phosphodiesterase to upregulate the levels of camp, which activates protein kinase a and/or gefii to facilitate insulin vesicle exocytosis (35,36). Tolbutamide, a katp channel blocker, depolarizes -cell membrane potential, as does kcl . In response to these stimuli, the insulin secretion in the f / f;cre islets was substantially increased compared with that of control littermates at every time point examined (fig . The biggest increase was in response to glucose, increasing as much as 369% (fig . This secretion increase was lower than what was detected during in vivo glucose challenge (fig . 3b), likely due to the synergetic effect of multiple hormones that regulate insulin secretion through go in vivo, but not in vitro . Importantly, these data suggest that go regulates insulin secretion through a mechanism that is shared by all these stimuli, most likely in steps that are distal to ca mobilization, as suggested for in vitro based studies (10). Go nullizygous islets secrete more insulin in response to multiple stimulations . A: perifusion assay results . The camp concentration is normalized against the od280 of islet extract (as an assay of protein content). Indeed, inactivation of go did not significantly affect camp production in isolated islets (fig ., similar increases in intracellular free calcium ([ca]i) concentration were seen in both f / f;cre mutant and control islets after elevated glucose stimulation (fig . Synchronous bursting and spiking activities were observed at 8, 10, and 15 mmol / l glucose, respectively (fig . The mean fold increase in [ca]i was also similar for various levels of glucose stimulation for both mutant and wild - type islets (fig . This result suggests that go has little effect on -cell electrical activity and suggests that go regulates insulin secretion downstream of elevated [ca]i in vivo . A: representative time courses of [ca]i activity estimated from the normalized fluo4/furared intensity ratio in both f / f (left) and f / f;cre islets (right) 10 min after glucose challenge . Shown are data from representative islets . Note that 2 mmol / l glucose does not elicit ca mobilization, whereas 8 mmol / l glucose induces synchronous bursting of ca activity in both sets of islets; 15 mmol / l glucose induces continuous spiking in both sets of islets . B: fold increases in [ca]i estimated from the normalized fluo4/furared intensity ratio for control islets (black) and mutant islets (gray) under varying glucose stimulation . We next examined how go affects insulin secretion . Because insulin vesicle docking on the cell membrane is necessary for insulin secretion, we used tem to investigate whether vesicle distribution in -cells is affected by loss of go . The size of each vesicle in -cells did not vary between f / f;cre and f / f control islets (fig . The density of granules in the f / f;cre and control -cell remained unchanged as well (fig . However, the number of secretory vesicles in direct contact with the cell membrane increased by about 100% in f / f;cre -cells as compared with that of controls (fig . 6c, d, and f). Because tem only allows us to examine vesicle docking on a thin section with limited depth, we used tirfm to verify the above findings . Tirfm uses evanescent light waves to selectively illuminate the -cell surface at a 100-nm depth . Thus, this technique allows us to exclusively visualize the granules that localize in the proximity of the cell membrane on a wide cell surface area . Isolated islet cells were fixed and stained with insulin antibodies and subjected to tirfm (fig . Consistent with the above tem - based finding, we observed a significant (p <0.01) increase in the number of insulin vesicles close to plasma membrane in go mutants (257/m) over that of the control cells (190/m) (fig . Note that the fold increase of docked vesicles revealed by tirfm (a 35% increase) is lower than that observed from tem - based analysis (100% increase). This is an expected result because tem identifies the vesicles that directly contact the plasma membrane, which is only a small portion of the vesicles that localize within 100 nm of the plasma membrane visualized through tirfm . Additionally, our vesicle density count with em and tirfm displayed a twofold difference (fig . 6e and h). This discrepancy could be due to the unequal vesicle distribution within the cytoplasmic compartment and cell membrane . Alternatively, it is possible that tirfm only visualizes high - insulin - content vesicles (due to antibody staining related issues), whereas em allows us to visualize all vesicles . Note that different -cells have different vesicle density or membrane - associated vesicles (c and d). E: vesicle density in -cells, presented as number of vesicles on two - dimensional views . Data are presented as number of vesicles over length of intercellular junctions (p <0.01). G: tirfm images showing the presence of insulin vesicles on the surface of fixed and insulin ab - stained -cells of control (f / f) and go deleted (f / f;cre) animals . Vesicles within the circled areas were counted and presented in h. tirfm visualizes vesicle movement in vivo in real time . We therefore recorded the vesicular dynamics close to the -cell membrane in wild - type and go mutant animals . Enhanced green fluorescent protein (egfp) fusion protein, which was previously shown to be packaged in normal insulin vesicles and to not interfere with insulin trafficking . As a result, the egfp - marked insulin vesicles could be followed in real time (29,37). Islet cells were stimulated with 22 mmol / l glucose (see research design and methods). 4), go mutant -cells release significantly more vesicles than control -cells (fig . It is possible that go inactivation could either shorten vesicle residence time on the plasma membrane before fusion or expedite transportation of vesicles from cytoplasm to plasma membrane . In order to differentiate between these possibilities, we counted the fusion events from predocked vesicles and newly arrived vesicles (newcomers or vesicles that appear close to cell membranes after the start of recording) during stimulation . Membrane - docked vesicles in go mutant -cells showed a trend of increased readiness for release (fig . 7b). Specifically, upon glucose stimulation, 23.1% of predocked insulin vesicles were released within 10 min in control -cells, whereas 35.7% of predocked vesicles were released within the same time frame in -cells without go (fig ., the fusion events contributed by newly arrived vesicles did not display a significant difference between the control and mutant -cells (fig . Overall, these data suggest that one of the possible go functions is to facilitate vesicle docking and, to a lesser extent, to increase the readiness of vesicle fusion to the plasma membrane (fig . A: the numbers of vesicle plasma membrane fusion events at several time points with 22 mmol / l glucose stimulation . The events are presented as fusions from predocked vesicles and fusions from newly arrived vesicles, respectively . B: the percent of predocked vesicles that are released within 10 min of glucose stimulation . C: the number of newly arrived vesicles that are released within 10 min of glucose induction . D: a simple model summarizing where go could exert its function in the vesicle - secretion process, including vesicle docking and possibly priming . We examined go protein expression in both embryonic and adult pancreata using a monoclonal antibody that recognized both go1 and go2 . Robust go production is detected in all hormone - expressing cells in all stages examined, including e11.5, e17.5, and 3-month - old adults (fig . 1). We do not detect go in exocrine acinar or pancreatic duct cells (fig . 1 and data not shown). Further rt - pcr analyses showed that both go1 and go2 mrna could be detected in adult islet cells, suggesting that both isoforms might be involved in islet cell function (fig . Expression patterns at three mouse stages, e11.5 (a), e17.5 (b e), and 3-month - old adult (f i), are shown . Three panels: go, hormone (green), and a merged image . Note that all hormone - expressing cells express go . (a high - quality digital representation of this figure is available in the online issue .) Go is not required for islet cell differentiation . A: a diagram showing the go (f) allele . Only some exons are shown (from 5 to 8). . Arrows p1p6 indicate the oligonucleotides used for detecting go mrnas in b, which shows rt - pcr detection of go mrna in 4-month - old adult islet cells . Rt reactions with insulin - specific oligos were used as controls (with or without reverse transcription). (p3 + p4) detects go2 mrna (cre refers to go; pdx1). C: body weights of f / f and f / f;cre animals at 12 weeks of age . D: total insulin content in 4- and 8-week - old mice (p28 and p56). (a high - quality color representation of this figure is available in the online issue .) We used a go conditional allele, in which two loxp sites flanked the fifth and sixth exons of go, common to go1 and go2 (go, fig . Deletion of the flanked exons produces a truncated mrna that only codes for the nh2-terminal 156 amino acids, which lacks all motifs that bind to adenylyl cyclase, phospholipase cs (plcs), and the subunits . We expect that this above manipulation results in a null go allele (go). Indeed, go animals display identical phenotypes as previously reported for null mutants (data not shown), whereas go mice showed a similar phenotype as wild - type littermates . Furthermore, the truncated protein did not perturb insulin secretion in a cultured -cell line (supplementary fig . 1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1719/dc1). Go;pdx1 (f / f;cre) adult animals were derived from standard genetic crosses . Pdx1 animals express cre in all undifferentiated pancreatic progenitors and inactivate go in all pancreatic progenitor cells of f / f;cre mice . In addition, no cre toxicity in pdx1 animals has been observed (22). Rt - pcr assays showed that the mrna sequence corresponding to the fifth and sixth exons of go was no longer detectable in islets of 4-month - old f / f;cre animals (fig . The f / f;cre animals were no different in body weight from their control littermates (go or f / f) at all ages examined: 6, 9, 12, and 20 weeks (fig . 2c). Additionally, no structural or behavioral (aggression, feeding, moving, and mating) defects were obvious in these animals . At postnatal day 1 (p1), the insulin contents in f / f;cre and f / f pancreata were not significantly different (supplementary fig . 2a, available in an online appendix), suggesting that go is not required for -cell differentiation . By p28, the insulin content in f / f;cre animals was reduced by 20% over that of control littermates (fig . 2d). At p56 (8 weeks), the insulin content of f / f;cre animals consistent with this finding, the -cell mass was reduced in f / f;cre animals at p56 as well (fig . We analyzed islet morphology and expression of several genes that are required for endocrine islet cell differentiation and function, including mafa, mafb, myt1, nkx6.1, and pdx1 (25) by immunofluorescence . These data suggest that go is not required for islet neogenesis, even though it is expressed in early ngn3-expressing endocrine progenitor cells (32). Gi and go inactivation by ptx uncouples the inhibitory effects of some neural hormones, such as adrenaline, on insulin secretion (12). Because both gi and go are expressed in islet cells and they both can be adp - ribosylated by ptx (14,33,34), it is not clear which g - protein is mediating the ptx effect on insulin secretion . The fasting blood glucose levels in f / f;cre and f / f animals were similar (fig . However, ipgtt showed that f / f;cre animals have significantly improved glucose clearance over control littermates (fig . Consistent with this observation, the fasting serum insulin levels are similar between f / f and f / f;cre animals . Fifteen minutes after glucose challenge, the serum insulin levels in f / f control animals increased by 2-fold but increased up to 10-fold in f / f;cre mice (fig . 3b). Because the insulin sensitivity in f / f;cre and control animals was similar (fig . 3c), the above findings demonstrate that losing go potentiates insulin secretion from -cells . We next tested whether gi proteins function to repress insulin secretion in the absence of go . If they do, we expect that ptx treatment of f / f;cre animals would further potentiate insulin secretion . Ptx injection into f / f animals resulted in a significant increase in glucose tolerance . Whereas ptx injection into f / f;cre animals had no significant effect (fig . 3d), suggesting that although gi proteins are expressed in islet cells and may be adp - ribosylated by ptx, go is the major mediator of ptx's effect on insulin secretion . Pancreas - specific go inactivation enhances glucose - clearing capability in adult animals . A: ipgtt results in 6- and 12-week - old animals . Go;pdx1 (f / f;cre) and go (f / f) animals were used . B: serum insulin levels 0 and 15 min after intraperitoneal glucose injection in 12-week - old f / f;cre and f / f males . Note the fold increases in both control (gray bars) and f / f;cre (black bars) animals after glucose stimulation . C: insulin sensitivity of 12-week - old f / f;cre and f / f animals . The y - axis is presented as the ratio of glucose levels of the assay point over that of the 0-min value . Islet perifusion assays were used to directly test how go inactivation affects insulin secretion in vitro . Islets from 2-month - old animals were assayed for insulin secretion in response to glucose, ibmx, tolbutamide, and kcl stimulation . Glucose induces insulin secretion through metabolism to alter the atp / adp ratio and other metabolites . Ibmx inhibits camp phosphodiesterase to upregulate the levels of camp, which activates protein kinase a and/or gefii to facilitate insulin vesicle exocytosis (35,36). Tolbutamide, a katp channel blocker, depolarizes -cell membrane potential, as does kcl . In response to these stimuli, the insulin secretion in the f / f;cre islets was substantially increased compared with that of control littermates at every time point examined (fig . The biggest increase was in response to glucose, increasing as much as 369% (fig . This secretion increase was lower than what was detected during in vivo glucose challenge (fig . 3b), likely due to the synergetic effect of multiple hormones that regulate insulin secretion through go in vivo, but not in vitro . Importantly, these data suggest that go regulates insulin secretion through a mechanism that is shared by all these stimuli, most likely in steps that are distal to ca mobilization, as suggested for in vitro based studies (10). Go nullizygous islets secrete more insulin in response to multiple stimulations . A: perifusion assay results . The camp concentration is normalized against the od280 of islet extract (as an assay of protein content). Indeed, inactivation of go did not significantly affect camp production in isolated islets (fig ., similar increases in intracellular free calcium ([ca]i) concentration were seen in both f / f;cre mutant and control islets after elevated glucose stimulation (fig . Synchronous bursting and spiking activities were observed at 8, 10, and 15 mmol / l glucose, respectively (fig . The mean fold increase in [ca]i was also similar for various levels of glucose stimulation for both mutant and wild - type islets (fig . This result suggests that go has little effect on -cell electrical activity and suggests that go regulates insulin secretion downstream of elevated [ca]i in vivo . A: representative time courses of [ca]i activity estimated from the normalized fluo4/furared intensity ratio in both f / f (left) and f / f;cre islets (right) 10 min after glucose challenge . Shown are data from representative islets . Note that 2 mmol / l glucose does not elicit ca mobilization, whereas 8 mmol / l glucose induces synchronous bursting of ca activity in both sets of islets; 15 mmol / l glucose induces continuous spiking in both sets of islets . B: fold increases in [ca]i estimated from the normalized fluo4/furared intensity ratio for control islets (black) and mutant islets (gray) under varying glucose stimulation . We next examined how go affects insulin secretion . Because insulin vesicle docking on the cell membrane is necessary for insulin secretion, we used tem to investigate whether vesicle distribution in -cells is affected by loss of go . The size of each vesicle in -cells did not vary between f / f;cre and f / f control islets (fig . The density of granules in the f / f;cre and control -cell remained unchanged as well (fig . However, the number of secretory vesicles in direct contact with the cell membrane increased by about 100% in f / f;cre -cells as compared with that of controls (fig . 6c, d, and f). Because tem only allows us to examine vesicle docking on a thin section with limited depth, we used tirfm to verify the above findings . Tirfm uses evanescent light waves to selectively illuminate the -cell surface at a 100-nm depth . Thus, this technique allows us to exclusively visualize the granules that localize in the proximity of the cell membrane on a wide cell surface area . Isolated islet cells were fixed and stained with insulin antibodies and subjected to tirfm (fig . Consistent with the above tem - based finding, we observed a significant (p <0.01) increase in the number of insulin vesicles close to plasma membrane in go mutants (257/m) over that of the control cells (190/m) (fig . Note that the fold increase of docked vesicles revealed by tirfm (a 35% increase) is lower than that observed from tem - based analysis (100% increase). This is an expected result because tem identifies the vesicles that directly contact the plasma membrane, which is only a small portion of the vesicles that localize within 100 nm of the plasma membrane visualized through tirfm . Additionally, our vesicle density count with em and tirfm displayed a twofold difference (fig . . This discrepancy could be due to the unequal vesicle distribution within the cytoplasmic compartment and cell membrane . Alternatively, it is possible that tirfm only visualizes high - insulin - content vesicles (due to antibody staining related issues), whereas em allows us to visualize all vesicles . Note that different -cells have different vesicle density or membrane - associated vesicles (c and d). E: vesicle density in -cells, presented as number of vesicles on two - dimensional views . Data are presented as number of vesicles over length of intercellular junctions (p <0.01). G: tirfm images showing the presence of insulin vesicles on the surface of fixed and insulin ab - stained -cells of control (f / f) and go deleted (f / f;cre) animals . We therefore recorded the vesicular dynamics close to the -cell membrane in wild - type and go mutant animals . Enhanced green fluorescent protein (egfp) fusion protein, which was previously shown to be packaged in normal insulin vesicles and to not interfere with insulin trafficking . As a result, the egfp - marked insulin vesicles could be followed in real time (29,37). Islet cells were stimulated with 22 mmol / l glucose (see research design and methods). 4), go mutant -cells release significantly more vesicles than control -cells (fig . It is possible that go inactivation could either shorten vesicle residence time on the plasma membrane before fusion or expedite transportation of vesicles from cytoplasm to plasma membrane . In order to differentiate between these possibilities, we counted the fusion events from predocked vesicles and newly arrived vesicles (newcomers or vesicles that appear close to cell membranes after the start of recording) during stimulation . Membrane - docked vesicles in go mutant -cells showed a trend of increased readiness for release (fig . 7b). Specifically, upon glucose stimulation, 23.1% of predocked insulin vesicles were released within 10 min in control -cells, whereas 35.7% of predocked vesicles were released within the same time frame in -cells without go (fig ., the fusion events contributed by newly arrived vesicles did not display a significant difference between the control and mutant -cells (fig . Overall, these data suggest that one of the possible go functions is to facilitate vesicle docking and, to a lesser extent, to increase the readiness of vesicle fusion to the plasma membrane (fig . A: the numbers of vesicle plasma membrane fusion events at several time points with 22 mmol / l glucose stimulation . The events are presented as fusions from predocked vesicles and fusions from newly arrived vesicles, respectively . B: the percent of predocked vesicles that are released within 10 min of glucose stimulation . C: the number of newly arrived vesicles that are released within 10 min of glucose induction . D: a simple model summarizing where go could exert its function in the vesicle - secretion process, including vesicle docking and possibly priming . Although the role of go in insulin secretion has been implicated for one - half century from ptx - based g - protein uncoupling studies (1113), the nonspecificity of ptx (which inactivates both gi and go) has made it impossible to investigate how go functions in vivo . Our findings suggest that go might regulate insulin granule dynamics distal to ca mobilization in vivo, a conclusion drawn from cell culture based studies (3842). Each -cell contains more than 10,000 vesicles (43,44), yet only a small portion of these vesicles can be readily released within the first phase of glucose induction (<10 min in all studied species) (2,7). Subsequently, insulin vesicles are transported from cytoplasm to the plasma membrane for docking, priming, and fusion to sustain the second phase of release . Thus, vesicle docking, although not the rate - limiting step for insulin secretion, likely plays an essential role in regulating insulin secretion . Consistent with this hypothesis, adult -cells that have lost the transcription factor gene foxa2 have more insulin vesicles docked on the cell membrane, and this phenotype is accompanied by excessive glucose - stimulated insulin secretion (45). Thus, understanding vesicle trafficking could provide key insights into the mechanisms that regulate insulin release in response to nutritional, neuronal, and hormonal stimuli . Both our tem- and tirfm - based studies show that loss of go results in more vesicles docking to the plasma membrane at the resting state . Furthermore, the docked vesicles in go nullizygous -cells appear more likely to fuse with the plasma membrane than docked vesicles in control cells . These data, combined with the finding that go inactivation does not significantly alter the transport of vesicles to plasma membrane, suggest that go could delay vesicle docking and possibly repress vesicle priming . Further supporting this notion is our finding that go does not appear to affect calcium flux, which seems to contradict some previously published findings (10). It is likely that only specific g - protein (g)-coupled receptor ligand coupling could affect channel activity via go, which cannot be activated in our in vitro assay . Alternatively, the in vitro assays may not be sensitive enough to detect the subtle channel activity alteration with or without go . For example, go could regulate the resting ca levels in -cells, which would be consistent with the finding that resting ca level affects the pool size of readily releasable granules (46). It would be interesting to analyze whether hormones, such as galanin, somatostatin, or adrenaline, can regulate specific channel activities in the presence or absence of go and how this might affect the resting ca levels in isolated islets . How go modulates the vesicle docking / priming process is not known . Because there are high levels of go protein in neuronal and neuroendocrine cells, it was proposed that one function of go was to act as a reservoir for the g subunits within cells . When stimulated, go will dissociate from the g to release g as an effector to regulate cell function . First, expressing a g binding protein, the ph domain of the g - protein linked receptor kinase 2 stimulates insulin secretion in response to secretagogues, similar to the consequences of g trimer formation (47). Second, introducing g proteins in neuronal cells mimics the effect of go protein activation, that is, dissociation of the g complex (48). In line with this possibility, loss of go could reduce cellular g subunits, which results in dysregulated vesicle trafficking and secretion (17). Unfortunately, it is currently unknown which specific - or -subunit interacts with go and has thus prevented us from directly examining this possibility . Alternatively, go proteins could directly interact with unknown effectors to regulate insulin secretion . Solving this issue will likely require a comprehensive understanding of all the protein / effectors that specifically interact with go under normal physiological conditions ., our analysis suggests that go modulates insulin secretion by regulating vesicle docking on the -cell membrane . Addressing the specific mechanism likely requires a comprehensive analysis of proteins that interact with go and how these proteins modulate vesicle trafficking, docking, priming, and fusion processes.
Nevertheless, the transition of sirnas from the laboratory to the clinical practice has encountered several obstacles . . The polyanionic phosphodiester backbone of sirna suffers from difficult cell uptake, and oligonucleotides may have off - target effects, either by stimulating the immune system or by entering other endogenous gene regulation pathways . Several chemical modifications have been proposed in the literature to address these drawbacks [24]. Most of these modifications are based on modified nucleosides and changes on backbone linkages [6, 7]. Thus, changes in sugar moiety influences sugar conformation, and, therefore, overall sirna structure . Modifications of the 2-oh by f or ome as well as lna [8, 9] are well tolerated and improve binding affinity and nuclease resistance . Base modifications that stabilize base pairs (5-bromouracil, 5-methylcytosine, 5-propynyluracil, and others) have also been proposed [7, 10]. Terminal conjugates, especially at the termini of the sense strand, have been modified with a large number of lipids to achieve improved cellular uptake . In addition to these modifications, sirna architecture is also crucial in the design of effective and specific sirna . In addition to the canonical sirna architecture of 21-nt antiparallel, double - strand rna with 2-nt 3-overhangs, several forms of sirna have been described . Blunt - ended sirna, 25/27 mer dicer - substrate or asymmetric sirna are among the sirna structures formed by two strands . This is the case in small hairpin rna (shrna), where the two strands are linked by a single loop, or rna dumbbells, made by closing the open end of the hairpin . Finally, sirna can also comprise three strands, namely, two 913 nt sense strands and the intact antisense strand . Some of these modifications have reduced off - target effects and increased potency (figure 1(a)). Another architecture not yet explored in sirna is the branched rna structure obtained from a central building unit and several branching points that enable the strand growth . Although the synthesis of these compounds is complex and tedious, commercially available synthons have improved the complexity and yields of these structures . The assembly of branched nucleic acids on a solid support can be achieved by convergent or divergent strategies . In the former, synthesis of branched oligonucleotides containing two or more identical strands can be achieved by branching derivatives 1,3-diaminopropanol, pentaerythritol, the commercially available symmetric doubler [1820], or by a ribonucleoside bisphosphoramidite as synthons . In contrast, in the divergent approach two or more distinct strands are prepared with synthons with orthogonal protecting groups or from commercial sources [20, 22]. In addition, 2-o - silylribonucleosides have been used to synthesize asymmetric double oligonucleotide strands . Initially, most of the interest in this area was focused on the study of branched oligoribonucleotides as splicing intermediates of eukaryotic mrnas [2426]. Moreover, branched oligonucleotides show high affinity for single - strand oligonucleotides to form alternated strand triplexes [2729]. Recently, branched oligonucleotides have been used as building blocks in the synthesis of new nanostructures [3034]. Multilabelled oligonucleotides containing branching points have been described to increase the sensitivity of hybridization experiments . Here, we synthesized branched rna structures (figure 1(b)) and evaluated their capacity to inhibit the tumour necrosis factor (tnf-) protein, which is involved in the apoptosis, inflammation, and immunity processes . We reasoned that branched sirna could provide a new rna architecture for rna interference activity . Given that the use of symmetric branching units is compatible with most of the modifications described to enhance the inhibitory capacity of sirna, the molecules described here may provide a starting point for further modifications . The following rna sequences were obtained from commercial sources (sigma - proligo, dharmacon): sense or passenger scrambled 5-cagucgcguuugcgacugg - dt - dt-3, antisense or guide scrambled 5-ccagucgcaaacgcgacug - dt - dt-3, antisense or guide anti - tnf-: 5-gaggcugagacauaggcac - dt - dt-3, and sense or passenger anti - tnf-: 5-gugccuaugucucagccuc - dt - dt-3. Rna monomers in capital letters, dt represents thymidine . The anti - tnf sirna was previously described to efficiently downregulate murine tnf mrna . Db stands for the symmetric doubler phosphoramidite obtained from commercial sources (glen research). Guanosine was protected with the dimethylaminomethylidene group, cytidine with the acetyl group, and adenosine with the benzoyl group . T - butyldimethylsilyl (tbdms) group was used for the protection of the 2-oh function of the rna monomers . The phosphoramidites were dissolved in dry acetonitrile (0.1 m), and a modified cycle was used with increased coupling time to 10 min . Oligoribonucleotide 1 was synthesized on a cpg solid support with a symmetric branching unit of two arms containing two dmt - protected hydroxyl groups, as described in . Oligoribonucleotides 2 and 3 were synthesized using standard low - volume polystyrene thymidine columns . After the solid - phase synthesis, the supports were treated with concentrated aqueous ammonia - ethanol (3: 1) for 1 h at 55c . After filtration of the supports, the solutions were evaporated to dryness . The residue was dissolved in 85 l of 1 m tetrabutylammonium fluoride (tbaf) in tetrahydrofuran (thf) for 12 h. then, 85 l of 1 m of triethylammonium acetate was added and the oligoribonucleotides were desalted on a nap-10 column using water as eluent . Column: nucleosil 12010 c18 (250 4 mm); 20-min linear gradient from 15% to 100% b (dmt on conditions); flow rate 3 ml / min; solution a was 5% acetonitrile in 0.1 m aqueous triethylammonium acetate (teaa) buffer and b 70% acetonitrile in 0.1 m aqueous teaa . Yields (0.2 mol scale synthesis) were between 510 od units at 260 nm . The thermal melting curves for duplexes of the oligoribonucleotides 13 and their unmodified rna complementary strands (guide strand) were performed following the absorption change at 260 nm . Samples were heated from 20c to 80c, with a linear temperature ramp of 0.5/min in a jasco v-650 spectrophotometer equipped with a peltier temperature control . Sample concentration of the samples was around 2 m . All the measurements were repeated three times and conducted in 15 mm hepes 1 mm mg(oac)2 and 50 mm koac ph 7.4 . Hela cells were cultured under standard conditions (37c, 5% co2, dulbecco's modified eagle medium, 10% fetal bovine serum, 2 mm l - glutamine, supplemented with penicillin (100 u / ml) and streptomycin (100 mg / ml)). Hela cells were transfected with 250 ng of a plasmid expressing murine tnf- using lipofectin (invitrogen), following the manufacturer's instructions . One hour after transfection cells were transfected with 100 nm double strand concentration of sirna against tnf-, using oligofectamine (invitrogen). Previously, sirna duplex annealing was performed by mixing modified (1, 2, 3) and unmodified passenger strands (unm) with the appropriate amount of the corresponding unmodified guide strand . Tnf- concentration was determined from cell culture supernatant by enzyme - linked immunosorbent assay kit (bender medsystems) following the manufacturer's instructions . The inhibitory capacity of the sirna duplexes is expressed as double strand concentration for comparative purposes . A 100 mm double strand concentration is equivalent to a 50 nm concentration of two - branched sirna (1 or 2) and to 25 nm of four - branched sirna (3). In order to prepare branched rna for rna interference, the potential steric hindrance of the branching unit with risc must be considered . As the passenger strand is removed from the sirna duplex upon binding to risc this position has been demonstrated to allow the introduction of a large number of modifications without affecting the inhibitory capacity of sirna [6, 7]. We thus designed branched oligonucleotide sequences 13 of the passenger strand of a sirna directed against tnf- (figure 2). Sequence 1 was synthesized using a controlled pore glass (cpg) solid support containing a symmetric doubler, as shown in figure 3 . Sequences 2 and 3 with two or four strands, respectively, were synthesized on a low - volume polystyrene support (lv200) functionalized with dimethoxytrityl- (dmt-) thymidine . The commercially available symmetric doubler phosphoramidite was used to introduce two and four branches on the 3-position of the starting thymidine (figure 3). The 2-oh function of ribonucleosides was protected with the t - butyldimethylsilyl (tbdms) group . Coupling yields, determined by the absorbance of the dmt cation released in each synthesis step, were more efficient (98%) on low - volume (lv200) polystyrene supports than on cpg support (95%). After assembly of the sequences, the dmt - containing oligonucleotides were released from the supports with ammonia, and the resulting compounds were treated with fluoride to remove the tbdms groups . Several peaks were observed for the synthesis of the two - branch rna sequences (1 and 2; figure 4). A fraction containing oligonucleotides with a single dmt group was eluted next, and the last fraction contained the desired sequence with two dmt groups . Mass spectrometry analysis (table 1) and electrophoresis analysis confirmed the mass and size of the desired branched oligoribonucleotides . Figure 5 shows the hplc profile of the mixture obtained in the synthesis of the four - branch rna sequence (3). In this case, three peaks in the dmt - containing area were observed . Although resolution of these peaks was not as good as in the previous case, the last eluting peak corresponded to the desired tetra - dmt compound (3). The purified compound had the expected molecular weight, was homogeneous by analytical hplc (figure 5), and showed the correct migration in polyacrylamide gel electrophoresis (page). The melting temperatures of the branched sirna duplexes formed by annealing of equimolar amounts of sequences 13 with unmodified passenger strand are shown in table 1 . Duplex 1 had the lowest melting temperature, which was 3.5c lower than the unmodified duplex (table 1). The small decrease in melting temperatures of the two - branched sirna structures is possibly due to a steric effect in the branching point that holds the two duplex strands in close proximity . The four - stranded architecture had a larger separation between strands as a result of the introduction of 3 branching units, thus the resulting duplexes showed greater similarity to the unmodified duplex . Thus we believe that the small destabilizing effect observed in the two - branched rna duplexes could be optimized in further experiments by adding a linker between the branching unit and the rna strands, as described by grimau et al . . Tumor necrosis factor (tnf-) was selected as a target for rna interference studies . This protein is a major mediator of apoptosis as well as inflammation and immunity, and it has been implicated in the pathogenesis of a wide spectrum of human diseases . Consequently, inhibition of this protein is of particular relevance . Modified oligoribonucleotides (13) were annealed with equimolar amounts of the unmodified guide, and the resulting duplexes were used to inhibit the expression of tnf- gene . Hela cells were transfected first with the murine tnf- plasmid using lipofectin, and 1 h later they were cotransfected with the sirna duplex using oligofectamine . After 24 h, cellular tnf- production was analyzed by enzyme - linked immunosorbent assay (elisa). The inhibitory capacity of the sirna duplexes is shown in figure 6 . To compare the efficiency of each sirna to inhibit tnf-, we normalized the data taking in account the number of strands of each sirna . Thus, a 100 nm double strand concentration is equivalent to 100 nm of unmodified sirna duplex, 50 nm of sirna 1, and 2, and 25 nm of sirna 3 . Figure 6 shows that the inhibitory capacity of the branched structures was maintained similar to that of the unmodified duplex . This result indicates that the branched sirnas described here are compatible with rna interference machinery, and thus the risc complex binds to branched rna structures in a similar way as to the linear rna duplexes shown in figure 1 . Two - stranded rna duplexes (1 and 2) were more efficient than four - stranded ones (3). In addition, sirna 1 showed slightly greater efficiency at inhibiting tnf- than sirna duplex 2 . This small difference may be related to the lower melting temperature of the former (table 1). For several years, research has focused on chemical modifications and delivery technologies to improve the pharmacokinetic properties of sirna . Many of the chemically modified sirna with interesting inhibitory capacity contain one or multiple modifications in the sugar, nucleobases, and phosphate linkages or at the 3- or 5-ends . In addition to these modifications, duplex architecture of sirna itself is also relevant, and several modifications have been reported to show satisfactory inhibitory capacity . Here we demonstrate that branched sirna is compatible with rnai and that, when transfected with cationic lipids, sirna has similar inhibitory capacity than unmodified duplex sirna . Although the potency of branched sirna containing two or four strands was not increased, we consider it a suitable starting point for further development . Given that the method described here is compatible with most of the rna modifications described to date, these compounds may be further functionalized to obtain more potent sirna derivatives . In addition, they offer an internal mid position that could be suitable for attachment to delivery systems . In this regard, optimization of the branching approach for the synthesis of asymmetric branched sirnas may lead to the development of sirna for the combined inhibition of multiple targets . These asymmetric sirna duplexes carrying two rna sequences attached or bound to an appropriate delivery system will insure the 1: 1 ratio of two rna sequences for the combined inhibition of two genes that may improve the treatment of a particular disease.
Photoinduced charge transfer (ct) via symmetry breaking (sb) plays a crucial role in photosynthetic reaction centers in living systems . Ct from one chromophore to another occurs upon photoexcitation, producing sb charge separation . Of great potential interest, but less well explored, are sbct processes in organic photovoltaics (opv) and related solar - harvesting systems . Among well - documented simple organic compounds exhibiting sb phenomena are 9,9-bianthryl derivatives; however, they do not absorb visible light, making them of limited use in systems harvesting the solar energy . Indeed, very few organic dyes that absorb in the visible region undergo sbct processes . Herein, we investigate the photophysics of zinc dipyrrin complexes (figure 1) that exhibit intense visible absorption in a range of organic solvents . These compounds have structural features related to 9,9-bianthryl (i.e., poorly coupled orthogonal chromophores) that are conducive to photoinduced sbct processes in weakly polar to polar solvents . Zinc dipyrrins and analogous compounds are attractive because, in addition to strong absorption in the visible region of the spectrum, their syntheses are easy and scalable . Moreover, the large body of work on boron dipyrrins (bdip) can be used to guide ligand design . Structures of homoleptic (zdip1zdip4) and heteroleptic (zdip2 and zdip3) zinc dipyrrin complexes . In opvs, the low dielectric constants of organic materials (s 3) lead to high exciton binding energies, and thus, a large energy offset between donor and acceptor is required to promote charge generation at the donor / acceptor interface (d / a). Use of organic dyes that undergo sbct processes might be a potential solution to reduce the energy cost for exciton dissociation to free charges at d / a . The polar environment at d / a may be sufficient to induce sbct in the chromophore, leading to spontaneous formation of internal ct excitons . Charge separation between electron donor and acceptor materials from these sbct excitons is expected to proceed with lower energetic requirements than for typical frenkel excitons found in organic materials . That being the case, the ability of zinc dipyrrin complexes to undergo sbct makes them a potential family of new materials for use in organic photovoltaics . Fluorescent metallodipyrrins have attracted considerable attention due to their potential use as probes for sensing metal ions (in particular, zn) in living systems . However, in spite of the increasing number of reported metallodipyrrins, their photophysics are not well - understood . Unlike the highly fluorescent bdips, homoleptic complexes mdn (d = dipyrrin or -extended dipyrrin ligands) generally exhibit low to moderate fluorescent quantum yields . In contrast, the heteroleptic complexes mdxn (x is an ancillary ligand) were shown to have quite high luminescent efficiencies (up to 80%). Several nonradiative deactivation pathways have been suggested for the photoinduced excited state of homoleptic zinc dipyrrins and -extended dipyrrins . Lindsey and co - workers reported that rotation of the phenyl ring at the meso - position on the dipyrrin ligand is a source of nonradiative deactivation of the excited state . Replacement of phenyl with the more bulky mesityl substituent to form zdip1 inhibits this rotation, leading to an improved quantum yield of 36% in toluene . Interestingly, a recent study on related mesityl - substituted zinc dipyrrins (zdip2 and zdip4) has reported that the quantum yields of the homoleptic complexes are strongly dependent on solvent polarity, decreasing from 2030% in toluene to 5% in dichloromethane . The authors proposed that this decrease in polar solvents is due to thermal promotion from a locally excited state on a single dipyrrin ligand to a nonemissive, charge - separated state (i.e., d d); however, no additional photophysical data was provided to support this hypothesis . Strong excitonic coupling between nonorthogonal ligands has also been suggested as another nonradiative deactivation pathway of zinc -extended dipyrrin complexes . However, excitonic coupling between the nearly orthogonal ligands in homoleptic zinc dipyrrin compounds such as zdip2 and zdip4 is negligible and thus cannot be used to explain the decrease in luminescent efficiency compared to their heteroleptic counterparts . In this paper, we show that sbct in polar solvents is an effective nonradiative decay pathway for the electronic excited states of homoleptic zinc dipyrrins . In nonpolar solvents such as cyclohexane, these complexes do not undergo sbct and thus exhibit even higher quantum yields than their heteroleptic analogs . The synthesis and characterization by nmr, mass spectroscopy, and elemental analysis of the zinc dipyrrin complexes described here are given in the supporting information (si) for this paper . Cyclic voltammetry (cv) and differential pulse voltammetry (dpv) were performed using an eg&g potentiostat / galvanostat model 283 . Freshly distilled thf (vwr) was used as the solvent under inert atmosphere with 0.1 m tetra(n - butyl)ammonium hexafluorophosphate (aldrich) as the supporting electrolyte . A glassy carbon rod, a platinum wire, and a silver wire were used as the working electrode, counter electrode, and pseudoreference electrode, respectively . Electrochemical reversibility was established using cv, while all redox potentials were determined using dpv and are reported relative to a ferrocenium / ferrocene (fc / fc) redox couple used as an internal standard . Visible spectra were recorded on a hewlett - packard 4853 diode array spectrophotometer . Steady - state emission experiments at room temperature and 77 k were performed using a photon technology international quantamaster model c-60se spectrofluorometer . Fluorescence lifetime measurements in cyclohexane, toluene, and thf were performed using an ibh fluorocube instrument equipped with a 405 nm led excitation source with the irf value of 0.4 ns . Fluorescence lifetime measurements in dichloromethane and acetonitrile were carried out using an excitation wavelength of 500 nm obtained from an optical parametric amplifier (coherent opa 9450) pumped by a 250 khz ti: sapphire amplifier (coherent rega 9050). The emission was collected at 520 nm for s1 state and 645 nm for the ct state using a r3809u-50 hamamatsu pmt with a becker and hickl spc 630 detection module (22 ps time resolution). Quantum efficiency measurements were carried out using a hamamatsu c9920 system equipped with a xenon lamp, calibrated integrating sphere, and model c10027 photonic multichannel analyzer . The error in the emission lifetime measurements is 5% and for the quantum yields is 10% . Pump and probe pulses were obtained from the output of a ti: sapphire regenerative amplifier (coherent legend, 1 khz, 4 mj, 35 fs). The excitation pulses centered at 500 nm were generated by pumping a type - ii opa (spectra physics opa-800c) with 10% of the amplifier 800 nm output and mixing the resulting opa signal output with the residual 800 nm pump in a type - ii bbo crystal . White light supercontinuum probe pulses spanning the visible (320950 nm) were obtained by focusing a small amount of the amplifier output into a rotating caf2 disk . The supercontinuum probe was collimated and focused with a pair of off - axis parabolic mirrors into sample, whereas the pump was focused before the sample position using a 25 cm caf2 lens . To avoid any contribution to the observed dynamics from orientational relaxation, the polarization of the supercontinuum was set at the magic angle (54.7) with respect to the pump polarization . The cross - correlation between pump and probe in a thin 1 mm quartz substrate gave a fwhm of 180 fs for 500 nm excitation . The supercontinuum probe was dispersed using a spectrograph (oriel ms127i) onto a 256-pixel silicon diode array (hamamatsu) for multiplexed detection of the probe . Samples containing zdip1, zdip2, or zdip3 dissolved in cyclohexane, toluene, dichloromethane, or acetonitrile were placed in a closed, capped 1 mm quartz cuvette . The concentration of each sample was adjusted to give an optical density between 0.11 and 0.18 at 500 nm . The solutions were deaerated by bubbling with n2 prior to analysis . During data collection, the samples were slowly oscillated perpendicular to the pump and probe to reduce photodamage to the sample by the pump . At early time delays, a strong nonresonant signal from the sample cell and solvent is observed . The solvent response is found to relax within 180 fs for cyclohexane, dichloromethane, and acetonitrile, while in toluene this signal was stronger and obscured the first 300 fs . To effectively remove this nonresonant signal, a second measurement of the neat solvent was performed in an identical cuvette under same excitation conditions . The transient signal resulting from the solvent was then subtracted from the zinc dipyrrin solution signal . The nonresonant solvent response did, however, give a useful measure of the temporal dispersion of the supercontinuum after propagating through the caf2 plate and sample . Transient absorption measurements were performed with pump fluences varying between 70 and 300 j / cm . Over this range, the signal was found to scale linearly with the pump energy . Samples were prepared in a nitrogen glovebox with dry solvents, such that the maximum absorbance was approximately od = 1 . These samples were sealed in 1 1 cm quartz cuvettes with kontes valves to keep the solution air - free . The third harmonic of a 10 hz q - switched nd: yag laser (spectra - physics quanta - ray pro - series, pulse width: 8 ns) was used to pump an optical parametric oscillator (spectra - physics quanta - ray mopo-700), tunable in the visible region . The excitation wavelength for each sample was chosen such that od (at excitation) = 0.30.4, and the laser power was attenuated to 3 mj / pulse using a half - wave plate and polarizer combination . For single - wavelength transient absorption kinetics measurements, probe light was provided by a 75 w arc lamp operated in either continuous or pulsed mode . Single wavelengths were selected by a double monochromator with 1 mm slits, detected by a photomultiplier tube, and amplified and recorded with a transient digitizer . Single - wavelength traces were acquired at approximately 5 nm increments over the range of 350595 nm, on a 2 s, 100 s, or 10 ms timebase window, averaging over 300 laser pulses . A reference wavelength (400 nm) was acquired as every third trace, to take into account the photodegradation of the sample . Kinetics traces at each wavelength were scaled on the basis of the intensity of the previous reference trace . Decays were fit to a single or double exponential with a long - time offset using matlab (version 2010b) curve fitting software . To generate transient absorption profiles at various time points, the od at that time point was taken from the exponential fit of each single - wavelength kinetics trace . The x - ray crystal structures of zdip3, zdip4, zdip2, and zdip3 were determined using a bruker apex ii ccd system equipped with a triumph curved - crystal monochromator and a mo k fine - focus tube (= 0.710 73). The frames were integrated with the bruker saint software package using a saint v7.68a algorithm . Representative synthetic schemes for preparing homoleptic (zdip1zdip4) and heteroleptic (zdip2, zdip3) complexes are presented in scheme 1 . Mesityl substituents on the meso - position of the dipyrrin ligand were chosen to eliminate aryl rotation as a potential nonradiative deactivation pathway . Dipyrrins dip1dip4 were prepared from the corresponding pyrrole and mesitylaldehyde and used directly in consecutive reactions with ddq and zn(oac)2 to form the homoleptic complexes zdip1zdip4 in total yields of 813% . The heteroleptic complexes were prepared from a reaction zn(-diketonate)2 and the corresponding dipyrrin . Of the three ancillary ligands examined, pentane-2,4-dione (acac), 1,1,1,5,5,5-hexafluoropentane-2,4-dione, and 2,2,6,6-tetramethyl-3,5-heptanedione (dpm), pure heteroleptic complexes were successfully isolated only using the dpm ligand . As seen for other heteroleptic zn complexes, zdip2 and zdip3 disproportionate to their respective homoleptic complexes (zdip2 and zdip3) in chloroform over the course of several hours, as observed by nmr measurements (see si). However, zdip2 and zdip3 are stable in the solid state and can be sublimed under vacuum without disproportionation . Single crystal x - ray analysis and high - resolution mass spectroscopy confirmed formation of zdip2 and zdip3. It should be noted that freshly prepared zdip2 and zdip3 solutions were used for each step of subsequent photophysical characterization to minimize the effects of disproportionation . Single crystal x - ray analysis was performed on zdip3, zdip4, zdip2, and zdip3; structures of representative homoleptic (zdip3) and heteroleptic complexes (zdip3) are shown in figure 2 . The structures of zdip3 and zdip4 are similar to those published for zdip1 and zdip2 . N bond lengths in the complexes range from 1.95 to 1.99, while the zn o bond lengths in zdip2 and zdip3 vary between 1.95 and 1.97 . The zinc center in all the complexes adopts a distorted tetrahedral configuration with the two ligands held nearly perpendicular to each other . The dihedral angles between mean planes of the two dipyrrin ligands in zdip1, zdip2, zdip3, and zdip4 are 85.0, 88.3, 76.7, and 83.4, respectively, whereas values for the related angles between the two ligands of zdip2 and zdip3 are 87.8 and 89.8, respectively . To evaluate the degree of distortion of the ligands, dihedral angles between the planes encompassing different groups of atoms as shown in figure 2 were measured (table 1). Compared to boron dipyrrin compounds, which are essentially flat, the dipyrrin framework in the zinc dipyrrins is considerably more flexible, as dihedral angles between planes 1 and 2 vary from 3 to 18. however, no clear correlation is apparent between the degree of alkylation and variation of the dihedral angles, indicating that the distortions are likely dictated by crystal packing forces . Ortep diagrams of (a) zdip3 and (b) zdip3 at 50% probability level . Planes containing different groups of atoms are indicated by the colored lines . In order to gain insight into the electronic structure of the zinc dipyrrin complexes, theoretical calculations were performed at the b3lyp / lacvp * * level of theory . For simplicity, unsubstituted homoleptic zdip and heteroleptic zdip are presented, as it was found that they possess the same essential electronic features as their alkylated / arylated analogs . Structures of the optimized complexes, highest and next highest occupied molecular orbitals (homo, homo1), and lowest and next lowest unoccupied molecular orbital (lumo, lumo+1), along with the corresponding energies, are shown in figure 3 . The homo (a2 symmetry in the d2d point group) and homo1 (b1) of zdip localize on both dipyrrin ligands, while the doubly degenerate lumos are localized on separate dipyrrin ligands (figure 3). Frontier orbitals of zdip solely populate the dipyrrin ligand, excluding any participation of the -diketonate ligand . Consistent with mo analysis, the calculated homo and lumo energies of the two complexes are similar . There is little - to - no contribution from atomic orbitals on zinc to the frontier orbitals in either complex . The minor difference in energy between the homo and homo1 in zdip (0.11 ev) is indicative of very poor electronic coupling between the two orthogonal dipyrrin ligands in the ground state . The transition dipole moment of the metallodipyrrin fragment lies in the plane of the dipyrrin ligand, along the long axis of the ligand . This places the transition dipole moments of the two dipyrrins in zdips orthogonal to each other, suggesting that there should be little or no excitonic or electronic coupling between the two dipyrrin ligands . Sham lumos (mesh) and energies for (a) zdip and (b) zdip and homos (transparent) for (c) zdip and (d) zdip. Zdip has d2d symmetry and zdip has c2v symmetry . Absorption spectra of (a) zdip2 in different solvents and (b) zdip2 and zdip2 in cyclohexane . The zinc dipyrrin complexes absorb strongly from 400 to 550 nm [= (1.011.17) 10 m cm]. Representative absorption spectra of zdip2 and zdip2 in different solvents are presented in figure 4, and the emission spectra in the same solvents are given for zdip2 in figure 5 . The photophysical properties of zdip1zdip4 and zdip2 and zdip3 at room temperature are summarized in table 2 and the si . The absorption spectra and the principal band in the emission spectra are nearly independent of solvent polarity, indicating little change in the dipole moment or polarizability upon excitation . All of the complexes display emission spectra with small stokes shifts indicative of a localized excited state . Representative spectra for zdip2 and zdip2 are shown in figure 6 . The luminescent quantum yields (f) range between 0.08 and 0.66 and the fluorescence decays are single exponential with lifetimes () that vary from 0.8 to 4.8 ns . The differences in f are due to significant variations in the rate constant for nonradiative decay (knr), as the rate constants for radiative decay (kr) are similar among all the derivatives . The data reveals an interesting effect of alkylation on knr: zdip2 zdip1 <zdip4 zdip3 (table 2). <zdip3. Comparing zdip1 and zdip2 shows that methylation at the -position of the dipyrrin only slightly decreases knr, suggesting that hindering excited state distortion toward a planar coordination geometry has little effect on nonradiative decay . Methyl substitution at the -position, adjacent to the mesitylene substituted meso - carbon, leads to a marked enhancement in the observed knr values for these complexes . The effect is also seen when comparing zdip2 to zdip3, where -methylation leads to a 6-fold increase in knr . The variation in knr for zdip1zdip4 is roughly correlated with the fwhm of emission spectra (figure 6, inset). The association of broader emission profiles with a faster knr suggests that structural distortion of the excited states increases internal conversion to the ground state . While one would have expected that libration of the mesityl group is more favorable in zdip1 and zdip2, thus increasing knr relative to their more sterically constrained analogs, zdip3 and zdip4, the opposite is true . A likely explanation for this is that steric interactions between the ortho - methyls on the mesityl group and the -methyls of the dipyrrin exacerbate out - of - plane distortions on the dipyrrin ligand in zdip3 and zdip4, which is not expected to be the case for zdip1 and zdip2 . Distortion of the dipyrrin from planarity will give both an increase in fwhm and knr, as observed here . While the absorption spectra for all the complexes are solvent - independent, the emission spectra of the homoleptic and heteroleptic derivatives exhibit distinct differences with respect to solvent polarity . Representative emission spectra of zdip2 measured in different solvents are shown in figure 5a, data for luminescent quantum yields from zdip1zdip4 and zdip2 and zdip3 are presented in figure 5b, and other photophysical data are given in table 2 and the si . With increasing solvent polarity there is little change in the emission maxima; however, the quantum yields for zdip1zdip4 sharply decrease and the excited state transients display multiexponential lifetimes with an increasing amplitude for a subnanosecond component . The subnanosecond component is faster than our instrument s response time (<22 ps). The multiexponential decay indicates that with increasing solvent polarity the majority of the s1 population is going to a second state and only partially relaxing to the ground state by the radiative process (figure s23 and table s14, si). For the least polar solvent, cyclohexane, single exponential decay is observed with lifetimes of 3.7, 4.8, and 1.4 ns for zdip1, zdip2, and zdip3, respectively . The radiative rate constants for emission in cyclohexane fall in a narrow range of 0.110.14 ns . In strong contrast, the luminescent quantum yields for zdip2 and zdip3 decline modestly in polar solvents and have radiative rate constants similar to those observed for zdip1zdip3 in cyclohexane (i.e., for zdip2 krad = 0.150.19 ns and for zdip3 krad = 0.11). For the mixed ligand complexes, we believe that the decrease in pl efficiency in polar solvents is due to their photoinstability . Both zdip2 and zdip3 disproportionate to zn(acac)2 and zn(dipyrrin) thermally; the process is likely driven optically and accelerated in polar media . The pronounced decrease in pl efficiency for zdip1zdip4 indicates that the locally excited state in the homoleptic derivatives equilibrates and deactivates to a weakly or nonemissive state in polar solvents . Moreover, a broad emission band emerges at low energy for zdip2zdip4 in polar solvents (figure 5a and si). The emission band red - shifts and becomes more pronounced with increased solvent polarity . The luminescence quantum yield and emission lifetime data (f <0.001, = 2.2 ns) indicate a very small radiative rate constant for this transition . However, the emission band is distinctly different from the phosphorescence of zdip2 recorded at 77 k in 2-methyltetrahydofuran (2-methf) (figure 5a, inset). To determine if the broad emission at 650 nm originates from an excimer or aggregate, emission intensities of zdip2 at 508 and 650 nm were measured at a range of concentrations (see the si). The intensities of the two bands vary linearly with concentration, suggesting that excimer or aggregate emission is not responsible for the weak red emission in these compounds . Thus, the low - energy emission band is assigned to a charge transfer transition similar to that reported for meso - coupled boron dipyrrin compounds . It is interesting to note that, in contrast to zdip2zdip4, no low - energy emission is detected from nonalkylated zdip1 in polar solvent (see the si). Likewise, no comparable emission feature is observed in zdip2 or zdip3 in the same solvents . (a) emission spectra of zdip2 in various solvents (inset is the phosphorescence spectrum in 2-methf at 77 k). (b) quantum yield of zdip1zdip4 and zdip2 and zdip3 plotted vs solvent from nonpolar to most polar . (inset) full width at half - maximum (fwhm) of emission plotted vs nonradiative rate constant knr for zdip1zdip4 and zdip2 and zdip3. The sharp decrease in luminescent efficiency from zdip1zdip4 in polar solvents, along with the simultaneous appearance of an additional broad peak at longer wavelengths in the emission spectra of zdip2zdip4, suggests a deactivation pathway to a weakly emissive state . This state most likely has ct character and is formed via a symmetry - breaking mechanism similar to that which occurs in other bichromophoric systems . Further support for the hypothesis that the observed photophysical properties of zdip1zdip4 are associated with sbct is the weaker dependence on solvent polarity for the heteroleptic complexes zdip2 and zdip3, where sbct is impossible . Cyclic voltammetry (cv) and differential pulse voltammetry (dpv) for zdip1zdip4 were carried out in dry thf under n2, and the results are presented in table 3 . All of the complexes exhibit two distinct reversible reduction peaks and one irreversible oxidation peak, with the exception of zdip4, where two quasireversible oxidation peaks are observed . The redox potentials are cathodically shifted by around 200 mv upon addition of two alkyl groups per ligand; however, the difference between the first oxidation and reduction potentials (electrochemical gap, eredox) remains relatively constant for all the derivatives . This data can be used to evaluate the thermodynamic requirements for zdip1zdip4 to undergo photoinduced sbct . Stabilization of the ct state in zdip1zdip4 is required to enable sbct, since eredox is greater than the optical s1 gap (e00). The required stabilization energies vary from 0.19 to 0.28 ev (see table 3). Thus, while nonpolar solvents disfavor sbct, polar solvents such as dichloromethane (s = 8.9) or acetonitrile (s = 37.5) provide a stabilization energy estimated to exceed 0.3 ev and can therefore promote sbct . Electrochemical values (0.02 v) were determined by differential pulsed voltammetry (dpv) vs fc / fc . The optical gap e00 is defined by the midpoint between absorption and emission spectra in thf . Triplet energies were measured in 2-methyltetrahydrofuran at 77 k. spectroelectrochemical measurements were also performed in order to identify absorption transitions characteristic of the ct state . The absorption profile for the one - electron - reduced form of zdip1 is broader than the neutral complex and has enhanced transitions between 370 and 430 nm along with a distinct peak at 517 nm (figure 7). Unfortunately, the irreversible nature of electrochemical oxidation in zdip1zdip3 precluded optical characterization of the cation . Likewise, the low stability of the zdip4 cation prevented characterization using spectroelectrochemical methods . Femtosecond and nano - to - microsecond transient absorption (ta) measurements were performed to confirm the presence of sbct and to study the kinetics of such processes . Femtosecond ta values of zdip1 in cyclohexane, toluene, dichloromethane, and acetonitrile are presented in figure 8 . Other femtosecond ta spectra of zdip2 and zdip3 in cyclohexane, toluene, and acetonitrile are shown in the si . In cyclohexane (figure 8a), excitation at 500 nm populates the s1 state, as observed by a ground - state bleach from 430 to 500 nm (compare to figure 4), stimulated emission (520600 nm, compare figure 6), and excited state absorption at 345 nm . The stimulated emission remains over the probing time (1.1 ns), resulting in a minimal shift of the bleach peak (figure 8a). Femtosecond transient absorption of zdip1 in (a) cyclohexane, (b) toluene, (c) dichloromethane, and (d) acetonitrile . Excitation at 500 nm and pump fluence of 160 j / cm were used for all, except panel c, which was performed at 70 j / cm . In polar solvents such as dichloromethane (figure 8c) and acetonitrile (figure 8d), stimulated emission and excited state absorption at 345 nm from s1 appear immediately following excitation, similar to what is observed in cyclohexane . However, over the course of 46 ps, the stimulated emission band disappears and new induced absorption bands at 370 and 517 nm grow in . In contrast to the ta spectrum in cyclohexane, the disappearance of the stimulated emission results in the shift of the bleach peak (figure 8c, d). The induced absorption at 517 nm is similar to that of the zdip1 anion [see figures 7 and s25 (si) for a detailed comparison]. Since the induced absorption peak at 370 evolves with similar kinetics to that at 517 nm, we assign the 370 nm peak to the new excited state as well . This state is assigned as the sbct species; note that the absence of characteristic sbct absorptions in cyclohexane indicates that stabilization by polar solvents is required to favor sbct over the local excited state, in agreement with the electrochemical analysis . The increase in the ground - state bleach during the first 10 ps (figure 8c, d) is a direct consequence of sbct . This is because only one of the dip ligands in the complex is bleached upon initial photoexcitation, whereas the second ligand is subsequently bleached upon sbct; thus, the bleach increases approximately by a factor of 2 . In a weakly polar solvent, toluene (figure 8b), both s1 stimulated emission and induced absorption at 370 nm are observed over the probing time of 1.1 ns, suggesting that the kinetic evolution of the transients is different from what was observed in either polar solvents or cyclohexane . The induced absorption at 370 nm indicates sbct of zdip1 in toluene; however, the induced absorption at 517 nm is hidden due to the overlap with the stimulated emission band . Additionally, the stimulated emission persists over the probing time (1.1 ns), which is much longer than that in acetonitrile or dichloromethane . The luminescent efficiency of zdip1 in toluene is also much higher than that in acetonitrile and dichloromethane (table 2). These observations can be explained by the presence of an equilibrium between the local excited state s1 and ct states in toluene . We propose that solvation by weakly polar toluene lowers the energy of the ct state close to that of the locally excited state . A similar equilibrium between locally excited and ct states of 9,9-bianthryl was reported in weakly polar media . In more polar solvents, large solvation energies further stabilize the ct states, shifting the equilibrium to formation of the charge transfer species . On the basis of the femtosecond ta measurements, a simplified jablonski diagram is proposed to explain the obtained results (figure 9). Global fitting of ta data from zdip1zdip3 in different solvents has been performed using the proposed scheme, and the results are presented in table 4 (a detailed description of the fitting scheme and the species - associated spectra are shown in the si). Sbct does not occur in nonpolar cyclohexane, and the kinetics of transient species was fitted to a monoexponential decay with the lifetimes of 4.5, 4.8, and 1.4 ns for zdip1, zdip2, and zdip3, respectively . These decay times are in good agreement with fluorescence lifetimes of the respective compounds in cyclohexane (table s14, si). A simplified jablonski diagram illustrating the dependence of the symmetry - breaking charge transfer process on solvent polarity . Krec is the total rate for recombination to either the triplet or ground state . Ta data of zdip1 in toluene were fitted with a different model to account for an equilibrium between the local excited state s1 and the ct state . The forward and backward rates (1/kct and 1/kcr) between the s1 and the ct states are 13 and 25 ps, respectively . Since these rates are 2 orders of magnitude faster than both the charge recombination rate of the ct state (1/krec = 3.5 ns) and the decay rate of the local excited state (= 3.0 ns), our assumption about a fast equilibrium is indeed reasonable . In polar dichloromethane, the fitting yields rates of 5.5 ps and 3.3 ns for the formation (1/kct) and the recombination (1/krec) of the ct state of zdip1, respectively . In acetonitrile, the rates for formation and recombination of the ct state are faster compared to those in dichloromethane (1/kct = 3.6 ps and 1/krec = 2.1 ns). Generally, the rate for formation of the ct state in polar solvents (1/kct = 1.15.5 ps) is 3 orders of magnitude faster than that for recombination (1/krec = 0.93.3 ns) (table 4). Interestingly, the femtosecond ta measurements of zdip2 and zdip3 in acetonitrile (figure 10 and the si) show faster rates for both formation and recombination of the ct state (1/kct = 1.1, 1.0 ps and 1/krec = 0.9, 1.4 ns for zdip2 and zdip3, respectively) compared to zdip1 . The two dipyrrin ligands of zdip2 and zdip3 are expected to have less torsional freedom around the zn center relative to zdip1 due to the presence of methyl groups at the -positions . This steric hindrance constrains the ligands to adopt a more orthogonal configuration . Without these steric impediments, this interaction will lower the energy of the excited state, making the s1 state less energetically favorable for forming the sbct state . (a) femtosecond transient absorption of zdip2 with 500 nm excitation in acetonitrile . (b) comparison of dynamics for formation of the ct state (monitored at 370 nm, 2) (filled) and ground state bleach (open) between zdip1 (red squares) and zdip2 (blue circles) in acetonitrile . While femtosecond ta measurements of zdip1zdip3 show that sbct occurs in weakly polar to polar solvents, questions about deactivation pathways of the ct state still remain open . Schutz and schmidt reported that the ct state of 9,9-bianthryl recombined radiatively to the ground state or nonradiatively to triplet states in polar solvents; the nonradiative ct s0 internal conversion was negligible . In contrast to 9,9-bianthryl, poor electronic coupling makes the ct state in zdip1zdip4 at best only weakly emissive, indicating nonradiative deactivation via either direct ct s0 internal conversion or ct t1 recombination . Direct internal conversion does occur as femtosecond ta measurements show partial recovery of the ground state bleach with a concomitant decrease of the ct induced absorption (figure 10b). On the other hand, energies for the triplet states of zdip2zdip4 measured at 77 k in 2-methf (1.75, 1.74, and 1.72 ev, respectively, see table 3) are lower than those for the ct states (1.92, 1.90, and 1.84 ev, respectively, as estimated from maxima of the ct emission peaks in dichloromethane). Deactivation of the ct states was further probed by performing nano - to - microsecond transient absorption measurements on zdip1 in different solvents; results are presented in figure 11 and the si . In all solvents, induced absorption peaks from the ct state are absent within the instrument response time (approximately 20 ns), and new induced absorption bands appear at 350450 nm (max = 420 nm) and 550600 nm . To elucidate the origin of these new induced absorption features, femtosecond ta of zdip1 was measured in dichloromethane / methyl iodide (ch2cl2/ch3i 1/4) (figure 11c), a solvent mixture that is expected to accelerate intersystem crossing from the excited singlet state to the triplet state . The induced absorption in this mixture matches well with the microsecond ta spectrum of zdip1 in acetonitrile (figure 11a). Thus, the induced absorption features from 20 ns to milliseconds observed in acetonitrile are assigned exclusively to the triplet state . Formation of the triplet state in cyclohexane was also observed; however, the signal intensity is much weaker than that observed in more polar solvents (figure 11b). We speculate that formation of the triplet state via s1 t1 intersystem crossing is less efficient in cyclohexane than via s1 ct t1 recombination, as observed in toluene and acetonitrile . By fitting the microsecond ta traces to a single exponential decay, triplet lifetimes (1/kt1) for zdip1 were determined to be 16, 50, and 33 s in cyclohexane, toluene, and acetonitrile, respectively . Nano - to - millisecond transient absorption of zdip1 in (a) acetonitrile and (b) different solvents at 0.5 s and (c) femtosecond transient absorption of zdip1 in ch2cl2/ch3i (1/4). Homoleptic zinc dipyrrins zdip1zdip4 exhibit photophysical properties that are strongly influenced by solvent polarity . These solvent - dependent properties are shown to occur by deactivation of the locally excited state via a symmetry - breaking charge transfer process . Transient absorption measurements revealed that sbct proceeds in polar solvents at a rate 2 orders of magnitude faster than charge recombination . This fast charge transfer rate (1.014 ps), in combination with slower charge recombination rate (1.03.5 ns), is a desirable property for materials used in opvs, as it allows sufficient time for charge separation at a donor / acceptor interface . The weakly emissive nature of the ct state of zinc dipyrrins is in contrast to the efficient ct emission observed for bianthryl and biperylenyl . In the latter complexes, significant electronic coupling exists between the two chromophoric units . For zdip1zdip4, poor molecular orbital overlap and weak dipolar coupling between the two nearly orthogonal dipyrrin ligands, as seen in the computational studies and crystal structure (figures 2 and 3), can be used to explain the decreased emissivity . It is interesting to note that no low energy emission was detected from nonalkylated zinc dipyrrin zdip1 in polar solvent in contrast to zdip2zdip4 (see the si). The sbct also sheds light on the origin of the low luminescent efficiencies exhibited by homoleptic zinc dipyrrin complexes in polar and weakly polar solvents, especially when compared to their heteroleptic counterparts . Our results suggest that metal complexes containing a single dipyrrin ligand are promising candidates as highly fluorescent probes in a range of applications; however, the instability to disproportionation is a drawback for the zn derivatives . Finally, this study demonstrates that sbct can occur in systems where the chromophores remain weakly coupled in the excited state . It is interesting to note that other molecules that have been shown to undergo sbct exhibit some degree of electronic overlap between chromophoric units, as indicated using calculated frontier orbitals . Additionally, the previous systems have a degree of rotational freedom around a -bond, leading to a twisted internal charge transfer mechanism, whereas the zdip1zdip4 complexes are capable of sbct, even when rotational motion is severely restricted between the orthogonal ligands . Similarly, weak intermolecular couplings are ubiquitous in the neat solid, where the dielectric constant should be comparable to that of toluene (s = 2.4). Thus, formation of ct states should be a relatively common occurrence upon photoexcitation of related chromophores in the solid state.
Smoking is the main preventable cause of early and untimely death and disabilities, which leads to approximately four million deaths every year.1 although smoking causes more mortality than other factors such as acquired immune deficiency disease (aids), alcohol drinking, driving accidents, murder, suicide, and fire, but still about one third of the adult population use tobacco products worldwide.2 according to the centers for disease control and prevention (cdc) in the united states of america, 80% of adults, started smoking before the age of eighteen and about three thousand people are smoking regularly at this early stage of life.3 if no considerations are taken in order to stop this harmful trend, in the near future more than five million children will die because of future tendency to smoke . Intervention and prevention in the age of less than 20 years old has a great importance.4 smoking in this age, causes more morbidity because of accumulation of narcotics over times, also intrigues other friends to smoke cigarettes . Tobacco use at this stage of age provides the chance of being attracted to other drugs and still it is difficult for them to give up smoking though they have realized the dangers of cigarette smoking.5 therefore, planning preventive actions is an important step to be considered . National surveys in iran showed that the mean age of the first attempt to smoke was in the teenage group.6 similar situation exists in various countries such as the united states (usa),7 and china.8 in spite of marked differences in the socio - cultural background of various countries, the knowledge, attitude and practice (kap) of youths about smoking is important about their smoking behavior . Therefore, in this study, we aimed to determine the kap of a sample of iranian college students and to compare it with their american and chinese counterparts . We used a questionnaire, with the original edition prepared and edited by torabi et al . (chancellor's professor, applied health science department, indiana university, usa) in 2002.9 for the purpose of accomplishing this survey, the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment, after consulting with dr . Torabi, the necessary modifications were taken into account . To be assured about the accuracy and effectiveness of the translation, we used the back translation method, i.e. The persian - translated questionnaire was again translated in to english, and unsuitable translations were corrected . This english edition has been used for gathering information from american and chinese college students . According to the obtained information, the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . Title of the noted study is consumption of alcohol, tobacco and other drugs in children and adults of indiana . The attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions, nine of them being demographic ones . Because of the increase in water - pipe smoking by young people, we added four related items to the practice questions, hence the number of practice questions increased to 21, and the number of total questions increased to 59 . Questions about awareness had multiple choices with only one right answer, but the applied scale in questions about attitude was based on likert system . Practice questions contained 5 questions about smoking cigarette, cigar, chewing tobacco, pipe, and waterpipe . For all of these noted tobacco products, the duration of consumption in the previous month and year, as well as quitting smoking were considered . We included this group to be comparable to studies conducted in the us and china . The male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . The outcomes of the studies were analyzed statistically by version 13.0 spss for windows (spss inc ., chicago, il) in order to obtain a reliable measurement for each scale . For reliability testing of knowledge and attitude scales, cronbach's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . Regarding to the understanding of each student who consumed cigarette, the score of one was given to correct answers and zero to wrong or blank answers . So, in the first stage, regarding the knowledge of students for each answer sheet, the total scores of knowledge questions could be a number between 0 and 11 . After changing the obtained numbers to the percentage, one could achieve the percentage between 1 and 100 percent . The perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . Number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . The scale of each answer could be between 18 and 90 . To change the number to the percentage scale, the outcome would reach between 20 and 100 percent . In order to determine the differences in terms of gender, the analysis of variance (anova) was used . We used a questionnaire, with the original edition prepared and edited by torabi et al . (chancellor's professor, applied health science department, indiana university, usa) in 2002.9 for the purpose of accomplishing this survey, the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment, after consulting with dr . Torabi, the necessary modifications were taken into account . To be assured about the accuracy and effectiveness of the translation, we used the back translation method, i.e. The persian - translated questionnaire was again translated in to english, and unsuitable translations were corrected . This english edition has been used for gathering information from american and chinese college students . According to the obtained information, the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . Title of the noted study is consumption of alcohol, tobacco and other drugs in children and adults of indiana . The attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions, nine of them being demographic ones . Because of the increase in water - pipe smoking by young people, we added four related items to the practice questions, hence the number of practice questions increased to 21, and the number of total questions increased to 59 . Questions about awareness had multiple choices with only one right answer, but the applied scale in questions about attitude was based on likert system . Practice questions contained 5 questions about smoking cigarette, cigar, chewing tobacco, pipe, and waterpipe . For all of these noted tobacco products, the duration of consumption in the previous month and year, as well as quitting smoking were considered . We included this group to be comparable to studies conducted in the us and china . The male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . The outcomes of the studies were analyzed statistically by version 13.0 spss for windows (spss inc ., chicago, il) in order to obtain a reliable measurement for each scale . For reliability testing of knowledge and attitude scales, cronbach's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . Regarding to the understanding of each student who consumed cigarette, the score of one was given to correct answers and zero to wrong or blank answers . So, in the first stage, regarding the knowledge of students for each answer sheet, the total scores of knowledge questions could be a number between 0 and 11 . After changing the obtained numbers to the percentage, one could achieve the percentage between 1 and 100 percent . The perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . Number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . The scale of each answer could be between 18 and 90 . To change the number to the percentage scale, the outcome would reach between 20 and 100 percent . In order to determine the differences in terms of gender, the analysis of variance (anova) was used . We used a questionnaire, with the original edition prepared and edited by torabi et al . (chancellor's professor, applied health science department, indiana university, usa) in 2002.9 for the purpose of accomplishing this survey, the questions were translated into persian and for assuring its comprehensiveness and proportionateness to iranian cultural environment, after consulting with dr . Torabi, the necessary modifications were taken into account . To be assured about the accuracy and effectiveness of the translation, we used the back translation method, i.e. The persian - translated questionnaire was again translated in to english, and unsuitable translations were corrected . This english edition has been used for gathering information from american and chinese college students . According to the obtained information, the antecedent of questionnaire in english edition goes back to the study which was done by indiana prevention resource center . Title of the noted study is consumption of alcohol, tobacco and other drugs in children and adults of indiana . The attitude scale of using tobacco which is used in this survey was first established by awaisu et al.10 the translated questionnaire contained 55 questions, nine of them being demographic ones . Because of the increase in water - pipe smoking by young people, we added four related items to the practice questions, hence the number of practice questions increased to 21, and the number of total questions increased to 59 . Questions about awareness had multiple choices with only one right answer, but the applied scale in questions about attitude was based on likert system . Practice questions contained 5 questions about smoking cigarette, cigar, chewing tobacco, pipe, and waterpipe . For all of these noted tobacco products, the duration of consumption in the previous month and year, as well as quitting smoking were considered . We included this group to be comparable to studies conducted in the us and china . The male - to female ratio and the proportion of junior to senior students were similar to the above mentioned surveys . The outcomes of the studies were analyzed statistically by version 13.0 spss for windows (spss inc ., chicago, il) in order to obtain a reliable measurement for each scale . For reliability testing of knowledge and attitude scales, cronbach's alpha reliability coefficients were calculated for the english version of the questionnaire to examine the internal consistency of these sections of the questionnaire . Regarding to the understanding of each student who consumed cigarette, the score of one was given to correct answers and zero to wrong or blank answers . So, in the first stage, regarding the knowledge of students for each answer sheet, the total scores of knowledge questions could be a number between 0 and 11 . After changing the obtained numbers to the percentage, one could achieve the percentage between 1 and 100 percent . The perception of each student involving in smoking cigarettes was achieved to add the whole numbers in the questionnaire . Number five showed the hatred of students towards smoking cigarettes and decreased from five toward one . The scale of each answer could be between 18 and 90 . To change the number to the percentage scale, the outcome would reach between 20 and 100 percent . In order to determine the differences in terms of gender, the analysis of variance (anova) this knowledge test is not a norm reliability test and its reliability was acceptable since it relied strongly on the criteria . In iran, from 1200 responses, 958 were complete to be used in the survey, which 456 of them were the responses given by male students (48.5%) and 51.5% by female students . In china, out of 1534 students participating in the survey, 39.7% were women and 60.3% were men . In the united states, from 597 participants, 62.1% were women and 37.9% were men . Overall, these three surveys comprised a total number of 3089 students consisting of 47.5% women and 52.5% men . Significant differences existed in the structure of the place of the residence in three countries . In iran, 73.7% of students were from urban areas, whereas 47% of american college students were from suburban areas . Among chinese students, regarding to marital status, the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . When the iranian participants were asked about their health status, 92.7% of them responded that they were in a good health condition; 5.9% were on average and 1.4% were of poor health conditions . In usa, 67% enjoyed a very good health condition and 30.5% of them were average . In china, 83.5% were of good health and 14.5% were among the average . When the iranian students were asked about their daily stress, 42.2% claimed that they have not much stress, 38.6% of them told they were fairly stressful and 14.5% of them were of the average, 10.4% had no stress and 8.8% of them suffered from sever stress . Internal consistency of the knowledge and the attitude of the three groups students were compared, and the results are summarized in table 1 . In all three surveys it was shown that almost a lower level of knowledge exists (about 70%), but concerning the attitude, in all three countries satisfactory attitude score was obtained . Internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students (both men and women). Similarly, table 3 shows the average number of attitude regarding to tobacco use among students (both men and women). The anova test showed that there were no significant differences between the sexes and the average knowledge . The result obtained for men was anova = 1.26412, p = 0.628, and anova = 1.26412, p = 0.96 . But the average meaningful knowledge, however, existed between iran and the other countries . (anova for all = 342.703, p <0.001). The average number of knowledge regarding to tobacco use among students (both men and women) the average number of attitude regarding to tobacco use among students (both men and women) in iran in both men and women this attitude is more than the americans and chinese . Anova test showed that among the sexes, the average of attitude could clearly be observed (in men, anova = 141.30, p <0.001 and anova = 284.14, p <0.001 for women). There is also a meaningful scale of attitude existed between the countries studied in this survey (anova = 394.86, p <0.001). It was shown that 70% of smoking participants started smoking between the ages of 19 - 22 . The survey carried out in china shows that among the cigarette addicts, most of the smokers had started trying cigarettes even before the age of thirteen . This percentage (37.6%) is much higher than in iran (20.1%) and the us (17.9%). In iran this statistics seems to be more expressive (chi - square = 157.7, p 0.001). The age of first cigarette smoking among students (both men and women) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students, american and iranian students put much more effort to stop smoking . In iran, from 1200 responses, 958 were complete to be used in the survey, which 456 of them were the responses given by male students (48.5%) and 51.5% by female students . In china, out of 1534 students participating in the survey, 39.7% were women and 60.3% were men . In the united states, from 597 participants, 62.1% were women and 37.9% were men . Overall, these three surveys comprised a total number of 3089 students consisting of 47.5% women and 52.5% men . Significant differences existed in the structure of the place of the residence in three countries . In iran, 73.7% of students were from urban areas, whereas 47% of american college students were from suburban areas . Among chinese students, regarding to marital status, the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . When the iranian participants were asked about their health status, 92.7% of them responded that they were in a good health condition; 5.9% were on average and 1.4% were of poor health conditions . In usa, 67% enjoyed a very good health condition and 30.5% of them were average . In china, when the iranian students were asked about their daily stress, 42.2% claimed that they have not much stress, 38.6% of them told they were fairly stressful and 14.5% of them were of the average, 10.4% had no stress and 8.8% of them suffered from sever stress . Internal consistency of the knowledge and the attitude of the three groups students were compared, and the results are summarized in table 1 . In all three surveys it was shown that almost a lower level of knowledge exists (about 70%), but concerning the attitude, in all three countries satisfactory attitude score was obtained . Internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students (both men and women). Similarly, table 3 shows the average number of attitude regarding to tobacco use among students (both men and women). The anova test showed that there were no significant differences between the sexes and the average knowledge . The result obtained for men was anova = 1.26412, p = 0.628, and anova = 1.26412, p = 0.96 . But the average meaningful knowledge, however, existed between iran and the other countries . The average number of knowledge regarding to tobacco use among students (both men and women) the average number of attitude regarding to tobacco use among students (both men and women) in iran in both men and women this attitude is more than the americans and chinese . Anova test showed that among the sexes, the average of attitude could clearly be observed (in men, anova = 141.30, p <0.001 and anova = 284.14, p <0.001 for women). There is also a meaningful scale of attitude existed between the countries studied in this survey (anova = 394.86, p <0.001). It was shown that 70% of smoking participants started smoking between the ages of 19 - 22 . The survey carried out in china shows that among the cigarette addicts, most of the smokers had started trying cigarettes even before the age of thirteen . This percentage (37.6%) is much higher than in iran (20.1%) and the us (17.9%). In iran, this percentage increases along higher age groups . In the us, this statistics seems to be more expressive (chi - square = 157.7, p 0.001). The age of first cigarette smoking among students (both men and women) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students, american and iranian students put much more effort to stop smoking . In iran, from 1200 responses, 958 were complete to be used in the survey, which 456 of them were the responses given by male students (48.5%) and 51.5% by female students . In china, out of 1534 students participating in the survey, 39.7% were women and 60.3% were men . In the united states, from 597 participants, 62.1% were women and 37.9% were men . Overall, these three surveys comprised a total number of 3089 students consisting of 47.5% women and 52.5% men . Significant differences existed in the structure of the place of the residence in three countries . In iran, 73.7% of students were from urban areas, whereas 47% of american college students were from suburban areas . Among chinese students, regarding to marital status, the majority of students were single although the number of single smoker students in china was higher than single smoking ones in iran and the us . When the iranian participants were asked about their health status, 92.7% of them responded that they were in a good health condition; 5.9% were on average and 1.4% were of poor health conditions . In usa, 67% enjoyed a very good health condition and 30.5% of them were average . In china, when the iranian students were asked about their daily stress, 42.2% claimed that they have not much stress, 38.6% of them told they were fairly stressful and 14.5% of them were of the average, 10.4% had no stress and 8.8% of them suffered from sever stress . Internal consistency of the knowledge and the attitude of the three groups students were compared, and the results are summarized in table 1 . In all three surveys it was shown that almost a lower level of knowledge exists (about 70%), but concerning the attitude, in all three countries satisfactory attitude score was obtained . Internal consistency for knowledge and attitude regarding to the country table 2 shows data regarding tobacco use among students (both men and women). Similarly, table 3 shows the average number of attitude regarding to tobacco use among students (both men and women). The anova test showed that there were no significant differences between the sexes and the average knowledge . The result obtained for men was anova = 1.26412, p = 0.628, and anova = 1.26412, p = 0.96 . But the average meaningful knowledge, however, existed between iran and the other countries . The average number of knowledge regarding to tobacco use among students (both men and women) the average number of attitude regarding to tobacco use among students (both men and women) in iran in both men and women this attitude is more than the americans and chinese . Anova test showed that among the sexes, the average of attitude could clearly be observed (in men, anova = 141.30, p <0.001 and anova = 284.14, p <0.001 for women). There is also a meaningful scale of attitude existed between the countries studied in this survey (anova = 394.86, p <0.001). It was shown that 70% of smoking participants started smoking between the ages of 19 - 22 . The survey carried out in china shows that among the cigarette addicts, most of the smokers had started trying cigarettes even before the age of thirteen . This percentage (37.6%) is much higher than in iran (20.1%) and the us (17.9%). In iran, this percentage increases along higher age groups . In the us, this statistics seems to be more expressive (chi - square = 157.7, p 0.001). The age of first cigarette smoking among students (both men and women) comparing 45.7% of american and 50.2% of iranian students with 23.6% of chinese college students, american and iranian students put much more effort to stop smoking . This survey has discussed the tendency toward smoking addiction among the students and their perception of this behavior and its outcome among the students in iran, the usa, and china . In this study students the knowledge and perception of each sex in one country and also between the three countries was carefully compared and evaluated . One of the problems the researchers are faced in this kind of survey is that students of the countries are of different cultures.1112 so, we cannot expect that the students studied in these three countries give a relatively appropriate result . In the case of representation, participants should be carefully chosen and the results must be studied with almost care . There is also another criticism raised in this form of survey . Since the questionnaires used in the three countries are the same and they are originally written in english, when being translated to persian and chinese the cultural differences might cause some problems . The young people studied in iran were all medical students, so there is a great possibility that their understanding of case and also their recognition of the harmful effects of smoking cigarettes might be much better than the students of other majors . This questionnaire has already been used in america and china.78 in case of iran, we were trying to translate it in to persian in a way that somehow agrees with our culture, but the problem still exists . The original questionnaire is in american english prepared by an iranian professor (dr torabi, chancellor's professor, applied health science department, indiana university, usa).10 we have already coordinated with him concerning the translation of the questionnaire . One of the changes that we made in persian version was adding waterpipe smoking to the text . This survey showed that male students in all stages of age and in all three countries smoke more than women.13 among the smoking products, cigarette is of high consumption . Overall, beside cigarettes, cigars and pipes have had more consumers during the last year in all countries.78 fortunately, chewing tobacco (being normal in western countries) is still unknown in our country.14 a very few people use this kind of tobacco . So, the whole survey shows that the number of male students smoking tobacco products is much higher than women . Studying carefully the true responses of students to the questions concerning the knowledge, reveals that women are little more concerned . The survey also shows that american students are more knowledgeable compared to iranian and chinese students . (knowledgeable college students are about 8.70%, in america, 6.97% in iran and finally 6.84% in china). These varieties might be the result of differences between the system of education and the policies that americans have been following.12 however, there are no noticeable differences regarding the meaningful perception in all three countries . Regarding the answers given by the students about their attitudes and perception, the survey showed that women's concern about smoking was more than men that is very important and meaningful according to the statistical analysis . The perception of iranian men and women was more than the same group of people in the united states of america and china.1112 the total percentage of perception among iranian college students is 87.85%, comparing to 65.60% in america and 64.4% in china . Iranian women's attitude toward smoking depends on the cultural condition and the government policies.14 that is why women in iran smoke much less (especially in public) compared to women in china . Regarding the cultural condition in china women are much more comfortable toward smoking in both outdoors and indoors.15 any way in all countries the consumption of cigarettes is important and statistically meaningful (p <0.001). So, among these countries giving up cigarette smoking is statistically significant (p <0.001). The current survey was planned to provide more information about the motives that provide certain conditions to start smoking . Regarding the proved dangers of smoking and the results gained in the survey, important and crucial steps have been taken by the policy makers of the country to educate effectively and show the disastrous outcome of smoking all over the country . Also, it is important to mention the fact that these kinds of surveys can submit valuable information to the health trainers, health policy makers, and ministry of health . In this way, the health ministry authorities can effectively understand the importance of using tobacco products and especially its cultural aspects . Therefore, it would enable them to think of a way which results in prevention or decrease of smoking in the country . Furthermore, we suggest to add some credit hours about smoking in universities educational curriculum.
They are caused by distinct viruses that belong to different families (varicella - zoster virus, a dna virus belonging to the family herpesviridae; morbilli virus, an rna virus belonging to the family paramyxoviridae). Owing to sustained high vaccination coverage with two doses of measles vaccine, at the age of one and seven years, measles are virtually eliminated in croatia . In the last decade only sporadic imported cases of measles are reported, with the exception of two small outbreaks, which occured in 2004 and 2008 following importation of measles . Since there is no universal chickenpox vaccination in croatia and the number of vaccinated persons upon request is very low, chickenpox is a common childhood disease with an annual incidence of 2530 cases per 1000 children under 15 years of age . The first symptoms of illness (fever and rash on the trunk and extremities) appeared during their stay in italy . Upon arrival to croatia, on 5 july, two of the children (15 and eight years of age) were admitted to hospital with suspected measles and chickenpox . Both children presented with fever up to 39c, dry cough and confluent macular rash wich was most prominent on the face and neck . The 15-year - old daughter had prominent retroauricular confluent rash and koplik spots on the buccal mucosa . Five days later, on 10 july an 18-year - old boy from the same family, presented at the infectology clinic with fever up to 39c and dense confluent macular rash, with numerous scabs on the face and entire body . An epidemiological investigation revealed that he had chickenpox with disease onset at the time of their stay in italy about 15 june . The epidemiological investigation focused on ten children from the same family . In six children (including the two hospitalized girls) blood samples were taken on 14 july and sent to the refence laboratory at the croatian national institute of public health (cniph) for measles and varicella - zoster serology testing . Based on clinical manifestations and results of serology testing, four of these six children had simultaneous infection with measles and varicella and two children had chickenpox . Four siblings who had no symptoms and had no history of measles immunization were vaccinated with a monovalent measles vaccine following the investigation . Serologic tests were performed at who national measles laboratory, department of virology (cniph). Specific igm and igg antibodies to measles and varicella - zoster virus (vzv) were detected using commercial enzyme - linked immunosorbent assay (virotech, russelsheim, germany). Vzv igg positive samples were tested for igg avidity (euroimmun, lubeck, germany). Serologic results are presented in table 1 . In two patients (patiens 1 and 2) varicella infection two patients (patients 3 and 4) had positive vzv igm antibodies with low igg avidity . For two patients (patients 5 and 6), paired samples were obtained three weeks later . In both patients, varicella infection was confirmed by low igg avidity in the second sample (table 1). Table 1serologic results in six patients with suspected measles and varicella infection.patientagesexmeasles virusvaricella - zoster virusigmiggigmiggigg avidity1i11fnegativenegativepositivepositivelow2i12mnegativenegativepositivepositivelow3i15fpositivenegativenegativepositivelow4i8fpositivenegativepositivepositivelow5i18mpositivenegativepositiveequivocal - iipositivepositivepositivepositivelow6i12fpositivepositivenegativenegative - iipositivepositivepositiveequivocallow*paired serum samples (obtained three weeks later). Paired serum samples (obtained three weeks later). The epidemiological investigation revealed that none of the ten children were vaccinated against measles (which is mandatory according to the childhood immunization programme in croatia) or varicella (which is not mandatory). The fact that none of the children were vaccinated represents a public health issue in a relatively small migrant population and indicates that vaccination coverage may be low in this population . Simultaneous infection and clinical illnesses of a viral and bacterial etiology is a well known fact, as well as co - infections but not simultaneous illness caused by two distinct viruses . However, clinical disease caused by two viruses at the same time is known to be a feature of only a few viruses, notably hiv, hepatitis b and hepatitis c viruses . Our literature search revealed only a few case report of children who had measles and chickenpox at the same time . All these cases occurred at the time when incidences of both measles and chickenpox were high in the populations . Due to very high incidence rates of chickenpox and still high incidence of measles in countries with low vaccination coverage, one would expect numerous cases of simultaneous disease in the absence of some kind of immunological interference . The rarity of reports on simultaneous measles and chickenpox illness indicates that recent infection with one virus results in suppression of clinically manifest disease caused by another virus if a person acquires the other infection soon afterward . The simultaneous illness described in our report is quite unexpected because measles is virtually eliminated in croatia and in earlier decades, when measles was a common childhood disease, we have not identified cases of simultaneous clinical illness caused by measles and chickenpox.
The model used here is of a population consisting of three types of female and male individuals denoted by t0, t1 and t2 . The population reproduces in discrete and non - overlapping generations and is assumed to be stationary in senses which are to be described . This paper concerns mainly the first result of bernstein which was a proof that mendel s coefficients of heredity (to be defined in section 2) were a necessary outcome if one assumed stationarity from the first generation of offspring, plus random mating, and that the union of two of the types, t0 and t2 always produced offspring of type t1 . S.n . Bernstein (see seneta (2001) for a biographical sketch) published condensed versions of his work in two short papers (bernstein 1923a, b). The second considers the case when there are an arbitrary number of types of individuals and another (non - mendelian) form of heredity . Sheynin (2004) provides translations into english of bernstein s publications in russian which concern the 1923 papers . The first of these (bernstein, 1922) gives background to the 1923 papers and refers to the studies on evolution by charles darwin (1859), experimental findings by mendel (1866, 1965) and biometrical work of francis galton and karl pearson . The second paper (bernstein, 1924) gives the solution in detail together with other models not considered here . A similar english version of the second paper (bernstein, 1942) was provided by emma lehner of the university of california, berkeley . Lehner states that the original paper of 1924 appeared in annales scientifiques de lukraine, vol . 1 (1924). Lehner s version contains only the first half of the bernstein (1924) paper . Ballonoff (1974) reproduced bernstein s two papers of 1923 (in the original french). Later (bernstein, 1976) he published an english translation of large parts of the 1924 paper which omitted some details of the proofs of theorems (in this he transliterated the author s name as bernshtein, but in the references of this paper, in the interests of consistency, it is given as this was accompanied by a short introduction in the same periodical (ballonoff, 1976). The following quotation from ballonoff (1976) suggests that ballonoff completely missed the main point of the bernstein (1923a) paper, despite the point being spelled out in the title of the paper . There are two major results of this paper, one already an accepted part of genetics theory, the other yet un - explored! The accepted result is the <<hardy - weinberg>> law for the equilibrium of mendelian genetic systems . The other result to which ballonoff refers is outside the scope of this paper . As noted above, the main point of bernstein s research was to establish mendel s first law . Lyubich has written a number of papers, including lyubich (1971 - translated into english by j. wiegold) and lyubich (1973), which were inspired by bernstein s work, culminating in a monograph (lyubich, 1992). By the 1920 s the importance of the short paper by hardy (1908) was recognised by geneticists, although the almost simultaneous independent presentation of the same idea, almost in passing by weinberg (1908) in a more ambitious paper on the inheritance of twinning in humans, had been overlooked till the 1940 s an interesting feature of bernstein s presentation is that he derives mendel s first law through hardy s formula without any reference to hardy . The only references in bernstein (1924) were to his own paper of 1922 and to the monograph of johannsen, given as 3 . Bernstein s (1922) paper is fairly general relating mainly to his desire to enhance biology by providing mathematical underpinnings to it . In his english version of bernstein (1924) sheynin s references include: johannsen, wm (1926) elemente der exakten erblichkeitslehre . Clearly there is a discrepancy in publication years between bernstein (1924) and johannsen (1926). The english - language version of bernstein (1924) given by sheynin contains the following: here, i shall not dwell on those fundamental considerations which convinced me in that, when constructing a mathematical theory of evolution, we ought to base it upon laws of heredity obeying the principle of stationarity . I only note that the mendelian law, which determines the inheritance of most of the precisely studied elementary traits, satisfies this principle (johannsen 1926, p. 488). The so - called mendelian law concerns three classes of individuals, two of them being pure races and the third one, a race of hybrids always born when two individuals belonging to pure races are crossing . We shall assume that bernstein had to hand, in essence, johannsen (1913), that is the 2 edition [2 auflage]. Johannsen was familiar with the hardy - weinberg distribution of genotype frequencies and gives it in his book (johannsen, 1913, p.486). Our next section gives basic concepts and notation as well as a summary of the hardy - weinberg formulae . This is followed by bernstein s (1924) question and a reference to his proof without the details . We then demonstrate that stasis (that is, constancy of type proportions over all generations, including the zeroth parental proportions) is possible under non - random mating . A model of assortative mating which is a special case of non - random mating, together with a numerical example, is next . The final section suggests that johannsen supplied all the background which bernstein needed and makes some general comments . The concepts and methods which we use have been described disparagingly by ernst mayr as beanbag genetics . Individuals are completely characterised by type of which there are three, namely t0, t1, t2 . Although they are not emphasized here, genes g and determine type according to the following correspondence: gg t0; g t1; gg~t2 . The effectively infinite population is reproduced sexually in discrete and non - overlapping generations . Taking account of gender there are 9 mating combinations as defined by the matrix: (1)[t0t0t0t1t0t2t1t0t1t1t1t2t2t0t2t1t2t2] a child which is one of the three types arises from each coupling and the aggregate of children form the new generation, later to become parents, in their turn . From his study of peas for each of the above couplings it gives the set of probabilities as to type of child . If we take the outputs, by column, of the 9 couplings, mendel s first law can be expressed in the form of the following matrix: (2)m=[11/201/21/4000001/211/21/21/211/2000001/41/201/21]where the order of columns is by mating couples: (3)[t0t0,t0t1,t0t2,t1t0,t1t1,t1t2,t2t0,t2t1,t2t2]and the rows are the proportions of offspring in the respective categories t0(gg), t1(g), t2(gg). The important point to realise about m is that it expresses probabilities relating to outcomes of single coupling events . To use the law to make predictions about populations requires a further step, namely a specification of the rule of formation of the aggregate of couples . This can be expressed in the form of a matrix of proportions of couples in the order given before . Following bernstein (1924), we use the symbols,, to denote the frequencies (proportions) of the respective types t0, t1, t2, the same in each gender . Then random mating is given by a vector whose form is, in accordance with the form (3): (4)u={2,,,,2,,,,2} then the composition of the population, that is the proportions of the three types, following one round of random mating under mendel s law is given by (5)t=(mu)={(+2)2,2(+2)(+2),(+2)2} the vector t is known as the hardy - weinberg distribution after the originators weinberg (1908) and hardy (1908). This can be expressed more compactly after introducing the following definitions: (6)g=+2; g=+2 the quantities g and, which are referred to as gene frequencies, give the proportions of genes of the two kinds in the population since they weight the type frequencies according to the number of genes, g or, in each type . Then (7)t={g2,2gg,g2} the important property of the hardy - weinberg formulation follows if we now subject the new array of type frequencies to another round of random mating . The coupling frequencies are given by the vector form: (8)(u)={(g2)2,2(g2)gg,g2g2,2ggg2,(2gg)2,2ggg2,g2,g2,2g2,gg,(g2)2} since g + = 1, the type frequencies among offspring are: (9)(mu)={g2,2gg,g2}that is, identical to t. so one round of random mating produces a set of type frequencies which are maintained indefinitely under random mating, often referred to as hardy - weinberg equilibrium . Notice that the mating vector u * has the property that frequency of matings of type t1 t1 is 4 times that of either t0 t2 or t2 t0, since the corresponding array is {g, 2g,}. We express this in self - evident notation to be formalized shortly as: f11=4f02 hardy (1908) pointed out that if the initial parental frequencies satisfy the relation (10)2=4then equilibrium frequencies will have been attained under random mating, even after the first round of mating . (10), hardy s equation, points out reasons why the hardy - weinberg law is so important . Firstly, a population can be characterised by a single gene frequency rather than a set of genotype frequencies, so it provides economy of description . But much more important is the stability behaviour, that is: there is no tendency for genetic variability to dissipate . Mayo (2008) concludes the summary of his review article on the hardy - weinberg law with bernstein (1924) repeated the steps above leading to the derivation of the hardy - weinberg proportions . He then turned the problem around by asking, if the population maintains constant proportions of types, namely {,,}, after an initial round of mating, and assuming mating is random, whether this implies that the heredity coefficients are necessarily those given by m. he began with a general form denoted by m where m is given by [pqrstuvwx1pp1qq1rr1ss1tt1uu1vv1ww1xxpqrstuvwx] however this form is too general to work towards constancy of type proportions from the first generation and bernstein modified it to (11)m=[pq0qtu0ux1pp1qq11qq1tt1uu11uu1xxpq0qtu0ux] that is he assumed that mating t0 t2 and the reciprocal mating t2 t0 always produce offspring of type t1 and the other reciprocal matings produce offspring identically . After one round of random mating the population structure is (12)t=mu bernstein (1924) then submits this population to a further round of random mating and he proves that m is the only set of heredity coefficients which reproduce t, this being the hardy - weinberg array (13)t=(mu)={(+2)2,2(+2)(+2),(+2)2}that is m m. thus (14)p=1, p=0, q=1/2, q=0,t=1/4, t=1/4, u=0, u=1/2,x=0, x=1 the argument by which he shows this is most readily seen in bernstein (1942). In this section we show that with two crucial assumptions, it is possible to derive mendel s heredity coefficients under non - random mating, and the assumption of stasis: that the proportions of types remain constant from an initial parental generation . Secondly assume that the matings vector is any probability distribution of the form: (15)u={a, b, c, d,4c, d, c, d, e} thus the mating matrix (15) has the property that the frequency of mating t1 t1 is 4 times that of both t0 t2 and t2 t0 . This last echoes the condition (10) of hardy . Applying m and gives offspring of types t0 and t2: (16)t0=pa+2qb+4tc+2ud+xe (17)t2=pa+2qb+4tc+2ud+xe however the parental distribution is (18){a+b+c, b+4c+d, c+d+e} equating the coefficients of {a, b, c, d, e} in eqs . (16) and (17) with their coefficients in the parental distribution (18) yields (14). The step of equating coefficients of {a, b, c, d, e} in eqs . (16) and (17) with their coefficients in the parental distribution (18) can be justified rigorously by a few steps of matrix theory from the fact that, for example, eq . (16) is to hold for fixed {p, q, t, u, x} but for all {a, b, c, d, e} for which eq . We now denote the proportions of couples in the various mating combinations by fij, (i = 0, 1, 2; j = 0, 1, 2), indicating the proportion of the mating ti tj . Stark (1976a, b) gives a mating system which can maintain a given departure from the hardy - weinberg form of genotype frequencies . In these citations mendel s first law was assumed to hold [that is, the heredity coefficients are given by eq . The proportions of mating couples are given by (19)fij = fifj(1+mdidj / v)where fi is the genotypic frequency of ti, i = 0, 1, 2 . = f1f2 + 4f0f2+f0f1 (22)d0=(2f2+f1), d1=f0f2, d2=2f0+f1 the terms d0, d1 and d2 are phenotypic values attributed to the respective types . The second is intermediate in value between the other two and separated from each by 1 . V is the variance of these values with respect to the distribution of type frequencies and m is the correlation between mates with respect to their phenotypic values which are standardised by dividing by their standard deviation . (19) satisfies f11 = 4f02, since f02 = f0(f1)f2/v and f11 is 4 times that expression, so that, by the general result of the previous section on mating matrices of type (15), genotype frequencies are maintained, verifying the earlier results of stark (1976a, 1976b). We give a numerical example of such a mating matrix which serves to illustrate various features of the model (and which allows for considerable flexibility as demonstrated here by incorporating a t0): (23)c=1625=[00.04160.06240.04160.24960.22080.06240.22080.1008]c is symmetric with elements adding to 1 and the middle element is 4 times the upper right hand (and lower left hand) element . Summing rows and columns of c gives the distribution of types in females and males, namely {13/125, 64/125, 48/125}. The important property of c is that, if mendelian heredity coefficients are applied to it, the offspring distribution is identical to the parental distribution . The offspring then become the next parents and so can continue the population in unchanged form . Example (23) conforms to (19) with {f0, f1, f2} = {13/125, 64/125, 48/125}, m = 1/4, d0 = 32/25, d1 = 7/25, d2 = 18/25, v = 256/625 . By contrast, if random mating is applied to frequencies {13/125, 64/125, 48/125}, the distribution of offspring is {81/625, 288/625, 256/625} and following a further round of random mating, this offspring distribution is reproduced . This is stationarity in the bernsteinian sense, imitating the conclusion of the hardy - weinberg law . Note that, considering these proportions as genotypic frequencies, gene frequencies remain constant at g = 9/25 and = 16/25 under both random and assortative mating, that is stasis in this gene frequency, rather than genotype frequency, sense is achieved under both systems of mating . Since the theme of bernstein (1923a) is the main focus of this paper, it is appropriate to discuss further bernstein s achievements and limitations, in respect of that paper . We believe that this paper encapsulates bernstein s most notable contribution to genetics although he wrote much more which he considered important . In his celebrated and contentious paper fisher (1936) writes in 1930, as a result of a study of the development of darwin s ideas, i pointed out that the modern genetical system, apart from such special features as dominance and linkage, could have been inferred by any abstract thinker in the middle of the nineteenth century if he were led to postulate that inheritance was particulate, that the germinal material was structural, and that the contributions of the two parents were equivalent . Bernstein (1923a) demonstrates that he not only anticipated fisher s assertion but showed how it could be realised mathematically . Fisher believed that mendel had a clear view of his own first law during the course of his experiment . In relation to (2008, p. ix) write it is our contention that this controversy should end . While fisher (1936) continues to fascinate, bernstein (1923a) and his other writings have been largely ignored . His two papers of 1923 and bernstein (1942) are listed in felsenstein (1981) and there is a pointer to holgate (1975) against the key word stationarity. There is no reference to bernstein in wright (1969) but many to fisher and j. b. s. haldane, as well as to other notable figures in the development of population genetics . The discipline of genetics in the soviet union experienced two periods of turbulence and isolation . The first was because of the first world war and the revolution and the second was when lysenko was given wide powers of control over teaching and research in biology and agriculture . Between these two periods, for reasons related to communist ideology and politics, there was a resurgence of lamarckism . In relation to the former period, dobzhansky (1980) noted that, after a period of about seven years, acquaintance with the experimental work of the morgan school, and with the findings of other geneticists in europe and in the united states, became possible only in about 1921 . Stark and seneta (2011) kolmogorov took a stand against lysenkoism in 1940 and how the publication of a new edition of bernstein s monograph on probability was stopped because it included material on mendelism . Both weismann and johannsen were included in the list of people who were the targets of lysenko s vitriol . Johannsen (1913) was the only source cited by bernstein (1924), apart from one paper of his own . When introducing mendel s first law johannsen (1913, p. 486) gave a table, here reproduced as table 1, which perhaps motivated bernstein as to how to approach his proof . In effect the table is a derivation of the hardy - weinberg formula through functional iteration . It can be said that johannsen supplied all the information that bernstein needed for his task . Dunn (1965) and grant (1975) and others pay tribute to johannsen s important role in the development of genetics . But bernstein had to work in isolation from some important developments in mathematical genetics, such as fisher (1918), haldane (1919) and wright (1921). In johannsen (1913) there are many references to galton, and pearson, as well as to darwin, but fewer to morgan . Johannsen, although a great supporter of statistical method in biology, was one of the leaders of a group of biologists opposing the views of galton, karl pearson and weldon on inheritance (guttorp and lindgren, 2009). The conflict with pearson started with johannsen s (1903) paper (see peters, 1959 for a version in english). However yule (1904), also a member of the english biometric school, came to johannsen s defence, calling his results one of the most important contributions to genetics . It may be of some relevance that bernstein was an admirer in the times of which we speak of karl pearson s grammar of science, in a russian translation of the second edition of 1900 (read, 1982, p. 24). Johannsen (1913, p. 711) cites weinberg (1908, 1909a, b). Hill (1984, p. 12) notes that weinberg s paper of 1908 was a small part of his work in genetics . It is this paper, with hardy (1908), which is nowadays associated with the discovery of the hardy - weinberg law . Hardy s (1908) paper is cited on p. 704 of johannsen (1913), although this page is not mentioned in the index, where only hardy, 486 occurs . There is no indication in bernstein (1924) that he had used the weinberg(1908) reference and no mention of hardy . Dunn (1965, p. 94) makes a remark which is important in the context of bernstein s place in genetics in the soviet union between the two world wars and beyond . He writes likewise, johannsen, in effect, cleared the air of the fear that acquired characteristics might, after all, be inherited . Weismann s arguments had in the long run been less effective than johannsen s simple experimental demonstrations, at least with those biologists who wanted to advance the study of heredity . Johannsen s conclusion that acquired modifications were not inherited was backed up a little later by castle and phillips (1909), using an argument of quite a different kind . While bernstein s model is ingenious and is supported by intricate calculations which are best displayed in bernstein (1942), he starts out by, in a sense, violating his main postulate, namely stationarity: he requires random mating in the first (and subsequent) generations which involves a change from the initial population, unless it is already in hardy - weinberg form . It is the assumption that stationarity is required only from the first generation onwards which causes considerable mathematical difficulty in arriving at mendel s coefficients of heredity . While this assumption does imitate the hardy - weinberg law in its formulation, it does not seem realistic in considering stability of population proportions starting from an arbitrary time point . We have, in contrast, shown relatively simply that mendel s set of heredity coefficients follow necessarily from (11), and (15) which embodies the property f11 = 4f02 . This last equation reflects the situation which exists in the hardy - weinberg approach after one generation of mating . Before 1900 there were several kinds of study and much speculation aimed at elucidating the phenomenon of inheritance . Essentially, mendel s was a study of individual hereditary events which could be collated to form the basis of a theory . The basic one of these was the hardy - weinberg model (weinberg (1908), hardy (1908)). This is idealistic in that the original formulation and current usage was and is based on the assumption of random mating . It has been shown by stark (1980, 2005, 2006a, b, 2007) and li (1988) that random mating with mendelian coefficients is a sufficient, but not a necessary, condition for hardy - weinberg equilibrium . Failure to appreciate this for example wikipedia (2011) states violations from the hardy - weinberg assumptions can cause deviations from the expectations...random mating...violations...will not have hardy - weinberg proportions . The novelty of bernstein s approach is that it starts from a view of a population and posits several conditions to derive a model of inheritance for single reproductive events.
Of late, preeclampsia, a leading cause for pregnancy related morbidity and mortality, has been classified as early onset (which develops before 34 weeks of gestation) and late onset (which develops after 34 weeks). Early onset preeclampsia is considered as a fetal disorder that is typically associated with reduction in placental volume, intrauterine growth restriction, abnormal uterine and umbilical artery doppler, low birth weight, multiorgan dysfunction, perinatal death, and adverse maternal and neonatal outcomes . Late onset preeclampsia is considered a maternal condition; a result of an underlying maternal constitutional disorder, and is more often associated with normal placenta, larger placental volume, along with a normal fetal growth . Preeclampsia when associated with placental hypoperfusion results in hypoxic response in developing fetus in the form of increased erythropoiesis and release of immature erythrocytes . Several studies have shown an increased number of nucleated red blood cell (nrbc) counts in the cord blood of newborns of preeclamptic mothers [3, 4]. Nucleated red blood cells were first noted in 1871 to be present in the peripheral blood of neonates . Until the sixth and seventh weeks of gestation, all fetal red blood cells are nucleated . By the twelfth week of gestation, although rarely found circulating in older children, they are commonly seen in the blood of newborns . The rise in number of circulating nucleated red blood cells can be the result of any stimulus which increases erythropoietic activity or a sudden release from the marrow storage pools . Any hypoxic event which induces a fetal compensatory response in the form of exaggerated erythropoiesis primarily in the bone marrow results in influx of immature red blood cells into fetal circulation . Nucleated red blood cells are a potentially useful tool in estimating the degree of intrauterine hypoxia . Elevated neonatal red blood cell counts have been shown in hypoxic conditions such as erythroblastosis fetalis, maternal diabetes mellitus, acute fetal distress, intrauterine growth restriction, premature rupture of membranes, and chorioamnionitis . This study was undertaken to assess the extent to which nrbcs were associated with adverse neonatal outcome in preeclampsia and healthy pregnant women and see if this count is a reliable marker to predict early neonatal outcome . Another aspect of the study was determination of the difference between the cord blood nrbc counts of preeclamptic and healthy pregnant patients . Maternal nrbc count was studied as in some studies it was postulated that disturbed fetomaternal cell trafficking in preeclampsia leads to raised maternal nrbcs . This was a prospective case control observational study done in the department of obstetrics and gynaecology at kasturba medical hospital, which is a teaching hospital for undergraduate and postgraduate students studying at manipal university, india . Singleton pregnancies with preeclampsia with gestational period 28 weeks were included as cases and low risk healthy pregnancies as controls for comparison . We wanted to know whether nucleated rbc counts in cord blood of preeclamptic mothers significantly differ from nrbc counts of healthy pregnant women . We referred to the published mean and standard deviation for nucleated rbc count per 100 wbc in umbilical blood of normal healthy neonates (8.6 7.01). Reported that 14 nrbcs/100 leucocytes in the cord blood demarcate hypoxic babies from healthy newborns and hence we hypothesized that mean nrbc count of 14 would be significantly different from the norm . With a desired level of power of 90% and level of significance of 0.05, the sample size was calculated using the following formula: (1)n=((z+z)10)2, where z = 1.96 (critical value that divides the central 95% of z distribution from 5% in the tails), z = 1.28 (critical value that separates the lower 10% of distribution from upper 90%), = 7.01 (standard deviation), and 1 2 = 14 8.6 = 5.4 (difference of two means). The minimum sample size required accordingly was 18 subjects in case and control groups; however, we decided to recruit 50 subjects for each group . Preeclampsia was diagnosed when the blood pressure was 140/90 mm hg and there was associated proteinuria of at least 30 mg / l (1 + on dipstick) in two random urine samples or 300 mg in 24 hours at 20 weeks of gestation . In the absence of proteinuria, preeclampsia was diagnosed when blood pressure was 140/90 mm hg in association with persistent cerebral symptoms, epigastric or right upper quadrant pain plus nausea or vomiting, fetal growth restriction or with thrombocytopenia, and elevated liver enzymes . Cases with fetal distress (taken as presence of late decelerations of the fetal heart, decreased fetal heart rate variability, fetal bradycardia, or persistent fetal tachycardia), prolonged rupture of membranes (more than 6 hours), multifetal gestation, diabetes mellitus, anaemia, rh isoimmunisation, and stillbirth were excluded as these conditions themselves may produce acute rise in fetal nrbc count . Immediately after delivery, 1 ml of umbilical cord blood was collected in a tube containing 1.5 mg ethylenediaminetetraacetic acid (edta). Using automated cell counter, total leucocyte count was obtained . For the purpose of making peripheral smear, a drop of blood sample was placed towards one end of a glass slide and spreader glass slide was placed at 45 degree inclination and in one uniform motion blood was smeared on the rest of the slide . Slide was allowed to dry and was then covered with leishman's stain . After 5 minutes slide was allowed to absorb stain for 15 minutes and was then washed in gentle stream of water . Under pathologist's supervision, smear was focused under high power microscope and rbcs (nucleated) were counted against 100 wbcs (figure 1). Corrected wbc count was obtained by using the following formula: (2)corrected wbc count = (uncorrected wbc count100)(nrbc per 100 leucocytes+100). We studied various neonatal outcome measures such as apgar scores, birth weights, need for assisted ventilation, nicu admission, and respiratory distress syndrome (rds). We defined low birth weight (lbw) as weight less than 2.5 kg at birth and intrauterine growth restriction (iugr) as birth weight less than tenth percentile for corresponding gestational age . Criteria for diagnosis of rds in the newborn included any of the following: (i) presence of rapid, noisy, or difficult breathing; (ii) respiratory rate> 60/min; (iii) cyanosis, chest retraction, or grunting; (iv) need for mechanical ventilation for more than 24 hours; (v) diffuse alveolar damage as shown by chest radiography; (vi) need for surfactant therapy . Statistical analysis was done using chi - square test to compare the categorical variables and independent student's t - test for comparing continuous variables with gaussian distribution, and mann - whitney test was used to compare continuous variables with non - gaussian distribution . Receiver operating curves were used to define cutoffs of nrbc count/100 leukocytes for each neonatal variable . A total of 100 women were studied out of which 50 preeclamptic women were cases and 50 healthy pregnant women were controls . Table 1 shows maternal and neonatal characteristics of both groups . The difference in mean maternal ages in preeclampsia and control groups was not statistically significant . The mean gestational age at the time of delivery in preeclamptic patients was significantly lower compared to normal controls . Both maternal and cord blood corrected wbc counts were matching in study and control groups . Neonatal characteristics revealed significant differences between occurrence of low birth weight babies (less than 2500 grams), intrauterine growth restriction (iugr), low apgar scores, need for assisted ventilation, and respiratory distress syndrome (rds). There was also increased need for admission to neonatal intensive care unit (nicu) for the babies born to preeclamptic mothers . We also studied the influence of gestational age on the cord blood nrbc count by regression analysis . The relation between these two variables was indicated by the following equation: (3)cord blood nrbc100 wbc = 292.567.389gestational age . The goodness of fit in linear regression analysis is explained by coefficient of regression (r) which varies between 0 and 1 . Values close to 1 indicate good correlation between two variables and those close to zero indicate poor correlation . The coefficient of regression in our study was 0.1413, indicating that only 14% of variation in nrbc count was explained by gestational age (figure 2). It was found that there was no significant difference in the cord blood nucleated red blood cells of the patients who had vaginal or caesarean delivery (mean cord blood nrbcs count (mean sd): vaginal delivery (12.8 32.7), caesarean delivery (30.08 76.1), p value: 0.20statistically not significant). Significantly higher counts of nrbc were found in the cord blood of preeclamptic women than in controls (table 2). Though variation is noted in the mean cord blood nucleated red blood cell count/100 leucocytes in both preeclampsia and control groups, the mean difference between the two groups was found to be statistically significant (p = 0.006), but not in maternal blood per se (p = 0.214). There were 26 women in preeclampsia group who delivered at term and their cord blood nrbc counts were compared with control group to eliminate the confounding effect of preterm gestation on elevated nrbc count . The difference of the counts between the term preeclampsia patients and the control group was still significant (12.8 14.3 versus 5.9 6.3, p = 0.02). Significant difference existed between cord blood nucleated red blood cell counts between neonates who had iugr, those admitted to nicu, those who needed assisted ventilation, or those who had rds and those who did not have these within the preeclampsia group (table 3). This means that the cord blood nrbcs are elevated more in babies manifesting hypoxia consequences . Out of the 33 non - iugr neonates of preeclampsia mothers, 2 (6%) had low apgar, 4 (12%) had rds, 4 (12%) needed assisted ventilation, and 8 were admitted to nicu in contrast to 17 iugr neonates of preeclampsia mothers, 10 (58%) of whom had rds, 5 (29.4%) had low apgar, 6 (35.3%) needed assisted ventilation, and 13 (76.5%) needed nicu admission . Using the roc curve, cord blood nrbc count cutoff was calculated for overall neonatal variables studied (figure 3). The graph was generated by comparing sensitivity (y - axis) against false positive rate (i.e., 1 specificity) in x - axis for various cut - off points of cord blood nrbc levels . The validity of a roc plot depends upon the area under the curve (auc) which in our study was 0.71 indicating fair association between test statistics and outcome status . One or more adverse neonatal outcomes could be predicted with cord blood nrbc count> 13/100 leucocytes with a sensitivity of 63% and a specificity of 89% . Table 4 shows various neonatal outcomes using cord blood nrbc cutoff of 13 . It can be seen that cord blood nrbc count 13 is less likely to be associated with adverse neonatal outcomes . It has been stated that the inability of cytotrophoblasts to differentiate correctly and subsequent failure to invade the uterus and its arterioles efficiently in preeclampsia lead to a relatively hypoxic placenta . So compensatory mechanisms like enhanced production of erythrocytes (nucleated rbcs) are activated to counteract this imbalance . Bayarm and coworkers from departments of pediatrics, uluda university, bursa, turkey, conducted a study on nrbcs in 43 preeclamptic and 25 healthy pregnant women . Preeclamptic women were further subgrouped based on the presence or absence of intrauterine growth restriction . A nucleated red blood cell count of 18.5 or above could predict fetal asphyxia with a sensitivity of 94.4% and a specificity of 80% . Similar observations were made in the present study, though the roc analysis gave a lower cutoff for nrbc . A raised nrbc count in newborn also serves as an indicator of perinatal asphyxia of both acute and chronic origins . In a turkish study, mean cord blood nrbc count/100 leucocytes was found to be 11.18 4.92 for acute fetal distress group (n = 11) and 24.43 20.05 for chronic fetal distress group (n = 11). Both values were significantly higher than the control group (p <0.05; p <0.005, resp . ). They also determined that a cutoff of 14 nrbcs/100 leucocytes in the cord blood discriminated the acidotic fetuses from the nonacidotic ones with sensitivity of 87% and specificity of 81% . Salafia et al . From department of pathology, montefiore medical center, bronx, new york, reported that the nrbc count was independent of gestational age in well - grown, nonacidotic, nondepressed preterm infants . In the present study too, the cord blood nucleated rbc count did not vary with the gestational age in the preeclampsia group or the control group or the two groups taken together . In the same study, they concluded that the number of cord blood nucleated red blood cells could be a useful marker in determining both the duration and degree of asphyxia injury . Ghosh and coworkers from all india institute of medial sciences, new delhi, india, demonstrated a statistically significant correlation between nrbc count/100 leucocytes in the cord blood and presence of iugr, umbilical artery ph at birth, and apgar score at 1 minute in their study comprising 75 subjects . They suggested that nrbc count/100 leucocytes in the cord blood can be used as a reliable index of early neonatal outcome . Maternal nucleated red blood cells have been studied in preeclampsia patients and have been shown to be raised compared to counts in maternal blood in controls suggesting disturbed fetomaternal cell trafficking in preeclampsia . However, in the present study, no significant difference was found between nucleated red blood cell count of preeclamptic mothers and that of healthy pregnant women . Studies incorporating advanced methods (such as fish, magnetic activating cell sorting protocol, etc .) Have demonstrated significant increase in the traffic of nucleated fetal cells in the preeclamptic subjects compared to the controls [13, 14]. A low first minute apgar score in newborn is also associated with high nucleated rbc level . It has been observed that caesarean delivery for fetal distress, iugr, oligoamnios, low apgar scores, and fetal academia (as indicated umbilical arterial ph <7) were associated with statistically significant increases in nucleated red blood cell counts [16, 17]. Neonates with higher nrbc counts were more likely to be admitted to the nicu according to the results of the present study . It also has been observed that nrbc levels tend to remain elevated for longer time during neonatal period among neonates with chronic fetal asphyxia compared to those babies who had acute fetal distress during delivery . In the present study, cord blood nrbc count was significantly raised in cases of iugr babies compared to appropriate for gestational age (aga) babies within the cases of preeclampsia . Thus, nrbc count may help to distinguish growth restricted babies from constitutionally small neonates . In the present study, it was found that adverse neonatal outcome can be expected when nrbc count/100 leucocytes in the cord blood of the neonate exceeds 13 with a sensitivity of 63% and a specificity of 89% . According to the results of the present study, it may be suggested that absence of elevated nrbcs may help in ruling out adverse neonatal outcome . At higher cut - off levels, cord blood nucleated red blood cells are significantly raised in preeclampsia and are associated with adverse early neonatal outcome . Neonates with elevated cord blood nrbc counts are more likely to have iugr, low birth weight, neonatal icu admission, respiratory distress syndrome, and assisted ventilation . Below the count of 13/100 leucocytes, repeat neonatal nrbc counts could have been done during the nicu stay . If repeated at regular intervals after birth, their persistence and trend could be studied in the preeclampsia cases and comparison could be made between neonates with adverse outcomes and healthy infants.umbilical venous blood gas analysis and fetal scalp ph were not done . Relation of cord blood nrbcs with fetal acidosis could not be studied.neonates with acute fetal distress during labour were excluded to eliminate bias as this event is associated with rise in nrbc count . However, it has been opined that such a rise is relatively less compared to those babies with chronic asphyxia, and if this subgroup of babies was included in the study, this phenomenon could be documented . This is of importance because cerebral palsy due to acute birth asphyxia is of medicolegal importance . If repeated at regular intervals after birth, their persistence and trend could be studied in the preeclampsia cases and comparison could be made between neonates with adverse outcomes and healthy infants . Umbilical venous blood gas analysis and fetal scalp neonates with acute fetal distress during labour were excluded to eliminate bias as this event is associated with rise in nrbc count . However, it has been opined that such a rise is relatively less compared to those babies with chronic asphyxia, and if this subgroup of babies was included in the study, this phenomenon could be documented . This is of importance because cerebral palsy due to acute birth asphyxia is of medicolegal importance.
Neurocysticercosis (ncc) is one of the most common helminthic infestations affecting the central nervous system (cns). The clinical manifestations of the disease may vary with the size and location of cysticerci and the intensity of the host's immune response . The other clinical conditions include headache, hydrocephalus, chronic meningitis, focal neurological deficits, and psychological disorders . Altered sensorium and raised intracranial pressure (icp) may require ventilatory support in an intensive care unit (icu). Here, a 41-year - old male, presented with history of fever, headache, and episodic seizures for one month . The neurological examination and routine investigations were normal, except for eosinophilia . A computed tomographic (ct) scan of the head showed numerous tiny hypodense foci scattered throughout the brain parenchyma . Cerebrospinal fluid serology was negative for antigens of herpes simplex, tubercular, and japanese encephalitis . The patient was managed with broad spectrum antibiotics and supportive treatments, but without any improvement . One week later magnetic resonance image (mri), revealed numerous tiny lesions scattered throughout the brain parenchyma, with relative predilection for a gray - white junction and basal ganglia . Similar lesions were also noted on the scalp, tongue, extraocular muscles, muscles of the face, neck, trunk, and limbs . The diagnosis of cysticercosis was confirmed after a skin and muscle biopsy was taken, which showed cysticercus surrounded by an intense inflammatory infiltrate . The patient was treated with pyrantal pamoate, mannitol, steroids, and sodium valproate . However, the sensorium deteriorated with a glasgow coma score (gcs) of e2v1m4 . Repeat mri of the brain done two weeks later showed similar findings without any parenchymal edema . In view of the prolonged mechanical ventilation oral albendazole was added to the treatment regimen . At present, after two months of treatment, the patient is on room air with a gcs of e4vttm2 . Magnetic resonance image (t2 weighted) of the brain shows numerous tiny lesions (neurocysticercosis) scattered throughout brain parenchyma with relative predilection for a gray - white junction and basal ganglia a 35-year - old male, non - vegetarian by diet, was diagnosed as having neurocysticercosis six months back, for which he received oral phenytoin 300 mg, daily . However, non - compliance with the treatment led to three episodes of generalized seizures and altered sensorium . In view of further neurological deterioration, a contrast - enhanced ct scan of the brain showed multiple ring enhancing lesions of ncc in the bilateral hemispheres, with left ventricular hydrocephalus [figure 2]. External ventricular drainage (evd) was done, but without much improvement in the sesorium . The patient underwent surgery the next day after admission, for endoscopic removal of the ventricular neurocysticerci followed by insertion of a ventriculoperitoneal shunt . The lungs were ventilated till the gcs improved to full score, following which the trachea was extubated . Postoperatively, the medical management included administration of phenytoin and steroids; cysticidal therapy was not given considering the presence of an intraventricular cyst . He was discharged after three weeks of hospital stay with advice for follow - up . Contrast - enhanced computed tomographic scan of the brain shows multiple ring enhancing lesions in the bilateral hemispheres, with the ventricular end of the shunt - catheter in situ a 41-year - old male, presented with history of fever, headache, and episodic seizures for one month . The neurological examination and routine investigations were normal, except for eosinophilia . A computed tomographic (ct) scan of the head showed numerous tiny hypodense foci scattered throughout the brain parenchyma . Cerebrospinal fluid serology was negative for antigens of herpes simplex, tubercular, and japanese encephalitis . The patient was managed with broad spectrum antibiotics and supportive treatments, but without any improvement . One week later magnetic resonance image (mri), revealed numerous tiny lesions scattered throughout the brain parenchyma, with relative predilection for a gray - white junction and basal ganglia . Similar lesions were also noted on the scalp, tongue, extraocular muscles, muscles of the face, neck, trunk, and limbs . The diagnosis of cysticercosis was confirmed after a skin and muscle biopsy was taken, which showed cysticercus surrounded by an intense inflammatory infiltrate . The patient was treated with pyrantal pamoate, mannitol, steroids, and sodium valproate . However, the sensorium deteriorated with a glasgow coma score (gcs) of e2v1m4 . Repeat mri of the brain done two weeks later showed similar findings without any parenchymal edema . In view of the prolonged mechanical ventilation oral albendazole was added to the treatment regimen . At present, after two months of treatment, the patient is on room air with a gcs of e4vttm2 . Magnetic resonance image (t2 weighted) of the brain shows numerous tiny lesions (neurocysticercosis) scattered throughout brain parenchyma with relative predilection for a gray - white junction and basal ganglia a 35-year - old male, non - vegetarian by diet, was diagnosed as having neurocysticercosis six months back, for which he received oral phenytoin 300 mg, daily . However, non - compliance with the treatment led to three episodes of generalized seizures and altered sensorium . In view of further neurological deterioration, a contrast - enhanced ct scan of the brain showed multiple ring enhancing lesions of ncc in the bilateral hemispheres, with left ventricular hydrocephalus [figure 2]. External ventricular drainage (evd) was done, but without much improvement in the sesorium . The patient underwent surgery the next day after admission, for endoscopic removal of the ventricular neurocysticerci followed by insertion of a ventriculoperitoneal shunt . The lungs were ventilated till the gcs improved to full score, following which the trachea was extubated . Postoperatively, the medical management included administration of phenytoin and steroids; cysticidal therapy was not given considering the presence of an intraventricular cyst . He was discharged after three weeks of hospital stay with advice for follow - up . Contrast - enhanced computed tomographic scan of the brain shows multiple ring enhancing lesions in the bilateral hemispheres, with the ventricular end of the shunt - catheter in situ a 41-year - old male, presented with history of fever, headache, and episodic seizures for one month . The neurological examination and routine investigations were normal, except for eosinophilia . A computed tomographic (ct) scan of the head showed numerous tiny hypodense foci scattered throughout the brain parenchyma . Cerebrospinal fluid serology was negative for antigens of herpes simplex, tubercular, and japanese encephalitis . The patient was managed with broad spectrum antibiotics and supportive treatments, but without any improvement . One week later magnetic resonance image (mri), revealed numerous tiny lesions scattered throughout the brain parenchyma, with relative predilection for a gray - white junction and basal ganglia . Similar lesions were also noted on the scalp, tongue, extraocular muscles, muscles of the face, neck, trunk, and limbs . The diagnosis of cysticercosis was confirmed after a skin and muscle biopsy was taken, which showed cysticercus surrounded by an intense inflammatory infiltrate . The patient was treated with pyrantal pamoate, mannitol, steroids, and sodium valproate . However, the sensorium deteriorated with a glasgow coma score (gcs) of e2v1m4 . Repeat mri of the brain done two weeks later showed similar findings without any parenchymal edema . In view of the prolonged mechanical ventilation oral albendazole was added to the treatment regimen . At present, after two months of treatment, the patient is on room air with a gcs of e4vttm2 . Magnetic resonance image (t2 weighted) of the brain shows numerous tiny lesions (neurocysticercosis) scattered throughout brain parenchyma with relative predilection for a gray - white junction and basal ganglia a 35-year - old male, non - vegetarian by diet, was diagnosed as having neurocysticercosis six months back, for which he received oral phenytoin 300 mg, daily . However, non - compliance with the treatment led to three episodes of generalized seizures and altered sensorium . In view of further neurological deterioration, a contrast - enhanced ct scan of the brain showed multiple ring enhancing lesions of ncc in the bilateral hemispheres, with left ventricular hydrocephalus [figure 2]. External ventricular drainage (evd) was done, but without much improvement in the sesorium . The patient underwent surgery the next day after admission, for endoscopic removal of the ventricular neurocysticerci followed by insertion of a ventriculoperitoneal shunt . The lungs were ventilated till the gcs improved to full score, following which the trachea was extubated . Postoperatively, the medical management included administration of phenytoin and steroids; cysticidal therapy was not given considering the presence of an intraventricular cyst . He was discharged after three weeks of hospital stay with advice for follow - up . Contrast - enhanced computed tomographic scan of the brain shows multiple ring enhancing lesions in the bilateral hemispheres, with the ventricular end of the shunt - catheter in situ neurocysticercosis (cyst lodged in the cns) is the most common form of human cysticercosis, with the brain parenchyma being the most common site . It is the main cause of acquired epilepsy and neurological morbidity in many of the developing countries . As per the estimation by the world health organization (who), over 50 000 deaths occur due to ncc each year, and many times an equal number of people have active epilepsy, with all the social and economic consequences that this implies . Mri is considered the best neuroimaging tool for the detection of degenerating and innocuous (viable) cysticerci, while ct is better for calcified lesions . The added advantage of mri is that it can differentiate the stages of the parasite, which ct fails to do . Enzyme - linked immunoelctrotransfer blot (eitb) with 100% sensitivity and an overall 98% specificity is used, currently, in most of the centers . Cases of human cysticercosis with such extensive dissemination, involving all possible sites, namely, the brain, spinal cord, eyes, muscles, and subcutaneous tissues simultaneously, are indeed very rare . We report case with extensive involvement of brain parenchyma, muscles of thorax, abdomen, and thigh (case 1). In this case, sudden neurological deterioration required tracheal intubation and mechanical ventilation . Although the patient received antihelminthic, antiepileptic, and steroid medications, there was no significant neurological improvement . After tracheostomy the patient was gradually weaned off the ventilator and was able to maintain a normal saturation of arterial oxygenation, on room air . Llompart pou et al . Reported a case, who came to the emergency department with complaints of severe headache for six hours . The cyst possibly caused obstruction to the flow of csf by migration through the aqueduct of sylvius . During a two - to - five day antiparasitic therapy, exacerbation of neurological symptoms occurred, which was attributable to local inflammation, following the death of the larvae . For this reason, both albendazole and praziquantel were generally given along with steroids for the control of cerebral edema that may occur after antiparasitic therapy . Seizure is the most common symptom occurring in 70 90% of the patients with ncc . However, non - compliance to treatment in our patient led to repeated episodes of seizures, with altered sensorium . In patients with intracranial hypertension secondary to ncc antiparasitic drug treatment is never the primary treatment modality, especially in the setting of elevated icp . For intraventricular cysts, hydrocephalus is managed with a ventriculoperitoneal shunt, but these shunts suffer frequent blockade by small cysts or inflammatory exudates, and require multiple revisions . Ncc is a preventable disease; however, control of intracranial hypertension or epileptic syndromes are the mainstay of management during acute presentations.
Targeted genetic engineering is driving progress in new areas of basic biological research, biotechnology, and gene therapy . Site - specific endonucleases, including zinc - finger nucleases (zfns), meganucleases, tal effector nucleases (talens), and crispr / cas systems, have dramatically enhanced the speed and efficiency with which researchers can introduce targeted genetic modifications into cells and organisms . Although site - specific nucleases are versatile and promote a broad range of genetic alterations, they rely on cellular dna repair mechanisms, such as error - prone non - homologous end joining (nhej) and homology - directed repair (hdr), to induce custom alterations . The lack of availability of dna repair pathways within certain cell types, however, may reduce the utility of this technology . In particular, poor induction of hdr via nuclease - induced dna double - strand breaks (dsbs) or nicks has been shown to be a major limiting factor for achieving high rates of site - specific integration . Additionally, off - target dsbs induced by site - specific nucleases are difficult to comprehensively characterize in the absence of an accompanying donor template and can be potentially toxic to cells and organisms . Thus, there remains a continued need for the development of new tools capable of achieving highly precise targeted modifications with minimal toxicity . Site - specific recombinases (ssrs, e.g., cre, flp, phic31, and bxb1) are a potentially powerful alternative to site - specific nucleases for targeted genetic engineering . Ssrs are highly specialized enzymes that promote high - fidelity dna rearrangements (e.g., integration, excision, or inversion) between defined segments of dna . The strict target specificities demonstrated by many ssr systems, however, have limited their adoption in disciplines that require tools with highly flexible recognition capabilities . To overcome this, various protein engineering strategies have been used to alter ssr target specificity . While these approaches permit the design of ssr variants with new properties, they nevertheless typically lead to the emergence of relaxed specificity, an undesirable byproduct that limits the utility and safety of these enzymes . Hybrid recombinases composed of catalytic domains derived from the resolvase / invertase family of serine recombinases (e.g., gin, hin, tn3, and) fused to custom - designed cys2-his2 zinc - finger or tal effector dna - binding domains represent a unique solution to this problem (figure 1a). In particular, zinc - finger recombinases (zfrs) are a flexible class of chimeric proteins capable of introducing targeted modifications into mammalian cells . Zfrs promote site - specific recombination between dna targets that consist of two inverted zinc - finger binding sites flanking a central 20-bp core sequence recognized by the recombinase catalytic domain (figure 1a). Unlike targeted nucleases and conventional ssr systems, zfr specificity is the cooperative product of modular site - specific dna recognition and sequence - dependent catalysis . As such, new zfrs with diverse targeting capabilities can be generated in a, we have demonstrated that tailored zfr variants can be rapidly assembled from a library of pre - selected gin recombinase catalytic domains (referred to here as gin,,,,, and) and zinc - finger modules . This customization strategy allows for the design of synthetic recombinases that have the capacity to recognize a broad range of user - defined dna targets and direct site - specific integration into endogenous genomic loci . Structure of a zinc - finger recombinase (zfr) and its dimer interface . (a) top: zfr monomers (left, red; right, yellow) consist of an activated serine recombinase catalytic domain fused to a cys2-his2 zinc - finger dna - binding domain . Zinc - finger proteins (zfps) can be replaced with tal effector dna - binding domains . Model shows the structure of an engineered zfr, generated from the crystal structures of the resolvase and aart zinc - finger protein (pdb ids: 1gdt and 2i13, respectively). Abbreviations are as follows: n indicates a, t, c, or g; r indicates g or a; y indicates c or t; w indicates a or t; zfbs indicates zinc - finger binding site . (b) interactions at the gin recombinase dimer interface from two vantage points . Left e helix colored red, right e helix colored yellow . Key residues shown as sticks (pdb i d: 3uj3). Despite their ability to specifically recognize dna segments up to 56 bp in length, we previously observed that custom - designed zfrs targeted integration with low specificity . One factor contributing to this is that the protein protein interactions that govern zfr - mediated recombination are not selective for the heterodimeric zfr species . Indeed, expression of any two zfr monomers required for genomic targeting inevitably leads to the formation of two side - product zfr homodimers capable of inducing off - target genomic modifications . Similar phenomena have been observed with zfns and talens, which rely on dimerization of the foki cleavage domain for dsb induction . To overcome this, numerous studies have utilized dimer interface redesign to generate enzyme variants with improved specificity . Most notably, structure - guided and selection - based approaches have yielded obligate heterodimeric variants of the foki cleavage domain capable of enhancing zfn and talen cleavage specificity . In addition, mutagenesis of the cre recombinase dimer interface has led to the isolation of mutants with improved recombination specificity, presumably due to destabilization of cre dimer binding cooperativity . Here, we employ rational design and directed evolution to redesign the serine recombinase dimerization interface and generate a new hybrid recombinase architecture that prevents formation of side - product recombinase homodimers by> 500-fold . We show that zfrs composed of these enhanced catalytic domains demonstrate substantially improved targeting specificity and efficiency, and enable the site - specific delivery of therapeutic genes into the human genome with low toxicity . In order to redesign the gin recombinase dimer interface and engineer zfrs that preferentially heterodimerize, we sought to identify the specific amino acid residues that govern recombinase dimerization . To accomplish this, we examined the crystal structures of the resolvase dimer and the activated, tetrameric configurations of the gin and sin recombinases . We focused our search on residues within the e helix a key mediator of dimer dimer interactions between recombinase subunits and identified five residues that likely associate with one another via hydrophobic interactions: met 100, phe 103, phe 104, val 107, and met 108 (all numbers hereafter according to the gin recombinase; figure 1b). In accordance with these structural observations, previous studies had revealed that introduction of cys residues at positions 100, 103, and 107 leads to spontaneous cross - linking of two recombinase monomers . On the basis of these data, we hypothesized that substitution of these residues with complementary charged amino acids would (i) disfavor association of homodimers by charge and steric repulsion and (ii) promote heterodimer formation through favorable electrostatic contacts . To evaluate the effect that charged substitutions within the dimer interface have on recombination, we created a collection of recombinase mutants based on the gin and catalytic domains that contained either arg (gin) or asp (gin) substitutions at positions 100, 103, and 107 and evaluated their ability to recombine dna as homodimers (i.e., arg - arg or asp - asp) and heterodimers (i.e., arg - asp). We determined recombination by split gene reassembly, a previously described method that links recombinase activity to antibiotic resistance . Notably, gin homodimers that contained substitutions at position 103 showed a> 10,000-fold reduction in recombination compared to wild - type enzymes (figure s1). The corresponding heterodimer pair demonstrated a> 100-fold increase in recombination compared to the inactivated recombinase mutants; however, no heterodimeric pair recombined dna as efficiently as the wild - type enzyme (figure s1). Furthermore, combining charge substitutions did not enhance the efficiency of heterodimer - mediated recombination, presumably due to suboptimal protein protein interactions between recombinase monomers (figure s1). In order to enhance zfr heterodimer - mediated recombination, we employed directed evolution to select new dimer interface residues that more effectively facilitate heterodimerization . We randomized position 103 and the residues surrounding this region (i.e., positions 100, 104, 107, and 108) within the gin catalytic domain (figure 1b) and held the complementary gin f103r monomer constant, as preliminary analysis indicated that arg at position 103 was 2-fold more effective at preventing homodimerization than asp (figure s1). We selected recombinase variants by split gene reassembly using cells that already harbored the gin f103r mutant expression plasmid (figure 2a). To ensure the formation of the intended heterodimeric species and reduce the possibility of homodimer - mediated survival, we fused the gin catalytic domain library and the gin f103r monomer to zinc - finger dna - binding domains with orthogonal specificities . After only four rounds of selection, the activity of the mutant zfr population increased by> 500-fold in comparison to the parental gin f103d mutant (figure 2b). We sequenced individual recombinase variants from the fourth round of selection and observed a striking degree of sequence similarity at positions 103 and 107, and significant diversity at positions 104 and 108 (figure 2c). Intriguingly, we found that only 5% of selected clones contained a negatively charged residue at any position targeted for randomization . In particular, 93% of selected clones contained the native phe residue at position 103 . A nearly identical library that contained a fixed asp substitution at gin position 103 yielded no enrichment following multiple rounds of selection in the presence of gin f103r (data not shown). Re - engineering the gin recombinase dimer interface (a) schematic representation of the split gene reassembly system used to evaluate heterodimer - mediated recombination . Expression of active recombinase variants leads to restoration of the -lactamase coding sequence and host cell resistance to carbenicillin, an ampicillin analogue . Base positions 3 and 2 of the left half - site are indicated . (b) selection of gin mutants that recombine dna when paired with gin f103r . Asterisk indicates the selection step in which incubation time was decreased from 16 to 4 h. (c) mutation frequencies (%) at positions targeted for randomization in the gin catalytic domain . (d) recombination by selected gin mutants on a symmetrical dna target upon forced homodimerization (red) or on an asymmetrical target when paired with gin f103r (orange). (e) recombination by various pairs of zfrs that contain the ykwt / r dimer interface, with wild - type control . (f) recombination specificity of the ykwt / r dimer interface compared to wild - type . Virtually all selected gin recombinase monomers demonstrated high - activity (> 25% recombination) in the presence of the complementary gin f103r monomer (figure 2d). Despite the absence of negative selection pressure, we found that the majority of the selected variants also showed a reduction in recombination upon forced homodimerization on a symmetric dna target (figure 2d). One selected mutant (gin m100y, f104k, v107w, and m108 t; hereafter referred to as ykwt) demonstrated a 2000-fold enhancement in recombination on an asymmetric dna target when paired with gin f103r compared to when used as a homodimer on a symmetric target (figure 2e). This obligate heterodimer also recombined dna 2-fold more efficiently than the counterpart zfr composed of the wild - type dimer interface (figure 2e). In order to determine whether the redesigned dimer interface negatively impacted zfr catalytic specificity, we next evaluated obligate heterodimer - mediated recombination on dna targets containing mutations within the 20-bp core site recognized by the gin catalytic domain . Substitutions were introduced at core positions 3 and 2 (figure 2a), as variations at these sites are highly tolerated by evolved serine recombinases with relaxed target specificity . In comparison to zfrs that contained the wild - type dimer interface, zfrs composed of the obligate heterodimeric architecture displayed a marked decrease in recombination on a non - cognate target harboring cc substitutions at positions 3 and 2 (figure 2f). Taken together, these data indicate that the serine recombinase dimer interface can be effectively redesigned to favor heterodimerization, and that zfrs composed of these enhanced catalytic domains display improved recombination efficiency and specificity in bacterial cells . We next investigated whether the redesigned gin recombinase dimer interface could improve zfr specificity in mammalian cells . To test this (l) and right (r) monomers of a zfr pair designed to target a 44-bp sequence from a non - protein coding region of human chromosome 4 (figure 3a). Importantly, this zfr pair provides an opportunity to directly assess the effectiveness of the redesigned dimer interface, as the left left homodimer side product of this zfr pair previously exhibited substantial recombination activity on the full - length zfr target site in mammalian cells . We measured recombination using a transient reporter assay that correlates zfr - mediated recombination with reduced luciferase expression in mammalian cells (figure 3a). We co - transfected human embryonic kidney (hek) 293 t cells with a luciferase reporter plasmid containing the full - length zfr target site and expression vectors for either the l or r zfr monomers . We then directly compared the fold reduction in luciferase expression to that of 293 t cells co - transfected with both l and r zfr monomers and reporter plasmid . Impressively, we found that zfr heterodimer pairs that contained the redesigned dimer interface demonstrated substantially improved specificity in comparison to the native zfrs, reducing off - target homodimer - mediated recombination by> 200-fold in both possible configurations (l / r and l / r, figure 3b). However, these obligate heterodimeric pairs recombined dna 2- to 5-fold less efficiently than the standard zfrs (figure 3c). Western blot analysis confirmed that the reduction in activity was not due to reduced levels of protein expression (figure s2). Zfr - mediated recombination leads to excision of the sv40 promoter and reduced luciferase expression in mammalian cells . (b) relative contribution to recombination from left right heterodimers and left left and the contribution of each homodimer to recombination was calculated by measuring the fold - reduction in luciferase expression in 293 t cells transfected with either l- or r - only zfr monomers, and dividing by the value obtained from cells transfected with both l and r zfr monomers . (c) recombination efficiency of wild - type and enhanced zfr heterodimers with and without the d12 g substitution . (d) crystal structure of the resolvase (gray surface) in complex with dna (orange sticks). Regions important for recombinase activity and specificity are highlighted and labeled . To further improve the recombination efficiency of the obligate zfr heterodimers, we searched our archive of evolved gin recombinase catalytic domains and identified four mutations that were frequently observed among hyperactivated variants: d12 g, n14s, k50e, and m70v . Analysis of the crystal structure of an activated mutant of the resolvase catalytic domain indicates that these residues lie near the active site serine and may enhance catalysis by optimally positioning dna for cleavage and strand exchange (figure 3d; only d12 g is shown). We introduced each mutation individually into both the l and r monomers of the obligate heterodimeric zfr architecture and evaluated their impact on site - specific recombination . Two of the four substitutions (d12 g and n14s) enhanced the catalytic activity of the obligate heterodimers (figure s3). In particular, inclusion of d12 g led to an increase in recombination efficiency that exceeded the standard zfr heterodimer and was similar to flpe, an evolved, highly efficient site - specific recombinase routinely used for cell - line engineering (figure 3c). Comparison of the relative non - specific contribution of each zfr homodimer to recombination revealed that the d12g / ykwt and d12g / f103r substitutions (hereafter referred to as enhanced zfrs; ezfrs) retained the ability to fully prevent recombination by illegitimate homodimers (figure 3b). We next examined the portability of the ezfr architecture by introducing each of the enhancing mutations into three different zfr pairs designed to target unique 44-bp sequences present on human chromosomes 1, 4, and x. importantly, these zfr pairs are composed of distinct combinations of gin recombinase catalytic domains, each with evolved recognition specificities . As such, this analysis served to evaluate the compatibility of the redesigned dimer interface with our collection of re - engineered gin catalytic domains . In comparison to wild - type zfrs, the ezfr pairs targeting human chromosomes x and 4 demonstrated increased recombination efficiency on their intended dna targets, while the ezfr pair designed to target chromosome 1 showed reduced activity (figure s4). However, when analyzed on a panel of non - cognate target sites in the context of the luciferase reporter assay, these ezfrs showed improved recombination specificity on the majority of substrates evaluated (28 out of 32) (figure s5). Taken together, these results demonstrate that dimer interface redesign improves the recombination specificity of custom - designed zfrs in mammalian cells but that context - dependent interactions between the recombinase dimer interface and target site might influence recombination efficiency . As the primary aim of this work was the improvement of zfr specificity in the context of targeted genome engineering, we next sought to evaluate whether the ezfr framework improved the specificity of targeted integration in mammalian cells . To this end, we co - transfected hek293 t cells with enhanced and standard zfr heterodimers designed to target the previously mentioned 44-bp sequence present on human chromosome 4 together with a 4.5-kb donor plasmid containing the cognate zfr target site and a puromycin - resistance gene (figure 4a). Side - product homodimers for this zfr pair have been observed to catalyze integration at the selected genomic target . Thus, this zfr pair allows for readout of the effectiveness of the ezfr architecture for preventing homodimerization (figure 4b). We evaluated zfr and ezfr - mediated integration by pcr, amplifying the 5 and 3 junctions between the donor plasmid and the chromosomal target 72 h after transfection . As anticipated, both ezfr configurations (l / r and l / r) catalyzed integration at the intended genomic locus (figure 4b). In contrast to the standard zfrs, no site - specific integration was observed after transfection with individual l or r ezfr monomers (figure 4b). We determined the rate of genome - wide integration of the ezfr heterodimers by puromycin - selection and found that each configuration displayed improved targeting efficiency, with the l / r configuration yielding a genome - wide integration rate near 0.8% (figure 4b). These efficiencies are similar to those reported for phic31-mediated site - specific integration in hek293 cells . We next investigated the specificity of ezfr - mediated integration by pcr analysis of individually expanded puromycin - resistant clones . In total, 10 of 16 (63%) and 4 of 16 (25%) clones were positive for targeted integration by ezfrs containing the l / r and l / r heterodimeric configurations, respectively (figure 4c). Compared to the standard zfr architecture (2 of 17 clones; 11%), the l / r ezfr heterodimers demonstrated a significant increase in targeted integration (= 9.23, p <0.03), while the l/ ezfr configuration did not (= 0.99, p> 0.8). Dna sequencing confirmed site - specific integration at the intended genomic locus . Site - specific integration into the human genome by enhanced zfrs . (a) schematic representation of sequence and location of the zfr target site on human chromosome 4 . (b) bulk pcr analysis of hek293 cells transfected with an empty donor plasmid containing only a puromycin - resistance gene and various zfr pairs designed to target human chromosome 4 . (c) clonal pcr analysis of puromycin - resistant cells transfected with empty donor and ezfrs in both orientations . We next evaluated the toxicity of the ezfrs by measuring their impact on cell viability . Surprisingly, we observed that the standard zfrs targeting human chromosome 4 induced high toxicity, leading to a 60% decrease in cell viability after 5 days at the highest concentrations tested (figure 5a). In contrast, ezfrs showed no apparent toxicity and demonstrated a viability profile similar to the rare - cutting and non - toxic i - scei homing endonuclease (figure 5a). Furthermore, western blot analysis revealed no difference in expression between zfr and ezfr variants (figures 5b and s2), indicating that the improved safety profiles of the ezfrs are not attributable to reduced expression levels . Together, these findings demonstrate that ezfrs promote targeted integration with improved specificity and demonstrate substantially lower toxicity than zfrs composed of the wild - type dimer interface . (a) cell viability of hek293 cells transfected with increasing amounts of expression vector of standard or ezfrs . (b) western blot of lysate from hek293 cells transfected with increasing amounts of expression vector of standard zfrs or ezfrs . Samples were taken 48 h after transfection and probed with horseradish peroxidase - conjugated anti - ha and anti--actin (loading control) antibodies . A potential application of zfr technology is the site - specific integration of therapeutic genes into the human genome . To explore the feasibility of this goal using ezfrs, we constructed a 6.25-kb donor plasmid containing (i) the zfr target site from human chromosome 4, (ii) a puromycin - resistance gene, and (iii) the cdna for one of two disease - associated genes: the human coagulation factor ix (fix) gene, whose deficiency leads to hemophilia b, and the human -galactosidase (gla) gene, which is necessary for lipid metabolism and whose mutation results in the metabolic disorder known as fabry s disease (figure 4a). The phic31 integrase has previously been used to deliver the human fix gene into animal models, but these studies were based on random integration into pseudo - recognition sites . Co - transfection of hek293 cells with donor plasmid and ezfrs with the l / r dimeric configuration led to efficient integration of each therapeutic factor into the intended target site on chromosome 4, and puromycin selection revealed a genome - wide ezfr - mediated integration rate of 0.3 and 0.4% for the fix- and gla - harboring donor plasmids, respectively (figure 6a). We evaluated ezfr - mediated integration specificity by pcr analysis of individual puromycin - resistant clones and found that 9 of 12 (75%) and 8 of 10 (80%) clones contained fix and gla cdna, respectively, at the intended genomic target site (figure 6b), which was verified by dna sequencing . Notably, the specificity of transgene insertion achieved by these ezfrs is similar to those reported for zfns, talens, and crispr / cas systems, indicating that ezfrs are effective tools for site - specific integration into the human genome . Lastly, toward characterizing the full integration landscape of the ezfrs, we computationally identified four potential off - target sites that contain up to three mismatches compared to the intended genomic target, and evaluated off - target integration from genomic dna isolated from puromycin - resistant cells that were negative for targeted integration . We observed no transgene insertions at any of the four pseudo - integration sites (figure s6). These findings indicate that more comprehensive genome - wide approaches are required to determine the full scope of ezfr - mediated off - target modifications . Targeted integration of the human coagulation factor ix and -galactosidase genes by enhanced zfrs . (a) bulk pcr analysis of hek293 cells transfected with ezfrs targeting human chromosome 4 and donor plasmids harboring either the human coagulation factor ix (fix) or -galactosidase genes (gla). (b) clonal analysis of puromycin - resistant cells transfected with ezfrs and donor plasmids containing the fix or gla genes . Advances in targeted genetic engineering are driving progress in many fields, including biotechnology and gene therapy . While site - specific nucleases have facilitated many of these achievements, their capacity for inducing off - target mutations and reliance on dna repair mechanisms could limit their effectiveness . In particular, the establishment of a new class of tools capable of specifically and safely delivering large payloads into the human genome would be broadly useful across diverse fields, including basic research, gene therapy and synthetic biology . Hybrid recombinases based on the serine resolvase / invertase family of enzymes are a class of reagents capable of delivering genetic payloads into the human genome with potentially few side effects . However, the specificity of these enzymes has proven low, primarily due to the formation of side - product homodimers capable of catalyzing off - target modifications . In this study, we have combined rational design and directed evolution to redesign the serine recombinase dimer interface to prevent formation of these deleterious homodimers, leading to the generation of a new class of hybrid recombinases that preferentially heterodimerize and catalyze site - specific integration into endogenous genomic loci with high specificity . This work expands upon our previous studies that focused on establishing a collection of re - engineered site - specific recombinases capable of targeting a broad range of genomic target sites . These results, and in particular our finding that ezfrs specifically introduce the human coagulation factor ix and -galactosidase genes into the human genome with minimal toxicity, support the continued development of this technology for potential therapeutic applications . However, further studies are required to evaluate the activity and flexibility of these enzymes in primary cells and their potential to modify genomic safe - harbor regions with large multi - gene payloads . Future efforts will also focus on establishing optimal delivery methods by evaluating zfr compatibility with integration - deficient lentiviral vectors or adeno - associated virus . In comparison to published results in similar cell lines, the ezfrs containing the l / r dimeric configuration directed site - specific integration with specificities comparable to zfns, talens and crispr / cas9 systems; however, the efficiency of ezfr - mediated integration remained lower than those typically observed with site - specific nuclease technologies . One reason for this is that zfr - mediated recombination is reversible, and as such, insertion events may be excised shortly after integration . The design of integration - competent / excision - defective zfr variants thus represents one potential solution for enhancing zfr - mediated integration efficiency . As proof - of principle of this concept, craig and co - workers recently reported the isolation of excision - competent / integration - defective variants of the piggybac transposase . To our surprise, zfrs composed of the wild - type dimer interface induced high levels of cellular toxicity . Because zfr - mediated recombination necessitates the formation of covalent protein dna linkages that may activate the nhej repair pathway, we suspect that the high levels of cell death induced by the wild - type zfr pair could be attributed to excessive amounts of cleavage at pseudo - recombination sites by zfr homodimers or zfr - mediated rearrangements spurred by the presence of excess homodimers . Thus, the dramatic reduction in toxicity observed with ezfrs can likely be attributed to the ability of the re - engineered dimer interface to prevent recombination by side - product homodimers . The improved efficiency and specificity demonstrated by ezfrs, as well as their ability to promote site - specific integration in the absence of dsbs, suggests that these tools might also be used to modify model organisms refractory to current genome engineering methods . Moreover, this new dimer interface is extensible and should be directly portable to a broad range of hybrid recombinases, including those based on tal effector dna - binding domains, and perhaps crispr / cas technology . Although it remains unknown whether the previously described tal effector architecture supports our expanded collection of gin recombinase catalytic domains, the dimer interface substitutions described here should nonetheless improve the specificity of any tal effector recombinase heterodimer that consists solely of the wild - type gin catalytic domain . These enhanced recombinases may also find utility in synthetic biology by enabling implementation of complex computational tasks using orthogonal custom recombinases . Finally, the recombinase dimer interface mutations described in this work may facilitate new advances in the understanding of site - specific recombination . In particular, studies focused on the residues targeted in this work may shed new light on the mechanisms that govern the conformational changes during target site cleavage, strand exchange, and religation . In summary, our findings provide a general means for improving the targeting efficiency, specificity, and safety of customizable recombinases and illustrate the potential of these enzymes for diverse genome engineering applications, including therapeutic gene transfer . The split gene reassembly vector (pbla) was derived from pbluescriptii sk () (stratagene) and modified to contain a chloramphenicol resistance gene and an interrupted tem-1 -lactamase gene under the control of a lac promoter . Briefly, a gfpuv stuffer was pcr amplified with the primers gfp - zfr--h1--p2-xbai - fwd and gfp - zfr--h1--p2-hindiii - rev and cloned into the spei and hindiii restriction sites of pbla to generate the pbla - zfr substrate used for selections . Briefly, the simian vacuolating virus 40 (sv40) promoter was pcr amplified from pgl3-prm (promega) with the primers sv40-zfr - bglii - fwd and sv40-zfr - hindiii - rev . Pcr products were digested with bglii and hindiii and ligated into the same restriction sites of pgl3-prm to generate the luciferase reporter vectors pgl3-zfr-1, 2, 3,..., 9 . Zfr donor plasmids (pdonor; previously pbabe - puromycin) were constructed as previously described with the following exceptions: cdna for the human coagulation factor ix (fix) and -galactosidase (gla) genes (genecopoeia) were pcr amplified with the primers psti - cmv - donor - fwd and bamh1-zfr - donor - rev . Pcr products were digested with psti and bamh1 and ligated into the same restriction sites of pdonor . Correct construction of each plasmid was verified by sequence analysis (tables s1 and s2). All primer sequences are provided in table s3 . To construct the zfr library, residues 1115 of the gin catalytic domain were pcr amplified from pbla - gin--h1 with the primers puc18-prim-2 and gin - dimer - lib - rev . Mutations were introduced at positions 100, 103, 104, 107, and 108 with the degenerate codon dnk (d: a, t, or g; n: a, t, c, or g; and k: g or t), which encodes all amino acids except pro, his and gln . Residues 115 through 144 of the gin catalytic domain and the h1 zinc - finger protein were pcr amplified from pbla - gin--h1with the primers gin - dimer - fwd and puc18-prim-1 and fused to the gin library by overlap pcr with the primers puc18-prim-1 and -2 . Fusion pcr products were digested with saci and xbai and ligated into the same restriction sites of pbla . Ligations were ethanol precipitated and transformed by electroporation into e. coli top10f (invitrogen) cells, which were modified to harbor the expression vector pprolar - gin--f103r - p2 (clontech laboratories, inc . ). After 1 h recovery in super optimal broth with catabolite suppression (soc) medium, cells were incubated with 100 ml of super broth (sb) with 30 g ml of chloramphenicol and cultured at 37 c with shaking . At 16 h, 30 ml of cells was harvested by centrifugation and plasmid dna was isolated by mini - prep (invitrogen); 3 g of plasmid dna was then used to transform e. coli top10f. After 1 h recovery in 5 ml soc, a portion of cells was plated on solid lysogeny broth (lb) with 30 g ml of chloramphenicol or 30 g ml of chloramphenicol and 100 g ml of carbenicillin, an ampicillin analogue . Recombination was determined as the number of colonies on chloramphenicol / carbenicillin plates, divided by the number of colonies on chloramphenicol - only plates . Colony number was determined by automated counting using the geldoc xr imaging system (bio - rad). The remaining recovery culture was incubated with 100 ml of sb medium with 30 g ml of chloramphenicol and 100 g ml of carbenicillin . At 16 h, cells were harvested, and plasmid dna was purified by maxi - prep (invitrogen). Selected zfrs were isolated by saci and xbai digestion and ligated into unmodified pbla for further selection . After each round of selection, sequence analysis (eton biosciences) was performed on individual carbenicillin - resistant clones . Recombination assays with individually selected zfrs was performed as described above . For zfr construction, the gin,,,,, and catalytic domains were pcr amplified from the previously described templates pbla - gin-,,,,, or as two fragments with the primers gin - hbs - d12g - koz and gin - ykwt - rev or gin - f103r - rev and gin - ykwt - fwd or gin - f103r - fwd and gin - agei - rev . Pcr products were fused by overlap pcr with the primers gin - hbs - d12g - koz and gin - agei - rev and cloned into the hindiii and agei restriction sites of pbh to generate the new superzif - compatible sub - cloning vectors: pbh - d12g - gin--, -, -, -, -, or -ykwt or f103r (ykwt denotes the mutations m100y, f104k, v107w, and m108 t). Previously constructed zinc - finger domains were ligated into the agei and spei restriction sites of the appropriate pbh - gin sub - cloning vector to generate pbh - ezfr - l - or - r-1, 2, 3, 4, or 5 (ezfr: enhanced zfr; l: left ezfr; r: right ezfr). Each ezfr gene was released from pbh by sfii digestion and ligated into pcdna 3.1 (invitrogen) to generate pcdna - ezfr - l- or - r-1, 2, 3, 4, or 5 . Human embryonic kidney (hek) 293 and 293 t cells (american type culture collection, atcc) were maintained in dulbecco s modified eagle s medium (dmem) containing 10% (v / v) fetal bovine serum (fbs; gibco) and 1% (v / v) antibiotic - antimycotic (anti - anti; gibco). Hek293 t cells were seeded onto 96-well plates at a density of 4 10 cells / well and established in a humidified 5% co2 atmosphere at 37 c . At 24 h after seeding, cells were transfected with 2550 ng of pcdna - ezfr - l-1 through 6, 2550 ng of pcdna - ezfr - r-1 through 6, 25 ng of pgl3-zfr, and 1 ng of prl - cmv (promega) using lipofectamine 2000 (invitrogen) according to the manufacturer s instructions . For cells transfected with only one zfr monomer, empty pcdna, cells were lysed with passive lysis buffer (promega), and luciferase expression was determined with the dual - luciferase reporter assay system (promega) using a veritas microplate luminometer (turner biosystems). Hek293 cells were seeded onto 24-well plates at a density of 1 10 cells / well and maintained in serum - containing media in a humidified 5% co2 atmosphere at 37 c . At 24 h after seeding, cells were transfected with 80 ng of pdonor, 10 ng of pcdna - ezfr - l-1, 10 ng of pcdna - ezfr - r-1, and 1 ng of pcmv - egfp (clontech) using lipofectamine 2000 according to the manufacturer s instructions . We note that ezfrs - l- and - r-1 target human chromosome 4 . At 24 h after transfection, transfection efficiency was determined by flow cytometry analysis of egfp expression (facscan dual laser cytometer; bd biosciences; facsdiva software). At 72 h after transfection, cells were harvested, and genomic dna was isolated using quick extract dna extraction solution (epicentre). Zfr targets and gapdh were pcr amplified from bulk genomic dna by nested pcr with the following primer combinations: gapdh - external - fwd and gapdh - external - rev, gapdh - internal - fwd and gapdh - internal - rev (control); zfr-1-external - rev and cmv - external, zfr-1-internal - rev and cmv - internal (forward integration); zfr-1-external - fwd and cmv - external, zfr-1-internal - fwd and cmv - internal (reverse integration). For pdonor vectors that harbored the human fix and gla genes, the following primers were used for internal pcr: zfr-1-internal - rev and fix - internal (fix forward integration); zfr-1-internal - fwd and fix - internal (fix reverse integration); zfr-1-internal - rev and gla - internal (gla forward integration); zfr-1-internal - fwd and gla - internal (gla reverse integration). For colony counting assays, at 72 h post - transfection, cells were split into 6-well plates at a density of 1 10 cells / well and maintained in serum - containing media with or without 2 g ml of puromycin . At 1418 days, cells were stained with 0.2% crystal violet staining solution, and genome - wide integration rates were determined by counting the number of colonies formed in puromycin - containing media divided by the number of colonies formed in the absence of puromycin . Colony counting was determined by automated counting using the geldoc xr system (bio - rad). For clonal analysis, at 72 after post - transfection, 1 10 cells were split onto a 100-mm dish and maintained in serum - containing media with 2 g ml of puromycin . Individual colonies were isolated with 10- 10-mm open - ended cloning cylinders (millipore) with sterile silicone grease (millipore) and expanded in 96-well plates in the presence of 2 g ml of puromycin . Genomic dna was isolated and used as the template for pcr as described above . Sequence analysis (eton biosciences) was performed across the 5 and 3 junctions for each amplicon . At 48 h post - transfection, zfr expression was analyzed by sds - page with a novex 420% tris - glycine gel (invitrogen). Samples were transferred onto a 0.2 m nitrocellulose membrane and incubated for 2 h in transfer buffer (25 mm tris - base, 0.2 m glycine, 20% methanol, ph 8.5). Membranes were washed with 1 tbs (50 mm tris - hcl, 150 mm nacl, 0.05% tween 20, ph 7.5) and visualized by automated chemiluminescence visualization using the gel doc xr imaging system . Zfr was detected by horseradish peroxidase conjugated anti - ha antibody (roche). -actin was used as an internal loading control and was detected with peroxidase - conjugated anti--actin antibody (sigma). Hek293 cells were seeded onto 24-well plates at a density of 1 10 cells / well . At 24 h after seeding, cells were transfected with 25500 ng of pcdna - zfr - l-1, 25500 ng of pcdna - zfr - r-1, 80 ng of pdonor, and 10 ng of pcmv - egfp . At 30 h post - transfection, cells were collected, and egfp fluorescence was measured by flow cytometry (facscan dual laser cytometer; bd biosciences; facsdiva software). For each sample, 50,000 live events were collected, and data were analyzed using flowjo (tree star, inc . ). At 5 days post - transfection, cells were again collected, and egfp fluorescence was measured via flow cytometry as before . Zfr - mediated toxicity was calculated by dividing the number of total viable cells (i.e., egfp - positive cells) measured at 5 days post - transfection by the number of egfp - positive cells at 30 h post - transfection.
Paragangliomas (pg) are the neoplasms arising from clusters of neuroendocrine cells in association with sympathetic and parasympathetic nervous system . These tumors include pheochromocytoma, carotid body tumor and other extra - adrenal paragangliomas (eap), which have a widespread distribution . The use of fine - needle aspiration (fna) in the diagnosis of paraganglioma has been controversial due to potential risk of hemorrhage within the lesion and precipitating a hypertensive crisis . However, when eap is not suspected clinically due to lack of typical presentation, fna is often used as a diagnostic modality for initial workup . The clinical differential diagnoses of eap in the head and neck region include lymphadenopathy, branchial cleft cyst, carotid artery aneurysm, carcinoma thyroid, and neural tumors . Eap poses a diagnostic challenge because of its widespread anatomic distribution, subtle clinical manifestations and a variety of morphologic patterns . The aim of this study is to describe the cytological findings of eap and discuss the differential diagnoses . Seven cases cytologically diagnosed as eap over a period of 10 years (2003 - 2012) were retrieved . Only one case presenting as superficial swelling in right upper cervical region was clinically suspected to be carotid body tumor . The superficial swellings were aspirated directly, and the retroperitoneal masses were aspirated under ultrasound guidance using 22-gauge lumbar puncture needle fitted to a 10 ml syringe . Smears were reviewed for cellularity, architectural pattern, cell shape, cytoplasm, nuclear features, and background . The age range was 25 - 75 years (mean 48.76 years), four patients were males and three were females . The sites involved were carotid body region (four cases), para - aortic region (two cases) and para - pharyngeal space (one case). Clinical details and radiologic findings in extra - adrenal paraganglioma (n = 7) the smears were cellular in five cases, while two cases were paucicellular [table 2]. The tumor cells were singly scattered and in clusters with focal acinar formations [figure 1a]. The cells were mostly round to oval, polygonal with plasmacytoid appearance at places [figure 1b]. The nuclei were single or multiple, pleomorphic with bland nuclear chromatin and prominent nucleoli in all cases . Irregular nuclear membranes and grooves were observed in three cases, with the presence of intranuclear inclusions in two and nuclear budding in one case [figure 1c]. Smears from extra - adrenal paraganglioma showing: (a) rich cellularity with the presence of cell clusters and dispersed cells (mgg, 200), (b) round to polygonal cells with nuclear pleomorphism, abundant granular cytoplasm and a few binucleated cells (arrow) (mgg, 400), (c) prominent intranuclear cytoplasmic inclusion (arrow) (mgg, 600) and a cell with nuclear budding (inset) (mgg, 600), (d) cell block preparation with clusters of tumor cells on the left side (h and e, 100) and immunohistochemistry for chromogranin with strong cytoplasmic positivity on the right side (ihc, 400) spectrum of cellular details in extra - adrenal paraganglioma (n = 7) histopathologic examination was done in five cases . Sections showed solid tumor with typical organoid pattern and pseudo - rosettes were seen in one case . The tumor cells were large polygonal cells with cellular and nuclear pleomorphism and abundant granular cytoplasm . All cases showed cytoplasmic immunopositivity for chromogranin and a final diagnosis of eap was made . Two cases were finally diagnosed as carotid body tumor on doppler ultrasound vascular imaging and computed tomography (ct) angiography . One case was clinically suspected to be carotid body tumor based on ultrasound findings prior to the fna procedure . The smears were cellular in five cases, while two cases were paucicellular [table 2]. The tumor cells were singly scattered and in clusters with focal acinar formations [figure 1a]. The cells were mostly round to oval, polygonal with plasmacytoid appearance at places [figure 1b]. One case with a para - aortic mass showed prominent spindling of tumor cells . The nuclei were single or multiple, pleomorphic with bland nuclear chromatin and prominent nucleoli in all cases . Irregular nuclear membranes and grooves were observed in three cases, with the presence of intranuclear inclusions in two and nuclear budding in one case [figure 1c]. Smears from extra - adrenal paraganglioma showing: (a) rich cellularity with the presence of cell clusters and dispersed cells (mgg, 200), (b) round to polygonal cells with nuclear pleomorphism, abundant granular cytoplasm and a few binucleated cells (arrow) (mgg, 400), (c) prominent intranuclear cytoplasmic inclusion (arrow) (mgg, 600) and a cell with nuclear budding (inset) (mgg, 600), (d) cell block preparation with clusters of tumor cells on the left side (h and e, 100) and immunohistochemistry for chromogranin with strong cytoplasmic positivity on the right side (ihc, 400) spectrum of cellular details in extra - adrenal paraganglioma (n = 7) sections showed solid tumor with typical organoid pattern and pseudo - rosettes were seen in one case . The tumor cells were large polygonal cells with cellular and nuclear pleomorphism and abundant granular cytoplasm . All cases showed cytoplasmic immunopositivity for chromogranin and a final diagnosis of eap was made . Two cases were finally diagnosed as carotid body tumor on doppler ultrasound vascular imaging and computed tomography (ct) angiography . One case was clinically suspected to be carotid body tumor based on ultrasound findings prior to the fna procedure . A substantial number of eaps are nonfunctional; hence, a correct clinical diagnosis is seldom made . Fna may be a first - line of investigation in such cases and help in arriving at a preliminary or final diagnosis . Eap are uncommon neoplasms, known to occur in a wide variety of anatomic sites . Among eap, carotid body region is the most common site as in the present series, followed by the retroperitoneum and mediastinum . Majority have a dispersed cell population with acinar / rosette formations of round to oval, plasmacytoid or spindle shaped cells with considerable anisokaryosis . Abundant cytoplasm with red granules, vacuolization and indistinct cytoplasmic borders cannot be overemphasized . The background is hemorrhagic in most of the cases . In our series, all cases showed clusters and singly scattered round to polygonal cells with pleomorphic nuclei and moderate to abundant cytoplasm with fine pink granularity . In addition, acinar formation, nuclear grooves, inclusions, membrane irregularity, budding, and nucleoli were also seen . Varma et al . Have reported nuclear budding in 8 out of 12 cytologically diagnosed cases of pg . The cytologic differentiation of paraganglioma from other entities is a herculean task due to varied anatomic locations and tremendous cellular pleomorphism noted in these tumors . In the neck region, it can be difficult to differentiate paraganglioma from thyroid neoplasms, neurogenic tumors and metastatic carcinoma . Acinar structures in paraganglioma can be mistaken for the follicles seen in follicular neoplasm of the thyroid . Occasional cases of paraganglioma may show intranuclear cytoplasmic inclusions as seen in papillary carcinoma thyroid . Presence of papillae and optically clear nuclei in papillary carcinoma thyroid are discriminating morphological features . Plasmacytoid appearance and intracytoplasmic reddish - pink granules in paraganglionic tumors may lead to a misdiagnosis of medullary carcinoma thyroid . A misdiagnosis of neurogenic tumor is not unlikely when there is predominance of ovoid or spindle shaped nuclei in some pg . The uniform and benign looking chromatin pattern of the nucleus in paraganglioma helps in distinguishing it from metastatic carcinoma . In the retroperitoneal location, differential diagnoses include epithelial tumors such as renal cell carcinoma, adrenal cortical tumor, carcinoid tumor, islet - cell tumor, chordoma, and metastatic adenocarcinoma . In renal cell carcinoma, the cytoplasm is less fragile with more prominent vacuolization and cell borders are well - defined . Adrenal cortical tumors show a lipid background and are comparatively less pleomorphic with tiny lipid droplets in the cytoplasm . The cytomorphologic features of chordoma are quite characteristic and show physaliferous cells in the background of chondroid matrix . Occasionally, hepatocellular carcinoma, neurogenic tumors, sarcomas and melanoma may also enter into the differential diagnosis . Radioimaging studies like ultrasound, ct scan with enhancement, magnetic resonance imaging and vascular imaging studies; like doppler ultrasound and selective digital subtraction angiography are useful adjuncts to cytomorphology . These studies help in exact localization of eap and also define their relation with major blood vessels . Cytomorphologic features like dispersed cell population with acinar pattern, plasmacytoid cells with salt and pepper nuclear chromatin and abundant granular vacuolated cytoplasm with indistinct cytoplasmic borders suggest a diagnosis of eap.
Probiotics are preparations of viable microorganisms, which upon ingestion exert health benefits on the host . They are among the new agents widely used for their therapeutic action [1]. The human body is home to hundreds of known bacterial species, comprising an estimated 100 trillion microorganisms . Probiotics have been deliberately incorporated during the processing of foods, because of their beneficial effects on human health [2]. Probiotics act by microbial adhesion to the target tissue, which compete with the pathogenic microbes on adhesion sites . They should adhere to saliva - coated surfaces and resist oral environmental conditions to exert probiotic effects on the oral cavity . Once adhered, they secrete antimicrobial substances such as bacteriocins, hydrogen peroxide and organic acids, which can modify the ph and the oxidation - reduction potential facilitating the elimination of pathogenic micro - organisms . In addition, probiotics can stimulate the non - specific immunity and modulate the cellular and humoral immune response . Modification of the oral cavity environment has already been used for the management of caries, periodontal disease, oral mucosal lesions, oro - pharyngeal cancers, halitosis and reduction in the levels of candida albicans . Oral health diseases are emerging as common health problems, amongst which dental caries is the most common infectious disease in humans [3]. In developing countries such as india, the major part of the population resides in rural areas lacking access to basic health facilities where oral health is largely neglected and dental caries is widely prevalent across all age groups especially in children . Therefore, there is a need to identify individuals at risk and to target preventive measures and active treatment for these individuals in the form of simple, cost - effective and easily available products . Clinical studies have demonstrated that ingestion of lactobacillus rhamnosus gg [4, 5], l. reuteri [6] and a lactobacilli mix, can reduce salivary mutans streptococci counts . Most of these studies have either used a dairy vehicle, lozenges [7] or chewing gums [8] as modes of administration, which are not commonly available in developing countries like india, facilitating a need to explore a cheaper and an easily available alternative for administering probiotics . The present study was conducted to investigate whether the oral administration of two commercially available probiotic preparations could reduce streptococcus mutans count and to evaluate the relative efficacy of them . The present study was carried out in two phases, which included an in - vitro study followed by an in - vivo study, which was a double blind, randomized, parallel and placebo controlled clinical trial conducted to assess and compare the effect of commercially available freeze dried powdered probiotics on mutans streptococci count . For the ratio of standard deviation to the difference of mean to be detected as 0.8, minimum sample size required for each group came to 10 subjects . It was further enhanced to 11 subjects in each group assuming 10% dropouts or attrition . Thus, the total number of subjects to be included in the study was 33 . In phase - one / in - vitro phase, the zone of inhibition of both probiotic groups, group a - l . Acidophilus, b. longum, b. bifidum and b. lactis (prowel, alkem laboratories, mumbai, india) and group b - lactic acid bacillus only (sporlac, uni - sankyo ltd ., hyderabad, india) against s. mutans of six different concentrations ranging from 1.5, 2 and 2.5 million spores was determined using the cup and plate method on mitis salivarius bacitracin agar with strain of lyophilized streptococcus mutans (hi media) atcc 25175 . The first step involved preparation of the media (mitis salivarius bacitracin agar) for primary isolation of the culture and muller hinton agar for antibacterial sensitivity . Then processing of the sample for antimicrobial susceptibility testing (cup plate method) for both the probiotic groups was performed; 50 l of the sample (s) and 50 l of the reference r (negative control) were dispensed in the well labeled c (control) and added to the plates as soon as possible, but no longer than 15 minutes after inoculation . After the loading of c, s and r on the plate, the plate was inverted and incubated at 35c for 16 to 18 hours . After incubation, the diameter of the zones of complete inhibition (including the diameter of the disk) was measured and recorded in millimeters . The measurements were made with a ruler on the under surface of the plate without opening the lid . The zones of growth inhibition were compared and recorded as susceptible, intermediate or resistant to each drug tested . The diameter was obtained by measuring the distance from the colony/ colonies closest to the center of the disk, then doubled and interpretation was recorded for each diameter . The colony / colonies inside the zone were picked, re - isolated, re - identified and retested in the disk - diffusion test to confirm the previous results . The presence of colonies with a zone of inhibition predicted the eventual resistance to the agent . Group c was instructed to use the placebo powder (calcium sandoz, novartis, mumbai, india). The study was conducted over a period of three weeks and examination and sampling of the subjects were done on days 0 (baseline), seven (one week), 14 (two weeks) and 21 (three weeks). The inclusion criteria were school children between 1215 years, healthy children without any systemic disorder, dmft score> 3, plaque index> 1, no history of oral prophylaxis within the past six months and no recent history of use of antimicrobial / antibacterial agents within the past three months . The exclusion criteria were schoolchildren whose parents / guardians did not give consent, subjects who were regularly using mouthwashes/ probiotic products, children who were absent on the day of examination, subjects who were using orthodontic appliances and habitual smokers . Before scheduling the present study the ethical clearance was obtained from the research review board, jaipur dental college to conduct the study and permission was obtained from the principal, govt . The participants whose parents signed a written informed consent form before being interviewed were included in the study . The trial was also registered retrospectively under clinical trials registry of india under reference no: ctri/2013/05/003677, dated 27/05/2013 . A detailed schedule of the study was prepared well in advance . During the morning hours the subjects were asked to refrain from oral hygiene measures for 24 hours before each recall visit . The examination was conducted in the play field of the school under natural light and the subjects were seated in such a way that maximum illumination was obtained . All the three powders were repackaged under sterile conditions into small transparent antistatic zip lock polythene pouches/ sachets and were individually color coded as red, blue and green . Red pouch contained group a probiotic powder containing freeze dried l. acidophilus, b. longum, b. bifidum and b. lactis (prowel, alkem laboratories, mumbai, india). Blue pouch contained group b probiotic powder containing freeze dried lactic acid bacillus only (sporlac, uni - sankyo ltd ., hyderabad, india) and green pouch contained placebo powder (calcium sandoz, novartis, mumbai, india). Six similar color coded pouches were further kept in a bigger zip lock polybag along with a stirrer, measuring jar with graduations up to 30 ml so that each study participant could use it for a period of one week until the next examination . The subjects were screened for the inclusion criteria and selected prior to the start of the study . Plaque scores were calculated for all 33 subjects by the first investigator and the subjects with varying baseline plaque scores were distributed amongst all the three groups equally using block randomization or strata of 11 subjects each . The second investigator who was blinded to the contents of the color - coded pouches carried out the allocation procedure . The color - coded pouches were distributed to the appropriate groups by the second investigator . The color - coded pouches were supplied in a regular, scheduled manner throughout the course of the study at weekly intervals . Repackaging was done just one day before the weekly recall examination to ensure the viability of the powder . The first investigator, who carried out the examination, was blinded to the allocation of study subjects to color groups and the subjects did not know which type of probiotic group they were using . The second investigator was not involved in recording of clinical parameters at any of the recall visits . The procedure of mixing the powder with water in the measuring jar using a stirrer was demonstrated after distributing the color - coded pouches . Subjects assigned to two test groups and a placebo group, were instructed to mix each sachet in 30 ml of water with the stirrer marked on the measuring jar and rinse their mouth once daily in the morning for three minutes . During the entire study period, participants were advised to exercise their usual oral hygiene practices and abstain from using any mouthwashes . Saliva samples were collected at baseline, one week, two weeks and three weeks . A pretested and validated questionnaire was used to record the information about oral hygiene practices, dietary habits, in - between meal snacking, existing dental problems and visit to a dentist . To minimize the effect of diurnal variations in the saliva, samples were collected in the morning, between 910 am and transferred immediately to microbiology laboratory within two hours . Saliva samples were vortexed and were then serially diluted in 10-fold steps in 0.05 m phosphate buffer . Aliquots of 500 l of the appropriate dilutions were cultured on the selective media for mutans streptococci, which has mitis salivarius bacitracin . All plates were cultured at 37c in a microaerophilic environment in 5% co2 for 48 hours . The streptococcus mutans count was estimated by counting the number of colony forming units (cfu) per ml of stimulated saliva . After the commencement of the study, dental health education and proper brushing techniques were taught to all the participants . A brief overview of the methodology followed is shown in fig brief overview of the methodology qualitative data collected were summarized as mean and standard deviation (mean sd). Where data had extreme values, which were found unduly affecting the mean sd, the median was used as a measure of central tendency and inter quartile range (25th and 75th percentile) as a measure of dispersion . The mean values were compared using one - way anova followed by post hoc lsd test for comparison of independent samples . For group comparisons, for all tests, a p - value of 0.05 or less was set at statistical significance . The study was carried out from 16 - 01 - 2013 to 06 - 02 - 2013 . No subject was lost to follow - up and no unintended / untoward effect was observed during the four - week follow - up period . For in - vitro study (table 1), the mean zone of inhibition for s. mutans was observed to be maximum at spore load of 2.5 million for group a probiotics . In group b, the mean zone of inhibition for s. mutans was maximum at spore load of 2 million . For probiotic group a (table 2, fig . 2), the s. mutans count significantly decreased from its baseline value to its first week (p<0.018) and second week value (p<0.018), but no significant reduction was observed at third week (p>0.054) and between first week and second week (p>0.054). However, significant reduction was observed between first week and third week (p<0.018) and between third week and second week (p<0.018). For group b (table 2, fig . 3) the s. mutans count decreased significantly from its baseline value to its first week value (p<0.018), second week value (p<0.018) and third week value (p<0.018). Also, significant reduction was observed between second week and first week (p<0.018), between third week and first week (p<0.018) and between third week and second week (p<0.018). Streptococcus mutans count during subsequent weekly intervals in group a streptococcus mutans count during subsequent weekly intervals in group b in vitro: zone of inhibition for streptococcus mutans (in mm) pairwise comparisons of streptococcus mutans levels among different time periods . Wilcoxon signed rank test for group c or the control group (table 2, fig . 4) the streptococcus mutans count did not show any significant reduction between baseline and the first week (p<0.020), second week (p<0.018) and third week (p<0.018). The present randomized control trial was conducted to evaluate the dental health outcomes following administration of commercially available freeze dried probiotic powder in the form of a mouthrinse at repeated weekly intervals in schoolchildren in jaipur . Based on the inclusion and exclusion criteria, 33 schoolchildren between 1215 years of age were included . Amongst the 33 study subjects, 19 male and 14 female subjects were randomly allocated to three groups each comprising of 11 subjects . In the present study, the examination period of 14 days was chosen to allow comparison with other studies [10, 11]. The period of seven days was chosen because most rapid changes in plaque formation take place during the first four to five days and the 21-day period was selected because the plaque becomes relatively stable by around the day 21 [12]. The two probiotic groups were selected based on the results of the in - vitro study (phase-1) that was conducted before the in - vivo phase . To ensure blinding and the viability of the powder, it was repackaged and color - coded as red, blue and green, the findings of the in - vitro study were in harmony with a study conducted by hasslf et al, in 2010 [13] where they compared the effect of eight commercial probiotic lactobacilli strains on growth inhibition of mutans streptococci and c. albicans and concluded that commercial probiotic lacto - bacilli inhibited the growth of reference strains and oral isolates of mutans streptococci and candida but the capacity differed significantly between the strains . Kll et al, in 2008 [14] used the deferred antagonism method to test the inhibitory capacity against mutans streptococci and c. albicans . The probiotic strains inhibited both the reference strains and the oral mutans streptococci isolates, except l. acidophilus . The results were consistent with the findings of simark - mattsson and coworkers in 2007 [15]. An in vitro study by ahola et al, in 2002 [5] showed that lactobacillus rhamnosus gg inhibited the colonization of streptococcal cariogenic pathogens and therefore reduced tooth decay incidence in children . The reason for this effect could be attributed to the production of bacteriocins by probiotic bacteria, which has an inhibitory effect on cariogenic pathogens [16]. An in - vitro study conducted by haukioja et al, in 2008 described two new possible mechanisms of probiotic action in the oral cavity and showed that lactobacillus and bifidobacterium strains used in commercial probiotic products may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle [16]. In the current study, short - term administration of probiotics resulted in significant reduction of streptococcus mutans counts, and thus signifies its preventive role against caries development . The results of the current study showed that the daily consumption of both the probiotics for two weeks reduced the salivary levels of s. mutans colony counts . For group a and group b, the s. mutans count decreased significantly from its baseline value to its first week value and even up to two weeks . The results were in accordance with the results of the study conducted by jindal et al, in 2011 [11] where subjects who used probiotics containing bacillus coagulans for 14 days, experienced a significant reduction in salivary s. mutans colony counts . Similar results were achieved by caglar et al, in 2008 [17] where salivary s. mutans levels in the probiotic test group significantly decreased . The difference in reduction of s. mutans count following 10 days of using the test medical device was significant compared to the control medical device . Several previous trials reported that the intake of lactobacilli - derived probiotic strains may reduce the counts of mutans streptococci in saliva [4]. Aglar et al, in 2008 [18] evaluated the effects of an ice - cream containing b. lactis on s. mutans and lactobacillus levels in the saliva and observed a significant reduction in s. mutans count, but not in lactobacillus . However, these findings were contradictory to the findings of lexner et al, in 2010 [19] as they could not observe any statistically significant difference in the microbial profiles or the estimated counts at baseline and follow - up, or between the two study groups after a short - term administration of milk supplemented with bacterium l. rhamnosus lb21 . Also, montalto et al, in 2004 [20] reported contradictory findings with an increase in lactobacillus in a fluid form or in capsules . However at the third week, the s. mutans count increased but was not significantly different between baseline and third week while in group b, it increased significantly compared to the baseline values . These findings were contradictory to the study by caglar et al, in 2006 [7] where statistically significant reduction of mutans streptococci levels was recorded after ingestion of probiotic bacteria via a straw and the tablets for three weeks, which was in contrast to the placebo controls . The variation in findings could be due to inadequate supervision and lack of interest in oral health education among rural schoolchildren . Continuous motivation, supervision and periodic reinforcement of oral hygiene practices among children are always necessary to counteract the effect of fading over a period of time, which could have been a confounding factor . It is recommended for the manufacturers to determine the physiological role, mechanisms of action, and extent of influence of probiotics on oral health using human studies targeting high - risk human populations for oral diseases and conducting further research to improve the consumer acceptance, stability, and efficacy of probiotic containing products by incorporating flavoring agents and making the products more palatable and more pleasing for the daily use . The results of the present study showed that both the probiotic groups showed short - term mutans streptococci inhibiting properties invivo . However, the trial findings cannot be generalized to the entire population as this study was conducted on rural schoolchildren . Further randomized controlled trials on different population groups are warranted to confirm or refute the long - term effects, means of administering probiotics and the dosages needed to achieve different preventive or therapeutic purposes.
Abdominal cocoon is a rare condition, which refers to partial or total encapsulation of abdominal viscera within a dense fibrous membrane . It has been referred to as peritonitis chronica fibrosa incapsulata by owtschinnikow . And abdominal cocoon is predominantly reported among females from the tropical and subtropical regions; however, cases of adult males were also reported . The correct diagnosis is not often made preoperatively, as ct and mri finding are nonspecific . A 39 year old male was admitted to our hospital with complaints of abdominal pain for years and weight loss of 10 kg within the last year . The pain was colicky, started in center of the abdomen, radiated to the periphery, aggravated by eating . There was no history of illnesses like: familial mediterranean fever, tuberculosis, sarcoidosis, systemic lupus erythematosus, protein s deficiency, malignancy, ascites or peritoneal dialysis . Abdomen ct scan showed clumping of ileal loops at the level of umbilicus, with a thin capsule surrounding it . The proximal loops appear mildly dilated and distal ileal loops collapsed . No evidence of vascular axis rotation (figs . Diagnostic laparoscopy showed that most of the small bowel was wrapped up in a grayish - pink membrane forming a cocoon (fig . The procedure was changed to laparotomy to avoid injury as it was very difficult to release the encased bowel laparoscopically . It seems from the diagnostic laparoscopy and laparotomy view that the membrane is originated from the mesothelial covering of the root of the mesentery . Excision of the whole membrane was done carefully and was sent for pathology examination . Encased small bowel loops were separated by sharp dissection (fig . Histopathology of the membrane, showed dense poorly cellular collagenised fibrous tissue, features consistent with sclerosing encapsulating peritonitis (abdominal cocoon; fig . Post operatively the patient showed improvement in his symptoms; with uneventful five months follow up . Sclerosing encapsulating peritonitis (cocoon abdomen) is a rare condition . With few cases described in literatures . Cocoon abdomen is mostly observed in young girls living in tropical and subtropical countries of the world, especially china, malaysia, singapore, pakistan, india, nigeria, kenya, saudi arabia, israel, and south africa . Although few cases have been documented in males . These include retrograde menstruation with a superimposed viral infection, retrograde peritonitis and cell - mediated immunological tissue damage incited by gynecological infection . However, since this condition has also been seen to affect males, premenopausal females and children, there seems to be little support for these theories . The secondary causes of abdominal cocoon include continuous ambulatory peritoneal dialysis, tuberculosis, systemic lupus erythematosus, sarcoidosis, familial mediterranean fever, gastrointestinal malignancy, protein s deficiency, liver transplantation, fibrogenic foreign material, luteinized ovarian thecomas, the use of povidone iodine for abdominal washout, placement of leveen shunt for refractory ascites, as well as the beta - adrenergic blocker (practolol). In our patient, the etiology appears to be primary, as all the known causes of secondary cocoon abdomen were ruled out . Patients with cocoon abdomen may present in one or multiple episodes with symptoms of acute or sub acute small bowel obstruction (that could sometimes resolve spontaneously). In this case, the patient has recurrent attacks of colicky abdominal pain and distension, which was sometimes resolved spontaneously . Abdominal mass may also be present due to an encapsulated cluster of dilated small bowel loops . A pre - operative diagnosis is usually very difficult and requires a high index of clinical suspicion . Most cases are diagnosed intraoperatively during laparotomy performed for intestinal obstruction, or during diagnostic laparoscopy . Abdominal ultrasonography may be of help in the diagnosis of cocoon abdomen by revealing an echogenic mass of dilated small - bowel loops surrounded by a thick rim of hypoechoic fibrous membrane . Barium follow - through may show delayed transit of contrast and clustering of the bowel loops in the pelvis . Ct findings may include clumping of small bowel loops in the center of the abdomen encased by a soft - tissue density mantle, peritoneal thickening and calcification, clumping of small bowel loops, and lobulated fluid collections . In this study preoperative diagnosis was possible by abdominal ct scan which showed clumping of ileal loops, surrounded by a thin capsule and small amount of fluid . Cocoon abdomen may be confused with congenital peritoneal encapsulation, which is characterized by a thin accessory peritoneal sac surrounding the small bowel, and is an incidental finding . Dissection (open or laparoscopic) of fibrotic membrane and adhesiolysis is usually sufficient to treat abdominal cocoon . Complications after surgery were reported; these include intra - abdominal infections, enterocutaneous fistula and perforated bowel . In this case follow - up has shown recurrence only in few cases, some of which resolved on conservative management . Primary sclerosing encapsulating peritonitis (cocoon abdomen) is rare condition; with unclear etiology . It affects young patients and its diagnosis needs a high index of suspicion, as signs and symptoms are nonspecific and imaging findings are not always conclusive . Careful excision of the accessory peritoneal sac and lysis of adhesions between bowels is the best treatment . The authors have no financial or personal relationships with other people or organisations to disclose . This study had been approval by the ethics committee in bahrain specialist hospital, manama, kingdom of bahrain . Analysis and interpretation: amer hashim al ani, najah al zayani, mohammad najmeddine, sunitha jacob and sunil nair . Manuscript drafted by: amer hashim al ani.
Focal gingival enlargements are quite frequent lesions in the oral cavity amounting to almost 3.1% of all oral tumors and for 9.6% of gingival lesions . In 1992, the world health organization classification about this kind of focal overgrowth had tapered toward one universal term peripheral cemento - ossifying fibroma . We, in our case report prefer to call it as peripheral ossifying fibroma (pof). Pofs are more frequent in younger ages, peak being in the second decade, although there are cases occurring in older age groups also there is uncertainty for diagnosing focal reactive overgrowths of the gingiva because of their nearly same clinical presentation . The typical appearance of pof is small gingival growth (epulis) initially which can attain large sizes so as to cause facial disfigurement . If the lesions grow to larger sizes, there can be a mild erosion of bone . The treatment of pof is straight forward with surgical excision with blade and scalpel commonly followed . A newer approach is the use of diode lasers for its removal, probably having an advantage of lesser bleeding and predicted results . Whatever may be the method of removal, it should be sent for histopathological diagnosis . Here a 60 year old female presented to us with a chief complaint of swelling on the right maxillary posterior region of jaw since 3 - 4 years . She also gave a history of the slow growth of the mass since last 3-years to reach the present size . The oral examination showed the presence of a solitary pedunculated mass of size 4 cm 2 cm 3 cm which was red in color with lobulated surface on the right maxillary posterior alveolar ridge . On palpation, the mass was firm, nontender, and not fixed to underlying structure . Orthopantomogram (opg) evaluation showed the presence of a unilocular radiolucent area extending superiorly into the corresponding maxillary sinus, posteriorly to maxillary tuberosity, and anteriorly until the distal end of canine . Dento - alveolar scan did not show the extension of mass into maxillary sinus, rather showed the inflamed lining of the sinus . A three dimensional construction image done after dento - alveolar scan confirmed the calcifications [figure 2c] within the mass which was seen as radio - opaque areas in opg . After clinical and radiographic investigations pof, peripheral giant cell granuloma (pgcg) and pyogenic granuloma (pg) (longstanding) were considered in differential diagnosis . The clinical presentation of intraoral mass (a) orthopantomogram showing the extension of mass . (b) dento - alveolar scan showing the extension of lesion and inflamed maxillary sinus lining . (c) three - dimensional construction showing calcifications within the mass a comprehensive explanation was given to the patient and sign was taken on the consent form . Excisional biopsy was carried out, and the tissue was sent for histopathological examination for confirmatory diagnosis . Macroscopically, the gross specimen was measuring 3.5 cm 2 cm 1 cm, creamish brown in color, firm to hard in consistency with lobulated, and pebbly surface . The specimen was cut into two halves, and most representative areas were taken for processing [figure 3]. The complete gross specimen the specimen was fixed in phosphate - buffered neutral formalin for 1 day, subsequently, five - micron paraffin sections were obtained and stained with hematoxylin and eosin (h and e). Microscopic analysis of the h and e section showed parakeratinized stratified squamous epithelium covering loosely arranged highly cellular connective tissue stroma [figure 4a]. Few vascular spaces of varied sizes and inflammatory infiltrate were also seen in underlying connective tissue . Based on these histologic features, (a) photomicrography showing parakeratinized stratified squamous epithelium covering loosely arranged highly cellular connective tissue stroma . Pof is clinically present as a soft nodular mass which may be either pedunculated or sessile present near the interdental papilla . Pof histopathologically is described as a lesion which has a fibrous stroma in which there is presence of mineralized tissues such as bone and/or cementum - like . However, this term is discarded as clinical and histopathological presentation is the same in those cases where there is no cementum . Some authors have reported the presence of odontogenic epithelium and also that the proliferating cells may be of myofibroblastic origin . Some researchers feel that it represents that some kind of chronic irritation is responsible for stimulation of cells in the periodontal ligament . The purpose of reporting this case was to give a brief review to the surgeon that there can be a number of lesions presenting with the same picture but have varying histopathological presentation . It is always better to be aware of the fact and plan the surgery accordingly . Pofs are common in the mandibular region and in the second decade of life usually . Irrespective of the type of mineralized component, the treatment of choice is surgical excision and there are very few cases of recurrence reported.
Worldwide, squamous cell carcinoma of the head and neck (scchn) is the sixth most common cancer and is diagnosed in more than 600,000 patients each year . A better understanding of its biology has been accompanied by the introduction of a large and rapidly expanding number of targeted agents into its management strategies . Planned and ongoing trials in scchn involving targeted agents are summarized in tables 1, 2, 3, and 4 . Potential targets include the epidermal growth factor receptor (egfr) and the erbb family, the vascular endothelial growth factor (vegf) and its receptor (vegfr), insulin - like growth factor 1 receptor (igf-1r), insulin receptor (ir), histone deacetylase (hdac), mammalian target of rapamycin (mtor), platelet - derived growth factor (pdgfr), heat - shock protein 90 (hsp90), nuclear factor - kappa b (nf-b), aurora a or b, phosphatidylinositol 3-kinase (pik3ca). The epidermal growth factor receptor (egfr) belongs to the erbb family of receptors which includes four members: egfr / erbb1, erbb2/her2/neu, erbb3, and erbb4 . Egfr consists of an extracellular n - terminal ligand - binding domain, a hydrophobic transmembrane domain, and a c - terminal intracellular domain, which includes the tyrosine kinase domain and the autophosphorylation sites . The epidermal growth factor receptor (egfr) is overexpressed in the vast majority of cases of squamous cell carcinoma of the head and neck (scchn). A high egfr expression and an increased egfr gene copy number are associated with an unfavorable prognosis [57]. Two of the potential egfr targeting strategies that are currently in use in the treatment of scchn are the monoclonal antibodies directed at the extracellular domain of the receptor and the small molecule and atp - competitive tyrosine kinase inhibitors (tkis). Cetuximab is the only targeted agent that got approval by the food and drug administration and the european medicines agency for the treatment of scchn [8, 9] and is still under active investigation in this disease (tables 1 and 2). Cetuximab is a chimeric human / murine igg1 antibody which binds with higher affinity to the egfr than the natural ligands egf and tnf-, thereby disrupting egfr signaling pathways . Another mechanism that contributes to the antitumor activity of cetuximab is depletion of the targeted receptors from the cell surface . The availability of egfr on the cell surface is reduced, and the receptor is downregulated [10, 11]. Finally, cetuximab's construction on an igg1 framework potentially allows the drug to mediate antibody - dependent cell - mediated cytotoxicity (adcc) via natural killer (nk) cells and macrophages . Cetuximab is administered once a week at an initial loading of 400 mg / m followed by a weekly dose of 250 mg / m . The recommended dose was used in the cetuximab studies in scchn studies mentioned below, unless stated otherwise . Bonner et al . [13, 14] conducted a multinational, randomized study comparing radiotherapy alone with radiotherapy plus cetuximab in 424 patients with stages iii or iv, nonmetastatic, measurable squamous cell carcinoma (scc) of the oropharynx, hypopharynx, or larynx . Investigators were required to select one of three radiotherapy - fractionation regimens before patient registration: 70.0 gy in 35 daily fractions of 2.0 gy / fraction, 5 fractions / week for 7 weeks, or two daily fractions of 1.2 gy / fraction up to 72.076.8 gy in 6064 fractions, 10 fractions / week for 66.5 weeks, or a concomitant boost regimen (72.0 gy in 42 fractions: 32.4 gy; 1.8 gy / fraction, 5 fractions / week for 3.6 weeks followed by a morning dose of 21.6 gy in fractions of 1.8 gy, 5 fractions / week for 2.4 weeks and an afternoon dose of 18.0 gy in fractions of 1.5 gy, 5 fractions / week for 2.4 weeks). Uninvolved nodal areas of the neck were treated with 50 to 54 gy, depending on the fractionation regimen used . Gross nodal disease received the same dose as the primary tumor . In the group assigned to receive radiotherapy plus cetuximab, cetuximab was initiated one week before radiotherapy at a loading dose of 400 mg / m, followed by a weekly dose of 250 mg / m for the duration of radiotherapy . The median duration of locoregional control (primary endpoint) was 24.4 months in patients treated with cetuximab plus radiotherapy and 14.9 months in patients treated with radiotherapy alone (hazard ratio (hr) for locoregional progression or death, 0.68; p = 0.005). The one-, two-, and three - year rates of locoregional control achieved with radiotherapy plus cetuximab (63, 50, and 47%), were significantly higher than those achieved with radiotherapy alone (55, 41, and 34%, resp . ). Median overall survival (os) for patients treated with cetuximab and radiotherapy was 49.0 months versus 29.3 months in the radiotherapy - alone group (hr for death: 0.73; p = 0.018)., the addition of cetuximab did not lead to an increased incidence of radiation dermatitis . However, as there is only one randomized phase iii trial with cetuximab - based bioradiation as opposed to the abundance of data supporting cisplatin - based concurrent chemoradiation (crt) [15, 16], the latter continues to represent the standard of care for medically fit patients with locoregionally (la) scchn, who can tolerate platinum - based therapy . The addition of cetuximab to cisplatin - based crt does not further improve the outcome . In radiation therapy oncology group (rtog) 0522, 895 evaluable patients with stage iii / iv nonmetastatic scchn were randomized between chemoradiation (72 gy in 42 fractions over 6 weeks plus cisplatin 100 mg / m on days 1 and 22) or the same regimen plus weekly cetuximab . At the time of the third interim analysis after 337 events and after a median followup of 2.4 years for the surviving patients, the conditional power of the trial becoming positive was below 10%, triggering early reporting . Over 90% of the patients received the planned two doses of cisplatin in both arms . The 2-year progression - free survival (pfs), (primary endpoint) was 64.3% with chemoradiation and 63.4% with chemoradiation plus cetuximab (hr: 1.05; 95% confidence interval (ci): 0.841.29; p = 0.67). The 2-year os was 79.7 and 82.6%, respectively (hr: 0.87; 95% ci: 0.661.15; p = 0.17). The estimated 2-year locoregional relapse rate was 19.8 and 24.5%, respectively (p = 0.92). The 2-year distant metastasis rate was 12 and 7.6%, respectively (p = 0.07). Overall, there was no difference regarding acute grade 3/4 acute toxicities between both arms . However, grade 3/4 mucositis (43 versus 33%) and in - field dermatitis (25 versus 15%) was more common with the addition of cetuximab . Grade 3/4 dermatitis outside the radiation field occurred in 19% of the patients treated with cetuximab . The tremplin trial is a randomized phase ii study in patients with scc of the larynx or hypopharynx suitable for total laryngectomy . After three 3 weekly cycles of tpf (docetaxel 75 mg / m and cisplatin 75 mg / m on day 1 followed by 5-fu 750 mg / m/day, days 15), patients who obtained at least a partial response (82% of the patients) were randomized to receive radiotherapy (70 gy in 35 fractions over 7 weeks) either with cisplatin 100 mg / m on days 1, 22, and 43 or with weekly cetuximab . The treatment compliance was better in the cetuximab arm with 71% of the patients receiving all planned cetuximab administrations . Forty - three percent of the patients received three cycles of cisplatin, and 83% received 2 cycles . There was no difference in grade 3/4 mucosal toxicity, but grade 3/4 in - field dermatitis was more frequently observed with cetuximab (57 versus 26%; p <0.001). Grade 1 renal dysfunction at last evaluation was observed in 22.4% of the patients treated with cisplatin . The larynx preservation rate 3 months after treatment (primary endpoint) was 95% with cisplatin versus 93% with cetuximab . The locoregional failure rate after a median followup of 36 months was 11.7% with cisplatin and 21.4% with cetuximab . However, more salvage laryngectomies were performed in the cetuximab arm, resulting in a similar ultimate locoregional failure rate in the two arms (10% versus 8.9%). The 2-year laryngoesophageal dysfunction - free survival was 79% with cisplatin versus 71% with cetuximab (p = 0.3). Randomized 110 patients with la scchn, who had received 2 cycles of carboplatin, paclitaxel, and cetuximab as induction chemotherapy, between weekly cetuximab in combination with either 5-fu, hydroxyurea, and hyperfractionated week - on week - off radiotherapy (7274 gy) (cetuxfhx), or cisplatin, accelerated radiation with concomitant boost (cetuxpx) (72 gy). After a median followup of 21 months, 2-year os rates were 89.5% with cetuxfhx and 91.4% with cetuxpx arm . Two - year pfs rates were 82.3% and 89.7%, respectively (p = 0.18). Grade 3 mucositis was present in 91.1% (cetuxfhx) and 94.3% (cetuxpx) of patients; grade 3 dermatitis in 82.1% and 50.9%, respectively . Ninety - five percent of patients completed therapy, demonstrating that cetuximab can be incorporated safely in both crt platforms . Argiris et al . Enrolled 32 patients in a phase i study combining pemetrexed, cetuximab and radiotherapy in poor prognosis head and neck cancer . Cohort a included patients who had not been previously irradiated, while cohort b included patients who had received prior irradiation . The maximum tolerated dose (mtd) of pemetrexed was 500 mg in cohort a and 350 mg / m in cohort b. grade 3/4 neutropenia was common (50% in cohort a and 33% in cohort b) and febrile neutropenia was the most frequent doselimiting toxicity . Grade 3/4 mucositis was observed in 8 of the 9 patients treated at the mtd in cohort a . In eastern cooperative oncology group (ecog) e2303, 63 patients with resectable stage iii / iv scchn, were treated with 6-week cycles of paclitaxel, carboplatin at an auc of 2 and cetuximab, followed by crt (weekly paclitaxel, 30 mg / m, carboplatin auc 1, and cetuximab). If at week 14, after a radiation dose of 50 gy, tumor was still present on biopsy, salvage surgery was performed . In case of a negative biopsy (91% of the patients), crt was continued to a total dose of 6872 gy . Jordan et al . Treated 152 t3-t4 scchn patients with three 3-week cycles of tpf (docetaxel 75 mg / m on day 1, cisplatin 35 mg / m on days 1 and 2, and 5-fu 750 mg / m/day, as a continuous infusion, days 15, with pegfilgrastim support) followed by chemoradiation (63 gy in 35 fractions of 1.8 gy over 7 weeks and weekly cisplatin, 40 mg / m) plus weekly cetuximab . The complete response rate in the 142 patients, who were evaluated after the completion of therapy, was 57% . Ferris et al . Treated 34 la scchn patients with three 3-weekly cycles of cisplatin 75 mg / m and docetaxel 75 mg / m plus weekly cetuximab followed by crt (70 gy in 2 gy fractions over 7 weeks, weekly cisplatin 30 mg / m) plus weekly cetuximab, followed by weekly cetuximab maintenance for 6 months . Mesia et al . Studied the role of cetuximab maintenance therapy in patients with la scc of the oropharynx . Patients in group a were treated concomitant radiotherapy, 69.9 gy in 28 days, plus weekly cetuximab . The locoregional control rate at 1 year (primary endpoint) was 56.8% with bioradiation and 60.5% with bioradiation followed by cetuximab maintenance . At 1 year, event - free survival rates were 55.6 and 60.9%, respectively, and os rates were 75.6 and 87%, respectively . In the extreme trial (erbitux in first - line treatment of recurrent or metastatic head and neck cancer), 442 patients with previously untreated r / m scchn were randomized to receive cisplatin 100 mg / m or carboplatin at an area under the curve (auc) of 5 mg / mg / min as an 1-hour infusion, followed by 5-fu 1000 mg / m day for 4 days as a continuous infusion every 3 weeks for a maximum of 6 cycles, either alone or in combination with cetuximab . After a maximum of six cycles of chemotherapy, patients in the cetuximab group who had at least stable disease received cetuximab monotherapy until disease progression or unacceptable toxic effects, whichever occurred first . The median overall survival (primary endpoint) was 10.1 months (95% ci: 8.611.2) in the cetuximab group and 7.4 months (95% ci: 6.48.3) in the chemotherapy - alone group (hr for death: 0.80; 95% ci: 0.640.99; p = 0.04). The median followup was 19.1 months in the cetuximab group and 18.2 months in the chemotherapy - alone group . Median pfs was 5.6 months and 3.3 months for the combined group and the chemotherapy alone group, respectively (hr for progression: 0.54; 95% ci: 0.430.67; p <0.001). The overall response rate (orr) was 36 and 20%, respectively (odds ratio (or): 2.33; p <0.001), and the disease control rate (dcr) was 80 and 60%, respectively (or: 2.88; p <0.001). Among the 100 patients who received cetuximab as maintenance treatment, the median pfs was 12 weeks from the start of maintenance treatment . The safety profile of the study treatment was consistent with that expected for the agents used, with no significant difference in the incidence of grades 3 or 4 adverse events between treatment arms . However, there were 9 cases of sepsis in the cetuximab group, as compared with 1 case in the chemotherapy - alone group (p = 0.02), and there were 11 cases of hypomagnesemia in the cetuximab group, as compared with 3 cases in the chemotherapy - alone group (p = 0.05). There were four grade 3 and two grade 4 infusion - related reactions after cetuximab . Ecog randomized 117 r / m scchn patients to receive cisplatin 100 m / m every 4 weeks plus either weekly cetuximab (group a) or placebo (group b). Median pfs was 4.2 months in arm a and 2.7 in arm b (hr: 0.78; 95% ci: 0.541.12; p = 0.09). Median os was 9.2 months in arm a and 8.0 months in arm b (p = 0.21). Hitt et al . Treated 46 patients with weekly cetuximab and paclitaxel 80 mg / m as first - line regimen for recurrence of metastatic scchn . Common grade 3/4 adverse events were acne - like rash (24%), asthenia (17%), and neutropenia (13%). In groupe d'oncologie radiothrapie tte et cou (gortec) 200803, 54 patients who had not received prior chemotherapy for r / m scchn were treated with docetaxel 75 mg / m and cisplatin 75 mg / m every 3 weeks and weekly cetuximab, up to 4 cycles, followed by cetuximab maintenance in the absence of disease progression . The orr and dcr were 51.9 and 96.1%, respectively . At time of reporting, high egfrviii and amphiregulin expression levels identify scchn patients who are less likely to benefit from combination treatment with cetuximab and docetaxel . Three phase ii trials examined the role of cetuximab in platinum - refractory or platinum - resistant disease [3033]. Responses (1013%) were observed irrespective of reintroducing the originally used platinum compound to which they had become refractory or resistant . The survival of around 6 months achieved with cetuximab in platinum - refractory disease was found similar to that seen in first - line therapy in r / m - scchn and represented an increase in survival of 2.5 months compared with platinum - refractory historical controls . Based on these results and particularly considering the fact that about 50% of the patients showed disease control, cetuximab monotherapy seems to be an option for patients with r / m scchn who have progressed on platinum - based chemotherapy . Randomized 61 patients, who had received 2 prior cytotoxic chemotherapy regimens for r / m scchn to receive cetuximab every 2 weeks at either 500 (a) or 750 mg / m (b). Cetuximab 500 mg / m every 2 weeks seemed to yield similar efficacy and tolerability as conventional weekly dosing for patients with r / m scchn . However, it is unclear how many of the patients in this study had platinum - refractory disease . There is no apparent efficacy advantage associated with dose escalation to 750 mg / m q2w . Randomly allocated 286 eligible incurable scchn in a 2: 1 ratio to receive either zalutumumab plus best supportive care (zalutumumab group) or best supportive care with optional methotrexate (control group). Eligible were patients with progressive disease according to recist confirmed by an independent review committee during or within 6 months after the failure of platinum - based chemotherapy (at least two cycles of cisplatin [60 mg / m per cycle] or carboplatin [250 mg / m per cycle] with an interval between the cycles of <4 weeks). Also eligible were patients with platinum intolerance which was defined as discontinuation or dose reduction of platinum - based chemotherapy due to adverse or toxic effects, irrespective of response . Patients were given an initial loading dose of 8 mg / kg followed up by two week doses of 4 mg / kg by intravenous infusion in 1 h. after the first three administrations, in patients with no rash or grade 1 rash, the dose was increased by 4 mg / kg every 2 weeks up to a maximum dose of 16 mg / kg . Treatment was withheld from patients with grade 3 rash until rash resolved to grade 1 . Seventy - two percent of the control patients received methotrexate from the initiation of the trial, and a further 6% started methotrexate during the trial . Median os (primary endpoint) was 6.7 months (95% ci: 5.87.0) in the zalutumumab group and 5.2 months (95% ci: 4.16.4) in the control group (hazard ratio (hr) for death, stratified by who performance status: 0.77; 97.06% ci: 0.571.05; unadjusted p = 0.0648). Progression - free survival was longer in the zalutumumab group than in the control group (hr for progression or death, stratified by who performance status: 0.63; 95% ci: 0.470.84; p = 0.0012). The most common grade 3/4 adverse events were rash (21% of patients in the zalutumumab group versus none in the control group), anemia (6% and 5%, resp . ), and pneumonia (5% and 2%, resp . ). Grade 3/4 infections occurred in 15% of the patients in the zalutumumab group and 9% in the control group . Its pharmacokinetic profile allows a convenient three - week administration . In the spectrum trial (study of panitumumab efficacy in patients with recurrent and/or metastatic head and neck cancer), 657 patients were randomized between cisplatin 100 mg / m on day 1, followed by 5-fluorourcacil 1000 mg / m/day for 4 days or the same chemotherapy plus panitumumab 9 mg / kg administered on day 1 . Patients were allowed to switch from cisplatin to carboplatin (auc 5) during treatment for specific cisplatin - related toxicities, such as grade 2 neurotoxicity or a drop in creatinine clearance to <50 ml / min . The median os in the combined arm was 11.1 months compared to 9.0 months in the chemotherapy alone arm (hr = 0.87; 95% ci: 0.731.05; p = 0.14). However, there was a statistically significant difference in response rate (36% versus 25%; p = 0.007) and pfs (median 5.8 months versus 4.6 months; hr = 0.78; 95% ci: 0.660.92; p = 0.004) in favour of the panitumumab - containing arm . Although the spectrum trial failed to meet its primary endpoint, the results are nevertheless consistent with the outcome of the extreme trial . Nimotuzumab is a humanized egfr targeting monoclonal antibody which was studied in multiple phase ii trials . A weekly fixed dose of 200 mg was established as recommended dose for use in combination with radiotherapy in la scchn . No grade 3/4 adverse events were reported in a pilot study of weekly nimotuzumab (200 mg), radiotherapy (66 gy in 33 fractions/2 gy per fraction over 6.5 weeks) and weekly cisplatin (40 mg / m) in 17 patients with la scchn . Enrolled 106 patients with inoperable la scchn in a double blind, randomized phase ii trial . The primary endpoint of the trial was the complete response rate, which was 59.5% with nimotuzumab versus 34.2% with placebo (p = 0.028). Patients included in group a were treated with radiation (6066 gy) with or without nimotuzumab . At 48 months of followup, os was 34% with rt plus nimotuzumab versus 13% with radiotherapy alone . Patients included in group b were treated with crt (6066 gy plus weekly cisplatin at a dose of 50 mg) with or without weekly nimotuzumab . At 48 months of followup, os rate was 47% with crt plus nimotuzumab versus 21% with crt (p = 0.01). Tyrosine kinase inhibitors which have been tested in scchn include gefitinib and erlotinib, which are reversible specific egfr tkis, lapatinib, a reversible dual egfr / her2 tki, afatinib, an irreversible dual egfr / her2 tki, and pf-00299804, a potent irreversible pan - her tki . Planned to randomize 330 patients to receive docetaxel 35 mg / m on days 1, 8, and 15 every 28 days either plus placebo or in combination with gefitinib 250 mg / day . The data monitoring committee recommended early stopping of enrollment after inclusion of 270 patients because there was <5% chance to meet the primary endpoint (overall survival). Eligible were patients who were previously treated with chemotherapy for r / m scchn (73% of the patients) and patients previously untreated for r / m scchn either with a poor performance status (ecog 2) or in case of relapse within 6 months after chemotherapy given as part the primary treatment with curative intent . Median os was 6.8 months with docetaxel plus placebo versus 6.2 months with docetaxel plus gefitinib (hr 0.99; 95% ci: 0.751.31; p = 0.97). The time to progression was significantly longer with the addition of gefitinib (median 3.5 months versus 2.1 months; hr 0.69; 95% ci: 0.490.99; p = 0.047). In the imex trial, 486 r / m scchn patients were randomly assigned to oral gefitinib 250 mg / day, gefitinib 500 mg / day, or methotrexate 40 mg / m intravenously weekly . Physicians and patients were blinded to the gefitinib dose . Patients were stratified into two groups: group a (n = 256) consisted of patients who had stable or progressive disease after at least two cycles of platinum - based chemotherapy for recurrent disease; group b (n = 230) consisted of patients who were considered unsuitable for platinum - containing chemotherapy . Neither gefitinib 250 mg / day nor gefitinib 500 mg / day improved os compared with methotrexate (hr: 1.22; 95% ci: 0.951.57; p = 0.12; hr: 1.12; 95% ci: 0.871.43; p = 0.39, resp . ). Median os was 5.6, 6.0, and 6.7 months in the gefitinib 250 mg / day, gefitinib 500 mg / day, and methotrexate groups, respectively . In group a, os was significantly longer with methotrexate (hr for death: gefitinib 250 mg versus methotrexate: 1.62; p = 0.01; gefitinib 500 mg versus methotrexate: 1.5; p = 0.02). Tumor hemorrhage - type events were more common with gefitinib (250 and 500 mg) than with methotrexate (8.9%, 11.4%, and 1.9%, resp . ). The most common adverse events were rash, diarrhea, cancer pain, nausea, and vomiting with gefitinib, and stomatitis, nausea, and constipation with methotrexate.the os with gefitinib in the imex trial was similar to what was observed in earlier uncontrolled phase ii studies with gefitinib or erlotinib [4346]. Dose escalation of gefitinib adaptive to skin toxicity grade did not improve response rate in a phase ii trial conducted by perez et al . . Argiris et al . Planned to randomize 330 patients to receive docetaxel 35 mg / m on days 1, 8, and 15 every 28 days either plus placebo or in combination with gefitinib 250 mg / day . The data monitoring committee recommended early stopping of enrollment after inclusion of 270 patients because there was <5% chance to meet the primary endpoint (overall survival). Eligible were patients who were previously treated with chemotherapy for r / m scchn (73% of the patients) and patients previously untreated for r / m scchn either with a poor performance status (ecog 2) or in case of relapse within 6 months after chemotherapy given as part the primary treatment with curative intent . Median os was 6.8 months with docetaxel plus placebo versus 6.2 months with docetaxel plus gefitinib (hr 0.99; 95% ci: 0.751.31; p = 0.97). The time to progression was significantly longer with the addition of gefitinib (median 3.5 months versus 2.1 months; hr 0.69; 95% ci: 0.490.99; p = 0.047). In the imex trial, 486 r / m scchn patients were randomly assigned to oral gefitinib 250 mg / day, gefitinib 500 mg / day, or methotrexate 40 mg / m intravenously weekly . Patients were stratified into two groups: group a (n = 256) consisted of patients who had stable or progressive disease after at least two cycles of platinum - based chemotherapy for recurrent disease; group b (n = 230) consisted of patients who were considered unsuitable for platinum - containing chemotherapy . Neither gefitinib 250 mg / day nor gefitinib 500 mg / day improved os compared with methotrexate (hr: 1.22; 95% ci: 0.951.57; p = 0.12; hr: 1.12; 95% ci: 0.871.43; p = 0.39, resp . ). Median os was 5.6, 6.0, and 6.7 months in the gefitinib 250 mg / day, gefitinib 500 mg / day, and methotrexate groups, respectively . In group a, os was significantly longer with methotrexate (hr for death: gefitinib 250 mg versus methotrexate: 1.62; p = 0.01; gefitinib 500 mg versus methotrexate: 1.5; p = 0.02). Tumor hemorrhage - type events were more common with gefitinib (250 and 500 mg) than with methotrexate (8.9%, 11.4%, and 1.9%, resp . ). The most common adverse events were rash, diarrhea, cancer pain, nausea, and vomiting with gefitinib, and stomatitis, nausea, and constipation with methotrexate . The os with gefitinib in the imex trial was similar to what was observed in earlier uncontrolled phase ii studies with gefitinib or erlotinib [4346]. Dose escalation of gefitinib adaptive to skin toxicity grade did not improve response rate in a phase ii trial conducted by perez et al . . Randomized 34 patients with resectable scchn to receive erlotinib daily for 2 to 8 weeks prior to surgery at standard (150 mg) or high doses (200 mg in never / former smokers, and 300 mg in current smokers). Response rates were documented in 18% and 29% of the patients in the low - and high - dose arms, respectively, suggesting that a subgroup of previously untreated scchn is highly sensitive to egfr tyrosine kinase inhibition.hayes et al . Randomly assigned 128 patients with la scchn to receive either cisplatin 100 mg / m on days 1, 22, and 43 combined with 70 gy of radiotherapy (arm a) or the same treatment plus 150 mg of erlotinib starting one week before crt and continued until the completion of radiotherapy (arm b). Serious adverse events were observed in 33% and 32% of the patients in arm a and b, respectively . Most common serious adverse events were nausea, vomiting, and dehydration and accounted for 30% of all saes reported, with no difference between arms . Gregoire et al . Enrolled 226 patients in a randomized phase ii trial testing the value of gefitinib during and/or after crt in la scchn . Patients received either placebo during crt followed by adjuvant placebo, or gefitinib 250 mg or 500 mg / day during crt followed by placebo, gefitinib 250 mg or 500 mg / day during and after crt, or placebo during crt followed by adjuvant gefitinib at a dose of 250 mg or 500 mg / day . There was no difference in 2-year local disease control rate (primary endpoint) between the 7 treatment arms . Evaluated the toxicity and recommended dose for adjuvant erlotinib after definitive crt for la scchn . No dose limiting toxicities were observed at a daily dose of 100 or 150 mg for 6 months . At the 150 mg dose, randomized 34 patients with resectable scchn to receive erlotinib daily for 2 to 8 weeks prior to surgery at standard (150 mg) or high doses (200 mg in never / former smokers, and 300 mg in current smokers). Response rates were documented in 18% and 29% of the patients in the low - and high - dose arms, respectively, suggesting that a subgroup of previously untreated scchn is highly sensitive to egfr tyrosine kinase inhibition . . Randomly assigned 128 patients with la scchn to receive either cisplatin 100 mg / m on days 1, 22, and 43 combined with 70 gy of radiotherapy (arm a) or the same treatment plus 150 mg of erlotinib starting one week before crt and continued until the completion of radiotherapy (arm b). Serious adverse events were observed in 33% and 32% of the patients in arm a and b, respectively . Most common serious adverse events were nausea, vomiting, and dehydration and accounted for 30% of all saes reported, with no difference between arms . Gregoire et al . Enrolled 226 patients in a randomized phase ii trial testing the value of gefitinib during and/or after crt in la scchn . Patients received either placebo during crt followed by adjuvant placebo, or gefitinib 250 mg or 500 mg / day during crt followed by placebo, gefitinib 250 mg or 500 mg / day during and after crt, or placebo during crt followed by adjuvant gefitinib at a dose of 250 mg or 500 mg / day . There was no difference in 2-year local disease control rate (primary endpoint) between the 7 treatment arms . Evaluated the toxicity and recommended dose for adjuvant erlotinib after definitive crt for la scchn . No dose limiting toxicities were observed at a daily dose of 100 or 150 mg for 6 months . At the 150 mg dose, encouraging preliminary results in r / m scchn after failure of a platinum - containing therapy were reported with afatinib, a dual egfr / her2 irreversible tyrosine kinase inhibitor, which was compared to single - agent cetuximab in a randomized phase ii study . Enrolled 67 patients into a randomized, placebo - controlled, phase ii trial, exploring the potential benefit of adding lapatinib to crt in patients with la scchn . Lapatinib (1500 mg / day) or placebo were started 1 week before crt (70 gy in 35 fractions over 7 weeks plus cisplatin 100 mg / m on days 1, 22, and 43) and continued during and after crt until disease progression . The addition of lapatinib did not impair the timely administration of crt and did not lead to an increase in mucositis and radiation dermatitis . The complete response rate at 6 months after treatment (primary endpoint) was 36% with placebo and 53% with lapatinib . Treated 107 patients with la scchn with either lapatinib or placebo for 26 weeks prior to crt . The overall response rate before crt in the 40 patients who received> 4 weeks of lapatinib was 17% . Siu et al . Enrolled 69 patients in a phase ii study with pf-00299804 at a dose of 45 mg qd, in previously untreated r / m scchn . Grade 3 adverse events were diarrhea (16%), fatigue (9%), acneiform dermatitis (7%), and hand - foot reaction (4%). A meta - analysis conducted by kyzas et al . Demonstrated that vegf protein overexpression, as detected with immunohistochemistry, is associated with a worse os in patients with scchn . Bevacizumab is a humanized igg1 monoclonal antibody that binds selectively to all isoforms of human vegf and neutralizes the biologic activities of vegf by blocking the binding of vegf to its receptors on the surface of endothelial cells . Combined pemetrexed 500 mg / m and bevacizumab 15 mg / kg given intravenously every 21 days until disease progression in 40 patients presenting with previously untreated r / m scchn . The median ttp (primary endpoint) was 5 months, and the median os was 11.3 months . In 37 evaluable patients, the orr was 30%, and the dcr was 86% . Grade 3 bleeding events occurred in 6/40 patients (15%) and was fatal in 2 (5%). Bevacizumab, 10 mg / kg, administered on day 1 of each 2-week cycle, can be safely combined with fhx crt regimen, consisting of five 2-week cycles of hydroxyurea 500 mg orally bid, 5-fu 600 mg / m/day administered as a continuous infusion, and radiotherapy, 1.5 gy bid for 5 days followed by 9 days without therapy (fhx), in patients with poor prognosis scchn . Salama et al . Conducted a randomized phase ii study evaluating the impact of adding bevacizumab (b) to the fhx regimen . Eligible were patients with t1 - 3, n0 - 1 and t4, n0 - 1 scchn . Two - year os was 89% in patients treated with fhx and 58% in patients treated with bfhx . These unexpected findings are not well understood and could be due to chance, given the small sample size . Harari et al . Demonstrated the safety and feasibility of combining crt (70 gy in 33 fractions and weekly cisplatin, 30 mg / m) with bevacizumab weeks 3, 1, 4, and 7 with dose escalation from 5 to 10 to 15 mg / kg in 10 patients with la scchn . Sorafenib and sunitinib are oral inhibitors of multiple kinases including the receptor tyrosine kinases of vascular endothelial growth factor (vegf) receptor . Elser et al . Treated 27 patients with r / m scchn or nasopharyngeal carcinoma (7 patients), who had received 1 chemotherapy for recurrent or metastatic disease with sorafenib 400 mg twice daily on a continuous basis . The treatment was well tolerated with few grade 3 or 4 toxicities . However, anticancer activity was modest . The same regimen was evaluated in the southwest oncology group study s0420, which enrolled 41 r / m scchn patients who had not received prior chemotherapy for r / m disease . However, the estimated median pfs was 4 months, and the estimated median os was 9 months . In the gortec 200601 study, 38 patients with r / m scchn received sunitinib 37.5 mg / day on a continuous basis . Forty - five percent and 3% of the patients had received one and two prior chemotherapy regimens for r / m disease, respectively . Local complications, including the appearance or worsening of tumor skin ulceration or tumor fistula, were recorded in 39.5% of the patients, and a fatal arterial bleeding in the head and neck region occurred in 10.5% of the patients . Fountzilas et al . Treated 17 r / m scchn patients with sunitinib 50 mg per day administrated in 4-week cycles followed by a rest period of 2 weeks as first - line treatment for r / m disease and observed no objective responses . Disease control rate was 18%, median ttp was 2.3 months, and median os was 4 months . The same intermittent sunitinib regimen was used by choong et al . Who observed an orr of 4.5% and a dcr of 27.3% . Sabichi et al . Combined paclitaxel, carboplatin auc 6, administered every 3 weeks, and sorafenib 400 mg bid, days 219, in patients with r / m scchn . Vandetanib is an inhibitor of vascular endothelial growth factor receptor-2 (vegfr-2), egfr, and rearranged during transfection (ret) tyrosine kinases . It restores hnscc cells' sensitivity to cisplatin and radiation in vitro and in vivo . As a single agent, it has antiproliferative effects on scchn cells in vitro and inhibits tumor growth in nude mice orthotopically injected with human scchn cells . Vandetanib can be safely combined with radiotherapy (2.2 gy / d, 5 days / week up to a total dose of 66 gy) or radiotherapy (2 gy / d, 5 days / week up to a total dose of 70 gy) plus weekly 30 mg / m of cisplatin . Heat - shock protein 90 (hsp90) is a protein which chaperones multiple client oncoproteins involved in tumor progression . Investigated the antitumor effect of the novel hsp90 inhibitor nvp - auy922 against oral scc (oscc). Nvp - auy922 inhibited the proliferation of oscc cells in vitro and induced a robust antitumor response and suppressed p - akt and vegf expression in an hsc-2 xenograft model . Several sirtuin family members (sirt1 - 7) function either as nicotinamide adenine dinucleotide (nad)-dependent deacetylases or as adp - ribosyl transferases and are involved in carcinogenesis . . Demonstrated that sirt3 is overexpressed in oscc in vitro and in vivo and that sirt3 downregulation inhibits oscc cell growth and proliferation and increased oscc cell sensitivity to radiation and cisplatin treatments in vitro . Histone deacetylases are enzymes involved in remodeling of chromatin by deacetylating the lysine residues and play a pivotal role in epigenetic regulation of gene expression . Hdac inhibitors have radio - enhancing properties [75, 76], increase the susceptibility of scc cell lines to cisplatin in vitro [77, 78], and inhibit tumor growth in xenograft models of scchn . . Demonstrated that the histone deacetylase inhibitor vorinostat in combination with the egfr tyrosine kinase inhibitor gefitinib induced synergistic inhibition of proliferation, migration, and invasion as well as induction of apoptosis in scchn cells, including cells resistant to gefitinib . High expression of aurora a or b is associated with a worse outcome in scchn [8186]. Combined a dual aurora a / aurora b inhibitor with cetuximab in scchn cell lines in vitro and observed at least an additive effect . Preclinical data strongly support the testing of mammalian target of rapamycin (mtor) in scchn [8890]. Activation of mtor is observed in the majority of scchn and is associated with a poor outcome . Patel et al . Found that inhibition of mtor diminished lymphangiogenesis in the primary tumors and prevented the dissemination of scchn cancer cells to the cervical lymph nodes in an orthotopic mouse model . Temsirolimus enhances the growth - inhibiting effects of the combination of bevacizumab, cetuximab, and irradiation in head and neck cancer xenografts . Aissat et al . Demonstrated that rapamycin displays antiproliferative effects and induces apoptosis in scchn cell lines and that combination of rapamycin with paclitaxel or carboplatin displayed synergistic and additive effects . Temsirolimus appeared to be a more potent radiosensitizer than cisplatin in mice bearing squamous cell carcinoma xenografts . In a pharmacodynamic evaluation of temsirolimus in scchn patients, ekshyyan et al . Found a significant inhibition of the mtor pathways in tumor cells and in peripheral blood mononuclear cells . Everolimus 10 mg / day, days 121 can be safely combined with cisplatin 20 mg / m, days 1, 8, and 15 of a 28-day cycle in patients with solid tumors . C - src is a nonreceptor cytoplasmic tyrosine kinase that regulates signals from cell surface molecules and that plays a key role in modulating multiple cellular functions by activating the signal transducer and activator of transcription (stat) family of transcription factors . Although preclinical data provided a rationale for testing c - scr inhibitors in scchn, the outcome with single - agent c - scr inhibitors in patients with r / m scchn was disappointing . Brooks et al . Treated 15 r / m scchn patients with dasatinib, a potent inhibitor of src - family kinases epha2, at a dose 100 mg twice daily . Saracatinib is also an orally available src kinase inhibitor which was administered to 9 r / m scchn patients at a daily dose of 175 mg . Eight patients had disease progression within the first eight - week cycle, and one patient was removed from the study after 11 days due to rapid clinical decline . The study was closed early due to lack of efficacy according to the early stopping rule . Nuclear factor kappa b is overexpressed in scchn, and nf-b expression is associated with a poor prognosis . Bortezomib is a small - molecule proteasome inhibitor which affects multiple signaling pathways including nf-b . Chung et al . Treated 25 r / m scchn patients with bortezomib 1.6 mg / m and docetaxel 40 mg / m on days 1 and 8 of a 21-day cycle and observed a orr and dcr of 5 and 52%, respectively . Were observed in the phase i portion of the study which included 10 patients up to the maximum planned dose of 15 mg / kg of bevacizumab, 46 additional patients were treated at that dose level . Forty - eight percent of the patients had received prior chemotherapy for recurrent / metastatic disease . The observed response rate was 15% with 4 complete responses allowing rejection of the null hypothesis that the response rate is 5% and the percentage of patients not progressing within two months is 30% . Higher ratios of phosphorylated over total vegf receptor-2 and egfr in pretreatment biopsies were associated with complete response (p = 0.036 and p = 0.036, resp .) And tumor shrinkage (p = 0.007 and p = 0.008, resp .) In a subset of 11 subjects with available tissue . The feasibility and efficacy of adding bevacizumab and erlotinib to concurrent crt in patients with la scchn was evaluated in a phase ii trial conducted by the sarah cannon oncology research consortium including 60 previously untreated patients . The treatment consisted of induction chemotherapy with 6 weeks of paclitaxel, carboplatin, infusional 5-fluorouracil, and bevacizumab, which was followed by radiation therapy, weekly paclitaxel, bevacizumab, and erlotinib . After a median followup of 32 months, the estimated 3-year pfs and os rates are 71% and 82%, respectively . Integrins promote and regulate endothelial cell proliferation, migration, invasion, and survival in tumors, securing vascularisation and vascular remodeling in tumors . Advantage is a phase i / ii trial evaluating cilengitide in combination with cetuximab, cisplatin and 5-fluorouracil in patients with r / m scchn . No dlts were observed in the phase i part of the study which tested cilengitide (500, 1000, and 2000 mg) twice weekly with standard doses of cetuximab, cisplatin and 5-fluorouracil . Cilengitide 2000 mg was considered safe and was selected for the subsequent randomised phase ii part assessing pfs . The road from preclinical evidence to clinical use is long and bumpy, and a large number of targeted agents are still at the start of the race . Some others reached the last stretch but stumbled on one of the last hurdles in phase ii, or even phase iii trials . Thus far, only the egfr - directed monoclonal antibody cetuximab has made it to the finish and is currently approved for the treatment of locoregionally advanced and recurrent / metastatic scchn, by the regulatory agencies of the united states and europe.
Obstruction of the appendiceal lumen with fecaliths or lymphoid hyperplasia is a well - known cause of appendicitis.1 in extremely rare cases, metastatic cancerous lesions of the appendix can cause appendicitis . We herein present a case of appendicitis derived from metastatic extrahepatic cholangiocellular carcinoma an 87-year - old female was admitted to the department of medicine with a chief complaint of abdominal pain with jaundice . She had a history of hypertension and type ii diabetes mellitus for which she was treated at the local clinic with regular medications . Laboratory findings revealed a white blood cell count within normal range (5,680 cells / mm with 74.7% segmented neutrophils), but c - reactive protein and the erythrocyte segmentation rate were elevated to 16.1 mg / l and 105 mm / hour, respectively . Liver function tests for aspirate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, alkaline phosphatase, and gamma - guanosinetriphospate were 284 iu / l, 403 iu / l, 9.1 mg / dl, 6.4 mg / dl, 540 iu / l, and 965 iu / l, respectively . Contrast - enhanced computed tomography (ct) revealed extrahepatic cholangiocellular carcinoma with direct hepatic invasion and lymph node metastasis in the para - aortic and peri - portal area . In addition, a dilated appendiceal lumen caused by a soft tissue mass at the base of the appendix was detected (fig . 1). Positron emission tomography - computed tomography (pet - ct) scan revealed an approximately 4.3 cm hypermetabolic mass in segment 4 of the liver with biliary dilatation and hypermetabolic nodes in the portocaval and para - aortic areas . The patient underwent an endoscopic retrograde cholangiopancreatography (ercp)-guided biopsy of the common bile duct (cbd) and ultrasonography - guided biopsy of the metastatic hepatic mass . The result of the hepatic mass revealed adenocarcinoma; however, the results of the cbd revealed chronic non - specific inflammation . Although the rebound tenderness was not severe, we were concerned that the degree of appendicitis might intensify as the metastatic mass in the appendix grew . Therefore, we decided to perform an elective laparoscopic appendectomy before the start of palliative chemotherapy . A mass - like lesion was detected between the proximal appendix and the cecum base (fig . The pathologic report indicated metastatic adenocarcinoma involving the subserosa and proper appendiceal muscle involving the serosa through the mucosa of the cecum (fig . Immunohistochemical staining revealed positive expression of cytokeratin 7 (ck7) but negative expression of cytokeratin 20 (ck20) and cdx 2 in tumor cells, which was suggestive of adenocarcinoma of upper gastrointestinal or pancreaticobiliary origin (fig . She later underwent a self - expandable metallic stent insertion in the biliary tract to relieve bile duct obstruction; however, she refused to undergo palliative chemotherapy . Follow - up ct after three months of laparoscopic appendectomy revealed more progressive disease with peritoneal carcinomatosis . Luminal obstruction of the appendix caused by fecaliths or lymphoid hyperplasia is a major cause of appendicitis . Unusual factors such as parasites, endometriosis, bacteria, and primary and metastatic tumors can also cause appendicitis in rare cases.1 primary appendiceal cancer is rare and is found in only 0.9 - 1.4% of all appendectomy specimens . In addition, metastatic appendiceal cancer is much more rare.2,3 in a recent literature review, only seven patients were found to have metastatic appendiceal cancer among 80,698 patients.1 the primary sites of metastatic appendiceal carcinomas were the lung, breast, cervix, prostate, stomach, liver, nasopharynx, mediastinum, and pancreas.3,4,5,6,7,8,9,10,11,12,13 as far as we know, primary cholangiocellular carcinoma very rarely metastasizes to the appendix.14 without imaging studies for patients with suspected appendicitis, it is very difficult to diagnose primary or metastatic carcinoma of the appendix.2,6,7,8,9,10,11 as imaging modalities are more frequently used, appendiceal masses are found before surgery more commonly, sometimes with unusual characteristics.3,4,5,13 in a recent report of a case of appendiceal metastasis from a hepatocellular carcinoma, ct scan was useful in the diagnosis of the metastatic lesion . It showed a hypervascular area on arterial phase and a hypo - dense area on delayed phase imaging, two imaging features that are similar to those observed in primary hepatocellular carcinoma.3 in our case, ct scan revealed an appendiceal mass with a dilated appendix and pet - ct revealed focal uptake of 18-fdg in the appendix base, which strongly suggested a malignant lesion . Although those examinations could not distinguish primary appendiceal carcinoma from a metastatic lesion of the appendix, they were helpful in the decision to perform an elective surgery . It is known that cancer cells spread to other sites via the lymphatics, a hematogenous route, or direct invasion . The common metastatic sites of cholangiocellular carcinomas are regional lymph nodes and adjacent organs; distant metastasis is uncommon.15 in particular, appendiceal metastasis from cholangiocellular carcinoma is an extremely rare event . It has been suggested that metastatic appendiceal carcinoma usually begins as serosal implants, which encroach progressively on the lumen.6,7 in this case, the pathologic results revealed that metastatic adenocarcinoma was involved through the serosa to the mucosa of the cecum . In addition, although there was no peritoneal seeding in the operative field, peritoneal carcinomatosis was confirmed by follow - up ct images three months after surgery . Thus, we think that serosal implantation with encroachment might be an adequate theory to explain the route of metastasis in the present case . In conclusion, although appendicitis caused by metastasis to the appendix from other primary cancer sites is extremely rare, physicians should be more cautious in recognizing the possibility when patients complain of right lower quadrant abdominal pain . Ct or pet - ct could be useful tools in detecting extraordinary appendicitis caused by metastatic extrahepatic cholangiocellular carcinoma.
Obesity is an epidemic in every country . In the u.s ., rates of obesity are observed over 20 percent in every state and exceed 2535 percent in 45 states . Medical care and cost may increase from $147 billion to nearly $210 billion per year in u.s . . The obesity epidemic long - associated with the western world is now extending to eastern nations like china and india . According to the overseas development institute (odi) about 1.46 billion asian indian people are now considered overweight or obese, with a national rate of about 11 percent . There are several approaches to manage weight by limiting the intake of food and absorption of food, suppressing appetite, and altering metabolism or increasing energy expenditure through physical activity and diet . Dietary supplements are also used for weight management and healthy metabolism and to support healthy lifestyles . Over a decade of basic and clinical research and safety data are available for dietary supplements, including herbal supplements, used for weight management and healthy metabolism . (synonyms: salacia prinoides) belongs to celastraceae (spike - thorn) family commonly called saptrangi, dimal, modhupal, ingli, cherukuranti, and nisul - bondi . The roots have biologically active compounds, such as triterpenes, phenolic compounds, glycosides, and coloring agents (figure 1) which show various medicinal properties . Salacia tends to contain salacinol, salaretin, mangiferin, kotalanol, triterpenes, 13 mrt, and ponkoranol [4, 5]. Recent preclinical and clinical studies have demonstrated that salacia roots modulate multiple targets: peroxisome proliferator activated receptor - alpha - mediated lipogenic gene transcription [6, 7], angiotensin ii / angiotensin ii type 1 receptor, alpha - glucosidase, aldose reductase, and pancreatic lipase . These multitarget actions may mainly contribute to salacia root - induced improvement of type 2 diabetes and obesity - associated hyperglycemia, dyslipidemia, and related cardiovascular complications seen in humans and rodents . The primary objective of this study was to compare the effect of 3 different doses of salacia chinensis extract (sce) on the glycemic and insulinemic response in normal healthy individuals . Most of the studies were conducted with healthy and type 2 diabetes with other salacia extract species at higher doses . Forty eligible subjects (n = 40) were healthy south asian indian volunteers, consenting adult human male and female subjects, aged 1855 (both ages inclusive), of bmi 24.5 to 29.5 kg / m . A full medical examination was performed on all subjects, including a physical examination, biochemical tests (routine blood and urine chemistry), and electrocardiogram . An oral glucose tolerance test was also performed in order to confirm normal oral glucose tolerance of each subject . The predose sample (1 4 ml) or placebo was administered within 1 hour sce administration to all the participants . The postdose blood samples (1 4 ml) were drawn at 30 min, 60 min, 90 min, 120 min, and 180 min in each period . Participants were required to avoid all dietary supplements, otc, or any foods that may interfere with postprandial glucose and insulin before and throughout the study period and were not allowed to drink alcoholic beverages or caffeine during the study period . Demographic data, medical history, physical and systemic examination, and vital parameters including respiratory rate, ekg, chest x - ray, hematology, biochemistry, serology, and urine analysis were collected or conducted . One capsule of 200 mg sce (r, t1) or one capsule of 300 mg sce (m, t2) or 500 mg sce (b, t3) or placebo (h, t4) were given orally to each subject with about 75 g of sucrose in about 250 ml of water at ambient temperature, in each study period, as per the randomization code list . Voucher specimen collected from the source populations is identified and authenticated by in - house botanist at omniactive ., india . 100 g of powdered material of salacia roots was extracted with 6 volumes of ethanol at 50 degrees . The ethanolic extract was filtered through bchner funnel and evaporated to dryness using rotary evaporator to get yield of 5% . Friedelane triterpenes, friedel-1-en-3-one, friedelan-1,3, dione 7-ol, friedelan-1,3-dione- 24-al, friedelan-1,3-dione-24-oic acid, 24,25-oxidofriedelan-1,3-dione, 7,24-oxidofriedelan-1,3-dione, and 25,26-oxidofriedelan-1,3-dione are isolated from root bark . Mangiferin, salacinol, kotalanol, salaprinol, ponkoranol, and leucopelargonidin monomer, its dimer, and tetramer are also reported from roots of salacia chinensis . Based on the totality of the evidence, on the basis of scientific procedures, history of exposure, and use, the consumption of salacia chinensis extract (sce) as a food ingredient at use levels of 50 mg / serving in certain specified foods resulting in a 90th percentile intake of 511 mg / person / day is considered safe and generally recognized as safe (gras). Blood samples were drawn for glucose and insulin measurements at a central laboratory at the following times: baseline (before product administration) and 30, 45, 60, 90, 120, 150, and 180 min . The serum samples were allowed to clot in serum separator tubes at room temperature and centrifuged at 1000 g for 15 min at room temperature . Glucose was measured with the use of an enzymatic method (hexokinase glucose) and insulin was measured with the use of a radioimmunoassay procedure . One capsule of salacia extract (salacia chinensis, sce), 200 mg capsules (r, t1), or one capsule of salacia extract (salacia chinensis, sce), 300 mg capsules (m, t2), or one capsule of salacia extract (salacia chinensis, sce), 500 mg capsules (b, t3), or one capsule of placebo (h, t4) was administered orally to each subject in sitting posture, with about 75 g of sucrose in about 250 ml at ambient temperature, in each study period, as per the randomization code list . The study protocol was reviewed and approved by the sri venkateshwara hospital ethics committee on 07 june 2015, and all enrolled subjects provided informed consent before the start of the study . The study was conducted in compliance with final protocol, the applicable harmonized tripartite guidelines for good clinical practice (gcp), the relevant sections of good laboratory practice (glp), local laws and regulations (icmr guidelines on biomedical research), schedule y (amended version, 2013) of cdsco (central drugs standard control organization), relevant sections of drugs and cosmetics (first amendment) rules 2013, cdsco bioavailability bioequivalence guidance, and the provisions of declaration of helsinki (brazil, october 2013). The number of subjects needed to detect difference in the acute study with 80% power at the 5% level of significance was 3540 subjects for a crossover design . Each variable was analyzed by using parametric or nonparametric (if declared nonnormal) period, treatment, and crossover analysis . The parametric analysis was performed by using repeated measures analysis of variance with variance components covariance structure including treatment and period as fixed effects and subject nested within site as random effect . The pairwise differences of least squares means of the treatments were tested with the use of tukey - kramer p value adjustments . A result was declared statistically significant if and only if a p value of an analysis <0.05 . A total of 40 subjects were enrolled and participated in the study, out of which 35 subjects completed four periods of the study . All enrolled subjects were healthy human adult male subjects of south asian race (indian). Of the 40 subjects enrolled, 38 subjects participated in period one, 37 subjects in period two, 36 subjects in period three, and 35 subjects in period four of the study . A total of 35 subjects completed the study (figure 2 and table 1). Baseline and postprandial values for serum insulin are seen in figure 3 for all available subjects . In addition, two doses of sce (300 mg and 500 mg) lowered serum insulin area under the curve (auc) for 0180 min postprandially in comparison with placebo (table 4). Baseline and postprandial values for both serum glucose and insulin are seen in figure 4 for all available subjects . In addition, a nonsignificant decrease of serum glucose area under the curve (auc) for 0180 min postprandial levels was observed in 200 mg sce in comparison with placebo (table 4). Fever, chills, headache, decreased hemoglobin, decreased hematocrit, increased serum sodium, increased platelet count, and decreased lymphocytes were observed and investigator declared that these are not related to product but they resolved with subjects . No symptoms related to gastrointestinal (gi) tolerance such as flatulence, belching, abdominal pain, nausea, and diarrhea after 24 h and 48 h of product administration were observed . This study presents the first published results on the effects of sce on postprandial blood glucose and insulin response in healthy people . The doses of the herbal extract had significant effects on postprandial insulinemia after administration with sucrose solution . The doses of extract for this study are lower than the 1000 mg dose found to be efficacious in other salacia extracts species studies [1315]. Salacia composition described here includes at least 12% of polyphenols, 2% of mangiferin, and 1% of 25,26-oxidofriedelane-1,3-dione by weight of the composition in the form of extract . The extract is a proprietary product with specific composition rich in oligomeric flavonoids (proanthocyanidines) [10, 16]. Postprandial hyperglycemia is a better predictor of progression to diabetes and key management marker for glycemic control . Reported that postprandial glucose (ppg) based on postlunch plasma glucose and extended postlunch plasma glucose was more reliable in predicting poor glycemic control than prebreakfast or prelunch plasma glucose . The degree of risk conferred by the 2 h ppg concentration was nearly twice that conferred by a1c level . Postprandial insulin levels are diagnostic markers to show insulin resistance and a predictive risk factor for cardiovascular risk . The delayed gastric emptying and a blunted response of gut hormones during feeding may potentially modulate satiety when treated by sce . Alpha - glucosidase is an intestinal enzyme which breaks down sucrose into glucose and fructose . Alpha - glucosidase inhibitors class delay and reduce the amount of glucose that is ready for absorption by interfering with the breakdown of the long - chain carbohydrates allowing the pancreas more time to secrete insulin to cover the meal . In the present study, during a sucrose load, sce reduced insulin in a dose - dependent fashion and glucose was also reduced nonsignificantly over placebo, but not dose - dependently . Insulin levels are reduced not only via a decreased glycemic stimulus but also by interference with other insulin releasing mechanism(s). For example, in the stop - niddm (study to prevent niddm) trial, the group randomly assigned to acarbose not only had a reduction in body weight, bmi, waist and hip circumferences, systolic and diastolic blood pressure, blood triacylglycerols, and 2 h postprandial glucose during a 3 y period following subjects with impaired glucose tolerance but also experienced a significantly reduced incidence of cardiovascular events and hypertension . A meta - analysis of 7 long - term studies showed that -glucosidase inhibitors significantly reduce the risk of myocardial infarction or any cardiovascular event . Sce showed significant inhibitory effects on -glucosidase, pancreatic lipase, and hmg - coa . Potential mechanism of action of sce observed in the current study might be due to its action as -glucose inhibition (figure 5). Lifestyle modifications consisting of diet and exercise can be effective for reducing macrovascular complications in patients with type 2 diabetes and for lowering relative risk of developing the disease in high - risk persons [22, 23]. Although diabetes and its encompassing symptoms are altered by diet and exercise, behavioral obstacles can prevent occurrence of changes . Several situational obstacles for adults with diabetes were identified for dietary adherence, such as resisting temptation, eating out, feeling deprived, planning meals, and social events [24, 25]. In the current study, sucrose loading increased blood glucose concentrations at 30 minutes after administration . Elevation of insulin levels following sucrose loading was also significantly inhibited by sce compared with placebo; the inhibition was significant at 30 and 60 minutes after administration of 300 and 500 mg sce . Slowdown of the postprandial hyperglycemic process, if possible, would offer an advantage to insulin - dependent diabetic individuals . Inhibition or delay of intestinal nutrient absorption is now being considered, at least in part, responsible for the hypoglycemic effect . No risk of lactic acidosis or other serious symptoms commonly seen with hypoglycemic ingredients were observed . Sce does not directly decrease glucose in the blood stream but was shown to inhibit intestinal absorption . Current limitations of the study include the facts that it includes healthy people and administered sucrose solution to study the effects on glucose and insulin postprandial response . They have healthy blood sugar levels and are not abnormal and blood sugar levels came back to normal in all groups at 180 min . Further short - term and long - term studies are required in healthy individuals and metabolic health conditions with and without meal . Historical and present uses of salacia in india and japan show that this herbal extract is used as a nutritional adjunct, either as a tea or supplement, taken with meals for its antidiabetic properties . Salacia reticulata [23, 2632] and salacia oblongata [33, 34] do lower postprandial glycaemia in patients with type 2 diabetes and metabolic risk factors [3537]. Salacia chinensis with a meal suppressed the increases of postprandial blood glucose at 30 minutes after meal . Aucglucose and aucinsulin in the subjects with a fasting blood glucose level between 100 and 125 mg / dl in healthy japanese volunteers . In another study, 1000 mg extract of salacia chinensis was given with carbohydrate - rich diet (approximately 600 kcal) and auc glucose decreased and no data are available on insulin . These two studies were reported with a meal and current study is with a sucrose loading as recommended by fda for alpha - glucosidase inhibitor products and sucrose (rather than starch) was the most appropriate carbohydrate load . As a disaccharide, sucrose cannot be systemically absorbed unless it is hydrolyzed to glucose and fructose by -glucosidase . After a dose of sce, the decrease in absorption of glucose produced from sucrose reflects the activity of -glucosidase and indirectly reflects the efficacy of sce . So administration of sucrose was recommended to provide a better baseline measure of -glucosidase activity . Administration of sucrose is more suitable than eating a meal since it produces a more reproducible change in serum glucose concentration . Studies on low dose sce were not reported in healthy people nor in disease condition . In a pilot study, the effects of s. chinensis were investigated in diabetic chronic kidney disease (ckd) patients . In this study, 30 stable diabetic ckd patients were randomized into 2 groups: groups a and b of 15 patients each . Group a was given s. chinensis 1000 mg twice daily, while group b received a placebo . There was stabilization of renal function as measured by serum creatinine and creatinine clearance in patients receiving s. chinensis compared to the placebo, suggesting that s. chinensis may retard the progression of chronic kidney disease . In a randomized double - blind, placebo controlled, crossover study, 30 healthy human subjects were given a placebo or 1000 mg of an s. chinensis hydroalcoholic extract as a one - time dose . The extract decreased postprandial plasma glucose levels after a carbohydrate - rich meal by about 13% at 90 min, while the plasma glucose area under the curve was decreased by about 34% . In a double - blind, placebo controlled, randomized trial, shivaprasad et al . Evaluated the efficacy and safety of s. reticulata leaves and root bark extracts in 29 subjects with prediabetes and mild to moderate hyperlipidemia . In this study, 29 subjects received either 500 mg / day of a s. reticulata extract or a placebo along with therapeutic lifestyle changes for the 6-week period . As compared to the placebo, improvements in lipid profiles and glycemic levels were observed in the s. reticulata - treated group at week 6 . A statistically significant reduction was observed in low - density lipoprotein cholesterol and fasting blood sugar levels at weeks 3 and 6 when treated with root bark extract . The leaves extract - treated group showed statistically significant reduction in fasting blood sugar levels at week 6 only . In a double - blind study, ozaki et al . Investigated the safety of drinks containing an aqueous extract of s. reticulata . In this study, a total of 54 subjects either healthy (n = 27) or with borderline blood glucose levels and mild type 2 diabetes (n = 27) were randomly assigned to untreated (placebo) or treated groups . The subjects consumed a drink containing the placebo or s. reticulata extract (450 mg; 3 times the recommended amount) at breakfast and dinner every day . The treated subjects showed no significant clinical changes or adverse effects, such as hyperglycemia or gastrointestinal symptoms, during the entire test period . The subjects with borderline blood glucose and mild type 2 diabetes in the treated group showed significant changes in the amount of hba(1c) and glycoalbumin during the test period compared to the placebo group . In another double - blind randomized placebo controlled trial, singh et al . Investigated the effects of a herbal tea containing s. reticulata in patients with type 2 diabetes mellitus . In this six - month study, 51 subjects with type 2 diabetes mellitus for longer than 6 months and with evidence of stable glycemic control over the preceding 6 months participated . The subjects were randomized to receive a standard preparation of s. reticulata tea for 3 months followed by placebo in similar tea bags for a further 3 months (n + 28) or in reverse order (n 23). Hba1c was measured at recruitment, at 3 months, and on completion of the study at 6 months . There were no significant differences between the two groups in age, body mass index, male / female ratio, glycemic control, and baseline laboratory tests . In a placebo controlled, crossover trial, kajimoto et al . Investigated the clinical usefulness of s. reticulata extract for prevention or treatment of type 2 diabetes . The study subjects were 20 individuals (10 males and 10 females, average age 58 15.5 years) with type 2 diabetes . The subjects were divided into two groups and were treated with either the extract containing diet (240 mg / day) or a control diet (placebo) for six weeks . The results indicated that the s. reticulata extract containing diet achieved significant reductions in fasting plasma glucose levels, in hba1c, and in bmi . In a randomized, double - masked, crossover design trial, heacock et al . Investigated the effect of different doses of s. oblonga extract on postprandial glycemic, insulinemic, and breath hydrogen responses in healthy adults . In this study, 39 nondiabetic subjects participated in four separate 3-hour meal tolerance tests . The volunteers, after fasting for 12 hours, consumed four test meals consisting of 480 ml of study beverage (14 g fat, 82 g carbohydrate, and 20 g protein) with 0, 500, 700, or 1000 mg of s. oblonga extract on four separate occasions . The results from this study showed that, compared with the control, the 1000 mg s. oblonga extract dose reduced the plasma glucose and serum insulin incremental areas under the curve (0 to 120 minutes postprandial) by 23% and 29%, respectively . The investigators stated that this type of dose - related' effect where the higher doses have greater effect tends to bolster the confidence researchers can place in the results of a study . In a randomized, double - masked, crossover design, collene et al . Evaluated the effects of an s. oblonga extract taken at a dose of 1000 mg daily . In this study, 43 healthy people were given a high carbohydrate beverage with or without addition of s. oblonga . The results showed that when the s. oblonga extract was included, the normal rise in blood sugar and insulin following consumption of the beverage was significantly decreased . As described in an honors thesis, washam investigated the effects of s. oblonga extract on the postprandial glycemic and lactate responses along with perceived gastrointestinal, satiety, and flatulence symptom severity following a solid, high starch meal . In this study, 14 nondiabetic individuals (8 males and 6 females) participated . The results of the study showed that s. oblonga extract lowered the postprandial glucose response to a higher starch meal (spaghetti noodles, meatless spaghetti sauce, and unsweetened caffeine - free tea; 480 mg of s. oblonga extract was added to the tea for the treatment meal). In a randomized, double - blind crossover study with s. oblonga, williams et al . Evaluated the effect of a s. oblonga extract on postprandial glycemia and insulinemia in 61 patients with type 2 diabetes following ingestion of a high carbohydrate meal . In a fasted state, subjects consumed one of 3 meals: a standard liquid control meal, a control meal + 240 mg s. oblonga extract, and a control meal + 480 mg s. oblonga extract . The results from the study showed that both doses of the s. oblonga extract significantly lowered the postprandial positive area under the glucose curve and the adjusted peak glucose response compared to the control meal with the higher dose performing better . Both doses of the s. oblonga extract also lowered the positive area under the insulin curve in comparison with the control meal with the higher dose performing better . So, the long - term benefits of this herbal extract on glycemic control was explored within this population to find its value in the realm of nutritive therapy . Sce markedly decreased digestion and absorption of sucrose by its inhibitory action on sucrase and then reduced increases in blood glucose and insulin without serious adverse effect . Therefore, sce might afford a safe and effective supplementary means for controlling metabolic health and healthy blood glucose and insulin levels.
The recent years have seen a renewal of peptides as candidate therapeutics for several reasons . First, recent advances in peptide chemistry and delivery have overcome the traditional limitations of peptides as drug candidates (1). Second, the shift of therapeutic strategies towards the network of protein interactions, particularly the search for protein protein interaction inhibitors, has pushed forward the limits of small chemical molecules, whereas advances in protein recombinant technologies provide evidence that larger therapeutics such as peptides or peptide derivatives could offer plausible alternatives (2). Another motivation also comes from the large reservoir of natural peptides that have diverse and specific biological activities, and among these, bacterial small proteins (3) and venom peptides (4) raise more interest . Finally, peptides are also described as promising candidates for the treatment of central nervous system disorders (5). To assist peptide lead identification and optimization, robust computational methods recent efforts from the community of computer scientists have tackled various aspects including the design of generic databases devoted to peptide protein interactions such as pepx (8), the problem of protein peptide docking (9), the search for peptidomimetics (10) and the development of fast peptide structure prediction methods (e.g. Pep - fold, bhageerath, pepstr, peplook, i - tasser, rosetta) (1116). In 2009, we introduced the pep - fold service (11) for de novo peptide structure prediction . Though this first rapid on - line version has been used by external users for structural characterization of peptides or protein fragments (17,18) and peptide or vaccine design (19,20), the maximal length of 25 amino acids limits the number of applications . In addition, like the bhageerath (12) and pepstr (13) servers, pep - fold was only available for linear peptides, whereas there are many natural cyclic peptides with disulfide bonds such as conotoxins or cyclotides (21) and disulfide bonds increase peptide stability (22). Recently, the peplook procedure (not available on - line) brought some improvements in this direction (14). Here, we introduce an improved version of the service open to the community that (i) extends the length of linear peptides to 36 amino acids and (ii) accepts cyclic peptides using disulfide bonds defined by the user . The 3d prediction scheme is very similar to that reported in (11) and (23). It is based on a hidden markov model derived structural alphabet (sa) (24), i.e. A kind of generalized secondary structure extending the number of states from 3 (helix, coil, strand) to 27 in our case . The first step predicts sa letters from the amino acid sequence . From the amino acid sequence, a psi - blast profile is generated and is used as input of a svm that returns a probability profile of each sa letter at each position of the sequence . The second step performs the 3d assembly of the prototype fragments associated with the letters selected . It relies on the sopep coarse grained force field (25), which uses a six bead representation (full backbone except the -hydrogen and one bead for each side chain). The 3d generation is achieved by an enhanced greedy procedure (26) that builds the peptide residue by residue . This build - up procedure works using a rigid assembly scheme and thus does not explore the full conformational space but only a discrete subset . The third step generates all - atom conformations from the coarse grained models returned by the 100 simulations and performs a clustering procedure . Figure 1.pep-fold 2012 flowchart . Two major improvements have been brought to this scheme in the new version of the service . First, the selection of the sa letters from the profile has been revisited so as to remove the letters with too low probabilities . As a result, whereas the initial pep - fold version used eight letters at each position, the new version uses 5.5 letters on average . We also removed the secondary structure constraints predicted by psi - pred because the sa profile contains this information . A second major modification involves the use of tm scores (27) to cluster the full generated structures and then the sopep energies or the predicted tmscores of apollo (28)an extension of (29) for the prediction gdt_ts from structural features to rank the clusters and the conformations within the clusters ., the disulfide bonds are not simply constrained to a typical bond distance because we grow the peptide from any position of the structure and thus all oxidized cysteines are not known in the early steps of the assembly . Rather, the interaction between two oxidized cysteines i and j is described by: (1) where denotes the distance between the side - chain centroids, = 3.36 is the distance where the energy is the lowest (15 kcal / mol), = 2.39 is the distance where is 0, and e0 is the energy value for . The left side term is identical to sopep former term and the right side results in a sharper behaviour preventing energy at longer distances . Finally, other minor modifications have been brought to the side chain positioning in the all - atom generation step it is now achieved using oscar - star (30)and the disulfide bond specifications are passed to the quick all - atom minimization based on gromacs (31). As described in figure 1, the service is now fully embedded in the mobyle framework (32), providing automated form generation, command execution and result display . The input fields include the primary sequence (only standard amino acids) and the specification of constraints (disulfide bonds, residue proximities). In addition to building models up to 36 amino acids, it is possible to treat sequences of 50 amino acids and specify the maximal 36-residue region subjected to 3d modelling . This facility is useful for peptides where the n- and c - terminal regions are known to be disordered and the user wishes to focus on the modelling of the structured core . To this end, our tests show that the prediction of the sa profile is best using the complete sequence rather than a truncated sequence . The output fields have also been modified as a result of the new clustering procedure . Although our results suggest that sorting the clusters using sopep remains best, they can also be sorted according to the predicted tm scores (27) obtained by apollo (28). Finally, the use of the mobyle framework comes with facilities to visualize the best models using the openastex (33) or jmol (34) applets and perform other analyses such as the secondary structure, side - chain conformations, etc . The modifications brought to pep - fold have a limited impact on the linear peptides with 925 amino acids . A table of the results on 24 peptides is presented on the on - line documentation of the service . We see that the sharper sa letter selection does not prevent pep - fold to generate near - native conformations, with the best rigid core (rc) conformations deviating on average by only 1.5 from the nmr structures, versus 1.7 using the earlier pep - fold service . Note that the pdb entry 1wz4 is excluded from analysis since its nmr structure displays a clash between the backbone oxygens of glu7 and asp11 . The updated pep - fold procedure also generates lowest - energy conformations deviating on average by 2.5 from the nmr rcs . The results on a set of linear peptides with 2536 amino acids are presented in table 1 . Averaged over the 13 systems, pep - fold returns lowest - energy conformations deviating from the first nmr model by 4.8 and 3.4 using the full structure (fs) and the rc . Looking at the five clusters of lowest energy, the average rmsd is 3.6 (fs) and 2.8 (rc). Overall, pep - fold generates near - native conformations (rc - d <4) for 11 among 13 structurally diverse proteins with secondary structure compositions varying from, 2 (2l0 g figure 2c), 2, 2,, to 3 . Of particular interest is the high quality prediction of the topology for the 36-residue 2ki0 protein with a rc - d of 2.0 based on the sopep energy (figure 2e) which is stabilized by long - range interactions in the amino acid sequence . Similarly, the updated pep - fold version returns the native 3 conformation of the 36-residue 1e0n protein (figure 2a), while the earlier version, using the 27 amino acids which are not random coil in the nmr structure, predicted a -strand packed against two helices (11). For the 28-residue 1psv and 31-residue 2gdl, the lowest - energy conformations differ by 7.4 and 4.8 rms from nmr using the rc . In figure 2b, interestingly, experimental and computational studies on homologous peptides have shown that the -hairpin is only marginally stable (35). In contrast, for the 2gdl target (figure 2 g) which displays an -coil- topology, the predicted -strand at the c - terminal is not compatible with the -helix structure observed by nmr . Pep - fold models are in cyan . From left to right, top to bottom: 1e0n (a), 1psv (b), 2l0 g (c), 1n0a (d), 2ki0 (e), 1kwd (f) and 2gdl (g) lowest - energy models and 1wm8 (h) best model . Table 1.results obtained for 13 linear peptides with 2536 residuespdb idtoplrcsopepbest5fs - drc - dfs - drc - drnk1by0a271:234.361.752.941.7411yyba271:206.491.473.41.4212kblb2293:4\|6:274.683.914.683.9111fsdab2281:264.143.883.913.6611psvab2282:257.197.444.614.642k76ba304:293.152.752.11.8812gdlaca318:8\|10:11\|14:15\|21:297.574.846.534.5732l0ga2325:324.533.322.11.6412bn6a2334:294.493.183.092.1411e0nb3351:252.162.161.681.6821wr3b3365:155.723.744.263.511wr4b3365:344.683.264.683.2612ki0bab365:11\|13:363.561.992.662.431mean4.83.43.62.8pdb i d, pdb identifier; top, secondary structure topology (a for helix, b for strand, c for coil); l, peptide length; rc, the definition of the rigid core (pdb positions start at 1); fs - d (rc - d), full structure (rigid core) rms deviation () for the model of lowest energy (sopep) and the best model among the five clusters of lowest energy (best5); rnk, the rank of the cluster containing the best model.the lowest - energy models for 1e0n, 1psv, 2l0 g, 2ki0 and 2gdl are shown on figure 2 . Pep - fold models . Pep - fold models are in cyan . From left to right, top to bottom: 1e0n (a), 1psv (b), 2l0 g (c), 1n0a (d), 2ki0 (e), 1kwd (f) and 2gdl (g) lowest - energy models and 1wm8 (h) best model . Results obtained for 13 linear peptides with 2536 residues pdb i d, pdb identifier; top, secondary structure topology (a for helix, b for strand, c for coil); l, peptide length; rc, the definition of the rigid core (pdb positions start at 1); fs - d (rc - d), full structure (rigid core) rms deviation () for the model of lowest energy (sopep) and the best model among the five clusters of lowest energy (best5); rnk, the rank of the cluster containing the best model . The lowest - energy models for 1e0n, 1psv, 2l0 g, 2ki0 and 2gdl are shown on figure 2 . Table 2 presents the results of 34 peptides containing one, two or three disulfide bonds using the peplook test set (14) except the peptides 2p7r, 1qvl and 1foz <8 amino acids, and the peptide 1ixu free of any disulfide bond in the nmr structure . For each peptide, we describe the models of lowest energy and of lowest rmsd (best) with respect to the nmr structure . We also give the number of ss bonds formed after the coarse - grained and the all - atom procedures . On average, the updated pep - fold generates best models deviating by only 2.7 and 2.5 rms and lowest - energy models deviating by 4.2 and 3.7 rms using the fss and rcs, respectively . This indicates that sopep is not optimal yet for recognizing near - native from higher rmsd conformations . Compared to the results of the earlier pep - fold version, which did not consider the disulfide bonds explicitly (11), we reach an improvement of 1.3 rms . Compared to peplook performances, the pep - fold best models are also closer by 1 rms to the nmr conformations . Whereas the server leads to good results for peptides containing 1 and 2 disulfide bonds (figure 2f and d for 1kwd and 1n0a) with 61.2% of the disulfide bonds formed at the coarse - grained level (table 1), the performances are degraded for the nine peptides with three disulfide bonds (figure 2h for 1wm8). For these peptides, the lowest - energy conformations deviate on average by 5.4 rms from the nmr structures and a similar behaviour is observed with peplook (5.6 rms). This discrepancy between prediction and experiment comes in part from the non - optimization of the sopep force field for cyclic peptides, but mainly we observe that the structures with three disulfide bonds are very constrained, leading to large deviations at the local level between the conformations observed in the nmr structures and the structural alphabet conformations predicted from the sequence free of any disulfide bond constraints . Similar results are obtained if we vary the number of sa letters from 5.5 to 8 during assembly . Table 2.results obtained for 34 cyclic peptides with disulfide bondspdb idl#ssrcsopepbestfs - drc - dcgaafs - drc - dcgaa1im72113:9::15:213.93.910002.52.510011jbl1612:143.33.310011.81.8001n0a1717:150.70.7010.40.4001n0c2412:11::14:242.32.310010.40.4011nim2411:233.81.8003.42.8011gnb1422:9::12:144.84.150143.85001b451323.63.310012.32.2001etl2223.13.110011.61.6001hje1923.63.65012.22.25021hp91222:113.53.55012.22.25011ien1324.84.85002.52.5001im11421.31.2011.21.25001kcn1625.75.610003.52.85011kwd3023:292.92.1100221.3011mii1121.81.85001.61.6001oig1522:7::11:146.56.15014.84.75011r8t2421:224.53.410012.62.25011rpc2123:216.96.35014.44001ter21275.950132.85001v6r2621:235.15.110023.93.9001wqc1322:131.71.310021.21.110021x7k2221:174.22.5004.22.5001xgb1623.5300335022ajw1322.40.95021.31002i282822.12.110011.51.5002oq92428:249.2410013.53.35012efz1234.24.2012.12.166.712nx71332:136.96.933.314.1433.311mmc1037.16.166.724.64.633.301orx2231:194.84.833.312.92.7001sp72831:10::12:284.54.366.722.6233.301v5a28355.166.713.73.8001wm81936:167766.723.63.666.722it72832:284466.713.73.766.71mean4.23.761.22.72.529.5pdb i d, pdb identifier; l, peptide length; ss, number of disulfide bond; rc, the definition of the rigid core (pdb positions start at 1); fs - d (rc - d), full structure (rigid core) rms deviation () for the model of lowest energy (sopep) and the model of lowest rmsd (best); cg, ss bonds formed in the coarse grained representation; aa, ss bonds formed in the final all - atom representation.our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation (1) lies within 0.7 . The discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied (2.0 0.2) in all - atom structures.the lowest - energy models for 1n0a and 1kwd, and the best rmsd model for 1wm8 are shown on figure 2 . Results obtained for 34 cyclic peptides with disulfide bonds pdb i d, pdb identifier; l, peptide length; ss, number of disulfide bond; rc, the definition of the rigid core (pdb positions start at 1); fs - d (rc - d), full structure (rigid core) rms deviation () for the model of lowest energy (sopep) and the model of lowest rmsd (best); cg, ss bonds formed in the coarse grained representation; aa, ss bonds formed in the final all - atom representation . Our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation (1) lies within 0.7 . The discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied (2.0 0.2) in all - atom structures . The lowest - energy models for 1n0a and 1kwd, and the best rmsd model for 1wm8 are shown on figure 2 . The modifications brought to pep - fold have a limited impact on the linear peptides with 925 amino acids . A table of the results on 24 peptides is presented on the on - line documentation of the service . We see that the sharper sa letter selection does not prevent pep - fold to generate near - native conformations, with the best rigid core (rc) conformations deviating on average by only 1.5 from the nmr structures, versus 1.7 using the earlier pep - fold service . Note that the pdb entry 1wz4 is excluded from analysis since its nmr structure displays a clash between the backbone oxygens of glu7 and asp11 . The updated pep - fold procedure also generates lowest - energy conformations deviating on average by 2.5 from the nmr rcs . The results on a set of linear peptides with 2536 amino acids are presented in table 1 . Averaged over the 13 systems, pep - fold returns lowest - energy conformations deviating from the first nmr model by 4.8 and 3.4 using the full structure (fs) and the rc . Looking at the five clusters of lowest energy, the average rmsd is 3.6 (fs) and 2.8 (rc). Overall, pep - fold generates near - native conformations (rc - d <4) for 11 among 13 structurally diverse proteins with secondary structure compositions varying from, 2 (2l0 g figure 2c), 2, 2,, to 3 . Of particular interest is the high quality prediction of the topology for the 36-residue 2ki0 protein with a rc - d of 2.0 based on the sopep energy (figure 2e) which is stabilized by long - range interactions in the amino acid sequence . Similarly, the updated pep - fold version returns the native 3 conformation of the 36-residue 1e0n protein (figure 2a), while the earlier version, using the 27 amino acids which are not random coil in the nmr structure, predicted a -strand packed against two helices (11). For the 28-residue 1psv and 31-residue 2gdl, the lowest - energy conformations differ by 7.4 and 4.8 rms from nmr using the rc . In figure 2b, interestingly, experimental and computational studies on homologous peptides have shown that the -hairpin is only marginally stable (35). In contrast, for the 2gdl target (figure 2 g) which displays an -coil- topology, the predicted -strand at the c - terminal is not compatible with the -helix structure observed by nmr . Pep - fold models are in cyan . From left to right, top to bottom: 1e0n (a), 1psv (b), 2l0 g (c), 1n0a (d), 2ki0 (e), 1kwd (f) and 2gdl (g) lowest - energy models and 1wm8 (h) best model . Table 1.results obtained for 13 linear peptides with 2536 residuespdb idtoplrcsopepbest5fs - drc - dfs - drc - drnk1by0a271:234.361.752.941.7411yyba271:206.491.473.41.4212kblb2293:4\|6:274.683.914.683.9111fsdab2281:264.143.883.913.6611psvab2282:257.197.444.614.642k76ba304:293.152.752.11.8812gdlaca318:8\|10:11\|14:15\|21:297.574.846.534.5732l0ga2325:324.533.322.11.6412bn6a2334:294.493.183.092.1411e0nb3351:252.162.161.681.6821wr3b3365:155.723.744.263.511wr4b3365:344.683.264.683.2612ki0bab365:11\|13:363.561.992.662.431mean4.83.43.62.8pdb i d, pdb identifier; top, secondary structure topology (a for helix, b for strand, c for coil); l, peptide length; rc, the definition of the rigid core (pdb positions start at 1); fs - d (rc - d), full structure (rigid core) rms deviation () for the model of lowest energy (sopep) and the best model among the five clusters of lowest energy (best5); rnk, the rank of the cluster containing the best model.the lowest - energy models for 1e0n, 1psv, 2l0 g, 2ki0 and 2gdl are shown on figure 2 . Pep - fold models . Pep - fold models are in cyan . From left to right, top to bottom: 1e0n (a), 1psv (b), 2l0 g (c), 1n0a (d), 2ki0 (e), 1kwd (f) and 2gdl (g) lowest - energy models and 1wm8 (h) best model . Results obtained for 13 linear peptides with 2536 residues pdb i d, pdb identifier; top, secondary structure topology (a for helix, b for strand, c for coil); l, peptide length; rc, the definition of the rigid core (pdb positions start at 1); fs - d (rc - d), full structure (rigid core) rms deviation () for the model of lowest energy (sopep) and the best model among the five clusters of lowest energy (best5); rnk, the rank of the cluster containing the best model . The lowest - energy models for 1e0n, 1psv, 2l0 g, 2ki0 and 2gdl are shown on figure 2 . Table 2 presents the results of 34 peptides containing one, two or three disulfide bonds using the peplook test set (14) except the peptides 2p7r, 1qvl and 1foz <8 amino acids, and the peptide 1ixu free of any disulfide bond in the nmr structure . For each peptide, we describe the models of lowest energy and of lowest rmsd (best) with respect to the nmr structure . We also give the number of ss bonds formed after the coarse - grained and the all - atom procedures . On average, the updated pep - fold generates best models deviating by only 2.7 and 2.5 rms and lowest - energy models deviating by 4.2 and 3.7 rms using the fss and rcs, respectively . This indicates that sopep is not optimal yet for recognizing near - native from higher rmsd conformations . Compared to the results of the earlier pep - fold version, which did not consider the disulfide bonds explicitly (11), we reach an improvement of 1.3 rms . Compared to peplook performances, the pep - fold best models are also closer by 1 rms to the nmr conformations . Whereas the server leads to good results for peptides containing 1 and 2 disulfide bonds (figure 2f and d for 1kwd and 1n0a) with 61.2% of the disulfide bonds formed at the coarse - grained level (table 1), the performances are degraded for the nine peptides with three disulfide bonds (figure 2h for 1wm8). For these peptides, the lowest - energy conformations deviate on average by 5.4 rms from the nmr structures and a similar behaviour is observed with peplook (5.6 rms). This discrepancy between prediction and experiment comes in part from the non - optimization of the sopep force field for cyclic peptides, but mainly we observe that the structures with three disulfide bonds are very constrained, leading to large deviations at the local level between the conformations observed in the nmr structures and the structural alphabet conformations predicted from the sequence free of any disulfide bond constraints . Similar results are obtained if we vary the number of sa letters from 5.5 to 8 during assembly . Table 2.results obtained for 34 cyclic peptides with disulfide bondspdb idl#ssrcsopepbestfs - drc - dcgaafs - drc - dcgaa1im72113:9::15:213.93.910002.52.510011jbl1612:143.33.310011.81.8001n0a1717:150.70.7010.40.4001n0c2412:11::14:242.32.310010.40.4011nim2411:233.81.8003.42.8011gnb1422:9::12:144.84.150143.85001b451323.63.310012.32.2001etl2223.13.110011.61.6001hje1923.63.65012.22.25021hp91222:113.53.55012.22.25011ien1324.84.85002.52.5001im11421.31.2011.21.25001kcn1625.75.610003.52.85011kwd3023:292.92.1100221.3011mii1121.81.85001.61.6001oig1522:7::11:146.56.15014.84.75011r8t2421:224.53.410012.62.25011rpc2123:216.96.35014.44001ter21275.950132.85001v6r2621:235.15.110023.93.9001wqc1322:131.71.310021.21.110021x7k2221:174.22.5004.22.5001xgb1623.5300335022ajw1322.40.95021.31002i282822.12.110011.51.5002oq92428:249.2410013.53.35012efz1234.24.2012.12.166.712nx71332:136.96.933.314.1433.311mmc1037.16.166.724.64.633.301orx2231:194.84.833.312.92.7001sp72831:10::12:284.54.366.722.6233.301v5a28355.166.713.73.8001wm81936:167766.723.63.666.722it72832:284466.713.73.766.71mean4.23.761.22.72.529.5pdb i d, pdb identifier; l, peptide length; ss, number of disulfide bond; rc, the definition of the rigid core (pdb positions start at 1); fs - d (rc - d), full structure (rigid core) rms deviation () for the model of lowest energy (sopep) and the model of lowest rmsd (best); cg, ss bonds formed in the coarse grained representation; aa, ss bonds formed in the final all - atom representation.our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation (1) lies within 0.7 . The discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied (2.0 0.2) in all - atom structures.the lowest - energy models for 1n0a and 1kwd, and the best rmsd model for 1wm8 are shown on figure 2 . Results obtained for 34 cyclic peptides with disulfide bonds pdb i d, pdb identifier; l, peptide length; ss, number of disulfide bond; rc, the definition of the rigid core (pdb positions start at 1); fs - d (rc - d), full structure (rigid core) rms deviation () for the model of lowest energy (sopep) and the model of lowest rmsd (best); cg, ss bonds formed in the coarse grained representation; aa, ss bonds formed in the final all - atom representation . Our criterion for evaluating a disulfide bond formed in the coarse grained structure is that rij in equation (1) lies within 0.7 . The discrepancy between the number of disulfide bonds in the atomistic and coarse grained structures comes from the fact that a very stringent condition on the s s distance must be satisfied (2.0 0.2) in all - atom structures . The lowest - energy models for 1n0a and 1kwd, and the best rmsd model for 1wm8 are shown on figure 2 . The first goal was to extend the length of linear peptides so as to develop a server able to explore systems with higher thermodynamic stabilities, and with new topologies such as the and 3 folds . Using a benchmark of 37 peptides with 936 amino acids, and a total of 100 simulations for each system, the updated pep - fold version fails to generate the native conformation for only one system, where a -strand is preferred over an -helix at the c - terminus . Note that our results are reproducible and do not change if we use 200 simulations . Preliminary simulations also indicate that pep - fold performs well for sequences upwards of 50 amino acids where the fs is modelled (data not shown), but opening the service for this length is under consideration given the computer resources needed . This increase in peptide size from 25 to 50 amino acids comes from the reduction of the sa letters considered at each position . The structure simulations of even larger systems within the framework of a greedy procedure remain to be evaluated . The second goal was to provide structural predictions for cylic peptides with disulfide bonds . For systems with one and two disulfide bonds, pep - fold performs better than peplook . For systems with more disulfide bonds, such as natural toxins, improvements in the sa letter prediction and atomistic construction one solution for construction, under investigation, is to enforce disulfide bond formation in the gromacs procedure and to relax the models by short molecular dynamics simulations . In summary, the updated version of pep - fold provides a fast and convenient on - line approach for the de novo design of peptides up to 36 residues, and it also supports the possibility to tackle peptide engineering by the insertion of disulfide bonds, a classical way to increase stability . The typical computational time for a 36-residue peptide is 40 min using 40 cores . The server also supports, using the same formalism as disulfide bonds, the possibility to specify additional constraints such as residue proximities . Clearly, our results open the door to further improvements such as the consideration of other covalent linking between side - chains and the modelling of ribosomal peptides at a genome scale can be considered . The institut national de la sant et de la recherche medicale recurrent funding, the centre national de la recherche scientifique recurrent funding, and the ia bioinformatics grant [bipbip]. Funding for open access charge: institut national de la sant et de la recherche medicale umr - s 973.
We defined broad consent as a process in which participants agree prospectively to have their samples, genomic data, and health information retained for use in any future research deemed appropriate by a biobank and/or relevant oversight bodies . Studies of broad consent may use an opt - in or an opt - out model . Categorical consent, by contrast, is a process in which participants agree prospectively to future use of their samples and data for particular types of research, usually by categories of disease (e.g., cardiac diseases, diabetes). Refers to the transfer of biospecimens with their associated genotypic and/or phenotypic information, data derived from biospecimens, and/or health information to researchers at institutions that are not directly affiliated with the biobanks or to other biorepositories . We systematically searched the literature on broad consent and data sharing for biobank research using the following databases: medline via the pubmed interface, web of science, national reference center for bioethics literature databases (ethxweb, genethx), and dissertation abstracts international . Search strategies used subject heading terms appropriate for each database and key words relevant to biobanking, consent, and data sharing (supplementary table s1 online). Searches were limited to the literature published since 1990 to capture current views about biobanking . We also manually searched the reference lists of included studies and of recent narrative and systematic reviews addressing the topic . Our initial searches were done between october and december 2013 and were updated in march 2015 . Two reviewers (n.a.s . And a colleague) initially screened titles and abstracts, and two investigators (n.a.g . And e.w.c .) Reviewed the full text of the included articles . Articles were included if they reported empirical data with sufficient detail to enable use and aggregation of the data and results about individuals in the united states regarding one or more of the following: participant perceptions of broad consent or data sharing for biobank research, preferences for different consent models for biobank research, information about people's opinions about participating in biobank research, or providing broad consent for biobank research . Disagreements between reviewers were resolved by discussion that included a third reviewer (a.h.m.a .) To reach consensus . We identified and screened a total of 3,205 citations and abstracts through the electronic database searches and manual review of articles and bibliographies (figure 1). After reviewing titles and abstracts, we excluded 2,714 studies that did not meet our criteria . We assessed the full text of the 491 remaining studies and excluded another 440 articles because they (i) did not address biobanking, consent, or data sharing (n = 403); (ii) were not conducted in the united states (n = 206); or (iii) were not obtainable (n = 1). Two investigators (n.a.g . And e.w.c .) Assessed the quality of studies using questions adapted from published criteria for the quality assessment of survey and focus group studies . Scoring criteria fell into the following broad domains: (i) description of the methods, (ii) participant recruitment from a representative pool and response rates, (iii) appropriateness of objective study questions, and (iv) data analysis lending to reproducible results . Articles that adequately defined criteria in all four domains were rated as good . Articles containing information that had adequate descriptions of the methods but did not fulfill the criteria for all of the other domains received a rating of fair . Articles that failed to adequately define their methods, thus preventing an evaluation of representativeness, bias, or reproducibility, received a rating of poor . Two investigators (n.a.g . And e.w.c .) Also characterized the studies as conducted in urban, rural, or combined settings . Data were extracted into summary tables (supplementary table s3 online) by outlining the study population and biobank focus, methods, quality assessment, urban / rural residency, and key outcomes related to consent and data sharing . We report the relevant findings based on the terminology, percentages, and number of significant digits as presented in the publications . We defined broad consent as a process in which participants agree prospectively to have their samples, genomic data, and health information retained for use in any future research deemed appropriate by a biobank and/or relevant oversight bodies . Studies of broad consent may use an opt - in or an opt - out model . Categorical consent, by contrast, is a process in which participants agree prospectively to future use of their samples and data for particular types of research, usually by categories of disease (e.g., cardiac diseases, diabetes). Refers to the transfer of biospecimens with their associated genotypic and/or phenotypic information, data derived from biospecimens, and/or health information to researchers at institutions that are not directly affiliated with the biobanks or to other biorepositories . We systematically searched the literature on broad consent and data sharing for biobank research using the following databases: medline via the pubmed interface, web of science, national reference center for bioethics literature databases (ethxweb, genethx), and dissertation abstracts international . Search strategies used subject heading terms appropriate for each database and key words relevant to biobanking, consent, and data sharing (supplementary table s1 online). Searches were limited to the literature published since 1990 to capture current views about biobanking . We also manually searched the reference lists of included studies and of recent narrative and systematic reviews addressing the topic . Our initial searches were done between october and december 2013 and were updated in march 2015 . Two reviewers (n.a.s . And a colleague) initially screened titles and abstracts, and two investigators (n.a.g . And e.w.c .) Reviewed the full text of the included articles . Articles were included if they reported empirical data with sufficient detail to enable use and aggregation of the data and results about individuals in the united states regarding one or more of the following: participant perceptions of broad consent or data sharing for biobank research, preferences for different consent models for biobank research, information about people's opinions about participating in biobank research, or providing broad consent for biobank research . Disagreements between reviewers were resolved by discussion that included a third reviewer (a.h.m.a .) To reach consensus . We identified and screened a total of 3,205 citations and abstracts through the electronic database searches and manual review of articles and bibliographies (figure 1). After reviewing titles and abstracts, we excluded 2,714 studies that did not meet our criteria . We assessed the full text of the 491 remaining studies and excluded another 440 articles because they (i) did not address biobanking, consent, or data sharing (n = 403); (ii) were not conducted in the united states (n = 206); or (iii) were not obtainable (n = 1). Two investigators (n.a.g . And e.w.c .) Assessed the quality of studies using questions adapted from published criteria for the quality assessment of survey and focus group studies . Scoring criteria fell into the following broad domains: (i) description of the methods, (ii) participant recruitment from a representative pool and response rates, (iii) appropriateness of objective study questions, and (iv) data analysis lending to reproducible results . Articles containing information that had adequate descriptions of the methods but did not fulfill the criteria for all of the other domains received a rating of fair . Articles that failed to adequately define their methods, thus preventing an evaluation of representativeness, bias, or reproducibility, received a rating of poor . Two investigators (n.a.g . And e.w.c .) Also characterized the studies as conducted in urban, rural, or combined settings . Data were extracted into summary tables (supplementary table s3 online) by outlining the study population and biobank focus, methods, quality assessment, urban / rural residency, and key outcomes related to consent and data sharing . We report the relevant findings based on the terminology, percentages, and number of significant digits as presented in the publications . A total of 51 publications comprising 48 studies were included in this review . Most studies involved surveys (n = 23), followed by focus groups (n = 8), mixed methods (n = 14), interviews (n = 1), and analyses of consent forms (n = 2) (supplementary table s3 online). Two publications used a mixed - methods approach that included qualitative studies that informed the development and implementation of a survey . Nineteen studies were of good quality, 27 of fair quality, and 2 of poor quality . Roughly one - third of the studies (n = 20) were written and published after the office of human research protections issued the anprm in july 2011 . The number of studies published per year from 2008 to 2014 ranged from five to seven, with no notable difference after the anprm was issued . Some of the studies published after 2011 mention the anprm . Although we examined studies published since 1990, no studies that met our inclusion criteria were published before 2001 . Of these, just over half (51.3%) of participants identified as white, and native - american, alaska native, native hawaiian, and pacific islander participants made up 2.2% of the sample . Twenty - eight studies were conducted primarily in urban settings, two were conducted in rural settings, nine were conducted in both urban and rural settings, and nine studies were conducted nationwide . Three papers each reported two unique studies; other studies were reported in multiple papers . For example, a retrospective analysis of signed informed consent forms found that 87.1% of 1,298 research participants at the nih authorized all future research . In a different large national study, the national health and nutrition examination survey (nhanes), 84.8% of 4,480 overall participants recruited in 1999 and 2000 agreed to dna specimen collection for inclusion in a national repository for genetic research . Many studies asked participants hypothetical questions about their willingness to provide broad consent for research . In indiana, 88.4% of 273 cancer patients agreed that they would be willing to permit their tissue sample to be used in research on any condition . After time for deliberation, 85% of 40 focus group participants in north carolina reported that they would agree to have blood and information stored indefinitely in a biorepository for future research . Similarly, 78% of 49 focus group participants in chicago were interested in participating in a biobank, and the majority stated they would give broad consent . Of 30 patients who were interviewed at a hawaiian cancer center, 77% endorsed broad consent . One representative nationwide survey found that 68% of 1,593 respondents were willing to give broad consent for research, although their enthusiasm waned if they had a moral objection to certain types of studies for which their samples might be used . Two studies examined patients' willingness to participate in biobanks managed by kaiser permanente: 69% of 500 kaiser patients in the northwest and 69% of 203 in colorado agreed to participate in a biobank . In a focus group study in boston, patients with breast cancer were generally positive about having their samples used for secondary studies that were not planned at the time they gave consent . Scott et al . Reported the results of a 1998 survey of blood donors that asked about their views regarding storage and use of the blood for research . Of the 49,775 respondents, 60.3% said that testing stored blood for any research was acceptable with the donor's permission, and 35.5% would not require permission for research use . These studies reported substantial acceptance for broad consent . Asking participants for their preference among different types of consent broad, study - by - study, or categorical consent revealed more mixed support for broad consent . For example, 47% of 931 veterans preferred to give broad consent over other types of consent for all research approved by an oversight board . After adjusting for missing data, a national survey of 4,569 adults found that 52% preferred broad consent, whereas 48% preferred study - by - study consent . In a survey of 751 iowans, 42% preferred broad consent and 29% favored study - specific consent, compared with 25% who favored categorical consent . In another study of 315 cancer patients at two hospitals in atlanta, 92 and 97% were willing to allow their samples to be used for research on other diseases; when asked to specify a preference, 56% preferred one - time broad consent and 11% preferred study - by - study consent over no consent or no preference . In a 2001 nationwide survey, 43% of 2,621 participants were willing to donate blood for genetic research and to allow it to be stored for future research . Similarly, only 39.3% of 30 patients who had already donated samples preferred broad consent over consent for specific studies . By contrast, 77.7% of 1,276 people recruited through a crowd - sourced internet marketplace were willing to donate to biobanks, even after receiving disclosures about potentially objectionable research; however, 40.8% of participants still felt that specific consent was necessary, even if it might inhibit research progress . A similar nationwide survey of 1,599 individuals conducted through a probability - based online panel of adults found a wide range of opinions, with broad consent and real - time study - by - study consent considered the several studies showed that participants preferred to give informed consent for each study rather than a broad consent, with preferences ranging from 42 to 72%: 42% of a national sample of 4,700 us adults (which rose to 48% after adjusting for missing data), 43 to 50% of 931 veterans nationwide, and 60.7% of adults recruited in new york . Of 393 parents, 72% reported that they would want to consent each time to allow their child's dried bloodspots to be used for research . In focus groups of 92 native hawaiians, respondents repeatedly expressed desire to re - consent, although some stated that they would be content if they trusted the researcher or the biobank's governance . In one study of 273 jewish individuals, 6075% believed that consent should be required regardless of whether the dna was collected in a research or clinical setting . In a focus group study of 178 alaska native participants, some indicated a preference to have consent options for a variety of specimen uses, storage duration, and destruction of the sample at the completion of the study . In the same group, some wanted re - contact each time, whereas others felt that a one - time consent was appropriate for new studies . In chicago, 239 postpartum women were asked about their willingness to enroll their children into a pediatric biobank: 48% of women would enroll their child, but 24% would not; of the latter, 82% of the participants were african american . In another focus group study, 11 of 15 participants preferred tiered consent over other methods to exert the greatest level of control regarding how they wanted their data to be shared; however, participants who were willing to provide broad consent also appreciated the option to opt in or opt out of dna data sharing . Some studies reported that most respondents favored an opt - in approach, whereas others found that opt - out was acceptable or even preferred by the majority . A majority of participants67% of 751 survey respondents and 63% of 57 focus group participants who were asked about biobank participation in iowa preferred opt - in, whereas 18% of survey respondents and 25% of focus group participants in the same study preferred opt - out . In a study of 451 nonactive military veterans, 82% thought it would be acceptable for the proposed million veterans biobank to use an opt - in approach, and 75% thought that an opt - out approach was acceptable; 80% said that they would take part if the biobank were opt - in as opposed to 69% who would participate if it were an opt - out approach . When asked to choose which option they would prefer, 29% of respondents chose the opt - in method, 14% chose opt - out, 50% said either would be acceptable, and 7% would not want to participate . In some cases, biobank participants were re - contacted to inquire about their thoughts regarding proposed changes to the biobank in which they participated . Thirty - two biobank participants who attended focus groups in wisconsin regarding proposed minimal - risk protocol changes were comfortable with using an opt - out model for future studies because of the initial broad consent given at the beginning of the study and their trust in the institution . A study of 365 participants who were re - contacted about their ongoing participation in a biobank in seattle showed that 55% thought that opt - out would be acceptable, compared with 40% who thought it would be unacceptable . Similarly, several studies explored perspectives on the acceptability of an opt - out biobank at vanderbilt university . First, 91% of 1,003 participants surveyed in the community thought leftover blood and tissues should be used for anonymous medical research under an opt - out model; these preferences varied by population, with 76% of african americans supporting this model compared with 93% of whites . In later studies of community members, approval rates for the opt - out biobank were generally high (around 90% or more) in all demographic groups surveyed, including university employees, adult cohorts, and parents of pediatric patients . Three studies explored community perspectives on using newborn screening blood spots for research through the michigan biotrust for health program . First, 77% of 393 parents agreed that parents should be able to opt out of having their child's blood stored for research . Second, 87 participants were asked to indicate a preference: 55% preferred an opt - out model, 29% preferred to opt - in, and 16% felt that either option was acceptable . Finally, 39% of 856 college students reported that they would give broad consent to research with their newborn blood spots, whereas 39% would want to give consent for each use for research . In a nationwide telephone survey regarding the use of samples collected from newborns, 46% of 1,186 adults believed that researchers should re - consent participants when they turn 18 years old . Some studies examined the differences in participants' willingness to provide broad consent for samples that were de - identified or anonymous as compared with identifiable . Respondents generally preferred to give consent if their samples were identifiable . In two studies involving 429 primarily native hawaiian participants, 78% of native hawaiians and 66% of whites indicated that they would require consent for research if the specimens were identifiable and collected in the clinical setting . For genetics research, 81% native hawaiians and 78% of whites indicated that they would require consent if the specimens were identifiable . In a us - wide telephone survey, 81% of 1,193 respondents stated that they would want to be informed about research being done with their sample if it were identifiable; additionally, 57% said they would require permission to use their samples if they were identifiable . For example, 65.8% of 504 adults who participated in a telephone survey across the united states reported that they would require consent for samples collected in the clinic if they were identifiable, compared with 27.3% who reported they would require consent if samples were anonymized . In the research setting, fewer people thought consent was required for identifiable (29.0%) or anonymized (12.1%) samples . In a study utilizing a hypothetical biobank scenario, 43% of 565 government and medical employees in new mexico indicated that they would donate their sample for future genetic testing if it could not be traced to them . Not all studies found that people were worried about identifiability . In one survey of 144 clinicians, 86% said that they would donate a dna sample to a hypothetical biobank in new york regardless of whether it was linked to or unlinked from their identity . In the study in new mexico, 36% of 565 respondents found it acceptable for broadly consented samples to be used by their local university, even if the samples were linked to them . Characteristics associated with favoring broad consent included being male, white / caucasian, older, and more affluent . By contrast, asians, black non - hispanics, african americans, and others (who represented 14.3% of the total) were less likely than whites to believe that research without explicit permission was acceptable . One study of consent forms showed that 75.0% of african americans gave broad consent compared with 88.4% of whites (p = 0.002). Similarly, in the nhanes data, 78.7% of african americans and 87.1% of whites consented to genetics research . One study reported that participants who were significantly more likely to prefer broad consent also believed that participating would make me feel like i was contributing to society (odds ratio = 1.85; p = 0.001), that the study would accelerate medical treatments and cures (odds ratio = 2.20; p = 0.001), and that participating in the cohort study would be easy (odds ratio = 1.59; p <0.001). Other investigators reported that the large majority (97.7%) of respondents said yes or maybe to the idea that it is a gift to society when an individual takes part in medical research . Many other studies cited the benefit of research to improve health as a reason to favor broad consent . Most studies of data sharing were conducted with studies of consent preferences; however, six studies were conducted with the primary goal of eliciting preferences on data sharing . Participants were generally willing to have their samples and information shared with other academic institutions . Willingness to share data with academic and medical researchers was acceptable for 92% of 4,659 us adults generally, and 80% of 931 us veterans specifically . More than 70% of 100 young adults in baltimore who were enrolled in a longitudinal study of prevention were willing to share results arising from their dna . Nearly three fourths of 40 community members in a focus group study in north carolina were comfortable with academic researchers having access to their samples . Many of 79 focus group participants in seattle endorsed the value of sharing, agreed that sharing locally and with close collaborators was acceptable, and were comfortable with nonprofit and public - interest organizations using data from their samples . In one focus group study of 48 primarily white and female participants in iowa, the majority cited positive reasons for donating their samples to help and to contribute to advancements in research, and that data sharing would not affect their decision to enroll in a biobank . In another focus group study of 100 african americans in north carolina, many recognized the benefits of data sharing but wanted the potential risks to be disclosed, and some wanted the data to be restricted . In another study of patients with inflammatory bowel diseases, 97.3% of 92 respondents were comfortable with sharing their biological sample with investigators in the united states, but 23.8% were uncomfortable with sharing with investigators outside the united states . Some participants expressed concern over sharing their data and information with federal repositories . In one study, 18.5% of 4,050 vanderbilt university faculty and staff were more likely to want to participate in their institution's biobank if the de - identified data were deposited into a national database; however, 12.1% were less likely to want to participate . In two large nationwide surveys, 80% of 4,659 adults were willing to have their data shared with government researchers; however, 75% of the same sample also were concerned about the government having [their] samples and information . Similarly, another study found that 71% of 931 veterans were willing to grant database access to government researchers, but half were concerned about the government having [their] samples and information . Other studies have shown that some people are concerned about government involvement in maintaining databases containing biomedical information . More than half of the 40 participants in a focus group study of north carolina community members were concerned about government researchers having access to their institution's biorepository . Despite concerns, 61% of 203 kaiser patients in colorado would still provide a sample even if the data would be submitted to a government database, and 82% of 500 kaiser patients in oregon agreed to have their information posted in a us government database . In a large metropolitan area in southwest florida, some of 95 focus group participants believed that biospecimens were already being collected from leftover tissue, and others suspected that tissues were already being shared with researchers in other countries who lack strict laws' governing research . In a focus group study of 178 alaska native participants, some cited mistrust of the government and police having access to their samples and wanted transparency from the researchers about how their samples were used . The majority of participants were willing to share with pharmaceutical company researchers, but the percentage was generally less than the percentage willing to share with academic researchers . Seventy - five percent of 4,659 us adults, 54% of 931 veterans, 55.2% of 1,599 adults responding to a nationwide survey, and 75.1% of members of the crohn's and colitis foundation of america partners cohort were willing to share with pharmaceutical company researchers . Focus group participants in florida voiced concern about providing blanket consent because they would not benefit financially from any resulting discoveries . Factors associated with views about data sharing . With the exception of gender, few demographic data (e.g., about race / ethnicity, socioeconomic status, education, and urban / rural residency) were available . Even when demographic information was obtained, investigators did not always report how these variables correlated with respondents' opinions . Therefore, it was largely not possible to draw meaningful conclusions about the associations between sociodemographic factors and views on data sharing . The willingness of patients with cancer to share seemed to be shaped by their devotion to the institution at which they were receiving care . For example, patients with cancer in indiana who agreed to participate in a biobank were less likely to be willing to allow their tissue samples to be used by researchers who were not affiliated with the local researchers (89.7%), compared with 96.3% who were willing to share with local university researchers (p <0.01). Half of 100 patients with breast cancer at md anderson cancer center preferred to allow only their physician (24%) or other researchers at their hospital (26%) to use their de - identified genetic data for research; fewer patients were willing to share their de - identified data with any cancer researcher (25%) or any researcher (18%). In another study, 95% of 315 patients with cancer in atlanta were willing to allow researchers to share samples with other local researchers, but only 85% and 92% of participants at two different sites were willing to have their samples shared elsewhere in the united states (p <0.05). A total of 51 publications comprising 48 studies were included in this review . Most studies involved surveys (n = 23), followed by focus groups (n = 8), mixed methods (n = 14), interviews (n = 1), and analyses of consent forms (n = 2) (supplementary table s3 online). Two publications used a mixed - methods approach that included qualitative studies that informed the development and implementation of a survey . Nineteen studies were of good quality, 27 of fair quality, and 2 of poor quality . Roughly one - third of the studies (n = 20) were written and published after the office of human research protections issued the anprm in july 2011 . The number of studies published per year from 2008 to 2014 ranged from five to seven, with no notable difference after the anprm was issued . Some of the studies published after 2011 mention the anprm . Although we examined studies published since 1990, no studies that met our inclusion criteria were published before 2001 . Of these, just over half (51.3%) of participants identified as white, and 13.6% were african american and 6.3% were hispanic / latino . Native - american, alaska native, native hawaiian, and pacific islander participants made up 2.2% of the sample . Many studies did not report other demographic data . Only 21 studies reported socioeconomic status, and 43 reported educational level . Twenty - eight studies were conducted primarily in urban settings, two were conducted in rural settings, nine were conducted in both urban and rural settings, and nine studies were conducted nationwide . Three papers each reported two unique studies; other studies were reported in multiple papers . For example, a retrospective analysis of signed informed consent forms found that 87.1% of 1,298 research participants at the nih authorized all future research . In a different large national study, the national health and nutrition examination survey (nhanes), 84.8% of 4,480 overall participants recruited in 1999 and 2000 agreed to dna specimen collection for inclusion in a national repository for genetic research . Many studies asked participants hypothetical questions about their willingness to provide broad consent for research . In indiana, 88.4% of 273 cancer patients agreed that they would be willing to permit their tissue sample to be used in research on any condition . After time for deliberation, 85% of 40 focus group participants in north carolina reported that they would agree to have blood and information stored indefinitely in a biorepository for future research . Similarly, 78% of 49 focus group participants in chicago were interested in participating in a biobank, and the majority stated they would give broad consent . Of 30 patients who were interviewed at a hawaiian cancer center, 77% endorsed broad consent . One representative nationwide survey found that 68% of 1,593 respondents were willing to give broad consent for research, although their enthusiasm waned if they had a moral objection to certain types of studies for which their samples might be used . Two studies examined patients' willingness to participate in biobanks managed by kaiser permanente: 69% of 500 kaiser patients in the northwest and 69% of 203 in colorado agreed to participate in a biobank . In a focus group study in boston, patients with breast cancer were generally positive about having their samples used for secondary studies that were not planned at the time they gave consent . Scott et al . Reported the results of a 1998 survey of blood donors that asked about their views regarding storage and use of the blood for research . Of the 49,775 respondents, 60.3% said that testing stored blood for any research was acceptable with the donor's permission, and 35.5% would not require permission for research use . These studies reported substantial acceptance for broad consent . Asking participants for their preference among different types of consent broad, study - by - study, or categorical consent revealed more mixed support for broad consent . For example, 47% of 931 veterans preferred to give broad consent over other types of consent for all research approved by an oversight board . After adjusting for missing data, a national survey of 4,569 adults found that 52% preferred broad consent, whereas 48% preferred study - by - study consent . In a survey of 751 iowans, 42% preferred broad consent and 29% favored study - specific consent, compared with 25% who favored categorical consent . In another study of 315 cancer patients at two hospitals in atlanta, 92 and 97% were willing to allow their samples to be used for research on other diseases; when asked to specify a preference, 56% preferred one - time broad consent and 11% preferred study - by - study consent over no consent or no preference . In a 2001 nationwide survey, 43% of 2,621 participants were willing to donate blood for genetic research and to allow it to be stored for future research . Similarly, only 39.3% of 30 patients who had already donated samples preferred broad consent over consent for specific studies . By contrast, 77.7% of 1,276 people recruited through a crowd - sourced internet marketplace were willing to donate to biobanks, even after receiving disclosures about potentially objectionable research; however, 40.8% of participants still felt that specific consent was necessary, even if it might inhibit research progress . A similar nationwide survey of 1,599 individuals conducted through a probability - based online panel of adults found a wide range of opinions, with broad consent and real - time study - by - study consent considered the several studies showed that participants preferred to give informed consent for each study rather than a broad consent, with preferences ranging from 42 to 72%: 42% of a national sample of 4,700 us adults (which rose to 48% after adjusting for missing data), 43 to 50% of 931 veterans nationwide, and 60.7% of adults recruited in new york . Of 393 parents, 72% reported that they would want to consent each time to allow their child's dried bloodspots to be used for research . In focus groups of 92 native hawaiians, respondents repeatedly expressed desire to re - consent, although some stated that they would be content if they trusted the researcher or the biobank's governance . In one study of 273 jewish individuals, 6075% believed that consent should be required regardless of whether the dna was collected in a research or clinical setting . In a focus group study of 178 alaska native participants, some indicated a preference to have consent options for a variety of specimen uses, storage duration, and destruction of the sample at the completion of the study . In the same group, some wanted re - contact each time, whereas others felt that a one - time consent was appropriate for new studies . In chicago, 239 postpartum women were asked about their willingness to enroll their children into a pediatric biobank: 48% of women would enroll their child, but 24% would not; of the latter, 82% of the participants were african american . In another focus group study, 11 of 15 participants preferred tiered consent over other methods to exert the greatest level of control regarding how they wanted their data to be shared; however, participants who were willing to provide broad consent also appreciated the option to opt in or opt out of dna data sharing . Some studies reported that most respondents favored an opt - in approach, whereas others found that opt - out was acceptable or even preferred by the majority . A majority of participants67% of 751 survey respondents and 63% of 57 focus group participants who were asked about biobank participation in iowa preferred opt - in, whereas 18% of survey respondents and 25% of focus group participants in the same study preferred opt - out . In a study of 451 nonactive military veterans, 82% thought it would be acceptable for the proposed million veterans biobank to use an opt - in approach, and 75% thought that an opt - out approach was acceptable; 80% said that they would take part if the biobank were opt - in as opposed to 69% who would participate if it were an opt - out approach . When asked to choose which option they would prefer, 29% of respondents chose the opt - in method, 14% chose opt - out, 50% said either would be acceptable, and 7% would not want to participate . In some cases, biobank participants were re - contacted to inquire about their thoughts regarding proposed changes to the biobank in which they participated . Thirty - two biobank participants who attended focus groups in wisconsin regarding proposed minimal - risk protocol changes were comfortable with using an opt - out model for future studies because of the initial broad consent given at the beginning of the study and their trust in the institution . A study of 365 participants who were re - contacted about their ongoing participation in a biobank in seattle showed that 55% thought that opt - out would be acceptable, compared with 40% who thought it would be unacceptable . Similarly, several studies explored perspectives on the acceptability of an opt - out biobank at vanderbilt university . First, 91% of 1,003 participants surveyed in the community thought leftover blood and tissues should be used for anonymous medical research under an opt - out model; these preferences varied by population, with 76% of african americans supporting this model compared with 93% of whites . In later studies of community members, approval rates for the opt - out biobank were generally high (around 90% or more) in all demographic groups surveyed, including university employees, adult cohorts, and parents of pediatric patients . Three studies explored community perspectives on using newborn screening blood spots for research through the michigan biotrust for health program . First, 77% of 393 parents agreed that parents should be able to opt out of having their child's blood stored for research . Second, 87 participants were asked to indicate a preference: 55% preferred an opt - out model, 29% preferred to opt - in, and 16% felt that either option was acceptable . Finally, 39% of 856 college students reported that they would give broad consent to research with their newborn blood spots, whereas 39% would want to give consent for each use for research . In a nationwide telephone survey regarding the use of samples collected from newborns, 46% of 1,186 adults believed that researchers should re - consent participants when they turn 18 years old . Some studies examined the differences in participants' willingness to provide broad consent for samples that were de - identified or anonymous as compared with identifiable . Respondents generally preferred to give consent if their samples were identifiable . In two studies involving 429 primarily native hawaiian participants, 78% of native hawaiians and 66% of whites indicated that they would require consent for research if the specimens were identifiable and collected in the clinical setting . For genetics research, 81% native hawaiians and 78% of whites indicated that they would require consent if the specimens were identifiable . In a us - wide telephone survey, 81% of 1,193 respondents stated that they would want to be informed about research being done with their sample if it were identifiable; additionally, 57% said they would require permission to use their samples if they were identifiable . For example, 65.8% of 504 adults who participated in a telephone survey across the united states reported that they would require consent for samples collected in the clinic if they were identifiable, compared with 27.3% who reported they would require consent if samples were anonymized . In the research setting, fewer people thought consent was required for identifiable (29.0%) or anonymized (12.1%) samples . In a study utilizing a hypothetical biobank scenario, 43% of 565 government and medical employees in new mexico indicated that they would donate their sample for future genetic testing if it could not be traced to them . Not all studies found that people were worried about identifiability . In one survey of 144 clinicians, 86% said that they would donate a dna sample to a hypothetical biobank in new york regardless of whether it was linked to or unlinked from their identity . In the study in new mexico, 36% of 565 respondents found it acceptable for broadly consented samples to be used by their local university, even if the samples were linked to them . Characteristics associated with favoring broad consent included being male, white / caucasian, older, and more affluent . By contrast, asians, black non - hispanics, african americans, and others (who represented 14.3% of the total) were less likely than whites to believe that research without explicit permission was acceptable . One study of consent forms showed that 75.0% of african americans gave broad consent compared with 88.4% of whites (p = 0.002). Similarly, in the nhanes data, 78.7% of african americans and 87.1% of whites consented to genetics research . One study reported that participants who were significantly more likely to prefer broad consent also believed that participating would make me feel like i was contributing to society (odds ratio = 1.85; p = 0.001), that the study would accelerate medical treatments and cures (odds ratio = 2.20; p = 0.001), and that participating in the cohort study would be easy (odds ratio = 1.59; p <0.001). Other investigators reported that the large majority (97.7%) of respondents said yes or maybe to the idea that it is a gift to society when an individual takes part in medical research . Many other studies cited the benefit of research to improve health as a reason to favor broad consent . We identified 23 studies of data sharing . The earliest publications about participants' preferences on data sharing date to 2006 . Most studies of data sharing were conducted with studies of consent preferences; however, six studies were conducted with the primary goal of eliciting preferences on data sharing . Participants were generally willing to have their samples and information shared with other academic institutions . Willingness to share data with academic and medical researchers was acceptable for 92% of 4,659 us adults generally, and 80% of 931 us veterans specifically . More than 70% of 100 young adults in baltimore who were enrolled in a longitudinal study of prevention were willing to share results arising from their dna . Nearly three fourths of 40 community members in a focus group study in north carolina were comfortable with academic researchers having access to their samples . Many of 79 focus group participants in seattle endorsed the value of sharing, agreed that sharing locally and with close collaborators was acceptable, and were comfortable with nonprofit and public - interest organizations using data from their samples . In one focus group study of 48 primarily white and female participants in iowa, the majority cited positive reasons for donating their samples to help and to contribute to advancements in research, and that data sharing would not affect their decision to enroll in a biobank . In another focus group study of 100 african americans in north carolina, many recognized the benefits of data sharing but wanted the potential risks to be disclosed, and some wanted the data to be restricted . In another study of patients with inflammatory bowel diseases, 97.3% of 92 respondents were comfortable with sharing their biological sample with investigators in the united states, but 23.8% were uncomfortable with sharing with investigators outside the united states . Some participants expressed concern over sharing their data and information with federal repositories . In one study, 18.5% of 4,050 vanderbilt university faculty and staff were more likely to want to participate in their institution's biobank if the de - identified data were deposited into a national database; however, 12.1% were less likely to want to participate . In two large nationwide surveys, 80% of 4,659 adults were willing to have their data shared with government researchers; however, 75% of the same sample also were concerned about the government having [their] samples and information . Similarly, another study found that 71% of 931 veterans were willing to grant database access to government researchers, but half were concerned about the government having [their] samples and information . Other studies have shown that some people are concerned about government involvement in maintaining databases containing biomedical information . More than half of the 40 participants in a focus group study of north carolina community members were concerned about government researchers having access to their institution's biorepository . Despite concerns, 61% of 203 kaiser patients in colorado would still provide a sample even if the data would be submitted to a government database, and 82% of 500 kaiser patients in oregon agreed to have their information posted in a us government database . In a large metropolitan area in southwest florida, some of 95 focus group participants believed that biospecimens were already being collected from leftover tissue, and others suspected that tissues were already being shared with researchers in other countries who lack strict laws' governing research . In a focus group study of 178 alaska native participants, some cited mistrust of the government and police having access to their samples and wanted transparency from the researchers about how their samples were used . The majority of participants were willing to share with pharmaceutical company researchers, but the percentage was generally less than the percentage willing to share with academic researchers . Seventy - five percent of 4,659 us adults, 54% of 931 veterans, 55.2% of 1,599 adults responding to a nationwide survey, and 75.1% of members of the crohn's and colitis foundation of america partners cohort were willing to share with pharmaceutical company researchers . Focus group participants in florida voiced concern about providing blanket consent because they would not benefit financially from any resulting discoveries . Factors associated with views about data sharing . With the exception of gender, few demographic data (e.g., about race / ethnicity, socioeconomic status, education, and urban / rural residency) even when demographic information was obtained, investigators did not always report how these variables correlated with respondents' opinions . Therefore, it was largely not possible to draw meaningful conclusions about the associations between sociodemographic factors and views on data sharing . The willingness of patients with cancer to share seemed to be shaped by their devotion to the institution at which they were receiving care . For example, patients with cancer in indiana who agreed to participate in a biobank were less likely to be willing to allow their tissue samples to be used by researchers who were not affiliated with the local researchers (89.7%), compared with 96.3% who were willing to share with local university researchers (p <0.01). Half of 100 patients with breast cancer at md anderson cancer center preferred to allow only their physician (24%) or other researchers at their hospital (26%) to use their de - identified genetic data for research; fewer patients were willing to share their de - identified data with any cancer researcher (25%) or any researcher (18%). In another study, 95% of 315 patients with cancer in atlanta were willing to allow researchers to share samples with other local researchers, but only 85% and 92% of participants at two different sites were willing to have their samples shared elsewhere in the united states (p <0.05). In 2013, nih funded the emerge consortium to perform a broad population - based survey to assess public opinion about broad consent for research and data sharing . This systematic literature review, which ultimately contained 48 studies involving 35,969 participants, was conducted to identify gaps and issues that needed to be addressed in this survey . The most notable finding is that many people do not favor broad consent for either research itself or for research and subsequent wide data sharing . While the majority often expressed support for broad consent when that was the only choice offered, only a minority of respondents favored broad consent when other options, such as tiered or study - by - study consent, were offered . Furthermore, earlier studies focused on the importance of obtaining consent for research, whereas later studies focused on the preferences for different consent options . Willingness to give broad consent increased if data were de - identified . While individuals were generally willing for data or biospecimens to be shared with other academic researchers, individuals were less willing for their data to be shared in federal databases or with commercial enterprises . What is equally striking are the large gaps in what is known about factors that affect people's decisions . While a few studies generally found that men were more likely to support broad consent, most investigators did not examine the impact of gender on attitudes . Although data about race / ethnicity are incomplete, it seems that minorities often have more concerns about broad consent, although existing evidence suggests that these concerns can be ameliorated in some cases by discussion and education . Much less is known about the impact of sociodemographic factors such as socioeconomic status, education, and whether people live in urban or rural environments on attitudes toward broad consent and data sharing . Building on these findings, the emerge cerc survey developed a sampling strategy, experimental study design, and survey questions to ascertain more uniformly the views of individuals throughout society in order to identify and address concerns . First, we used broad search terms to capture the existing literature on broad consent and data sharing . Thus, while we used multiple approaches (e.g., searching multiple sources, reviewing reference lists, and searching the unpublished, gray literature, such as dissertations and reports) to comprehensively identify studies, we may not have identified all salient research . Second, we adapted existing metrics of quality scores to our study . For many studies, we were unable to ascertain the appropriateness of study questions or an analysis plan, thus limiting our ability to thoroughly assess the quality of the studies . Third, the studies that have been conducted to date have a number of limitations, which in turn limit the generalizability of this literature review . Many of the surveys focus what people say they think, rather what they actually do, even though opinions may differ from action . Definitions of consent were not always consistent and have changed over time, which not only limits our ability to compare studies but also may affect our evaluation of older studies given today's ethical standards for biobanking governance . However, all studies were sufficiently focused on broad consent for research or for data sharing to permit some comparison . Most of the surveys heavily oversampled whites, whereas the qualitative studies disproportionately involved minority participants . Studies that incorporated an educational component may have influenced respondents compared with those studies that did not involve education around biobanking practices . This review also was limited to the united states, which is warranted given the different policy preferences in other countries . The ultimate goal of this literature review and the emerge cerc survey is to obtain a more comprehensive understanding of public opinion about broad consent for data sharing and use . The studies included here typically noted a general acceptance for broad consent and endorsement of data sharing, but with notable privacy and governance concerns, especially by minority participants . The policy question will be what to do if some people, particularly from certain demographics, express a desire for more granular control over the use of data obtained from them in light of the policy trend toward requiring individual consent for broad data use and sharing . At a minimum, it suggests the need to engage those who are skeptical, even if it is decided that the public good of research to improve health outweighs honoring individual objections in some cases or the risk that some people will choose not to participate.
The purposes of endodontic treatment are to remove all necrotic and vital tissue and microorganisms from the root canal system and obturate the canal space tightly to avoid microbial growth13 . In this context, the filling materials should also be free of microorganisms to avoid canal recontamination . Currently, gutta - percha is the most commonly used root canal core filling material . The sap of the isonandra gutta tree is the raw material for gutta - percha . Chemically, it is defined as the trans - form of polyisoprene and is harder, more brittle and less elastic than the more familiar natural rubber . The familiar consistency of endodontic gutta - percha cone is achieved by the addition of wax and resins as softeners (1 - 4%), metal sulfate for radiopacity (1 - 15%), and zinc oxide filler as the main component (59 - 76%)2 . Gutta - percha cone possesses some antimicrobial activity owing to its zinc oxide component8 . In spite of this antimicrobial activity, two studies have reported that 8% and 20% of gutta - percha cones taken out of their sealed package yielded bacterial growth when cultured on agar plates, respectively7,10 . Contrary to these findings, others have not detected microbial contamination on gutta - percha cones taken from the manufacturer's sealed package3,6,12 . However, it is expected that contamination would increase upon opening of the package and starting to use the gutta - percha cones in the clinical environment . While some studies have investigated the microbial contamination of gutta - percha packages that have been opened and used in a clinic for a certain period4,12, the present study, differently, recorded the number of root canals filled as an indicator to determine the frequency of exposure to the potential contaminants . This study examined the degree of microbial contamination on gutta - percha cones at the time of package opening and during clinical use, and also verified whether a correlation existed between the number of filled root canals and the number of the contaminating microorganisms . Twelve sealed packages of #15 - 40 gutta - percha cones (sure - endo, sure - dent co., seoul, korea) were opened under aseptic laboratory conditions and two gutta - percha cones from each size was randomly drawn . Twelve cones chosen in this manner from each package (two of each #15, #20, #25, #30, #35 and #40 cones) were added to tubes containing 750 l of sterile 0.9% saline and glass beads (figure 1). The tubes were vortexed for 30 s, 10-times serially diluted to 10, and samples of 250 l were cultured on tryptone soy agar plates (tsa; oxoid, basingstoke, uk). The plates were incubated at 37c for 3 days and microbial colonies were counted under stereomicroscope at 6x magnification (olympus sz- pt, tokyo, japan). Tubes containing only glass beads and saline were sampled . As a positive control group, guttapercha cones rolled between ungloved fingers for 30 s were sampled as described above . In a separate test, the whole gutta - percha contents of three sealed packages were sampled . However, in this test, the gutta - percha cones removed from the packages were added to tubes containing 1.5 ml saline, and volumes of 500 l were pipetted and cultured . The initially sampled packages were distributed randomly to 12 final year dental students (one package per student). The students were also asked to record the number of root canals they filled with the given gutta - percha packages . The packages were collected at the end of the first and the third day and sampled as described above . At each laboratory differences between the number of root canals filled and also between the number of microorganisms cultured at the end of the first and third days were analyzed statistically by the wilcoxon signed - rank test . The correlation between the number of microorganisms cultured and the number of filled root canals was analyzed using the spearman's rank correlation coefficient (spearman's rho). Differences between the ratio of the contaminated packages at the first and third days were analyzed using the z - test . Twelve sealed packages of #15 - 40 gutta - percha cones (sure - endo, sure - dent co., seoul, korea) were opened under aseptic laboratory conditions and two gutta - percha cones from each size was randomly drawn . Twelve cones chosen in this manner from each package (two of each #15, #20, #25, #30, #35 and #40 cones) were added to tubes containing 750 l of sterile 0.9% saline and glass beads (figure 1). The tubes were vortexed for 30 s, 10-times serially diluted to 10, and samples of 250 l were cultured on tryptone soy agar plates (tsa; oxoid, basingstoke, uk). The plates were incubated at 37c for 3 days and microbial colonies were counted under stereomicroscope at 6x magnification (olympus sz- pt, tokyo, japan). Tubes containing only glass beads and saline were sampled . As a positive control group, guttapercha cones rolled between ungloved fingers for 30 s were sampled as described above . In a separate test, the whole gutta - percha contents of three sealed packages were sampled . However, in this test, the gutta - percha cones removed from the packages were added to tubes containing 1.5 ml saline, and volumes of 500 l were pipetted and cultured . The initially sampled packages were distributed randomly to 12 final year dental students (one package per student). The students were also asked to record the number of root canals they filled with the given gutta - percha packages . The packages were collected at the end of the first and the third day and sampled as described above . At each laboratory differences between the number of root canals filled and also between the number of microorganisms cultured at the end of the first and third days were analyzed statistically by the wilcoxon signed - rank test . The correlation between the number of microorganisms cultured and the number of filled root canals was analyzed using the spearman's rank correlation coefficient (spearman's rho). Differences between the ratio of the contaminated packages at the first and third days were analyzed using the z - test . The positive controls showed microbial growth (84, 120 and 135 cfu; n = 3). The samples of the whole content of the packages displayed microbial growth (12, 15 and 18 cfu; n = 3), confirming that the package contents were not sterile . Initial samples of the packaged gutta - percha cones showed either no microbial growth (7 of 12 packages) or as few as 3 cfu of microbial growth (5 of 12 packages; table 1). Because the baseline cultures could yield up to 3 cfu, this number was considered as the threshold, and further cultures not exceeding 3 cfu were considered to have got no extra contamination . Descriptive statistics for the first and third day numbers of root canal fillings and cultured microorganisms are shown in table 2 . The number of root canals filled at the end of the first day and the third day were 3.9 1.1 and 11.3 2.9 (mean sd), respectively . Difference between the number of filled root canals at the first and the third day was significant (wilcoxon test; p = 0.001). The number of microorganisms recovered at the first and third day cultures were 8 9.9 cfu and 4.5 8.3 cfu (mean sd), respectively . Differences between the numbers of microorganisms found at the first and third day cultures were not significant (wilcoxon test; p = 0.304). No significant correlation was found between the number of filled root canals and cultured microorganisms at either the first day (correlation coefficient, significance; r = 0.481, p = 0.113) and the third day (r = - 0.034, p = 0.917). At the end of the first day, microbial contamination was found in five packages (42%). At the end of the third day, microbial contamination was found in three packages (25%). Difference between the ratio of the contaminated packages at the first day and the third day was not statistically significant (z - test; p = 0.386). The microbial status of the packages used by the students and the number of root canals filled using these packages have been shown in table 1 . The major findings of this study were that the contents of the gutta - percha packages were not sterile and that further microbial contamination could occur upon clinical use of the packages . While the number of microorganisms was quite small at the beginning, clinical use of the gutta - percha packages led to increased microbial contamination . However, no significant correlation existed between the number of filled root canals and the number of microorganisms cultured . For example, contamination was found in four packages (packages d, e, f and j; table 1) in the first day cultures . However, the third day cultures of the same packages revealed no contamination despite the larger number of filled root canals . Similarly, the third day culture of one package (the h package) yielded fewer microbial colonies than the first day culture of the same package . One explanation may be that the microbial contamination did not affect the whole content of the package evenly; it is possible that while some of the gutta - percha cones in the package became contaminated during clinical use, other cones remained free of contamination . The findings of this study on the initial microbial status of the gutta - percha cones are in agreement with those of montgomery7 and namazikhah, et al.10, who found that some of the gutta - percha cones taken from the manufacturer's sealed package harbored microorganism, but differ from those of doolittle, et al.3, klager and dupont6 and pang, et al.12, who found that none of the gutta - percha cones taken from the manufacturer's sealed package were positive for microbial growth . In addition, the brands of the gutta - percha packages tested in these experiments are different . There may be differences in the manufacturing technology among the manufacturers in terms of aseptic production and packaging . The latter statement is supported by gomes, et al.4, who found that the contents of freshly opened gutta - percha packages of one brand could be negative while the contents of another brand could be positive for microbial growth . It was expected that the gutta - percha cones would become contaminated upon opening of the packages and starting their clinical use . As expected, the contamination occurred in seven of the twelve packages within a total of three days . In a previous study12, 29 of 150 gutta - percha cones (19.4%) that had been in clinical use for three months prior to the microbiological sampling this finding is consistent with presented study, confirming that the clinical use of the guttapercha package is associated with the risk of contamination . However, five of the packages (packages a, b, g, i and k; see table 1) remained at or below the threshold limit (3 cfu) throughout the study . The finding that packages used by certain students remained non - contaminated throughout the study may be explained by the awareness of these students of the importance of maintaining aseptic conditions during treatment . Unexpectedly, no strong correlation was found between the number of the root canals filled and the number of the cultured microorganisms . Additionally, it may be speculated that the antimicrobial effects of gutta - percha cones1,7 could have suppressed the activity of the contaminating microorganisms and kept their cultivable numbers under a certain level . Thus, the number of the cultured bacteria did not parallel the number of the root canal fillings . This lack of correlation has also been reported in a previous study where the number of contaminated guttapercha cones remained almost constant within up to 2.5 years of clinical use4 . Current root canal obturation techniques involve the use of a sealer along with gutta - percha . Thus, microorganisms contaminating the gutta - percha cones can be eliminated after coating the cones with the sealer . In a previous study10, no microorganism was cultured from gutta - percha cones coated with an epoxy resin - based sealer whereas some of the noncoated cones yielded microbial growth . In addition to the antimicrobial effect of the sealer, several chemicals such as 70% alcohol, sodium hypochlorite, glutaraldehyde, benzalkonium chloride, paraformaldehyde, formocresol, polyvinylpyrrolidone - iodine have been proposed for guttapercha disinfection6 . Among these chemicals, sodium hypochlorite, with its rapid antimicrobial action, is a safe and effective medication4,9,11,12 . The findings of the present study are in line with those of previous studies that have addressed the risk of microbial contamination on gutta - percha cones and sought solutions to overcome this problem3,4,6,7,912 . Our results indicate the need for maintaining careful handling of the gutta - percha packages: the package should be kept closed when not used and a sterile instrument should be used for taking out guttapercha cones from the package . It is concluded that gutta - percha cones taken directly from the manufacturer's sealed package harbored cultivable microorganisms . While the numbers of these microorganisms were quite low at the time of opening of the package, clinical use of the packages increased the number of microorganisms contaminating the gutta - percha cones.
Although the pathogenesis of menire's disease remains unknown it is likely to be multifactorial . A candidate is the gene encoding aquaporin 5 (aqp5) as aquaporins play an important role in maintaining inner ear fluid homeostasis . Furthermore, aqp5 is preferentially localized within the external sulcus cells and the spiral prominence of the cochlea implying a physiological role of aqp5 in auditory function [1, 2]. Following sequencing of the aqp5 promoter region of 50 healthy white caucasians we have described a novel, functional, and common single nucleotide (-1364a / c) polymorphism . Substitution of c for a at position -1364 was associated with increased binding of transcription factors, as shown for nuclear extracts from hela cells, but reduced transcriptional activation of the aqp5 gene in response to serum and camp . This latter finding is of special interest since stimulation of the vasopressin v2-receptor by arginine - vasopressin (adh) results in increased aqp5 mrna concentrations and increased translocation of aqp5 to the plasma membrane . This is mediated via an increase in camp concentration and subsequent activation of the protein kinase a pathway . Thus, the c - allele may facilitate the binding of an inhibitory transcription factor . These results were corroborated by showing that the c - allele was associated with decreased aqp5 mrna transcripts in human right atrial muscle and decreased aqp protein expression in red cell membranes . Furthermore, the c - allele was associated with suppression of the renin - angiotensin - aldosterone system (raas) in response to a high salt diet, while the raas suppression in individuals carrying the aa genotype was significantly blunted . Thus, there are good reasons to hypothesize that the aqp5 promoter polymorphism could impact upon key mechanisms of md . Accordingly, we tested the hypothesis that genotypes of the -1364a / c aqp5 promoter polymorphism are associated with an increased risk for md, fmd, or eh . After approval by the local ethics committee and written informed consent data and blood samples from 102 caucasian patients with menire's disease (grade 14 according to aao - hns guidelines) were included into the study cohort (62 females, 40 males; mean age: 59.1 years 14.3 (max 96, min 25), see table 1)). The dna of 102 patients, 39 with menire's disease (md), 54 with familial menire's disease (fmd), and 9 with endolymphatic hydrops (eh) was genotyped for the aqp5 promoter-1364a / c polymorphism . Familial menire's disease (fmd) was assumed when two or more family members were affected by menire's disease . Endolymphatic hydrops (eh) was defined as fluctuating low - tone hearing loss with no signs of vestibular affection . The group consisted of patients referred to the department for diagnosis and/or therapy of menire's disease or endolymphatic hydrops by smaller departments or local ent colleagues . As patients were usually referred to the department when standard conservative treatment was not successful, the groups of patients with grade 2 (n = 27) and grade 3 (n = 53) disease are the largest of our cohorts (see table 2(b)). The control cohort consisted of 292 randomly chosen healthy white, caucasian blood donors (age: 56.7 years 4.1) of either gender (110 female, 182 male) who were recruited at the local department for transfusion medicine, university hospital essen . Dna was extracted from whole blood using the qiaamp kit (qiagen, hilden, germany). For genotyping the aqp5 promotor-1364 a / c polymorphism by pyrosequencing, pcr was performed with the forward primer aqp5-se 5-gaaactgcaggatgagagaaat-3, and the biotinylated reverse primer aqp5-as 5-tctctgttctccacctctcca-3 resulting in a 120 nt fragment . After denaturation at 94c, 40 cycles of dna amplification were done using taq pcr mastermix (eppendorf, hamburg, germany) at 94c for 40 s, 53c for 40 s, and 72c for 40 s. the biotinylated strand was captured on streptavidin sepharose beads and annealed with a sequencing primer aqp5-seq 5-cagagagactaagacagca-3. Pyrosequencing was performed using psq hs 96 gold snp reagents and the psq hs 96 (biotage, uppsala, schweden). All statistical analyses were done using spss 11.0 (spss, chicago, il, usa). The hardy - weinberg equilibrium was tested by a goodness - of - fit -test . The -test was applied to compare genotype distributions and allele frequencies between patients and controls . Genotype frequencies were not different between controls (aa: 69%, ac: 30%, and cc: 1%) and patients with md aa: 65.7% (23 md, 37 fmd, and 8 ed); ac = 23.5% (12 md, 11 fmd, and 1 ed); cc = 3.9% (1 md, 3 fmd) (table 2(a)). Subgroup analysis, however, revealed the cc genotype to be more frequent in patients with fmd than in controls (p = 0.042). However, neither the a - allele nor the c - allele was associated with fmd or md . Allergies, metabolic disorders (diabetes mellitus, impairment of cholesterol metabolism), hypertension, and depression were significantly more frequent in the group of patients with md than in patients with fmd . However, there was no significant association between these disorders and specific genotypes of the aqp5 promoter polymorphism in these patients . Additionally, severity of md was not associated with the aqp5 promoter-1364a / c polymorphism, as depicted in table 2(b). However, this calculation has to be interpreted carefully as the different grades are only a snapshot in the course of disease in patients presenting for diagnosis or therapy . The present investigation suggests for the first time that genotypes of the -1364a / c aqp5 promoter polymorphism are neither associated with the incidence nor the severity of md, fmd, or eh . Although the cc genotype was more frequent in patients with fmd than in controls (p = 0.042), we believe that this association is fortuitous because neither the a - allele nor the c - allele was associated with fmd or md . Comorbidities such as allergies, metabolic disorders, hypertension, and depression were more frequent in patients with md than in those with fmd . With regard to the relatively small number of patients in each group these results have to be interpreted carefully . However, it is well known that depression and allergies might act as cofactors or so - called triggers evoking menire attacks [8, 9]. The preferential presence of aqp5 within the external sulcus cells and the spiral prominence of the cochlea implies a physiological role of aqp5 in auditory cell function and inner ear water homeostasis [1, 2] and the -1364a / c aquaporin 5 gene promoter polymorphism impacts upon aqp5 expression in different cohorts . Nevertheless, the -1364a / c aquaporin 5 gene promoter polymorphism was not associated with the incidence of md, fmd, or eh . We speculate, therefore, that aqp5 expression in the apical turns of the cochlea only may play a limited role in inner ear fluid homeostasis in patients with menire's disease . This speculation is supported by observations of klar et al . Who defined a new candidate region for an increased risk for fmd on chromosome 12 . Although this region on chromosome 12p12 is close to the aqp 2, 5, and 6 loci, there was no overlapping or linkage disequilibrium between these loci . Reported normal hearing in aqp5 knockout mice and suggested redundant or alternative mechanisms for regulation of water homeostasis in the inner ear . However, according to the findings of hirt and coworkers, there should be further attention paid to the special differential localization and role of aqp5 and aqp 4 in the outer sulcus cells, as it suggests a possible water shunt in the perilymph endolymph barrier which could have an impact on dysfunctional water regulation such as endolymphatic hydrops or sensorinureal hearing loss . Limitations of this report should be mentioned . The study population was relatively small and it was hardly possible to build matched pairs as far as comorbidities and age are concerned . Different selection criteria of blood donors and an unrecognized selection bias, inherent to many genetic association studies, cannot be excluded, too . However, the low incidence of md, fmd, and eh in the general population suggests an interaction of extrinsic (e.g., allergies, inflammation, metabolic and endocrinological disorders, trauma, and migraine), genetic, and developmental intrinsic factors . This investigation represents a further step towards identifying candidate genes which could impact on the onset or severity of md and eh . However, in this survey on caucasians neither comorbidities nor the aqp5 polymorphism was associated with the incidence or severity of md, fmd, or eh.
This prospective, multicenter, open - label, surveillance study was conducted between august 2006 and march 2009 at 26 clinical practice sites in korea . At the time all patients provided voluntary, signed informed consent before the commencement of any study - related procedures . The main inclusion criteria for enrollment were age> 18 years; a diagnosis of moderate to severe dry eye disease (based on clinicians' standard clinical practices); symptomatic dry eye disease; and nonresponsiveness to conventional treatment such as artificial tear drops, gels, ointments, and punctal plugs . The main exclusion criteria were use of systemic or topical csa in the previous 90 days; anticipated use of any temporal punctal plugs during the study; women who were pregnant, planning a pregnancy, or lactating; end - stage lacrimal disease or dry eye disease caused by destruction of goblet cells; active ocular infections; and suspected hypersensitivity to any of the ingredients in the csa formulation . The study comprised a baseline visit and three follow - up visits after 1, 2, and 3 months of treatment . Irvine, ca, usa) was applied every 12 hours to each eye as monotherapy or adjunct therapy . New treatments, including any eye drops or punctal plug, were not added after enrollment . At each study visit, patients were examined according to the clinicians' standard clinical practices . In addition, symptoms of ocular discomfort (i.e., stinging / burning, itching, sandiness / grittiness, blurred vision, light sensitivity, pain, and soreness), schirmer scores, use of artificial tears, global evaluation of improvement, and adverse events were recorded at each visit . There was an optional measurement of conjunctival staining (oxford score) performed at baseline and after three months of treatment . Clinicians' also assessed global evaluation of improvement in symptoms of dry eye disease from baseline at each follow - up visit according to the following categories;> 90% improvement, 75% to 90% improvement, 25% to 75% improvement, condition unchanged, and condition worsened . Patient satisfaction was assessed through the completion of a four - question survey at the final study visit . The exit survey comprised the following questions: how are your chronic dry eye symptoms? ; with restasis, how does your chronic dry eye condition now affect your normal daily activities? ; overall, how satisfied are you with restasis? ; how quickly did restasis start working to relieve your chronic dry eye symptoms? Scoring for each question was based on a scale bar from 0 (no symptom) to 10 (maximum symptom experienced). The primary effectiveness outcome measures included changes in symptom scores (rated on a scale of 0 [no symptoms] to 4 [always have symptoms]) and schirmer scores (mm, with or without anesthesia). The secondary effectiveness outcome measures included change in conjunctival staining (rated on a scale of 0 to 5), use of artificial tears, global evaluation of the treatment, and patient satisfaction with the treatment . All data are summarized using frequency distributions and/or descriptive summary statistics (mean and standard deviation [sd]). All statistical analyses included data for the treated eye or the mean data for both eyes (if patients received treatment for both eyes). Schirmer scores (with or without anesthesia) for all visits were compared using a repeated measures analysis of variance . For all other effectiveness variables, patients with missing baseline values were excluded from the analyses, and missing post - treatment data were inferred by carrying forward the subsequent observation . This prospective, multicenter, open - label, surveillance study was conducted between august 2006 and march 2009 at 26 clinical practice sites in korea . At the time all patients provided voluntary, signed informed consent before the commencement of any study - related procedures . The main inclusion criteria for enrollment were age> 18 years; a diagnosis of moderate to severe dry eye disease (based on clinicians' standard clinical practices); symptomatic dry eye disease; and nonresponsiveness to conventional treatment such as artificial tear drops, gels, ointments, and punctal plugs . The main exclusion criteria were use of systemic or topical csa in the previous 90 days; anticipated use of any temporal punctal plugs during the study; women who were pregnant, planning a pregnancy, or lactating; end - stage lacrimal disease or dry eye disease caused by destruction of goblet cells; active ocular infections; and suspected hypersensitivity to any of the ingredients in the csa formulation . The study comprised a baseline visit and three follow - up visits after 1, 2, and 3 months of treatment . Irvine, ca, usa) was applied every 12 hours to each eye as monotherapy or adjunct therapy . New treatments, including any eye drops or punctal plug, were not added after enrollment . At each study visit, patients were examined according to the clinicians' standard clinical practices . In addition, symptoms of ocular discomfort (i.e., stinging / burning, itching, sandiness / grittiness, blurred vision, light sensitivity, pain, and soreness), schirmer scores, use of artificial tears, global evaluation of improvement, and adverse events were recorded at each visit . There was an optional measurement of conjunctival staining (oxford score) performed at baseline and after three months of treatment . Clinicians' also assessed global evaluation of improvement in symptoms of dry eye disease from baseline at each follow - up visit according to the following categories;> 90% improvement, 75% to 90% improvement, 25% to 75% improvement, condition unchanged, and condition worsened . Patient satisfaction was assessed through the completion of a four - question survey at the final study visit . The exit survey comprised the following questions: how are your chronic dry eye symptoms? ; with restasis, how does your chronic dry eye condition now affect your normal daily activities? ; overall, how satisfied are you with restasis? ; how quickly did restasis start working to relieve your chronic dry eye symptoms? Scoring for each question was based on a scale bar from 0 (no symptom) to 10 (maximum symptom experienced). The primary effectiveness outcome measures included changes in symptom scores (rated on a scale of 0 [no symptoms] to 4 [always have symptoms]) and schirmer scores (mm, with or without anesthesia). The secondary effectiveness outcome measures included change in conjunctival staining (rated on a scale of 0 to 5), use of artificial tears, global evaluation of the treatment, and patient satisfaction with the treatment . All data are summarized using frequency distributions and/or descriptive summary statistics (mean and standard deviation [sd]). All statistical analyses included data for the treated eye or the mean data for both eyes (if patients received treatment for both eyes). Schirmer scores (with or without anesthesia) for all visits were compared using a repeated measures analysis of variance . For all other effectiveness variables, the changes from baseline data were compared using a paired sample t - test . Patients with missing baseline values were excluded from the analyses, and missing post - treatment data were inferred by carrying forward the subsequent observation . For all analyses, statistical significance was set at p 0.05 . A total of 392 patients were enrolled in the study and included in the safety and tolerability analysis; 362 (92.3%) patients completed the study and were included in the effectiveness analysis . The main reasons for study discontinuation were loss to follow - up (n = 21 patients) and adverse events (n = 6 patients). Patients were predominantly female, and slightly less than half were 50 years of age or older (table 1). Most patients had been diagnosed with moderate to severe dry eye disease for one to five years, and approximately one - quarter had undergone a previous ocular surgery . Treatment with csa 0.05% significantly improved symptom, schirmer, and staining scores and reduced the use of artificial tears . There were significant reductions from baseline in all mean ocular symptom scores (stinging / burning, itching, sandiness / grittiness, blurred vision, light sensitivity, pain or soreness) after one month of treatment (p <0.0001) (table 2) and which persisted for up to three months of treatment (p <0.0001). There were significant increases from baseline in mean schirmer scores both with and without anesthesia at each study visit (p <0.0001) (fig . 1). There were no significant differences between schirmer scores with and without anesthesia . The baseline conjunctival staining score (oxford score sd) was 3.2 2.2, while the corresponding score after three months of treatment was 2.8 2.8 . After three months of treatment, the mean percentage (sd) reduction from baseline in conjunctival staining score was -12.8 64.7% (p <0.01), and there was a significant reduction from baseline in mean (sd) use of artificial tears (5.6 5.7 and 4.0 4.5 drops / day for baseline and three months, respectively; p <0.0001). According to clinicians' global evaluations of treatment with csa 0.05%, the majority of patients experienced an at least 25% to 50% improvement at each visit (fig . A greater percentage of patients experienced a 75% to 90% improvement after three months of treatment than after either one or two months of treatment . Dry eye disease worsened in three, five, and two patients after one, two, and three months of treatment, respectively . According to the exit survey findings, patients generally reported that csa 0.05% treatment provided relief from dry eye symptoms, and that their experience with the treatment was positive (fig . Specifically, most patients (57.2%) reported that they had no or mild symptoms of dry eye disease (score of 0 to 4 out of 10, 214 / 374 patients) (fig . Most patients (55.2%) also reported that their dry eye disease had no or little effect on their normal daily living (scores 0 to 4 out of 10; 206 / 373 patients) (fig . The majority of patients (72.0%) were satisfied or very satisfied with csa 0.05% treatment (scores of 5 to 10 out of 10; 270 / 375 patients) (fig . Relief from dry eye symptoms began three to five weeks after commencing csa 0.05% treatment (209 / 356, 58.7%) (fig . 3d). Treatment with csa 0.05% was well - tolerated, and very few patients discontinued the study because of adverse events . The most common adverse events (incidence> 5%) were ocular pain (43 / 392 patients, 11.0%) and ocular irritation (23 / 392 patients, 5.9%). Six patients (1.5%) discontinued the study due to adverse events, including three patients (0.8%) with ocular irritation, two patients (0.5%) with ocular pain, and one patient (0.3%) with ocular hyperemia . Our study findings suggest that csa 0.05% is an effective and tolerable treatment for moderate to severe dry eye disease in korean clinical practice . We found that treatment with csa 0.05% for three months led to improvements in both objective (schirmer and conjunctival staining scores) and subjective (symptom scores and artificial tear use) measures of dry eye disease . Favorable tolerability was indicated by the very low percentage of patients who discontinued the study, the absence of serious adverse events, and high patient satisfaction with treatment . The results of our study extend the efficacy findings of earlier clinical trials and clinical practice studies of csa 0.05% in korean patients with moderate to severe dry eye disease and support the use of csa 0.05% for treatment of moderate to severe dry eye disease in korean clinical practice . Consistent with previous studies, we found that csa 0.05% was an effective treatment for dry eye disease as indicated by changes in symptom, schirmer, and conjunctival staining scores, artificial tear use, and clinicians' global evaluations of treatment . The statistically significant reductions in symptom scores in our study are similar to those found in phase ii and phase iii clinical trials of csa 0.05% . Likewise, the significant improvements in mean schirmer scores in our study are similar to those found in korean patients after short - term (six to eight weeks) and longerterm (up to three months) csa 0.05% treatment . Importantly, our study included a much larger number of patients (n = 362) in the effectiveness analyses compared with those in the two previous clinical practice studies performed in korea (n = 23 and n = 26). The improvements in schirmer scores (with and without anesthesia), conjunctival staining score, artificial tear use, and the clinicians' global evaluations of treatment in our study are in agreement with those determined in a randomized controlled trial of csa 0.05% . The significant reduction in artificial tear use in our study is also consistent with findings from a previous study, in which the majority (> 60%) of patients reported decreased artificial tear use after 60 days of csa 0.05% treatment . Together, these findings confirm that csa 0.05% reduces the symptoms of dry eye disease . Therefore, we speculate that csa 0.05% partially resolves the pathophysiological changes that cause dry eye disease . However, additional studies are needed to confirm this conclusion . We found that treatment with csa 0.05% was generally well - tolerated, and that most patient experiences with csa 0.05% were positive . The most common adverse events in our study (ocular irritation and pain) are known adverse reactions to csa 0.05% [20 - 22]. The proportion of patients who discontinued treatment because of adverse events (1.5%) in our study is similar to the proportion (2.2%) in a phase ii trial of csa 0.05% . Moreover, the patient - reported experience with csa 0.05% in our study is similar to that in clinical practice studies performed in the united states . Our findings regarding patientreported rating of symptoms (i.e., no or mild symptoms) and the effect of dry eye on daily activities after three months of csa 0.05% treatment are similar to those of a clinical practice study in which patients reported a 30% reduction in symptom severity and improvement in their abilities to perform daily activities after two months of treatment with csa 0.05% . Likewise, our patientreported satisfaction findings are similar to those of a self - reported compliance study in which compliant patients had a mean satisfaction score of 7.7 (0 = not at all satisfied, 10 = very satisfied) after two months of treatment with csa 0.05% . First, interpretation of our findings is limited by the single - arm, open - label study design . However, the clinical practice setting enhances the external validity of our findings, as does the prospective, multicenter, surveillance design . Second, as our patients were not grouped according to the cause of dry eye disease, we were unable to determine which patients may (or may not) respond to csa 0.05% treatment . Third, we cannot exclude the possibility that improvements in dry eye disease were caused by the emulsion vehicle or that subjective improvements were a reflection of patients' providing socially desirable answers . However, the results of two randomized controlled trials have shown that csa treatment results in significantly greater improvements in dry eye disease than does treatment with the emulsion vehicle . In addition, the fact that there was agreement between clinician and patient evaluations of treatment (i.e., treatment resulted in improvement) would argue against the possibility that patients provided socially desirable answers . Finally, we did not assess patient adherence to study medication, document the type of artificial tear use, or use a validated questionnaire for assessing patient satisfaction with treatment . Despite these limitations, the use of multiple objective (i.e., schirmer and conjunctival staining scores) and subjective measures (i.e., symptom scores and artificial tear use) has enabled us to provide clinically relevant evidence of the effectiveness of csa 0.05% for treatment of dry eye disease . In conclusion, the effectiveness and tolerability findings of our study support the use of csa 0.05% for the treatment of moderate to severe dry eye disease in the korean clinical practice setting.
Although extensive research has been done in the last 2 decades, the regulation of c - myc activation and c - myc - dependent signal transduction pathways has not yet been fully elucidated . In this article we review current knowledge about htert transcription, which has been proved to be significantly up - regulated by c - myc, and, based on previous findings, we postulate that e2f1, another c - myc - upregulated gene, may act as a negative regulator of htert expression for its function in repressing htert transcription . The e2f1-mediated feedback inhibition of htert transcription, if existing, might be an important mechanism for normal cells to control the transmission of c - myc signals upon oncogenic stress . As a transcription factor, the c - myc oncoprotein has been estimated to regulate up to 15% of all human genes . These genes are essential to almost every aspect of cell behavior, including cell growth and proliferation, differentiation, and apoptosis . In light of these functions, it is not surprising that the activation of c - myc and c - myc - mediated downstream signal transduction is tightly regulated in normal cells . A great deal of recent work has determined that dysregulation of c - myc is closely associated with tumor initiation and progression . Supporting the role of c - myc in human tumors is the observation of sustained c - myc activation and its association with poor prognosis . In addition, studies using transgenic mice, in which the c - myc oncogene is constitutively expressed in a given cell type by means of a tissue - specific promoter, have supported the view that c - myc activation, as an initial event, is important for the formation of certain tumors . Telomere maintenance by telomerase has been proposed as an essential prerequisite for cell immortalization and tumorigenesis . Human telomerase reverse transcriptase, htert, is the catalytic subunit of telomerase, and its upregulation is the rate - limiting step for telomerase activity . Given the fact that ectopic expression of htert leads to immortalization of many human somatic cells, it is almost inevitable that in normal cells the transcription of htert gene is tightly repressed . Elevated expression of htert has been detected in over 85% of human tumors . Thus, the question emerged as to how the transcription of htert gene is triggered in the process of tumorigenesis . Over the past 10 years, mounting evidence indicates that upregulation of htert is one of the important links in c - myc signal transduction pathway . The contribution of elevated c - myc activity to cell immortalization was already recognized nearly 30 years ago when c - myc was classified as an oncogenic protein that cooperated with ras to transform primary embryonic rat cells . Consistent with this, some years later htert was identified as a direct transcriptional target of c - myc, and the transcriptional induction is independent of additional protein synthesis . This finding defines a pathway of oncogenic immortalization in which c - myc activation increases the expression of htert, and increased htert expression leads to cell immortalization . E2f1 is also a transcription factor and displays multiple activities that could be involved in either suppressing or promoting tumor development . Until now, few cellular genes have been demonstrated to have such a dual role in tumorigenesis . Generally, e2f1 is required for cell proliferation, whose activation promotes cell proliferation and growth by binding to the promoter region of several genes, including those that are involved in cell cycle regulatory activities and dna replication, and due to this it is considered as an oncogene in tumor progression . However, it is now clear that in normal cells elevated e2f1 expression participates in many aspects of the apoptotic process, either by acting alone or in cooperation with other factors, such as p53, to protect against tumorigenesis in the face of oncogenic transformation . Furthermore, e2f1 has been determined to be able to suppress htert expression by directly binding to the htert promoter [1416], indicating an additional mechanism for normal cells to control the activation of htert, in which e2f1 also functions as a tumor suppressor gene . Both c - myc and e2f1 are important regulators of cell signaling . It has been revealed that there is a close crosstalk between c - myc and e2f1 signaling pathway, which is believed to control cell fate decisions . It is known with certainty that c - myc and e2f1 can activate each other s transcription, thereby establishing a positive feedback circuit . In the absence of additional regulatory mechanisms, this circuit might be expected to overactivate both c - myc and e2f1, leading to cellular confusion . Obviously, this does not happen, indicating the possibility of another mechanism for regulation of c - myc and e2f1 interaction in cells . Recent evidence indicated that in noncancerous cells the mir-17 - 92 cluster is another important regulator involved in the c - myc and e2f1 networks [2022]. E2f1 are inhibited at the post - transcriptional level by members of the mir-17 - 92 cluster such as mir-20a and mir-17 - 5p, while, as transcription factors, both c - myc and e2f1 can induce the expression of micrornas within the mir-17 - 92 cluster . Since both mir-17 - 92 and e2f1 are transcriptionally activated by c - myc, this establishes an unusual network in which c - myc activates the transcription of e2f1 while simultaneously inhibiting its translation, suggesting that mir-17 - 92 is the negative control mechanism that prevents a runaway c - myc / e2f1 positive feedback . By using a mathematical model, it is additionally thought that the interactions between mir-17 - 92 and the transcription factors c - myc and e2f1 might represent a molecular switch for cell proliferation and apoptosis via determining the levels of these 2 proteins . Besides the above description, because c - myc and e2f1 have some common regulatory properties in cell biology, including proliferation and apoptosis, it is also inevitable that there are other types of interaction between these 2 signal transduction pathways . A study using k5 c - myc - transgenic mice showed that e2f1 is not required for proliferation, apoptosis, or tumorigenesis induced by c - myc . However, inactivation of e2f1 was found to not only increase the incidence of tumor formation, but also to promote tumor initiation, indicating that e2f1 activation is a control mechanism serving to protect normal cells from the c - myc - induced tumorigenesis . Surprisingly, in this model system, inactivation of e2f1 increased the level of apoptosis induced by c - myc overexpression . Because the apoptotic process was not impaired in this model, the accelerated tumor progression caused by e2f1 inactivation cannot result from the lack of e2f1-mediated apoptosis, suggesting that e2f1 has other functions to suppress the oncogenic transformation by c - myc . Observations that support this hypothesis also include: 1) c - myc - mediated proliferation and lymphomagenesis are compromised by e2f1 loss; and 2) e2f1 blocks and c - myc accelerates hepatic ploidy and tumorigenesis in transgenic mouse models . These results indicate that e2f1 can counteract c - myc oncogenic activation, especially in the process of tumor initiation . In normal cells, the activity of c - myc is tightly controlled to maintain cellular homeostasis through multiple levels of regulation . In theory, as a transcription factor, oncogenic c - myc activation can regulate up to 15% of cellular genes . However, only a fraction of these genes is responsive to c - myc activation in all cells analyzed, indicating the existence of some suppressors that function to limit the transcriptional function of c - myc . Interestingly, e2f1 is found to be able to bind the htert promoter and repress its expression . Therefore, these processes create a negative feedback loop that limits the c - myc - induced htert transcriptional expression . E2f1 plays a dual role in signaling both cell proliferation and apoptosis, which is dependent on the expression level of the protein itself . Thus, it may be hypothesized that under mild c - myc activation, concomitant e2f1 upregulation could limit the expression of htert, which normally participates in the process of cell proliferation . Whereas, under severe c - myc activation, the level of e2f1 protein is greatly increased, which leads to cell apoptosis (figure 1). As noted above, mir-17 - 92 is within the regulatory loop between c - myc and e2f1 . If mir-17 - 92 is significantly upregulated in response to c - myc activation, it can abrogate the increase in e2f1 expression induced by c - myc at the post - transcriptional level, and thus attenuate the inhibitory effect of e2f1 on the transcriptional expression of htert . Mutants of the htert promoter region might be another mechanism by which it can escape from the negative regulation of e2f1 . Overall, the inhibition of htert transcription by e2f1 might be an important mechanism for normal cells to counteract the oncogenic activity of c - myc . If the negative regulating function of e2f1 is removed, htert is easily activated by oncogenic c - myc activation, and thereby results in cell immortalization and tumorigenesis . Precise regulation of c - myc and its signaling pathway is critical for cellular physiology, while its perturbations have detrimental consequences to cells and lead to various diseases, especially tumors . Evidence from many studies clearly shows that c - myc - dependent htert transcription is a major mechanism for maintaining expression of telomerase and, apparently, contributes to tumor initiation . However, not all increased c - myc expression in normal cells will result in activation of oncogenic sequences including htert . E2f1 was determined to be transcriptionally activated by c - myc and has been shown to crosstalk extensively with c - myc signaling . E2f1 was also found to be able to inhibit htert gene transcription [1416]. Therefore, we speculated that e2f1 may be a potential negative regulator of c - myc - induced htert activation, which exhibits a negative feedback regulation in normal cells upon c - myc oncogenic activation . E2f1 has the ability to induce 2 seemingly contradictory processes of cell proliferation and apoptosis . Under normal conditions, notably, e2f1-induced apoptosis has a physiological role and is not just an accidental result of ectopic expression . We postulate that in normal cells an accompanying increase of e2f1 expression suppresses the c - myc - dependent htert transcription, and once the accompanying e2f1 expression had exceeded a certain threshold, cells enter spontaneously into apoptosis . At present, there are still no direct experimental data to support this hypothesis . Therefore, specific studies designed to provide information about the possible feedback regulation are needed . Also, the integration of the mir-17 - 92 cluster into the feedback regulation awaits further study and might prove to be a comprehensive mechanism to initiate tumorigenesis . Since mutants of htert promoter region can make cells escape from the surveillance mediated by e2f1, c - myc activation, even when the activation is mild, will lead to the transcriptional activation of htert and consequently lead to cell tumorigenesis under such conditions . Because htert activation constitutes a critical step in the process of tumorigenesis, a better understanding of htert regulation may provide not only a rationale for the molecular basis of tumor initiation, but also a path to the development of therapies based on the manipulation of htert activity . In this article, we postulate e2f1 as a negative regulator of c - myc - induced htert expression, and suggest possible explanations for ways by which cells escape this negative regulation . If this hypothesis were confirmed, it would indicate several important molecular targets for cancer prevention.
We reviewed final reports of nontyphoidal salmonella outbreaks investigated by cdc from 1984 to 2002 . We excluded outbreaks that occurred in a healthcare setting or outside the united states, including on cruise ships . When antimicrobial susceptibility was not recorded in the final report, we searched cdc microbiology records for susceptibility test results on isolates collected as part of the outbreak . Outbreaks were only included if susceptibility data were available for> 1 isolate . Because different laboratories performed susceptibility testing, the antimicrobial agents tested and the methods used varied between outbreaks . We classified outbreaks as resistant when the outbreak strain was resistant to 1 antimicrobial agent; other outbreaks were considered pansusceptible . Outbreaks caused by resistant strains were additionally classified as r - type ac / kssut when the outbreak strain was at least resistant to ampicillin, chloramphenicol or kanamycin, streptomycin, sulfamethoxazole, and tetracycline . Outbreaks were additionally classified as resistant to a clinically important agent when the outbreak strain was at least resistant to ampicillin, trimethoprim - sulfamethoxazole, aminoglycosides, fluoroquinolones, or a third - generation cephalosporin . Data from the final investigative reports were used for the analysis . When analyzing data according to outbreaks, we calculated the medians for percentage hospitalized and percentage who died and compared medians with the wilcoxon rank - sum test . When analyzing data according to ill persons, we pooled data from the reports, calculated proportions, and compared proportions with chi - square or, when appropriate, fisher exact test . The denominators for percentage hospitalized and died varied depending on the number of persons in whom outcome data were ascertained . Data were analyzed by using sas v.9 (sas institute, cary, nc, usa). From 1984 to 2002, cdc investigated 48 community outbreaks of nontyphoidal salmonella strains in the united states . Of these, 47 (98%) had a final report available for review (table a1). We restricted our analyses to the 39 (83%) outbreaks in which data about antimicrobial susceptibility were available . The largest outbreak occurred in 1985, in which culture - confirmed s. typhimurium infection associated with milk consumption developed in 16,659 persons (11). Strains from 11 (28%) outbreaks were resistant, and 28 (72%) were pansusceptible . The 11 outbreaks caused by resistant strains involved 18,698 persons . Of these 11 outbreaks, 7 (64%), involving 17,182 persons, had strains that were at least r - type ac / kssut, and 9 (82%), involving 17,919 persons, had strains that were resistant to a clinically important agent . The hospitalization rates were higher for each type of outbreak caused by resistant salmonellae compared with outbreaks caused by susceptible salmonellae (p<0.01 for each comparison) (table). To account for differences in the size of outbreaks, we compared the median proportion of persons hospitalized and compared hospitalization rates after excluding a large salmonella outbreak that occurred in 1985 . The median proportion hospitalized for each type of outbreak caused by resistant strains (26%) was> 2.5 times higher than the median proportion hospitalized for outbreaks caused by pansusceptible strains (10%, p<0.05 for all resistance patterns). The difference in hospitalization rates between outbreaks caused by resistant and susceptible strains was similar after we excluded the large 1985 outbreak of resistant s. typhimurium, in which the percentage hospitalized was 22% . The results also remained similar after excluding s. enteritidis outbreaks, in which rates of hospitalization and isolate resistance were low . * ac / kssut, ampicillin, chloramphenicol, kanamycin, streptomycin, sulfamethoxazole, tetracycline . Mortality data were available for 24 outbreaks involving 21,927 persons . A greater proportion of persons died in resistant outbreaks than in pansusceptible outbreaks, but the difference was not significant (0.1% in outbreaks caused by resistant strains vs. 0.06% in outbreaks caused by pansusceptible strains, p = 0.57) (table). Three (38%) outbreaks were due to s. enteritidis and 2 (25%) to s. typhimurium . In the 6 outbreaks for which hospitalization data were available, 70 (20%) of 353 persons were hospitalized . In the 4 outbreaks for which mortality data were available, 7 (0.4%) of 1,708 persons died . Outbreaks caused by antimicrobial - resistant, nontyphoidal salmonellae were associated with an increased rate of hospitalization compared with outbreaks caused by pansusceptible salmonellae . Persons who take antimicrobial drugs for reasons unrelated to gastroenteritis have an increased risk of developing antimicrobial - resistant salmonella infections; such patients may be taking antimicrobial drugs because they have medical conditions that increase their risk for hospitalization (79). We doubt that this explains the differential hospitalization rate observed in our study, because the outbreaks we studied occurred in diverse community settings . A second explanation for the higher hospitalization rate is that persons with resistant salmonella infections may fail empiric antimicrobial treatment, and their physicians subsequently hospitalize them for inpatient therapy . A third explanation is that resistant salmonellae may be more virulent because of some unknown factor . In the united states, resistant salmonellae are more often associated with hospitalization and bloodstream infection compared to pansusceptible salmonellae (13). In canada and denmark, studies have also found excess death rates associated with resistant salmonella infection (14,15). In england and wales, a study found no association between resistance and bloodstream infection, but that study had substantial limitations, including the failure to use pansusceptible salmonellae as a referent group and the failure to adjust for confounders, such as age (16). Since the review of salmonella outbreaks published in 1984, several changes have occurred in the epidemiology of antimicrobial - resistant salmonellae . First, s. typhimurium dt104 has emerged as a cause of antimicrobial - resistant salmonella infections . Five outbreaks in this study were caused by s. typhimurium with r - type ac / kssut; isolates with this resistance pattern are often dt104 (2,17). The hospitalization rate was higher in these 5 outbreaks (median proportion hospitalized: 17%) than in 3 outbreaks caused by pansusceptible s. typhimurium, which suggests that resistance or related factors, rather than just serotype, contribute to the differential rates of hospitalization . Second, a strain of s. newport resistant to at least 9 agents and with diminished susceptibility to ceftriaxone recently emerged in the united states; 1 outbreak in this review was caused by this strain and had a hospitalization rate greater than that seen in most other resistant outbreaks (18). Third, s. enteritidis has emerged as a frequent cause of foodborne outbreaks (19). S. enteritidis strains are infrequently resistant to antimicrobial agents, and in this review, s. enteritidis outbreaks were associated with low hospitalization rates . Excluding s. enteritidis outbreaks did not change the results of our analysis . The previous analysis by holmberg et al . Found that the rate of death was significantly greater in outbreaks caused by resistant strains (4%) compared with outbreaks caused by pansusceptible strains (0.2%) (10). Because this study covered a more recent period, improvements in medical care may explain the overall lower death rates . Studies with larger sample sizes are needed to determine whether antimicrobial resistance is associated with increased fatality rates . State health departments investigate hundreds of salmonella outbreaks annually . Occasionally, state health departments request assistance from cdc in investigating these outbreaks . The reasons for such requests vary but may be related to size, severity, setting, or other features of the outbreak . While concern about a high hospitalization rate might encourage a state health department to request assistance from cdc in a salmonella outbreak, susceptibility results are usually not known initially and are unlikely by themselves to influence the request for assistance . Another limitation is that our study was based on final reports, not raw data . As a result, we were unable to adjust for the different patient populations affected, including age, medical condition, and other factors that could affect hospitalization . Excluding outbreaks that occurred in healthcare settings may have reduced some of this bias . Adjustment for serotype, which could also affect hospitalization rates, was also not possible because of the small sample size . Despite these limitations, we believe this study adds to the weight of evidence about the health effects of antimicrobial - resistant salmonellae . The evidence now includes data from outbreaks and sporadic illness in the united states from 1970 to 2002 . Data from sporadic illness and 1 outbreak is also available from denmark (14,20). Across these diverse studies, such data about the adverse human health effects of resistant salmonellae should be incorporated into programs that promote appropriate antimicrobial use in humans and animals.
In spite of the absolute number of incident tb cases falling globally, tuberculosis (tb) continues to be the leading cause of mortality worldwide and has also been considered to be an occupational disease in the health care setup . One of the major problems in the current treatment of tuberculosis is the noncompliance to prescribed regimens, primarily because treatment of tb involves continuous, frequent multiple drug dosing . Adherence to treatment and the outcome of therapy could be improved with the introduction of long - duration drug formulations releasing the antitubercular agents in a slow and sustained manner . Polymer - based drug delivery systems like polymeric nanoparticles have achieved a potential position in the controlled release of therapeutic agents . Polymeric nanoparticles are solid colloidal particles with diameters ranging from 1 to 1000 nm . They consist of macromolecular materials in which the active ingredient is dissolved, entrapped, encapsulated, and adsorbed or chemically attached . The fate of nanoparticles in the gastrointestinal tract has extensively been investigated [57]. Sustained release cross - linked polymeric nanoparticles enable improvement of drug bioavailability by offering protection to the drugs in gastrointestinal environment and enhancement of solubility because of nanonization . This approach may help in overcoming the first pass effect by getting absorbed from the intestinal tract and entering into the blood streams . Here, the uptake of polymeric nanoparticles may occur by transcytosis via m cells and intracellular uptake and transport via the epithelial cells lining of the intestinal mucosa via peyer's patches . The selection of polymer to develop polymeric nanoparticles is dependent on many factors like size of nanoparticles required, inherent properties of the drug, surface characteristics, biodegradability, biocompatibility, toxicity, and drug release desired profile . Chitosan is the most extensively studied polysaccharide to develop polymeric nanoparticles . As a biodegradable polymer, chitosan is a popular choice in the application as a drug delivery carrier due to its biocompatibility, chemical versatility, and low cost . In the present study the main objective of the present study was to formulate and optimize oral sustained release chitosan nanoparticles of rifampicin by design of experiment (doe). Chitosan (cs) (degree of deacetylation: 93%) was purchased from yarrow chem products (mumbai, india). Sodium tripolyphosphate (tpp) was sourced from sigma - aldrich (mumbai, india). A 2 full - factorial experimental design was used to optimize formulation and process parameters for the preparation of chitosan nanoparticles . In order to optimize, the concentration of chitosan (x1), speed of homogenization (x2), and concentration of tripolyphosphate (tpp) (x3) were selected as independent variables . Eight formulations of drug loaded polymeric nanoparticles (cn1 to cn8) were prepared according to the design as shown in table 1 . The particle size, percentage of encapsulation efficiency, and percentage of drug loading were taken as response parameters . The rifampicin loaded chitosan nanoparticles were prepared by modified ionic gelation method . In this method, first o / w emulsion was prepared and then ionic gelation was done by polyanionic molecule as previously reported by ajun et al . . Chitosan solutions (25 ml) of different concentrations (1% w / v, 2% w / v) were prepared by dissolving chitosan in 1% acetic acid under stirring at room temperature . After dissolving completely, tween-80 (2% v / v) was added as a surfactant . Subsequently, rifampicin (62.5 mg) was dissolved in dichloromethane (2.5 ml), and then this oil phase was added dropwise to the aqueous phase . This addition was accompanied by stirring at different speeds (19,000 rpm, 26,000 rpm) with the help of high - speed homogenizer (d-8si, art - miccra, germany). Stirring was continued for 5 minutes after the complete addition of the oil phase to the aqueous phase . Later cross - linking of the particles was induced by the drop wise addition of tripolyphosphate (tpp) solutions (10 ml) of different concentration (0.1% w / v, 0.2% w / v) into o / w emulsion under magnetic stirring at 500 rpm . To ensure complete evaporation of dichloromethane nanoparticles were isolated by centrifugation at 13,500 rpm for 20 minutes at 20c using cooling centrifuge (sigma 3k30, germany), and the supernatant was used for the measurement of free rifampicin by uv spectrophotometer (uv 1800, shimadzu, japan). The particle size of the formulations was determined by laser scattering technique using malvern nano s90 (malvern instruments, uk) after appropriate dilution with double distilled water . Light scattering was measured at 25c and with an angle of 90. the particle size distribution is reported as a polydispersity index (pdi). The values close to zero indicate the homogenous nature of the dispersion and those greater than 0.5 indicate the heterogeneous nature of the dispersion . The surface characteristics of samples were studied by scanning electron microscopy (sem) from 1700x to 5200x magnifications . The powder sample was dispersed in the double distilled water and dispersion drop was put on the slide . The aluminum stubs were placed in the vacuum chamber of a scanning electron microscope (xl 30 esem with edax, philips, the netherlands). The samples were observed for morphological characterization using a gaseous secondary electron detector (xl 30, philips, eindhoven, the netherlands) with working pressure: 0.8 torr, acceleration voltage: 30.00 kv . The entrapment efficiency and drug loading of selected formulation were calculated by the following equation: (1)% drug encapsulation efficiency = dadsda100,% drug loading= dadsna100, where da is the total amount of drug added in system, ds is the amount of drug in supernatant after the centrifugation, and na is the total amount of nanoparticles obtained . The amount of drug in supernatant was calculated from concentration values obtained from the calibration curve on spectrophotometric analysis of the samples at 475 nm (shimadzu uv 1800, japan). Usa) was used for the analysis of the effect of each variable on the designated response . The statistical significance of the difference in particle size, percentage of drug encapsulation, and percentage of drug loading was tested by one - way analysis of variance (anova) using the following polynomial equation (2): (2)y = b0+b1x1+b2x2+b3x3+b1b2x1x2+b1b3x1x3+b2b3x2x3+b1b2b3x1x2x3, where y is the measured response, b0 is the arithmetic mean response, b1 is the main effect of chitosan concentration (x1), b2 is the main effect of speed of homogenization (x2), and b3 is the main effect of tpp concentration (x3); b1b2, b1b3, b2b3, and b1b2b3 are the interactions of the main factors . The significant response polynomial equations generated by design expert were used to validate the statistical design . Response surface plots were generated to visualize the simultaneous effect of each variable on each response parameter . A checkpoint analysis was performed to confirm the utility of the established polynomial equation in the preparation of rifampicin loaded chitosan nanoparticles . Three checkpoint values of independent variables (x1, x2, and x3) were taken and the values of dependent variables were calculated by substituting the values in the respective polynomial equation (7). Rifampicin loaded chitosan nanoparticles were prepared experimentally by taking the amounts of the independent variables (x1, x2, and x3). Differences of theoretically computed values of dependent variables and the mean values of experimentally obtained value of dependent variables were compared by using student t's test method . The desirability function was used for optimization of the formulation . During the optimization of formulations, the responses have to be combined in order to produce a product of desired characteristics . Optimized nanoparticles should have low - particle size and high percentage of entrapment efficiency and percentage of drug loading . The individual desirability for each response was calculated using the following method [14, 15]. The percentage of drug encapsulation efficiency and percentage of drug loading values were maximized in the optimization procedure, as optimized nanoparticles batch should have high percentage of drug encapsulation efficiency and percentage of drug loading . The desirability functions of these responses were calculated using the following equation: (3)id1 or id2=yiyminytargetymin,id1 or id2=1 for yi> ytarget, where id1 is the individual desirability of percentage of drug encapsulation efficiency and id2 is the individual desirability of percentage of drug loading . The values of ytarget and ymin for percentage of drug encapsulation efficiency are 49.36 and 20.17, the values of ytarget and ymin for percentage of drug loading are 45.17 and 23.05, and yi is the individual experimental result . The particle size value was minimized in the optimization procedure, as optimized nanoparticles batch should have low particle size . The desirability functions of this response were calculated using the following equation: (4)id3=ymaxyiymaxytarget, id3=1 for yi <ytarget, where id3 is the individual desirability of particle size . The values of ymax and ytarget for particle size were 383.3 and 180.5, and yi is the individual experimental result . The overall desirability values were calculated from the individual desirability values by using the following equation: (5)od=(id1id2id3idn)1/n, where n = 3 (number of desirable responses of the experiment). In vitro drug release study of polymeric nanoparticles of the best two batches according to desirability function was performed by the dialysis bag diffusion technique . Polymeric nanoparticles equivalent to 25 mg rifampicin were filled in dialysis bag (mwco 1214 kda, pore size 2.4 nm) and immersed in a receptor compartment containing 150 ml of phosphate buffer solution at three different ph values, 6.8, 5.2, and 7.4, in the presence of ascorbic acid (0.2% w / v). Ascorbic acid was used to prevent the degradation of rifampicin in the dissolution medium due to atmospheric oxygen . The system was stirred at 100 rpm and maintained at a temperature of 37 0.5c . The ph values were selected to simulate intestinal fluid ph (6.8), physiological ph (7.4), and endosomal ph of macrophages (5.2). At predetermined time intervals, five milliliter of samples was withdrawn and diluted appropriately, and the absorbance was measured by uv / visible spectrophotometer (uv 1800, shimadzu, japan) at 475 nm . Various kinetic models zero order, first order, higuchi, hixson crowell and korsmeyer - peppas, and weibull models were applied to obtain the drug release mechanism from the chitosan nanoparticles [1719]. Particle size was varied in the range of 180.5 (cn3) nm to 383.3 (cn6). The drug loaded nanoparticles exhibited relatively narrow particle size distribution as indicated by relatively low pdi values in the range of 0.202 to 0.472 . Morphology of chitosan nanoparticles under scanning electron microscope (sem) is shown in figure 1 . It also shows that there is only little aggregation between the prepared chitosan nanoparticles . Percentage of drug encapsulation efficiency and percentage of drug loading for respective batches are shown in table 1 . Higher drug encapsulation efficiency and drug loading were observed for the batch cn8, and cn5 has the lowest drug encapsulation efficiency and drug loading . A statistical design was utilized in order to derive the relationship between the response variables and the independent variables . The analysis of variance (anova) results (p value) of the effect of the variables on particles size, percentage of drug encapsulation efficiency, and percentage of drug loading can be seen in following full - model polynomial equation: (6)y1=249.61 + 31.99x1(p<0.0001)22.89x2(p<0.0001)+38.39x3(p<0.0001)8.66x1x2(p<0.0001)+10.76x1x3(p<0.0001)12.86x2x3(p<0.0001)8.14x1x2x3(p<0.0001),y2=29.84 + 9.92x1(p<0.0001)2.48x2(p<0.0001)+4.41x3(p=0.0105)1.77x1x2(p= 0.0551)+1.28x1x3(p=0.1539)+3.61x2x3(p<0.0007)+1.93x1x2x3(p=0.0389),y3=30.56 + 8.40x1(p<0.0001)2.82x2(p=0.0008)+3.89x3(p<0.0084)1.21x1x2(p=0.2164)+1.67x1x3(p=0.0941)+4.02x2x3(p<0.0006)+1.59x1x2x3(p=0.1108). The terms of full - model polynomial equation having insignificant p value (p> 0.05) have negligible contribution to obtained dependent variables and thus are omitted to get reduced model equation . Equations (7) representing the quantitative effect of the formulation and process variables on the particle size, percentage of drug encapsulation efficiency, and percentage of drug loading are described as follows: (7)y1=249.61 + 31.99x122.89x2 + 38.39x38.66x1x2 + 10.76x1x312.86x2x3 8.14x1x2x3; r2=0.999,y2=29.84 + 9.92x12.48x2 + 4.41x3 + 3.61x2x3 + 1.93x1x2x3; r2=0.925,y3=30.56 + 8.40x12.82x2 + 3.89x3 + 4.02x2x3; r2=0.892 . Response surface graphs were generated using the above polynomial equations, which represent the simultaneous effect of any two variables on response parameters by taking one variable at a constant level . Coefficients with one factor in polynomial equations are attributed to the effect of that particular factor, while the coefficients with more than one factor are attributed to the interaction between those factors . A positive sign of the polynomial terms indicates a positive effect, while a negative sign indicates a negative effect of the independent factors . Polynomial equation (7) represents the effect on particle size, percentage of drug encapsulation efficiency, and percentage of drug loading, respectively . The higher coefficient value of the main effects and interaction terms in the polynomial equation indicates that the effect of independent parameters on particle size is much higher than the effect on percentage of drug encapsulation efficiency and percentage of drug loading . It can also be concluded that the concentration of chitosan and concentration of tpp have positive effect; however, the speed of homogenization has a negative effect on all dependent variables . This can also be seen in the response surface methodology indicating the effect of independent parameters on particle size (figure 2), drug encapsulation efficiency (figure 3), and drug loading (figure 4). The increase in the particle size with an increase in the concentration of chitosan is due to the fact that at higher concentration of chitosan, viscosity is much higher and hence it affects the shear capacity of homogenizer and stirrer as well . The reason for the increases in the particle size with an increase in the concentration of tpp would be due to the stiffness of the cross - linkage between tpp and chitosan; as the tpp concentration increases, there would be more tripolyphosphoric ions to cross - link with amino groups on chitosan chains . However, the increase in homogenization speed would decrease particle size, probably due to the fact that at the higher speed, smaller emulsion droplet was formed, resulting in smaller sized particles . Increase in the encapsulation efficiency and drug loading with increase of chitosan concentration would be due to the fact that the higher amount of chitosan has higher ability of ionic gel formation which prevents the rifampicin movement to the external phase and increases in the drug encapsulation efficiency hence the drug loading . Drug loading and encapsulation efficiency increase with the increase in tpp concentration indicating the better cross - linking density of chitosan matrix . In addition, at higher speed of homogenization there is a reduction in drug encapsulation efficiency and drug loading . It would be due to diffusion of the drug to the outer phase during emulsification by size reduction using high speed homogenizer . In order to validate the equation that describes the influence of the factors on the particle size, percentage of drug encapsulation efficiency, percentage of drug loading of nanoparticles, three additional checkpoint experiments (batch cp1, batch cp2, and batch cp3) were taken and table 2 shows the actual and predicted values of independent parameters . The t - test was applied between the actual and predicted values of independent parameters and it was observed that p value> 0.05 . Therefore, it is concluded that the polynomial equations are valid to prepare chitosan nanoparticles of desired characteristics . Therefore, this batch was considered as the best batch and the values of independent variables of this batch were considered to be optimum values to prepare chitosan nanoparticles . Release studies were carried out by using three different release medium, phosphate buffers at ph 7.4, ph 6.8, and ph 5.2 in order to simulate the physiological condition, intestinal condition, and the macrophage environment, respectively, shown in figure 5 . At ph 7.4, in both of the batches, about 5% to 8% of the drug similarly, at ph 6.8 and ph 5.2, in both of the batches, about 8% to 13% of the drug was immediately released in 1 hour . This finding indicates that some of the drug is localized on the surface of the nanoparticles due to the partition of the drug into the surface - active agent layer adsorbed at the surface of the emulsion droplets . After this initial burst, drug release is almost constant, and around 90% of the drug was released from the chitosan nanoparticles in the range of 28 hours to 34 hours . It is concluded that rifampicin release of the chitosan nanoparticles is ph dependent: it is faster at a lower ph than around neutral ph (ph 5.2> ph 6.8> ph 7.4). This is the consequence of the higher solubility of chitosan at lower ph, where the d - glucosamine residues are ionized resulting in an extensive polymer swelling and faster drug release . Moreover, rifampicin solubility is ph dependent: it increases as the ph increases . When comparing the drug release profiles from cn8 and cn4 chitosan nanoparticles, decrease of the release rate is obtained from the cross - linked nanoparticles . This is due to the higher amount of tpp, and hence high degree of cross - linking in the case of cn8 compared with that of the cn4 . The higuchi model was best fitted as a release kinetic of rifampicin from chitosan nanoparticles . Optimization of formulation and process parameters for the development of chitosan nanoparticles is a prerequisite to obtain the drug loaded chitosan nanoparticles with desired characteristics . Chitosan nanoparticles were modified by various factors to control particle size, percentage of drug loading, and encapsulation efficiency . The result shows that concentrations of chitosan, concentration of tpp, and homogenization speed are significantly affecting the particle size, drug loading, and drug encapsulation efficiency . Though rifampicin is a poorly water soluble drug, it can be loaded successfully to a hydrophilic matrix of chitosan nanoparticles using modified emulsion ionic gelation method . Release of rifampicin from chitosan nanoparticles was concentration independent and sustains for a longer period of time . Thus, in vivo study can further explore the potentiality of this system for improving patient compliance by reducing the dosing frequencies in tuberculosis.
Kala - azar occurs in 2 foci (figure 1) and is caused by l. donovani . In the northern focus (upper nile, jonglei, and unity states), phlebotomus orientalis is the vector; in the southern focus (parts of eastern equatoria state), p. martini is the vector (3,4). Although studies in eastern sudan have found domestic animals infected with the parasite (5,6), whether these animals play a role as disease reservoirs has not yet been proven; thus, transmission is still thought to be anthroponotic . Shaded areas represent those counties where primary cases were reported from january through june 2007 . (adapted from world health organization, southern sudan health update, july august 2007 .) The disease was first reported from southern sudan in 1904, and the first epidemic was documented in 1940 with a death rate of 80% (7). Beginning in 1984, an epidemic (unrecognized until 1988) devastated the western part of upper nile state, ultimately causing 100,000 deaths in a population of 280,000 over a 10-year period (3). Passive case - detection data on kala - azar in southern sudan, collected by the world health organization (who) since 1989, indicate a cyclical pattern of kala - azar with considerable variation in the caseload from year to year (figure 2). The dynamics presented in figure 2 also suggest that southern sudan is currently between epidemics and provide a warning that cases may rise dramatically in coming years . In 2006, a total of 1,117 cases were reported, 65.4% of which were primary cases; the remainder were either relapses or cases of post kala - azar dermal leishmaniasis . From january through june 2007, the 5 locations accounting for 74.2% of the primary cases in 2007 were malakal (n = 83), ulang (n = 72), nasir (n = 63), and kiechkuon (n = 25) in upper nile state and lankien (n = 79) in northern jonglei state . Since 2002, the case - fatality rate recorded at healthcare facilities has been 4%6% . Total annual number of kala - azar cases in southern sudan reported to the world health organization, 19892006 . These data likely underestimate the actual number of cases because healthcare providers do not always provide complete reports and many kala - azar patients never visit healthcare facilities . Epidemiologic modeling of data from upper nile state estimated that those who visited healthcare facilities from october 1998 through may 2002 represented only 55% of cases and that 91% of kala - azar deaths were undetected (8). Health coverage is so minimal that some patients must walk for several days to access even the most basic healthcare services . Despite the availability of different rapid diagnostic tests, most facilities used clinical diagnosis alone until recently . Only a few had the supplies and equipment to confirm suspected cases through microscopic examination of lymph node aspirate, which has a sensitivity of only 53%65% (1). Nongovernment healthcare providers and government of southern sudan administered healthcare facilities thus had to confirm suspected kala - azar by direct agglutination test before they could receive a supply of the first - line treatment sodium stibogluconate from who through pharmaciens sans frontires . The use of the direct agglutination tests was required because of concerns about the sensitivity and specificity of the rk39 dipsticks in east africa; a recent study suggests that these concerns were well founded (9). Also, many facilities that had received dipsticks were not using them . Until 2004, many healthcare facilities did not have the equipment or skills to conduct direct agglutination tests, and blood samples had to be sent to kenya for analysis, which often led to treatment delays as long as 18 days . Now some healthcare facilities can analyze these samples internally and start treatment within 24 hours . Confirmed cases are treated with sodium stibogluconate at a dose of 20 mg / kg / day for 30 days . Recently, because of an increasing number of patients in upper nile state who were nonresponsive to sodium stibogluconate, mdicins sans frontires tested a combination of sodium stibogluconate and paromomycin, which would reduce treatment duration (from 30 to 17 days) and cost . Patient survival and initial cure rates were better than those for patients who received sodium stibogluconate monotherapy (10). However, completion of multicountry phase iii trials being conducted by the drugs for neglected diseases initiative (www.dndi.org) is eagerly awaited before the combination can be considered as an alternative . Amphotericin b, a second - line drug for treatment of kala - azar, is not yet available in southern sudan s facilities except in those run by mdicins sans frontires . Much remains unknown about the epidemiology of kala - azar in southern sudan (11). In the absence of detailed information on risk factors (cultural, demographic, epidemiologic, clinical, and geographic), use of long - lasting insecticide - treated nets seems a suitable method of prevention . Results from studies in northern sudan showed that insecticide - treated nets provided 27% protection from kala - azar (12). Whether similar protection can be achieved in southern sudan s disease - endemic areas requires confirmation because effectiveness is dependent on human and vector behavior (13). The return of stability to southern sudan has opened up new challenges and opportunities for kala - azar control . Large - scale population movement of susceptible or infected populations into kala - azar endemic or nonendemic areas respectively, poses a major epidemic risk . The healthcare systems are weak and rely on support from faith - based and nongovernment organizations, which need to be coordinated to ensure consistency in diagnosis, treatment, and prevention . As health infrastructure and human resources are being built up, kala - azar will need to be addressed as an integral part of multifunctional healthcare delivery by government staff, but this requires training and the provision of essential supplies . Kala - azar falls under the mandate of the director general of preventive medicine within the ministry of health government of southern sudan . The ministry of health, with support from who and in conjunction with nongovernment organizations working on kala - azar, has embarked on a number of activities to strengthen case - management . Laboratory technicians in most referral facilities have now been trained on the direct agglutination test; case - management guidelines have been updated; the essential drugs list is being reviewed and expanded to include alternatives for second - line treatment; and rk39 dipsticks are being distributed to peripheral health facilities to complement clinical diagnosis . With the revision of diagnosis and treatment guidelines, facilities are now able to obtain sodium stibogluconate by providing pharmaciens sans frontires with a positive rapid diagnostic test result, but they are encouraged to also take a blood sample for direct agglutination testing, as this is still considered more reliable (8). Meanwhile, the uk - based malaria consortium is providing long - lasting insecticide - treated nets to areas in jonglei and eastern equatoria, where malaria and kala - azar are co - endemic . A strong presence of international donors and the southern sudan government s desire to quickly reconstruct the healthcare sector provide ample opportunity to reduce the incidence of kala - azar.
A recent study found that survival after inhospital cardiac arrest has improved markedly over the past decade, from 13.7% in 2000 to 22.3% in 2009 . While encouraging, it remains unknown whether this survival trend has occurred uniformly across most hospitals or driven by large improvements at a smaller number of hospitals . It is possible that some hospitals have achieved larger gains in survival over time compared with other hospitals . Identifying topperforming sites that have achieved large gains in inhospital cardiac arrest survival and the associated hospital factors is the critical next step to inform ongoing quality improvement efforts for inhospital resuscitation . To address these gaps in knowledge, we used data from get with the guidelines (gwtg)resuscitation, a large national registry of inhospital cardiac arrests, to characterize hospitallevel trends in inhospital cardiac arrest survival over the past decade . Additionally, we determined whether some hospitals have achieved greater improvements in survival compared with others, and evaluated hospital factors associated with the extent of hospital rates of survival improvement . Gwtgresuscitation, formerly known as the national registry for cardiopulmonary resuscitation, is a large, prospective, hospitalbased clinical registry of patients with inhospital cardiac arrest in the united states . Briefly, cardiac arrest in the registry is defined as the absence of a palpable central pulse, apnea, and unresponsiveness . Consecutive patients with a cardiac arrest, without donotresuscitate orders, and who received cardiopulmonary resuscitation (cpr) are identified and enrolled by specially trained personnel using an online, interactive case report form . Multiple case finding methods are used to ensure that all cases within a hospital are captured . These include centralized collection of cardiac arrest flow sheets, review of hospital page system logs, and routine checks of code carts, pharmacy tracer drug records, and hospital billing charges for use of resuscitation medications . Data are collected according to the utsteinstyle definitions, which are a template of uniform reporting guidelines developed by international experts . Prior to data collection and entry, moreover, the data submission software performs internal data checks to ensure data completeness and to alert the data entry person for outlier responses . Within gwtgresuscitation, we identified 122 746 patients at 590 hospitals during 20002010 who were 18 years of age or older and had an index cardiac arrest with an identifiable initial rhythm (asystole, pulseless electrical activity, ventricular fibrillation, or pulseless ventricular tachycardia) (figure 1). From this sample, we excluded patients who were missing information on survival (n=197) and calendar year of the arrest (n=55). We also excluded 43 hospitals (5348 patients with cardiac arrest) that were missing information on hospital characteristics . Finally, given that the estimate of survival improvement from hospitals with low cardiac arrest volume or few years of available data would be unreliable, we restricted our sample to only those hospitals that participated in the registry for 5 years and had a mean annual case volume of 10 cases in accordance with previous studies . As a result our final sample consisted of 93 342 patients from 231 hospitals . In an effort to better understand which hospital characteristics are associated with survival improvement, we merged data from the 2008 american hospital association annual survey with gwtgresuscitation to obtain information on hospital characteristics . These included information on a hospital's geographic region (north midatlantic, south atlantic, north central, south central, and mountain / pacific), location (rural, urban), ownership (nonprofit, public, private), teaching status (fellowship program, residency program, or nonteaching), and bed number (250, 250 to 499, 500). Patientlevel data available from gwtgresuscitation included demographics (age, sex, race), initial cardiac arrest rhythm (asystole, pulseless electrical activity, ventricular fibrillation, pulseless ventricular tachycardia), location of cardiac arrest (intensive care unit, telemetry unit, nonmonitored unit), time of day (work hours: 7 am to 10:59 pm versus after hours: 11 pm to 6:59 am) and day of week (weekday versus weekend) of cardiac arrest, and use of a hospitalwide cardiopulmonary arrest alert (ie, code blue). Moreover, we used information on comorbid conditions (congestive heart failure; myocardial infarction; diabetes mellitus; renal, hepatic, or respiratory insufficiency; neurological status prearrest [as determined by admission cerebral performance category {cpc} scores]; baseline evidence of motor; cognitive, or functional deficits [central nervous system depression]; acute stroke; pneumonia; hypotension; arrhythmia; sepsis; trauma; metabolic or electrolyte abnormality; cancer), and therapeutic interventions in place at the time of cardiac arrest (use of mechanical ventilation, antiarrhythmic drugs, intravenous vasopressors, dialysis, pulmonary artery catheter, intraaortic balloon pump). The main independent variable was calendar year, and the primary outcome was hospital rate of survival to discharge . We first conducted bivariate analyses to evaluate unadjusted trends in patient and hospital characteristics over time by using the maentelhaenszel test for categorical variables and linear regression for continuous variables . To evaluate temporal trends in hospitallevel survival rates such models account for clustering of patients within a hospital and avoid overestimation of significance of statistical associations . The initial model included hospitallevel random intercepts and slopes by calendar year, which was modeled as a continuous variable . From this model, empirical bayesian estimates of hospitalspecific intercepts and slopes were obtained, which are shrunken toward the mean for smallvolume hospitals, reflecting the lack of information available for those hospitals and thus mitigating issues of outliers . Slopes estimates were exponentiated to represent the relative change in odds of survival per year for each hospital . 1) were considered to have improved trends in survival, with the magnitude of the slope (and or) quantifying the extent of annual survival improvement . Finally, we repeated these analyses including adjustment for patient and hospital characteristics, to obtain adjusted annual trends in hospital rates of survival . We examined hospital variation in survival trends using a cumulative frequency plot of the hospitalspecific or . Next, we categorized hospitals into quartiles using the hospitalspecific estimates of survival improvement from the model just described . Finally, to explore which hospital characteristics were associated with a higher rate of survival improvement, we included interaction terms between each hospital characteristic (as described earlier in the hospital and patient variables section) and calendar year in our multivariable model . Data were complete for all covariates, except race (6.5%), admission cpc score (15%), hospital location of arrest (2.1%), and time of cardiac arrest (1.1%). Missing data on covariates first, to exclude the possibility that hospitals with the largest temporal improvement in survival to discharge were not confounded by higher rates of neurological disability, we examined whether hospitals with the greatest survival gains were also the same hospitals with the largest temporal improvement in favorable neurological survival . Information on discharge neurological status in gwtgresuscitation was collected using cpc scores . Because data on discharge cpc scores were missing in 2241 (12%) survivors, we used multiple imputation to assign discharge cpc scores for survivors for whom that information was missing . We defined favorable neurological survival as survival to discharge with a cpc of 1 (ie, none or mild neurological disability) and calculated adjusted hospitallevel rates of favorable neurological survival using the hierarchical model just described . We then examined pearson's correlation between hospital trends in favorable neurological survival and survival to discharge . Second, to ensure that our findings were not influenced by temporal changes in hospital discharge practice patterns (eg, early discharge to hospice of patients with poor likelihood of survival), we repeated our analyses of hospitallevel trends after classifying patients who were discharged to hospice as having died . We then examined whether this substantially altered our classification of hospitals with the greatest improvement in survival . All statistical analyses were prespecified and conducted using sas version 9.1.3 (sas institute), iveware (university of michigan), and r version 2.6.0 (free software foundation). The institutional review board at university of iowa approved the study and waived the requirement for informed consent . Gwtgresuscitation, formerly known as the national registry for cardiopulmonary resuscitation, is a large, prospective, hospitalbased clinical registry of patients with inhospital cardiac arrest in the united states . Briefly, cardiac arrest in the registry is defined as the absence of a palpable central pulse, apnea, and unresponsiveness . Consecutive patients with a cardiac arrest, without donotresuscitate orders, and who received cardiopulmonary resuscitation (cpr) are identified and enrolled by specially trained personnel using an online, interactive case report form . Multiple case finding methods are used to ensure that all cases within a hospital are captured . These include centralized collection of cardiac arrest flow sheets, review of hospital page system logs, and routine checks of code carts, pharmacy tracer drug records, and hospital billing charges for use of resuscitation medications . Data are collected according to the utsteinstyle definitions, which are a template of uniform reporting guidelines developed by international experts . Prior to data collection and entry, moreover, the data submission software performs internal data checks to ensure data completeness and to alert the data entry person for outlier responses . Within gwtgresuscitation, we identified 122 746 patients at 590 hospitals during 20002010 who were 18 years of age or older and had an index cardiac arrest with an identifiable initial rhythm (asystole, pulseless electrical activity, ventricular fibrillation, or pulseless ventricular tachycardia) (figure 1). From this sample, we excluded patients who were missing information on survival (n=197) and calendar year of the arrest (n=55). We also excluded 43 hospitals (5348 patients with cardiac arrest) that were missing information on hospital characteristics . Finally, given that the estimate of survival improvement from hospitals with low cardiac arrest volume or few years of available data would be unreliable, we restricted our sample to only those hospitals that participated in the registry for 5 years and had a mean annual case volume of 10 cases in accordance with previous studies . As a result in an effort to better understand which hospital characteristics are associated with survival improvement, we merged data from the 2008 american hospital association annual survey with gwtgresuscitation to obtain information on hospital characteristics . These included information on a hospital's geographic region (north midatlantic, south atlantic, north central, south central, and mountain / pacific), location (rural, urban), ownership (nonprofit, public, private), teaching status (fellowship program, residency program, or nonteaching), and bed number (250, 250 to 499, 500). Patientlevel data available from gwtgresuscitation included demographics (age, sex, race), initial cardiac arrest rhythm (asystole, pulseless electrical activity, ventricular fibrillation, pulseless ventricular tachycardia), location of cardiac arrest (intensive care unit, telemetry unit, nonmonitored unit), time of day (work hours: 7 am to 10:59 pm versus after hours: 11 pm to 6:59 am) and day of week (weekday versus weekend) of cardiac arrest, and use of a hospitalwide cardiopulmonary arrest alert (ie, code blue). Moreover, we used information on comorbid conditions (congestive heart failure; myocardial infarction; diabetes mellitus; renal, hepatic, or respiratory insufficiency; neurological status prearrest [as determined by admission cerebral performance category {cpc} scores]; baseline evidence of motor; cognitive, or functional deficits [central nervous system depression]; acute stroke; pneumonia; hypotension; arrhythmia; sepsis; trauma; metabolic or electrolyte abnormality; cancer), and therapeutic interventions in place at the time of cardiac arrest (use of mechanical ventilation, antiarrhythmic drugs, intravenous vasopressors, dialysis, pulmonary artery catheter, intraaortic balloon pump). The main independent variable was calendar year, and the primary outcome was hospital rate of survival to discharge . We first conducted bivariate analyses to evaluate unadjusted trends in patient and hospital characteristics over time by using the maentelhaenszel test for categorical variables and linear regression for continuous variables . To evaluate temporal trends in hospitallevel survival rates such models account for clustering of patients within a hospital and avoid overestimation of significance of statistical associations . The initial model included hospitallevel random intercepts and slopes by calendar year, which was modeled as a continuous variable . From this model, empirical bayesian estimates of hospitalspecific intercepts and slopes were obtained, which are shrunken toward the mean for smallvolume hospitals, reflecting the lack of information available for those hospitals and thus mitigating issues of outliers . Slopes estimates were exponentiated to represent the relative change in odds of survival per year for each hospital . 1) were considered to have improved trends in survival, with the magnitude of the slope (and or) quantifying the extent of annual survival improvement . Finally, we repeated these analyses including adjustment for patient and hospital characteristics, to obtain adjusted annual trends in hospital rates of survival . We examined hospital variation in survival trends using a cumulative frequency plot of the hospitalspecific or . Next, we categorized hospitals into quartiles using the hospitalspecific estimates of survival improvement from the model just described . Finally, to explore which hospital characteristics were associated with a higher rate of survival improvement, we included interaction terms between each hospital characteristic (as described earlier in the hospital and patient variables section) and calendar year in our multivariable model . Data were complete for all covariates, except race (6.5%), admission cpc score (15%), hospital location of arrest (2.1%), and time of cardiac arrest (1.1%). Missing data on covariates first, to exclude the possibility that hospitals with the largest temporal improvement in survival to discharge were not confounded by higher rates of neurological disability, we examined whether hospitals with the greatest survival gains were also the same hospitals with the largest temporal improvement in favorable neurological survival . Information on discharge neurological status in gwtgresuscitation was collected using cpc scores . Because data on discharge cpc scores were missing in 2241 (12%) survivors, we used multiple imputation to assign discharge cpc scores for survivors for whom that information was missing . We defined favorable neurological survival as survival to discharge with a cpc of 1 (ie, none or mild neurological disability) and calculated adjusted hospitallevel rates of favorable neurological survival using the hierarchical model just described . We then examined pearson's correlation between hospital trends in favorable neurological survival and survival to discharge . Second, to ensure that our findings were not influenced by temporal changes in hospital discharge practice patterns (eg, early discharge to hospice of patients with poor likelihood of survival), we repeated our analyses of hospitallevel trends after classifying patients who were discharged to hospice as having died . We then examined whether this substantially altered our classification of hospitals with the greatest improvement in survival . All statistical analyses were prespecified and conducted using sas version 9.1.3 (sas institute), iveware (university of michigan), and r version 2.6.0 (free software foundation). The institutional review board at university of iowa approved the study and waived the requirement for informed consent . Table 1 describes characteristics of the study population . The mean age (sd) was 65.9 (15.9) years . The initial cardiac arrest rhythm was asystole or pulseless electrical activity in nearly 4 in 5 patients . More than 80% of inhospital cardiac arrests occurred in a monitored setting (intensive care unit [48.0%], telemetry unit [15.7%], or other closely monitored hospital locations [17.6%]), and> 30% of arrests each occurred during nighttime and on the weekend . Approximately, 80% of all patients had a witnessed arrest, and this was substantially higher for cardiac arrests in the intensive care unit (94.5%) compared with those in monitored units (68.7%) and nonmonitored units (48.3%). In general, patients had a high burden of comorbidities heart failure (18.1%), respiratory insufficiency (42.5%), renal insufficiency (33.4%), diabetes mellitus (30.4%), septicemia (15.9%), as well as use of mechanical ventilation (31.0%) and intravenous vasopressors (27.4%) before the cardiac arrest . Characteristics of study patients * cns indicates central nervous system; cpc, cerebral performance category; ed, emergency department; pea, pulseless electrical activity, vf, ventricular fibrillation; vt, ventricular tachycardia . For illustrative purposes, trends in baseline characteristics are presented as 3 time periods (20002003, 20042006, and 20072010). However, the p value for trend is for temporal changes in these characteristics by calendar year . Data were missing for the following variables: race 6023 (6.5%), time of cardiac arrest 1031 (1.1%), hospital location 1981 (2.1%), witnessed arrest 13 (0.01%), and cpc category on admission 13 969 (14.9%). Temporal trends in patient characteristics are also described in table 1 . Between 2000 and 2010, there was a significant decline in cardiac arrests due to ventricular fibrillation and pulseless ventricular tachycardia (p<0.001). Significant temporal trends were also noted in demographics (decrease in age, p<0.001), comorbidities (decrease in prevalence of heart failure and myocardial infarction and increase in prevalence of respiratory insufficiency, renal insufficiency, diabetes, septicemia, and malignancy; p<0.001 for all comparisons), and acute illness severity (increase in intensive care unit location, in patients receiving mechanical ventilators and vasopressors before the cardiac arrest; p<0.001 for all comparisons). The median duration of hospital participation in gwtgresuscitation was 7 years, and 73 (31.6%) hospitals participated for 8 years . Most hospitals were either nonprofit (70.1%) or government owned (16.9%) with few forprofit hospitals (13%). Most hospitals (44.2%) were intermediate in size (200 to 499 beds), whereas 35.5% were small hospitals (<250 beds) and 20.3% were large hospitals (500 beds). A majority of hospitals were academic teaching hospitals with a residency (30.3%) or a residency and fellowship (23.8%) program . Characteristics of study hospitals (n=231) * at baseline (ie, during the first year of participation in gwtgresuscitation), the mean unadjusted hospital survival rate for inhospital cardiac arrest was 18.2% . Survival rates improved at 218 (94%) hospitals (ie, hospitals with estimate of slope> 0), with a mean 4% improvement in survival per year (or 1.04, 95% ci 1.03 to 1.05, p<0.001; figure 2a and table 3). Hospitals in the top quartile had a mean yearoveryear survival improvement of 7%, while hospitals in the second and third hospital quartile had a mean yearoveryear survival improvement of 5% and 3%, respectively . The mean yearoveryear change in survival for hospitals in the lowest hospital quartile was 1%, suggesting little to no improvement in survival over time . Unadjusted and adjusted relative rate of annual survival improvement, by hospital quartiles * hospitals were divided into quartiles using hospitalspecific odds ratios for annual survival improvement obtained from the multivariable hierarchical regression model . Values in the table are the mean and the range of the hospitalspecific odds ratio for annual survival improvement . After adjustment for patient and hospital characteristics, the mean relative improvement in inhospital cardiac arrest survival rates was 7% per year (adjusted or 1.07, 95% ci 1.06 to 1.08, p<0.001; table 3). Compared with a mean adjusted hospital survival rate of 18.1% during the 20002003 period, hospital survival rate increased to 21.4% in 20072010, which translated into a 3.3% absolute improvement in survival during this period (figure 3). Notably, there was marked variation in annual survival improvement across sites (figure 2b). Hospitals in the top quartile achieved a mean yearoveryear adjusted survival increase of 11%, whereas the hospitals in the bottom quartile experienced only a mean annual improvement of survival of 3% (table 3). Change in inhospital cardiac arrest survival rates from 20002003 to 20072010 . The mean adjusted hospital survival rate increased from 18.1% in 20002003 (range 9.1% to 29.8%) to 21.4% in 20072010 (range 13.9% to 32.2%). There was a significant interaction between academic status and rate of survival improvement across hospitals . Compared with minor teaching hospitals (or 1.04, 95% ci 1.02 to 1.06), hospital rate of survival improvement was greater at major teaching (or 1.08, 95% ci 1.06 to 1.10) and nonteaching hospitals (or 1.07, 95% ci 1.05 to 1.09, p value for interaction=0.03; table 4). However, other hospital characteristics, including bed size, geographic status, ownership status, and rural versus urban location, were not associated with a hospital's rate of survival improvement for inhospital cardiac arrest . Association between hospital structural characteristics and improvement in cardiac arrest survival in sensitivity analyses, there was a moderately strong positive correlation between hospital trends in favorable neurological survival and survival to discharge (pearson's correlation coefficient=0.62; p<0.0001), which confirms that hospitals with the highest survival gains were largely the same hospitals with the greatest improvements in favorable neurological survival . Moreover, after reclassifying 833 patients (4.5% of survivors) who were discharged to hospice as having died, we still found an average 6% peryear improvement in survival . There was excellent agreement when we compared hospital classification in quartiles of survival improvement by using this approach with our primary analysis (=0.80, 95% ci 0.74 to 0.87), which suggests that our findings were robust to hospital discharge patterns . At baseline (ie, during the first year of participation in gwtgresuscitation), the mean unadjusted hospital survival rate for inhospital cardiac arrest was 18.2% . Survival rates improved at 218 (94%) hospitals (ie, hospitals with estimate of slope> 0), with a mean 4% improvement in survival per year (or 1.04, 95% ci 1.03 to 1.05, p<0.001; figure 2a and table 3). Hospitals in the top quartile had a mean yearoveryear survival improvement of 7%, while hospitals in the second and third hospital quartile had a mean yearoveryear survival improvement of 5% and 3%, respectively . The mean yearoveryear change in survival for hospitals in the lowest hospital quartile was 1%, suggesting little to no improvement in survival over time . Unadjusted and adjusted relative rate of annual survival improvement, by hospital quartiles * hospitals were divided into quartiles using hospitalspecific odds ratios for annual survival improvement obtained from the multivariable hierarchical regression model . Values in the table are the mean and the range of the hospitalspecific odds ratio for annual survival improvement . After adjustment for patient and hospital characteristics, the mean relative improvement in inhospital cardiac arrest survival rates was 7% per year (adjusted or 1.07, 95% ci 1.06 to 1.08, p<0.001; table 3). Compared with a mean adjusted hospital survival rate of 18.1% during the 20002003 period, hospital survival rate increased to 21.4% in 20072010, which translated into a 3.3% absolute improvement in survival during this period (figure 3). Notably, there was marked variation in annual survival improvement across sites (figure 2b). Hospitals in the top quartile achieved a mean yearoveryear adjusted survival increase of 11%, whereas the hospitals in the bottom quartile experienced only a mean annual improvement of survival of 3% (table 3). The mean adjusted hospital survival rate increased from 18.1% in 20002003 (range 9.1% to 29.8%) to 21.4% in 20072010 (range 13.9% to 32.2%). There was a significant interaction between academic status and rate of survival improvement across hospitals . Compared with minor teaching hospitals (or 1.04, 95% ci 1.02 to 1.06), hospital rate of survival improvement was greater at major teaching (or 1.08, 95% ci 1.06 to 1.10) and nonteaching hospitals (or 1.07, 95% ci 1.05 to 1.09, p value for interaction=0.03; table 4). However, other hospital characteristics, including bed size, geographic status, ownership status, and rural versus urban location, were not associated with a hospital's rate of survival improvement for inhospital cardiac arrest . Association between hospital structural characteristics and improvement in cardiac arrest survival in sensitivity analyses, there was a moderately strong positive correlation between hospital trends in favorable neurological survival and survival to discharge (pearson's correlation coefficient=0.62; p<0.0001), which confirms that hospitals with the highest survival gains were largely the same hospitals with the greatest improvements in favorable neurological survival . Moreover, after reclassifying 833 patients (4.5% of survivors) who were discharged to hospice as having died, we still found an average 6% peryear improvement in survival . There was excellent agreement when we compared hospital classification in quartiles of survival improvement by using this approach with our primary analysis (=0.80, 95% ci 0.74 to 0.87), which suggests that our findings were robust to hospital discharge patterns . Among hospitals participating in a large national quality improvement registry, we found hospitallevel survival rates for inhospital cardiac arrest have significantly improved during the past decade . Importantly, there was marked variation in the extent of survival improvement at participating hospitals . We found that a quarter of hospitals achieved an average 11% relative improvement in survival per year, while another quarter of hospitals experienced little to no improvement . Many of the individual hospital structural characteristics were unrelated to the extent of variation in hospitallevel trends, which suggests that unmeasured hospital processes of care are likely driving the survival improvement at topperforming sites . Recently, we reported that inhospital cardiac arrest survival has improved during the past decade at the patient level . While it is possible that the improved survival trends that we observed are limited to hospitals committed to quality improvement due to participation in gwtgresuscitation, 2 subsequent studies have confirmed similar trends in other nationally representative databases . Here, we extend the findings of our previous work by showing that survival improvement varied markedly across sites even after accounting for differences in patient characteristics between sites . Given that treatment of inhospital cardiac arrest is time sensitive and requires a coordinated effort among a diverse group of providers, marked variation in survival improvement across sites likely reflects differences in how individual hospitals approach resuscitation care and organize quality improvement efforts . Patients and their appropriate triage to telemetry or intensive care units, timely recognition of cardiac arrest, prompt mobilization of a resuscitation team to the patient bedside, rapid evaluation of the patient and initiation of resuscitative efforts, conduct of an organized and coordinated acute resuscitation response, and highquality postresuscitation care . Hospitals that consider cardiac arrest to be a priority, examine their performance through ongoing data collection and feedback, identify areas of weakness, and invest resources and personnel to improve processes of care are likely to have achieved greater gains in cardiac arrest survival . In prior work in acute myocardial infarction, a number of institutional attributes organizational values and goals, senior leadership, staff engagement, communication, coordination, problem solving, and learning from previous mistakes were also found to distinguish topperforming hospitals from lowperforming hospitals . It is likely that similar organizational factors underlie hospitals' ability to improve systems of care for resuscitation . Among hospital structural characteristics, only teaching status was found to be significantly associated with extent of survival improvement . Survival improvement was comparable at major teaching hospitals (hospitals with residency and fellowship programs) and nonteaching hospitals but was lower at minor teaching hospitals (hospitals with only residency programs). This relationship may be due to the presence of lessexperienced trainees (eg, residents) who may be first responders during acute resuscitation and primary providers during postresuscitation care . The presence of advanced trainees (eg, fellows) at major teaching hospitals appears to mitigate this relationship, as survival improvement at major teaching hospitals was similar to that of nonteaching hospitals where experienced physicians provide patient care . Although this may be a plausible explanation, we did not have data on composition of code teams or inpatient staffing patterns to confirm whether this is accurate . The general lack of association between hospital structural characteristics and survival improvement is not entirely unexpected . Chan et al found wide variation (2% to 55%) in rates of delayed defibrillation for cardiac arrest due to ventricular fibrillation and pulseless ventricular tachycardia . In that study, hospital performance on defibrillation times was unrelated to measured structural characteristics (except bed size). Based on these findings, we posit that hospital processes of care, rather than structure, are more important in achieving improved inhospital cardiac arrest survival over time . In fact, singlecenter studies have demonstrated the value of innovative process redesign such as debriefing after a cardiac arrest event, use of simulation or routine mock codes, and implementation of efforts to improve quality of cpr, as well as devices capable of providing audiovisual feedback during resuscitation, in improving resuscitation performance and outcomes . It is possible that topperforming hospitals have implemented similar and other novel strategies to realize their cardiac arrest survival gains . Evaluating important resuscitationrelated processes of care as potential drivers of inhospital cardiac arrest survival improvement is a critical next step . Currently, neither gwtgresuscitation nor other existing hospital databases collect information on these innovative resuscitation processes of care and treatment strategies . Therefore, to better understand the organizational and process variables that underlie hospital resuscitation performance, detailed site surveys within large registries like gwtgresuscitation or studies using mixed methods (qualitative and quantitative) may be needed . In fact, bradley and colleagues used a similar approach to identify best practices associated with shorter doortoballoon times for primary percutaneous coronary intervention for stelevation myocardial infarction another timesensitive condition . This approach has been widely successful in significantly reducing doortoballoon times nationally and serves as powerful example to guide future quality improvement work for other timesensitive conditions such as inhospital cardiac arrest . First, it is possible that hospital cardiac arrest survival rates have improved because of a decrease in overall risk among patients who undergo resuscitation . Although mean patient age has decreased over time, there has been a temporal increase in the prevalence of comorbidities (sepsis, renal insufficiency, respiratory insufficiency, and malignancy), mechanical ventilation, and vasopressor use before arrest, suggesting that illness severity has increased over time . This finding was also noted in another study, which found an increase in the mean charlson comorbidity score among patients with an inhospital cardiac arrest (2.5 in 20002001 to 2.7 in 20082009; p<0.001). Second, although clinical practice guidelines support the use of therapeutic hypothermia for preventing neurological damage and improving survival following an outofhospital cardiac arrest, 2 recent studies from gwtgresuscitation found therapeutic hypothermia to be rarely used after inhospital cardiac arrest (<3%). Therefore, it is unlikely that the observed variation in survival improvement between hospitals is explained by differences in use of therapeutic hypothermia . Third, we assumed that improvement in survival occurred in a linear fashion in this study . Although we did not test this assumption, our prior work using data from gwtgresuscitation was most consistent with a linear temporal trend in survival . Important processes of care (eg, quality of cpr, innovative resuscitation strategies, quality improvement programs, etc) that may be associated with survival improvement were not available . Second, we had only a single year of american hospital association data to define hospitallevel characteristics, and it is possible that structural characteristics of hospitals changed over time . Third, although we adjusted for a number of patient and hospital factors in our multivariable models, the possibility of residual confounding remains . We addressed this by requiring that study hospitals be restricted to those that participated in gwtgresuscitation for at least 5 years . Finally, hospitals participating in gwtgresuscitation are more likely to be committed hospitals that are engaged in quality improvement . Moreover, we also excluded hospitals that were low volume (annual cardiac arrest volume <10) or participated infrequently (<5 years) in the registry . For both these reasons, our findings may not be generalizable to all us hospitals . During the past decade, survival after inhospital cardiac arrest survival has improved at nearly all hospitals participating in a large national quality improvement registry . However, marked differences in the extent of survival improvement were observed . Most structural hospital characteristics were not associated with the extent of hospital improvements in cardiac arrest survival . Future studies are needed to identify hospital process of care (best practices) that have achieved the largest improvement in inhospital cardiac arrest survival . The american heart association, which sponsors the get with the guidelinesresuscitation, had no role in the design and conduct of the study; the collection, management, analysis, and interpretation of the data; the preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication . In addition to study authors saket girotra and paul s. chan, the american heart association get with the guidelines resuscitation adult trask force include comilla sasson, md, ms and steven bradley, md, mph, university of colorado; michael w. donnino, md, beth israel deaconess medical center; dana p. edelson, md, ms, university of chicago; robert t. faillace, md, scm, geisinger healthcare system; romergryko geocadin, md, johns hopkins university school of medicine; raina merchant, md, mshp, university of pennsylvania school of medicine; vincent n. mosesso, jr ., md, university of pittsburgh school of medicine; joseph p. ornato, md and mary ann peberdy, md, virginia commonwealth university.
Members of high - risk families should be given recommendations which may improve prophylaxis, early diagnosis and treatment [1 - 4]. During the last 10 years now it is possible to identify several genes involved in the hereditary forms of some types of cancers including breast cancer . Hereditary forms of breast cancer are mostly caused by mutations in such genes as brca1 and brca2 and are thought to account for 5 - 10% of all cases of breast cancer . Because of high penetration of mutated genes as well as autosomal dominant trait, female family members are frequently affected with breast cancer, therefore taking the family history is a major, effective and cheap way of diagnosing hereditary forms of breast cancer . Primary tumour growth is different than sporadic ones, and it seems that early detection of hereditary forms may be more difficult . Affected patients with the hereditary form of breast cancer are also at increased risk of the development of the second primary breast cancer . The purpose of this work was to evaluate in lithuanian breast cancer patients such clinical factors as: frequency of diagnosis of primary breast cancer in clinical stage i - iv and frequency of the second primary breast cancer in groups with a hereditary form . In order to identify patients suspected for hereditary breast cancer specific pedigree criteria in 2001 in vilnius university institute of oncology there were 521 patients treated for breast cancer . Patients suspected for site specific hereditary breast cancer were identified according to the following pedigree criteria: mother and daughter affected by breast cancer; one of them developed breast cancer under the age of 50 years . Every case of hereditary breast cancer was analysed retrospectively by stage according to tnm classification and compared with the control group which was selected from sporadic cases diagnosed in the same cancer centre and matched for the same age of diagnosis . The relative risk was used as an estimate of the relative effectiveness for the recruitment cases by stage . Relative risk was calculated by stage as a ratio at each category to the total number of observation . The frequency of the development of the second primary tumour in the hereditary breast cancer group was calculated . In a group of 521 patients treated with breast cancer in vilnius university institute of oncology in 2001, there were 26 patients from 25 families with family history indicating diagnosis of site specific hereditary breast cancer . All these patients were affected with primary breast cancer under the age of 50 years, which is in concordance with the early age of onset observed in patients with positive breast cancer family history . We evaluated the frequency of breast cancers diagnosed in clinical stages i - iv in hereditary cancer patients . To avoid possible differences in diagnostic effectiveness between diagnostic centres in lithuania, a control group was selected from sporadic breast cancer cases diagnosed in the same cancer centre and matched for the same age of diagnosis . Comparison of breast cancer frequencies diagnosed in clinical stages i - iv is shown in table 1 . We observed much lower frequency of hereditary cases diagnosed in stage i than sporadic ones (11% vs. 23%, rr = 0.49). In contrast hereditary breast cancer cases were diagnosed more often than sporadic tumours, in advanced stages (stage ii in 61% vs. 44%, rr = 1.39; and stage iii in 27% vs. 17%, rr = 1.62, respectively). We have not observed any hereditary breast cancer patient diagnosed in clinical stage iv, which can be caused by the low number of studied cases . The low rate of hereditary cancers diagnosed in stage i and high rate of stage ii and iii may suggest that growth of these tumours may be accelerated and standard surveillance is less effective than in a group of patients without family history . Similarly to other observations, cancer family history is correlated with an increased risk of contralateral breast cancer . In our group of 26 patients, experience of many foreign hereditary cancer centres proved that hereditary cancer patients as well as their family members must be followed in an institution prepared for biological genetic testing and specialist surveillance . A better understanding of the process of coupling in healthy people with a genetic risk of cancer including genetic consultations is relevant here to make a decision about prevention or screening [7 - 11]. Breast cancer in a study group and in a population by cancer stage *) matched by age and territory with the study group, 2000 the results of this study show the need to create a lithuanian specialist centre for hereditary cancer diagnosis, treatment, and prophylaxis, which will coordinate and connect work between different specialists . This study was supported by a grant of ec project qlri - ct-1999 - 00063 " development of network of cancer family syndrome registries in eastern europe ".
Duchenne muscular dystrophy (dmd) is an inherited neuromuscular disorder which affects boys . Until recently the mean age of death was around 19 but there have been significant improvements in clinical management and they can now expect to live to around 25 years . To investigate, from their own perspectives, how the well - being of young men living with dmd, and that of their families, can be maximised, particularly at the transition to adulthood, and from children's to adult services . It was an example of inclusive research, involving young men and a national family support group, as well as clinical services . Postal survey of family carers in three regions followed by interviews with 40 young men age 15 + with dmd and their families about the issues they faced at transition to adulthood . Family carers generally considered that transition planning at school leaving was poor or non - existent and that they lacked information about services . Once they had left school or college, the majority of young men lacked meaningful day time activities and friendship networks . Much effort has gone into producing policies and processes, with an uncritical assumption that this will lead to better outcomes . The study (20072009) was funded by the english department of health's long - term neurological conditions research programme.
The p53 tumor suppressor protein plays an important role in preventing malignant development (vousden and prives, 2009), functioning primarily as a transcription factor to regulate the expression of a large number of genes that induce cellular responses such as cell - cycle arrest and apoptosis (beckerman and prives, 2010). While effective in preventing cancer development, these activities of p53 must also be tightly controlled to allow normal growth and development . Numerous mechanisms through which p53 is regulated have been described, including the control of translation, protein stability, subcellular localization, and interaction with other components of the transcriptional machinery (hollstein and hainaut, 2010). In many cancers, the function of p53 is ablated through point mutations that lead to the expression of a mutant p53 protein (joerger and fersht, 2007). These tumor - associated point mutations occur predominantly in the central dna binding domain of p53 and result in a diminished ability of p53 to bind consensus sites in the promoters of p53-regulated genes . While some of these mutations result in amino - acid substitutions of residues within p53 that directly contact the dna (contact mutants), other mutations result in the misfolding of the p53 protein . The p53 dna binding domain shows a low thermodynamic stability in vitro, and mutations in this region can lead to further instability, causing the protein to become denatured at 37c (joerger and fersht, 2007) and a potential to form p53 protein aggregates within the cell (xu et al ., the net effect of these tumor - associated point mutants is both the loss of wild - type p53 activity and a gain of function that contributes to the invasive behavior of cancers (muller et al ., 2011). The mechanisms underlying this gain of function are still under investigation but at least partially reflect the ability of the mutant p53 proteins to modulate the activity of other transcription factors such as p63, p73, and srebp (freed - pastor and prives, 2012). Molecular chaperones are a group of proteins that assist in protein folding (hartl et al ., 2011). They not only prevent misfolding and aggregation of proteins but can also target misfolded proteins for degradation . Probably the best - understood chaperones are the heat shock proteins hsp70 and hsp90, which play a role in conformational maturation and help to target improperly folded proteins for ubiquitination and proteolysis, and the ring complex chaperonins, which enclose proteins within their structure for folding of newly synthesized peptides (mayer, 2010). Chaperonins are double - ring oligomers, each ring enclosing a cavity where protein folding takes place through an energy - consuming process (douglas et al ., 2011; the cytosolic group ii chaperonin cct (also known as tric) consists of a double ring, each one containing eight subunits (cct in mammals and cct18 in yeast). Like other chaperonins, cct has two main conformations that are controlled by atp hydrolysis . The open conformation recognizes unfolded peptides, and atp binding and hydrolysis induce the closed conformation, which results in the folding of the protein (douglas et al . Although the mechanism of substrate - cct recognition and binding remains under investigation, each of the subunits can recognize different polar and hydrophobic motifs within substrate proteins (yam et al . Potentially up to 15% of all newly synthesized polypeptides can associate with the cct complex, although only a few proteins have so far been shown to depend on this chaperonin for folding and function (thulasiraman et al . Cct plays an important role in the folding of newly synthesized proteins (frydman et al ., 1994; yam et al ., 2008a) but can also prevent the aggregation of proteins with polyglutamine regions (kitamura et al ., 2006; tam et al ., 2006) and so potentially contributes to the suppression of misfolding diseases such as huntington, parkinson, and alzheimer . These activities are executed in conjunction with other chaperones or cochaperones (siegers et al ., 1999). Both wild - type and mutant p53 have been shown to be regulated by binding to hsp70 and hsp90 (blagosklonny et al . However, a role for the chaperonins in the control of p53 has not been investigated . We show here that p53 is a client of the cct complex and that failure to interact with this molecular chaperone can promote oncogenic functions of p53 in the absence of classic tumor - derived dna binding domain mutations . We carried out a proteomic analysis to identify proteins that interact with wild - type p53 and a tumor - derived point mutant, 175h . P53 itself, parc, and cullin-7 (andrews et al ., 2006; nikolaev et al ., 2003) were most frequently identified in immunoprecipitation with wild - type p53 (figure 1a), validating this approach . Interestingly, peptides from seven of the eight cct subunits were also found in complex with p53 (figure 1a), consistent with our recent study identifying cct subunits as part of a p53 interactome (coffill et al ., 2012). To further study the consequences of the interaction of p53 with cct subunits in cells, we utilized several cell lines that each expressed broadly equal endogenous levels of various cct subunits (figure s1a available online). Coprecipitation of endogenous wild - type p53 with endogenous cct and cct from hct116 cells (figure 1b) or u2os cells (where p53 was stabilized by treatment of cells with the mdm2 inhibitor nutlin 3; figure 1c) demonstrated the ability of these proteins to interact in cells . To compare the binding of wild - type and mutant p53 directly in the same cells, we turned to h1299, a p53 null tumor cell line transiently transfected with p53-expression constructs (figure 1d). These results indicated that wild - type p53, 175h, and 273h all efficiently interacted with endogenous cct . Similarly, wild - type p53 and 248w were coprecipitated with cct in hct116 cells (figure s1b). The specificity of the binding was confirmed by showing that the coprecipitation of p53 was dependent on cct expression (figure 1e). Cct is a large, multisubunit structure (960 kda), and we used gel filtration to determine whether p53 could associate with the whole cct complex (figure 2a). Endogenous wild - type p53 largely comigrated with cct and cct in high - molecular - weight complexes, consistent with an interaction with the whole cct complex (figure 2a). Immunoprecipitation of pooled fractions confirmed that the majority of the cct was present in fractions 813 and coprecipitated with p53 (figure 2a). Similar results were observed in lysates from h1299 cells transiently transfected with wild - type p53 (figure s2). Depletion of cct and cct resulted in the release of some p53 from the larger complexes, supporting the interaction of p53 with cct (figure 2b), although the retention of some p53 in larger complexes following cct depletion likely reflects p53 s interaction with other proteins (collavin et al ., quantification of p53 levels in each fraction indicated that approximately 12% of the total p53 was released from the larger complexes, suggesting this proportion of p53 is bound to cct under these conditions . Cooperation between various chaperone complexes in protein folding has been described, and p53 is known to associate with several other chaperones, including hsp70 (blagosklonny et al ., 1996; walerych et al ., 2009). Hsp70 contributes to the interaction between vhl and cct (melville et al ., 2003), and we also found that hsp70 slightly increased the binding of p53 to cct (figure 2c). To test directly whether p53 interacts with the cct complex, we mixed bacterially expressed wild - type p53 with purified bovine cct . Following separation by native electrophoresis, the high - molecular - weight band corresponding to cct was excised and subjected to sds electrophoresis, which revealed the presence of cct subunits and p53 (figure 3a), confirming the presence of a stable cct: p53 complex . First, over 1,000 end - on views of the isolated apo - cct particles were processed, and the average image revealed the typical presence of the doughnut - shaped structure with an empty cavity surrounded by eight similar masses (figure 3b). The averaging of almost 2,000 cct: p53 particles revealed a similar structure except for the presence of mass inside the cavity that interacted mainly with one cct subunit (figure 3c; figures s3a previous studies have shown that the atp - dependent conformational change leading to closure of the cct complex also results in its protection against proteinase k digestion (meyer et al ., 2003). We were also able to confirm a dose - dependent degradation of the cct complex following incubation with proteinase k, which was prevented by the addition of atp (figure 3d). Importantly, this protection from degradation also applied to the p53 protein bound to cct, as the closed conformation of the chaperonin was able to protect the trapped p53 from protease attack (figure 3e). Taken together, these data demonstrate that p53 interacts with the cct complex and can be trapped in the interior of the chaperonin cavity in an atp - hydrolysis - dependent manner . To determine which regions of p53 were important for the cct interaction deletions or point mutations within the dna binding domain or the c terminus of p53 did not affect binding to cct (figures 4a and 4b). Interestingly, several of the c - terminal truncations also removed the oligomerization domain and the nuclear import signals from p53, suggesting that neither of these is required for cct binding in cells . As shown previously, cct localized predominantly to the cytoplasm, although some nuclear staining was also detected (figures s4a s4c) (joly et al . Both endogenous and transfected wild - type p53 were found predominantly (although not exclusively) in the nuclear fractions (figures s4b and s4c). However, despite detecting both p53 and cct proteins in the nucleus, coimmunoprecipitation showed that the endogenous p53/cct interaction occurs in the cytosol (figure s4b). While alterations within the dna binding and c - terminal domains of p53 did not affect the cct interaction, deletion of the n - terminal 43 amino acids of p53 resulted in the loss of interaction with cct (figure 4b). The n terminus of p53 is responsible for many protein interactions, including binding to mdm2 and the components of the transcriptional machinery (residues 1729; kussie et al ., 1996). While mutation in the mdm2 binding and transactivation domains (deleted in 1318 and 1928, mutated in 22/23) slightly reduced but did not abolish cct binding, deletion within the n - terminal 13 amino acids of p53 (213 and 413) resulted in substantial loss of interaction with cct (figure 4c). Deletion of these residues also resulted in the loss of binding of p53 to cct (figure s4d). The failure of these mutants to interact with cct proteins was not a result of lack of cytoplasmic localization, since 213 was clearly detected in both nuclear and cytoplasmic fractions (figure s4c). Importantly, this interaction was also seen in a reciprocal immunoprecipitation, where wild - type but not 413 p53 pulled down endogenous cct (figure 4d). The loss of cct binding to 213 and 413 p53 was also confirmed using in vitro translation in reticulocyte lysates, which contain endogenous cct (figure 4e). The n - terminal region of p53 shows moderate conservation between different species and contains polar and hydrophobic amino acids (figure 4f), which may mediate the interaction with cct (gmez - puertas et al ., 2004). To assess the contribution of hydrophobic bonds to the p53-cct interaction, we examined the effect of chaotropic salts containing cations that can weaken hydrophobic bonds (feldman et al . The disruption of the p53-cct interaction following incubation with licl and mgso4 suggests a possible contribution of hydrophobic interactions . Taken together, these results show that the first 13 amino acids of p53 are necessary for the p53-cct interaction . To further assess whether cct impacts mdm2 binding by p53, we examined the effect of cct depletion on the endogenous p53/mdm2 interaction (figure s4f). Importantly, the binding of mdm2 to p53 was not affected by depletion of cct and cct, an effect that is most clearly seen following stabilization of the p53 protein using the proteasome inhibitor mg132 (figure s4f). Furthermore, the cct nonbinding n - terminal deletion of p53 (413) retained the ability to bind mdm2, an activity that was lost in the more extensive n - terminal truncation 39 (figure s4 we also examined the impact of loss of cct binding on the interaction between p53 and hsp70 (figure 4 g). Again, deletion of the extreme n terminus of p53 (413) that abolished the cct interaction did not prevent binding to hsp70, although a more extensive deletion (39) reduced hsp70 binding . Taken together, these results show that cct binding is not required for p53 to bind mdm2 or hsp70 . The interaction of p53 with the cct complex suggests that this chaperonin may play a role in promoting the correct folding, and thus activity, of p53 . The conformation of p53 can be assessed using two antibodies: ab1620, which recognizes a domain only present in the correctly folded protein and therefore indicates functional wild - type p53 (milner et al ., 1987), and ab240, which recognizes a cryptic epitope (amino acids 211220) only exposed in the unfolded or denatured conformation (gannon et al ., 1990). The relative amounts of p53 recognized by ab1620 and ab240 provide an indication of the extent of p53 folding . To determine whether cct contributes to p53 folding, we used small interfering rna (sirna) to deplete u2os or mcf7 cells (that express endogenous wild - type p53) of cct and cct (figure 5a). Immunoprecipitation of p53 with the conformation - specific antibodies showed a clear decrease in ab1620-reactive p53 and an accumulation of ab240-reactive p53 in cells depleted of cct complex components (figure 5a). A similar response was seen in a2780 cells (figure s5a) and in cells where p53 protein was induced in response to doxycycline treatment (figure 5b). In these cells, both wild - type p53 and the 273h mutant, which retains predominantly wild - type conformation, became misfolded (and ab240 reactive) following cct depletion (figure 5b). During the course of these studies, we noted that depletion of both cct subunits and frequently led to an increase in p53 protein levels in cells expressing endogenous wild - type p53 cells (figure 5a; figure s5b), although this was not evident in cells expressing transfected mutant p53 (figure 1e). The increase in p53 protein levels following cct knockdown was accompanied by a slight increase in p53 half - life in both hct116 and u2os cells (figure s5b), and depletion of cct in otherwise unstressed cells resulted in a reduction in extent of ubiquitination of endogenous p53 (figure s5c). Previous studies have shown that cct depletion can result in cell - cycle arrest (grantham et al ., 2006; liu et al ., 2005), indicating that the incorrect folding of cct client proteins is likely to promote a stress response that could indirectly signal to activate p53 . Following transient expression of p53, both wild - type and transactivation domain mutant 22/23 retained predominantly wild - type (ab1620) conformation, while depletion of cct resulted in an increase in misfolded p53 detected by ab240 (figure 5c). By contrast, p5343, lacking the cct binding and transactivation domain, adopted a more unfolded conformation that was not further affected by cct depletion (figure 5c). To more closely define the effect of cct binding, we examined the smaller n - terminal deletions of p53 . Using standard extraction procedure (lysing cells and immunoprecipitating at 4c), we found variable results with the cct nonbinding p53 mutants (213 and 413), which showed a slightly higher ratio of ab240 reactivity in some assays but mainly ab1620 reactivity similar to wild - type p53 in others (for example, compare control lanes in figures 5d and 5e). Previous studies have shown that p53 has a low thermodynamic stability in vitro, which is further reduced by mutations within the dna binding domain (bullock et al . We therefore considered whether the p53 mutants that are unable to bind to cct might show a greater sensitivity to temperature - induced denaturation . To test this, we examined the conformation of p53 in cell extracts following incubation at 37c (figure 5d). As previously described (bullock et al ., 2000), the 175h mutant was highly unstable compared to the wild - type p53 protein, which became predominantly ab240 reactive only after 8 min at 37c . Interestingly, both p53213 and p53413 were clearly less stable than the wild - type protein in this assay (figure 5d). To extend these studies, we incubated p53-transfected cells at 39c for 2 or 5 hr immediately before lysis and carried out conformation - specific immunoprecipitation (figure 5e). These studies again showed that p53213 was less stable than wild - type p53, shifting significantly to the unfolded, ab240-reactive conformation after only 2 hr heat shock, at which time point the wild - type protein remained predominantly ab1620 reactive . To look directly at the conformation of p53 in cells, we used an immunofluorescence assay in which we compared the signal from total p53 (as detected using a p53 polyclonal antibody) with the signal using ab240 (figure 5f). To validate the assay, we showed that heat shock induced the expected increase in ab240 reactivity in cells expressing wild - type p53, consistent with the results shown in figure 5e . Turning to p53 mutants, both wild - type and transactivation domain mutant 22/23 showed low ab240 reactivity in cells incubated at 37c, while the 175h mutant reacted strongly with this antibody (figure 5 g), as expected . In this assay, deletion within the n - terminal 13 amino acids (213 and 213, 22/23) resulted in a significant acquisition of ab240 reactivity . Taken together, these results show that deletion of the cct binding domain in the n terminus of p53 results in a protein that is structurally less stable than wild - type and more sensitive to unfolding under temperature stress . Wild - type p53 showed a shift to the misfolded ab240 reactive conformation following treatment of cells with an hsp70 inhibitor alone or in combination with heat shock (figure s5d). A similar shift in the conformation of wild - type p53 following treatment with the hsp70 inhibitor was seen in cells using the immunofluorescence assay (figure s5e). These results indicate that both cct complex and hsp70 can promote the folding of p53 and that impeding either chaperone impacts the conformation of p53 . P53 has two transactivation domains: ad1, which is located to residues 1428, and ad2, which is located to residues 3861 (candau et al ., 1997; the transcriptional activity of the n - terminal p53 mutants described here was therefore assessed using a p53-responsive promoter (pg13 luciferase; figure 6a). As expected, deletion of residues within ad1 (p5339 and 1928) significantly impaired the transcriptional activity of p53, which was largely retained in a mutant deleted of residues outside this region (p532840 and p534149; figure 6a). However, the cct binding mutants p53213 and p53413 (which would not be predicted to impinge on ad1) showed reduced activity, consistent with the misfolding of these proteins . As seen in earlier assays, the levels of transfected p53 protein were not increased following cct depletion (figure 6b), indicating that ectopic overexpressed protein is less sensitive to the endogenous p53 degradation machinery . However, the activity of wild - type p53 and transcriptionally active mutants that retained the n - terminal cct binding domain (p532840, p534149) was reduced following knockdown of cct and cct subunits (figure 6b), consistent with a loss of wild - type conformation . By contrast, the lower level of transcriptional activity retained by p53213 and p53413 was not further compromised by inhibition of cct complex (figure 6b), supporting our observation that these mutants no longer interact with, and are therefore not subject to, regulation by cct . The modulation of p53 s transcriptional activity by cct was also apparent when examining the activation of endogenous p53-dependent target genes . P53-induced expression of both puma and p21 was clearly impeded by the depletion of cct, both at the protein and messenger rna (mrna) level (figures 6c and 6d; figures s6a and s6b). Interestingly, however, mdm2 expression was not affected by cct depletion, suggesting that the ability of p53 to induce the expression of mdm2 is less dependent on p53 conformation . Expression of p53413 also showed a decreased ability to activate expression of p21 and puma (figure 6d). As seen with the p53 reporter assays, depletion of cct reduced the transcriptional activity of wild - type p53 but did not impact the lower activity of the 413 mutant (figure 6d). Interestingly, depletion of the cct complex did not result in a profound decrease of endogenous p53 activity, either under basal conditions or following activation of p53 by treatment of the cells with nutlin 3 or low levels of actinomycin d (figures s6c and s6d). Although depletion of cct resulted in an increase in endogenous p53 stability (figure s5), no corresponding increase in transcriptional activity was detected either . Taken together, these results suggested that the response of endogenous p53 to cct knockdown is complex and that the outcome is a balance between increased protein levels (due to reduced degradation of p53 as part of a general stress response) and decreased conformational integrity (due to defects in the protein folding process). Tumor - derived p53 point mutations such as 175h and 273h acquire an ability to promote matrigel invasion, invasion into organotypic assays, and random migration (adorno et al ., 2009; expression of wild - type p53 in these cells inhibits proliferation and enhances cell death and cannot be used as a control for invasion . We therefore used the transcriptionally defective p53 22/23 mutant (which shows impaired growth inhibitory activity), which did not result in enhanced invasion above that seen with the empty vector control (figure 7a). Interestingly transient expression of non - cct binding mutants p53213 22/23, p53413 22/23, or p5339 promoted invasion, albeit to a lesser extent than the tumor - derived 175h or 273h mutant (figure 7a; figure s7a). Notably, loss of the n terminus in 175h- or 273h - expressing cells did not impact invasive capacity (muller et al ., 2009). Further investigation of the ability of the non - cct binding mutants of p53 to promote activities associated with tumor - derived mutants, such as random motility or invasion into organotypic assays, required cell lines that stably express these p53 proteins . Despite attempts to generate h1299 cell lines stably expressing p53 22/23, p53213 22/23, p53413 22/23, and p5339, only p5339-containing cells retained expression over time, although this was lower than 273h (figure 7b; figure s7b). These results are likely to reflect the retention of some residual growth inhibitory activity in the p53 22/23 mutant . Compared to empty vector transfected h1299 cells (ctrl), p5339 cells induced random motility and invasion into matrigel and organotypic assays to a similar or slightly lesser extent than 273h (figures 7b7d). Furthermore, despite the loss of expression of transiently transfected p53 22/23 and p53413 22/23 during the course of the experiment, both p53413 22/23 and 175h 22/23 cells showed some invasion into organotypic plugs (figure s7b). Finally, we examined whether depletion of the cct complex would have an effect on cell invasion using rpe cells (expressing wild - type p53) and variants stably expressing 273h or 175h, mutants that promoted the invasive ability of these cells (figure 7e; figure s7c). Depletion of cct complex by sirna in either 175h- or 273h - expressing cells resulted in decreased invasion (figure 7e; figure s7c) that is likely to reflect the misfolding of other cct client proteins like actin and tubulin, as previously described (matus et al .,, we were unable to achieve a complete depletion of cct complex proteins, indicating that only cells with partial knockdown continued proliferation, consistent with our observation that cct complex depletion resulted in a stress response that activated p53 (figure 6). Interestingly, cells that survived with a partial depletion of cct showed a reproducible increase in invasion (figure 7e), consistent with a role of cct in maintaining wild - type conformation and function of p53 . Depletion of wild - type p53 in these cells resulted in enhanced invasion (figure s7d), so it was not possible to demonstrate p53 dependence of the increased invasion in cct - depleted wild - type p53-expressing cells . Importantly, however, depletion of both p53 and cct decreased invasion (figure s7d), similar to the effect seen in mutant p53-expressing cells . These results show that despite a general inhibition of invasive behavior in response to cct depletion, wild - type p53-expressing cells show enhanced invasion that correlates with the misfolding of the p53 protein . Correct protein folding is essential for function, and this process is assisted by molecular chaperones . The chaperonin cct assists in specialized folding of selected proteins that are not fully folded by the nonspecialized chaperones alone . In this study, we demonstrate that p53 is a substrate for cct and provide evidence that this chaperonin regulates p53 folding and activity . Many previous studies have shown that tumor - derived p53 point mutants result in the failure of p53 to adopt the wild - type conformation, leading to the concept that p53 can either be in the wild - type or mutant (i.e., misfolded) conformation . Misfolding of p53 due to cancer - associated dna binding domain mutations, which enhances the thermoinstability of p53 (bullock et al ., 1997), results in the loss of wild - type dna binding activity and the acquisition of transforming functions, including the ability to promote invasion . Our studies suggest that cct plays a specific role in maintaining a pool of wild - type, functional p53 in the cell and that failure of p53 to bind to cct results in the accumulation of misfolded p53, which then acquires activities characteristic of tumor - derived mutant p53s . While the most straightforward model for the role of cct is to assist in the correct folding of newly synthesized p53 (mayer, 2010), it is also possible that cct binding to p53 prevents its aggregation, as has been shown for other proteins . Cct would function to allow the correct folding of p53 that occurs either spontaneously or through a cooperation with other chaperones, for example with hsp70 . In either case, the consequence of loss or modulation of the cct - p53 interaction would be the accumulation of misfolded p53 with invasive capacity . Based on these results, we suggest that the regulation of wild - type p53 folding may be part of the normal biology of p53 . Regulation of the interaction of p53 with cct could determine the generation of folded or unfolded p53, without the requirement for mutations in the dna binding domain . Our study shows that the effect of loss of cct binding on p53 conformation is subtle, supporting in vitro studies in which wild - type p53 can spontaneously fold into an active conformation . Nevertheless, the role of cct in protein folding becomes more apparent under stress, as shown in the heat shock experiments described here . It seems possible that the role of cct will also be evident under other physiological stress conditions, such as hypoxia, reactive oxygen species, and loss of cell adhesion . Several recent studies have shown that cct activity can be controlled by phosphorylation by rsk and s6k (abe et al ., 2009) or deacetylation by sirt1 (liu et al ., 2010). In addition, phosphorylation of serine 6 and 9 in p53 has also been identified (meek and anderson, 2009), so further studies will determine whether the p53/cct interaction can be actively modulated through changes in posttranslational modifications of either protein . Active control of cct binding in this way could allow for the expression of wild - type p53 in either folded or unfolded conformations resulting in distinct activities . According to this model, dna binding domain point mutations that are selected in cancers would force p53 to constitutively adopt the unfolded conformation . The activity of mutant p53 would therefore be the inappropriate manifestation of a normal state of wild - type p53 rather than the acquisition of a completely new activity . The accumulation of unfolded p53 may lead to the formation of aggregates, which would not be reversible (ano bom et al ., however, in vitro studies have shown that there is an initial reversible step in the denaturation of p53 (wilcken et al ., 2012), and in cells, temperature - sensitive p53 mutants can shift from mutant to wild - type forms (friedlander et al . Understanding when wild - type p53 adopts the denatured or misfolded conformation, how this is regulated, and the functional consequences of this conformational switching in vivo will be an interesting future challenge . H1299, hct116, u2os, mcf-7, and rpe cells were cultured as described in the supplemental experimental procedures . Generation of stable cells lines expressing mutant p53 175h and 273h was performed as described elsewhere (muller et al ., 2009). All p53 mutant constructs have been described previously or created by site - directed mutagenesis as detailed in the supplemental information . Details of immunoprecipitation and in vitro binding assays are described in the supplemental information . Samples were applied onto carbon - coated copper grids previously glow - discharged and stained with 2% uranyl acetate . Micrographs were taken and individual particles and image classification were performed as described in the supplemental information . Pg13 luciferase and renilla luciferase constructs were transfected in combination with different p53 constructs and analyzed as described in the supplemental information . Matrigel assays were performed as described previously (muller et al ., 2009). Invasion toward a gradient of 10% fetal calf serum and a mixture of growth factors was measure by confocal microscopy in serial sections of 10 m each, and quantification of invading cells was performed by imagej software . Organotypic invasion assays and migration assays (wound scratch) are detailed in the supplemental information.
Acquired immunodeficiency syndrome (aids) caused by human immunodeficiency virus (hiv) is known to lead to profound immunosuppression . In the majority of the hiv - positive patients, the cause of morbidity and mortality is not the virus itself but the opportunistic infections that result from an immunocompromised state.1 according to the centre for disease control and prevention (cdc), more than 20% of the aids - defining diseases reported, are fungal diseases affecting any anatomic site.2 a common opportunistic invasive mycosis encountered is invasive aspergillosis . This clinical entity is generally seen in advanced stages of aids and most commonly involves the lung.3, 4 although infection by aspergillus species was initially included by cdc in the list of aids - defining illnesses, it was deleted in the subsequent revisions, the primary reason for this was that aspergillosis is a relatively uncommon and unusual entity in aids patients.5 this can be explained by the fact that while hiv infection is characterized by depletion of cd4 population of t - cells, it is the depletion of neutrophils and macrophages that play a central role in the pathogenesis of aspergillosis.6 the disease is, therefore, more commonly seen in disease states associated with neutropenia or a defective cell - mediated immune response such as patients on cytotoxic chemotherapeutic agents, immunosuppressants, high - dose / prolonged steroids, broad - spectrum antimicrobials, and in patients with hematological malignancies.7,8 however, reports have suggested a resurgence and a possible rising incidence of pulmonary aspergillosis in patients with hiv / aids.3,9,10 hiv - positive patients with pulmonary aspergillosis, particularly the invasive form, generally have very low cd4 counts and often other risk factors are concomitantly present.11 the relative lack of any pathognomic sign or symptom, the frequent absence of roentgenographic features in immunocompromised patients as well as the non - specific nature of computerized tomography findings often preclude an early diagnosis of pulmonary aspergillosis . Moreover, the prognosis of hiv - associated pulmonary aspergillosis, particularly the invasive form is generally poor, with a median survival of 3 months following diagnosis.12 the poor prognosis of pulmonary aspergillosis in aids also requires that to improve the survival of patients, diagnosis is made both as rapidly and at a stage as early as possible so that appropriate and effective antifungal regimen can be instituted promptly . The diagnostic yield of conventional diagnostic modalities including culture and histopathology is generally low.13 this calls for non - culture based techniques for diagnosis such as detection of aspergillus galactomannan antigen and molecular methods such as polymerase chain reaction . However, molecular assays are not available routinely in most laboratories and are therefore not considered standard for diagnosis.14 in contrast, assays for detection of galactomannan are widely available commercially.15 information on hiv / aids associated pulmonary aspergillosis is generally scarce especially from an indian perspective, and this paucity of data prompted us to undertake the present evaluation . This study was undertaken as an attempt to understand the role of pulmonary aspergillosis as opportunistic mycoses in hiv / aids patients with pneumonic involvement, and to explore the diagnostic utility of serum galactomannan assay as an adjunct to microscopy and culture for the diagnosis of invasive pulmonary aspergillosis . The present prospective, cross - sectional study was conducted at a tertiary care health facility in north india . The study population comprised 71 hiv - positive subjects presenting to the antiretroviral center or admitted in the ward with signs and symptoms suggestive of a lower respiratory tract infection . The participants of this cohort were recruited during a 3-year period from october 2008 to september 2011 . At the time of enrollment, a detailed history and clinical examination were performed in all patients, the details of which were noted in a pre - designed proforma . A special attempt was made to explore the risk behavior of subjects and to elucidate the possible modes of hiv transmission . After a complete physical examination, routine laboratory investigations, cd4 counts, and chest radiographs were obtained . Written informed consent was obtained from each patient before enrollment, and the study had the approval and ethical clearance from the institutional review board . Sputum samples were collected in sterile wide - mouthed screw capped containers and immediately transported to the laboratory for processing . An aliquot of each sputum sample was transferred to an eppendorf containing 10% potassium hydroxide (koh) and incubated overnight at 37c . Subsequently, a wet mount was prepared and examined under 40 objective of the microscope for any fungal elements . In addition, the specimens were inoculated on sabouraud dextrose agar . For each sputum sample, the cultures were examined every second day for up to 6 weeks before they were reported as sterile . Any suspected fungal growth during this period was examined for gross colony morphology including color, texture, topography and the underside or reverse . Further confirmation of the isolates was done by slide culture technique, preparing lactophenol cotton blue mount and examining with the aid of 10 and 40 objective of the microscope . Identification of aspergillus species was done based on their characteristic morphological features and employing tests as per standard protocol.16 any positive result on microscopy and/or culture was confirmed by subsequent sampling and repeat demonstration / isolation . Apart from the sputum sample, 2 - 5 ml blood sample was collected from each case in a sterile centrifugation tube by venepuncture following universal precautions . Following clot retraction, serum was separated by centrifugation and stored in a labeled eppendorf . Galactomannan testing in serum samples was done employing the commercially available immunoenzymatic sandwich microplate assay, the platelia aspergillus eia (biorad laboratories). With each run of samples appropriate negative control, cutoff control and positive control sera after the validity of the test run was verified, the presence or absence of galactomannan antigen in the test sample was determined by calculating an index for each patient specimen, which was defined as a ratio of the optical density (od) value of the specimen to the mean od of the wells containing cutoff control serum . Data entry was performed using microsoft excel sheet and all the entries double - checked for any possible keyboard errors . Data were analyzed using the epi info software, version 3.5.3, cdc, atlanta, ga, usa . Descriptive statistics were calculated with arithmetic mean and standard deviation for central tendencies and median for non - normal / skewed distributions . The age of the study population ranged from 17 to 61 years with a mean age of 33.01 9.15 years . The study group comprised 52 (73.2%) males, 16 (22.6%) females, and 3 (4.2%) transgenders . 57 (80.3%) of the 71 participants were married while 14 (19.7%) were unmarried . The commonly reported modes of hiv transmission were sexual in 61 (85.9%), intravenous drug abuse in 11 (15.5%), and blood transfusion in 4 (5.6%) of the total 71 study subjects . The chief presenting complaints were fever (49 of 71; 69%), cough (43 of 71; 60.6%), and dyspnea (27 of 71; 38%). While 50 (70.4%) of the study participants were ambulatory and recruited from the antiretroviral center, 21 (29.6%) 42 (59.2%) participants had active pulmonary tuberculosis and 14 (19.7%) had a history of prior pneumocystis pneumonia or had present active disease . The main roentgenographic features noted were a normal chest radiograph in 37 (52.1%), pleural effusion in 22 (31%), infiltrates in 6 (8.5%), opacity in 3 (4.2%), consolidation in 1 (1.4%), hyperinflation in 1 (1.4%), and pneumothorax in 1 (1.4%). The mean and median cd4 count of the study population was 194.70 116.79 and 178 cells/l, respectively, with a range of 16 - 445 cells/l . A summary of the sociodemographic and cd4 profile of the study group is given in table 1 . Sociodemographic and cd4 profile of the study group (n=71) aspergillus species were recovered from 12 (16.9%) sputum samples . Direct microscopy for fungal hyphae was positive in 7 (9.9%) samples (all positive for aspergillus species on culture). The platelia aspergillus eia test for serum galactomannan antigen was positive in 36 (50.7%) patients . A summary of the modalities employed and their diagnostic yield is given in table 2 . Diagnostic yield of various methods employing a positive microscopy (dichotomously branched septate hyphae seen in koh wet mount) and culture as a diagnostic criterion, pulmonary aspergillosis was diagnosed in 7 (9.9%) patients, 5 of whom showed a positive antigenemia and were thus considered to be suffering from invasive forms of pulmonary aspergillosis . The prevalence of pulmonary aspergillosis was highest in individuals 21 - 40 years of age (13.3%), and none of the cases occurred in individuals above 41 years of age . The mean age of the patients with pulmonary aspergillosis was 29.57 6.02 years . Among the patients with pulmonary aspergillosis (7); the prevalence of pulmonary aspergillosis was more in females (18.7%; 3 of 16) as compared to males (7.7%; 4 of 52). 2 (28.6%) of the 7 patients with pulmonary aspergillosis had active pulmonary kochs; 2 (28.6%) had concomitant pneumocystis pneumonia and one (14.3%) had dual infection (both tuberculosis and pneumocystis pneumonia). While 3 (42.8%) of these patients had a normal chest radiograph on presentation, 2 (28.6%) presented with infiltrates and 2 (28.6%) with pleural effusion . 2 (28.6%) of the seven patients with pulmonary aspergillosis had a cd4 count less than 100 cells/l . The mean cd4 count of the patients with pulmonary aspergillosis was 155.86 119.33 cells/l (median = 117 cells/l; range = 18 - 329 cells/l). A comparison of the cd4 counts of patients with and without pulmonary aspergillosis is given in table 3 . A comparison of cd4 profile of hiv positive patients with and without pulmonary aspergillosis the common aspergillus species isolated from the sputum samples of these patients were aspergillus flavus (4; 57.1%), aspergillus fumigatus (2; 28.6%), and aspergillus niger (1; 14.3%). A line listing of the patients with pulmonary aspergillosis along with their demographic, clinical and microbiological profile is summarized in table 4 . Alhough aspergillus species were initially considered uncommon pathogens in hiv disease, several series of patients with advanced hiv disease and aspergillus respiratory infections, particularly the invasive forms, have been reported.3,9,10,17 employing a positive direct microscopy, culture and/or serum galactomannan antigen as a diagnostic criterion, 7 cases of pulmonary aspergillosis, including 5 cases of invasive disease were identified in this study cohort of hiv - positive patients . In another study conducted by the present authors to describe the clinicomycological profile of hiv - positive patients with suspected fungal infections, 4 of 60 patients were diagnosed as probable cases of invasive aspergillosis (positive antigenemia), while 1 patient, was diagnosed as a proven case of invasive pulmonary aspergillosis (positive direct microscopy, culture, and antigen detection).18 on the other hand in a study population comprising 160 confirmed hiv - positive patients with a lower respiratory tract infection, khan et al . Diagnosed pulmonary aspergillosis in only 4.19 the varying prevalences in different studies can be explained by the different inclusion criteria employed, differences in the study population, the diagnostic modalities employed, and the defining criteria used . While the prevalence of pulmonary aspergillosis was highest in individuals 21 - 40 years of age, it dropped dramatically among patients above 40 years . A plausible hypothesis is the influence of age and hormonal factors in aspergillosis.20 moreover, the high - risk behavior of patients in this age category that predisposes them to hiv / aids is an important influencing factor . Evidence from previous studies suggests that fungal pathogens such as aspergillus species be considered a possible differential diagnosis in hiv - positive patients with cd4 counts below 100 cells/l.21 while some studies have attributed the increased prevalence of pulmonary aspergillosis in this group to neutrophil depletion seen in advanced stages of hiv infection, we did not detect neutropenia in any of our patients with a positive microbiological diagnosis of pulmonary aspergillosis.8 the increased prevalence of aspergillosis in these patients can be explained by the various qualitative or functional abnormalities seen in neutrophils and macrophage lineages in hiv - positive patients with very low cd4 counts.22 impaired neutrophil oxidative burst mechanisms in these patients have been previously reported.22 a study conducted among hiv - infected children found that in study participants with low cd4 counts the antifungal activity of neutrophils against aspergillus hyphae was impaired in vitro.23 five of the 7 patients with pulmonary aspergillosis in our study had a positive history of tuberculosis and/or pneumocystis carinii pneumonia . An association between hiv - associated aspergillosis and a history of other aids - defining opportunistic infections has also been noted in several previous reports.24 while p. carinii pneumonia and its complications damage the lung architecture and provide a favorable niche for the colonization and growth of aspergillus species, the high dose steroid therapy for its treatment may further deteriorate the immune status of the patient and make them more susceptible to this fungal infection.24 - 26 similarly, tuberculosis of the lung also often leaves behind a scarred pulmonary parenchyma vulnerable to fungal colonization . Thus, the reasons for development of pulmonary aspergillosis in hiv - positive patients are several and include advent of highly active antiretroviral therapy that has prolonged the survival of patients in advanced stages of aids, i.e., with very low cd4 counts, qualitative neutrophil and macrophage dysfunction, prior or co - existing opportunistic lung infections such as tuberculosis and p. carinii pneumonia and increased use of corticosteroids and broad - spectrum antibiotics for the prophylaxis and treatment of opportunistic infections.3,9,10,22 - 28 since the sample size of the study group as well as the number of cases that were eventually diagnosed with pulmonary aspergillosis was small, meaningful statistical correlation with any clinical sign and symptom, risk factors and cd4 profile could not be established . With regard to the diagnosis of pulmonary aspergillosis, we found that the yield was highest with platelia aspergillus eia test for serum galactomannan antigen . The test was positive in 26 patients with respiratory disease but without any culture and/or direct microscopic evidence of aspergillus species in sputum samples . While some of these cases could correspond to real circulation of aspergillus antigens implying that the test picked up more positive samples than the conventional methods, the possibility of false - positive results can also not be ruled out . A false positive rate of up to 14% has been reported in a study.29 the reasons for false positive results are many and include consumption of milk preparations containing high concentrations of galactomannan, patients receiving piperacillin / tazobactam or some batches of amoxicillin / clavulanic acid parenteral preparations, patients on dialysis, those with chronic graft - versus - host disease and cross - reactivity against penicillium species, alternaria, paecilomyces, fusarium.13,30 - 35 the specificity of antigen detection can be increased by serial sampling . False positivity rates were shown to decline from 54.2% to 11.2% if positivity for at least two samples was taken as a defining criterion.36 however, since all the positive results in our study were confirmed by subsequent repeat sampling and testing, false positive results seem to be less likely and it is probably the low sensitivity of the traditional methods (microscopy and culture), that a number of positive cases were missed . Our study highlights that aspergillus species account for a significant proportion in the etiology of lower respiratory tract infection in hiv - positive patients . The relative lack of any pathognomic sign and symptoms and the need for early diagnosis keeping in view the poor prognosis associated with this condition calls for a combination of modalities (microbiological, histopathological, and serological) to make a prompt diagnosis and institute appropriate and effective antifungal therapy . Mere isolation of aspergillus species from respiratory specimens does not differentiate colonization from infection, making it imperative to document tissue invasion to attribute any clinical significance to the isolate . However, the serious underlying condition of the patient often does not permit invasive diagnostic interventions such as biopsy for this purpose . These shortcomings are overcome by the platelia aspergillus eia assay, which in addition to its high diagnostic yield offers several other advantages . Serum galactomannan can be detected at an early stage of infection, usually, several days before the clinical signs and chest radiographic abnormalities become evident and before a positive culture can be obtained and thus allows for an earlier diagnosis.37,38 moreover, the test is specific, non - invasive and, can be repeated at frequent intervals and without removing patients from protective isolation . First, keeping in view the serious underlying condition of the patients and for ethical reasons, confirmation of the results by tissue diagnosis could not be accomplished, therefore, false results could not be identified, and the true diagnostic performance of the platelia aspergillus eia could not be estimated . The second limitation was the relatively small sample size that precluded any meaningful statistical analysis . Our study highlights that pulmonary aspergillosis should be considered a possible differential of pulmonary infection in hiv - positive patients with low cd4 counts . The dismal prognosis associated with the disease the diagnostic dilemma encountered with the currently used modalities prompts their use in combination and also necessitates the need to include screening for serum galactomannan antigen as a possible diagnostic tool.
Struma testis is a rare entity, and there are only few reports on the malignant transformation of a testicular teratoma to papillary thyroid carcinoma in the literature . In this report, we describe the malignant transformation of struma testis with distant lung metastasis associated with trisomy 17 and a coexisting papillary microcarcinoma in the thyroid . A 56-year - old man presented after a left orchiectomy for an undescended left testicle . Pathologic examination identified a monodermal teratoma composed of thyroid parenchyma and associated with a 1.7-cm papillary thyroid carcinoma . Total thyroidectomy revealed a 0.5-mm focus of papillary thyroid cancer, and removal of the lung mass confirmed metastatic papillary thyroid cancer . Array - comparative genomic hybridization of both tumors showed trisomy 17 in the struma testes and the lung metastasis . The patient responded well to radioactive iodine ablation and has no evidence of cancer 3 years later . To our knowledge, this is the first case of papillary thyroid cancer in struma testes metastatic to the lung . Surgery to remove cancer foci, followed by radioactive iodine ablation, resulted in an excellent response in our patient . Interestingly, trisomy 17, which has so far been observed only in noninvasive thyroid nodules, was associated with pulmonary metastasis in our patient . Papillary thyroid carcinoma is the most common type of differentiated thyroid carcinoma, which is usually confined to the thyroid and tends to metastasize to regional lymph nodes . Cases identifying thyroid tissue in an ovarian teratoma and struma ovarii are well described in the literature . It is estimated that thyroid tissue presents in 15% of ovarian teratomas and that it is the dominant tissue, classified as struma ovarii, in 5% of the cases [1, 2]., we describe a rare case of struma testis characterized by malignant transformation and pulmonary metastasis associated with trisomy 17, with a coexisting papillary microcarcinoma of the thyroid . A 56-year - old caucasian male with a history of an undescended left testicle was admitted to a community hospital with right lower quadrant pain . Although he had refused orchiectomy in the past, he consented to it on this admission . The left testicle was noted to be in the left pelvic sidewall and was resected without complication . Prior to admission, he was asymptomatic and denied weight loss, dysphagia, odynophagia, and voice hoarseness . He did not have a history of smoking, exposure to radiation, or a family history of thyroid cancer . The evaluation for testicular tumor markers included normal serum -fetoprotein and normal serum -human chorionic gonadotropin . Lactate dehydrogenase was slightly elevated (680 units / l, normal range 313618). A chest x - ray on the day of admission revealed a well - demarcated 3-cm round density in the lower right perihilar region . A ct scan of the chest with intravenous contrast showed a well - circumscribed soft tissue mass arising from the inferior right hilar area, with a size of 2.2 2.4 2.7 cm, as well as a 0.7-cm lesion in the right superior hilar area and multiple smaller nodules in the lower lobes of both lungs . The pathology report identified the testicular tissue as a monodermal teratoma composed of thyroid parenchyma and associated with the development of papillary thyroid carcinoma with a predominantly follicular growth pattern (fig . Immunoperoxidase stains demonstrated that the neoplastic cells were strongly positive for cytokeratin cam 5.2 and thyroid transcription factor 1 . -fetoprotein, vimentin, -human chorionic gonadotropin, placental alkaline phosphatase, s100, cd30, inhibin, and calretinin stains were negative in the malignant cells . A video - assisted thoracoscopic wedge biopsy of the right lower lobe nodule revealed metastatic papillary thyroid carcinoma . Due to concerns about the effectiveness of i-131 ablation in the presence of a right lower lobe mass the pathology report of all specimens confirmed papillary thyroid cancer, with a maximum tumor size of 2.1 cm in the right lower lung lobe (fig . Interestingly, the patient had one microscopic focus (0.5 mm) of papillary carcinoma with follicular architecture (fig . A pretreatment stimulated (thyroid - stimulating hormone 95 miu / ml) whole - body scan using 1.5 mci of i-123 showed a residual uptake in the neck (2.5%) in addition to foci of increased uptake in the lungs bilaterally . At the same time, the serum thyroglobulin (tg) level was 16.8 ng / ml, and that of tg antibodies (tgabs) <0.9 iu / ml . To determine whether the papillary microcarcinomas of the lung and testis have a similar origin, we performed braf and ras (hras, nras, and kras) mutational analyses, which were negative, and comparative genomic hybridization (array - comparative genomic hybridization, a - cgh) from both the lung and testis revealed trisomy 17 and monosomy x, indicating their common origin (fig . The patient was treated with 200 mci of i-131, and the posttherapy whole - body scan did not show any new areas of increased uptake . He was started on levothyroxine 175 g daily and did very well clinically and biochemically . Nonstimulated (thyroid - stimulating hormone 0.111 miu / ml) tg and tgabs were undetectable . One year after the i-131 ablation, thyrogen - stimulated tg was <0.2 ng / ml, with tgabs at <20 iu / ml (normal range 0.0020), and a whole - body scan using 1.55 mci of i-123 did not show any iodine concentration areas consistent with metastases . A ct scan of the neck and chest revealed a decrease in the size of the unresected lung nodules . We presented a unique case of a metastatic papillary thyroid carcinoma from struma testis and raised the question whether the testicular and lung lesions could be metastases from the papillary thyroid microcarcinoma . First, normal thyroid tissue was present adjacent to the malignant papillary thyroid component in the teratoma, making it most likely that the teratomatous thyroid tissue underwent malignant transformation . Second, it is generally accepted that papillary microcarcinomas exhibit benign behavior and have an overall excellent prognosis . In fact, there is an up to 35% incidental prevalence of papillary microcarcinoma at autopsy [5, 6]. Furthermore, distant metastases from papillary microcarcinomas are reported to be 0.6% in a large meta - analysis . The sites of distant metastasis from papillary microcarcinoma included pulmonary, intratracheal, and cervical lymph nodes . However, no cases of papillary microcarcinoma metastasizing below the diaphragm in general, and to the testis specifically, have ever been reported . On the contrary, there is a case report of a malignant metastatic testicular teratoma that was found initially in the thyroid . Furthermore, molecular genetic analysis of the three tumors was performed in order to answer the question of whether the papillary microcarcinoma could have been the primary tumor . First, braf v600e and ras (hras, nras, and kras) mutational analyses were performed and were negative . The dna extracted from the testis and lung separately was hybridized with normal dna controls onto an a - cgh designed by nimblegen and analyzed using genoglyphix software (perkin elmer, signature genomics, spokane, wash . The thyroid microcarcinoma could not be evaluated, because no malignant tissue remained for sampling . Have demonstrated a strong association between isolated trisomy 17 and follicular thyroid nodules, which have nuclear features of papillary thyroid carcinoma, suggesting a link between papillary thyroid carcinoma and trisomy 17 . However, all nine cases studies were noninvasive . In our case, the a - cgh indicates that the testis and lung have the same genomic profile and are therefore most likely of the same origin (i.e. Teratomatous transformation in the struma testis which metastasized to the lung). The first case was a 65-year - old male who was found to have a cystic structure in the testes described as viable thyroid tissue with areas of complex papillary arrangements . The second case of malignant struma testis was a 68-year - old euthyroid male with a 5-cm mass in the upper pole of the right testis . Gross and microscopic examination revealed a replacement of testicular parenchyma with a microcystic tumor that had the appearance of normal thyroid tissue with histologically, solid, trabecular, follicular, and papillary areas . For the first time, we report a case with struma testis that underwent malignant transformation with distant lung metastasis as confirmed by a - cgh, and without regional para - aortic lymph nodes, and a coexisting papillary microcarcinoma in the thyroid . Our approach to the management of this tumor seems to be rewarding as the patient is cancer free 3 years later . Trisomy 17, which has typically been associated with noninvasive thyroid nodules with features of focal papillary carcinoma changes, was detected in both testicular and pulmonary tissues, confirming the invasive nature of the tumor . In recent years, increasing focus on the cytogenetic and molecular analysis of tumor cells has allowed a better understanding of thyroid nodules as well as of thyroid cancer . Our case provides evidence of a papillary thyroid cancer in struma testes metastatic to the lung, confirmed by cgh studies . Surgery to remove cancer foci, followed by radioactive iodine ablation, resulted in an excellent response in our patient . In contrast to previous studies, our case report has shown that trisomy 17, which has so far been observed only in noninvasive thyroid nodules, was associated with pulmonary metastasis . The authors declare that there are no conflicts of interest regarding the publication of this paper.
Laryngeal mask airway (lma) is characterized by simple operation, a small airway, and cardiovascular reactions, and is favored in clinical anesthesia, even sometimes for obese patients . However, lma as a relatively unstable artificial airway and may induce hypoventilation, upper airway obstruction, gastroesophageal reflux and aspiration, and other abnormalities, even with accurate positioning . These abnormalities are more likely to appear in the presence of abnormal respiratory dynamic parameters, such as high airway resistance or poor chest - lung compliance, caused by various factors . The main factors affecting respiratory dynamic parameters include individual physiological and pathological factors such as, age, body mass index (bmi), respiratory system diseases, and anesthesia - related factors such as surgical method, surgical position, ventilation mode, and ventilation tools [26]. With increased bmi, pulmonary compliance decreases due to excessive adipose tissue, leading to restrictive changes of lung functions, such as decreased vital capacity, functional residual capacity, expiratory reserve volume and inspiratory capacity, forced expiratory volume in 1 second (fev1), and diffusion capacity for carbon monoxide of the lung . In the surgical process, any position that compresses or limits thoracic activity or diaphragmatic contraction and leads to reduced thorax - lung compliance may also affect pulmonary ventilation . Therefore, this study aimed to compare the differences in respiratory dynamic indexes in patients with lma ventilation in supine and lithotomy surgical positions, and also to observe the correlation between bmi and respiratory dynamic indexes with these 2 positions, to provide evidence for secure implementation of lma in overweight patients with specific surgical positions . This study was approved by the ethics committee of shanghai general hospital affiliated to shanghai jiaotong university . Patients undergoing elective surgery in orthopedics and urology between july and november 2013 were selected . Inclusion criteria were: patients were grade i or ii according to american society of anesthesiologists (asa) marking system, and aged 2065 years old . Patients were divided into a supine position group (sp group) and a lithotomy position group (lp group) according to the surgical positions . The sample size was calculated based on pilot experiments, with a calculation formula: n=(z/2)/e, 95% confidence interval . Exclusion criteria were: significant abnormalities in liver, kidney, heart, or lung functions, history of gastroesophageal reflux, or respiratory tract infection within the previous 3 weeks . Patients fasted for 8 h prior to surgery, and were given an intravenous supplement of 500 ml 5% glucose if necessary . Electrocardiogram, heart rate (hr), non - invasive blood pressure (nibp), pulse oxygen saturation (spo2), and respiratory dynamic indexes were continuously monitored using a multi - function monitor (datex - ohmeda s/5 am, ge company, ct). Prior to the induction of general anesthesia, patients received intravenous infusion of 10 ml / kg sodium lactate ringer s solution . Patients received intravenous injection of midazolam 1~2 mg, fentanyl 2~4 g / kg, and propofol 1.5~2.0 mg / kg . After the patients lost consciousness, they were given an intravenous injection of succinylcholine 1~2 mg / kg, and an lma (traditional type, tuoren co., china) of appropriate size (3~5) was implanted 1 min later . Intravenous injection of rocuronium 0.2~0.4 mg / kg was given after the lma was positioned . After implantation of the lma, patients were given mechanical ventilation using volume - controlled mode (datex - omeda aespire, ge) until the end of surgery . The inspiration: expiration ratio was set at 1:1.5, the tidal volume was 6~10 ml / kg, positive end - expiratory pressure was set at zero, and the partial pressure of carbon dioxide in end - expiratory gas (petco2) was maintained at 35~40 mmhg . The air capsule of the lma was inflated with 20~30 ml of air, during which the chest fluctuations were observed and breath sounds in the lungs and neck were auscultated to identify the position of the lma . Finally, examination using fiberoptic bronchoscopy was performed to identify the position of the glottidis rimae and to exclude airway obstruction . The lma position was scored using the marking system described by brimacombe, and then the test of airway sealing pressure was performed . Cases with disqualifying scores or test results were switched to endotracheal intubation and were excluded from the study . Prior to skin incision, patients received intravenous injection of fentanyl 0.05~0.10 mg and sevoflurane was inhaled continuously with a volume fraction of 1~4% in 100% oxygen in 1 l / min fresh airflow during the operation . The inhalational concentration was maintained at about 1.0 mac, and additional intravenous injection of fentanyl 1~2 g / kg was administered when the elevations of hr and nibp were greater than 20% of their baseline amounts . Patients with systolic pressure <90 mmhg or with a reduction of mean arterial pressure (map) of 30% were given an intravenous injection of ephedrine at a dose of 3~6 mg / time and patients with hr<50 beats / min received intravenous injection of atropine 0.2~0.5 mg / time . Patients who showed resistance to the ventilator during the operation were given an additional injection of intravenous rocuronium with a dose of 10 mg / time . The main outcome measures were: respiratory dynamic parameters, including inspiratory plateau pressure (pplat), mean airway pressure (pmean), peak airway pressure (pip / ppeak), positive end - expiratory pressure (peep), peak inspiratory flow (fimax), peak expiratory flow (femax), airway resistance (raw), and partial pressure of carbon dioxide in end - expiratory gas (petco2). These were recorded prior to receiving anesthesia induction (t0) and at 1 (t1), 5 (t2), 10 (t3), 15 (t4), 30 (t5), and 45 (t6) min after implantation of lma . Arterial blood gas was monitored at 15 min after anesthesia induction (i - stat blood gas monitor, abbott company), including arterial partial pressure of carbon dioxide (paco2) and arterial partial pressure of oxygen (pao2). Physiologic dead space in percent of tidal volume (vd / vt), static compliance (cst), expiratory resistance (re), and work of breathing (wob) were calculated using the formula provided by casati et al . . Secondary outcome measures were: hr, nibp, and spo2 at 1 (t1), 5 (t2), 10 (t3), 15 (t4), 30 (t5), and 45 (t6) min and at 5 min after retraction of the lma (t7). Air leakage of the lma, aspiration, bucking during the operation, pharyngeal pain, trachyphonia, and muscular soreness after the operation and other adverse events were observed . Because the surgical position cannot be set with blind method, all the respiratory dynamic and hemodynamic indexes were objective data; therefore, all the intraoperative observation indexes were non - blind, and patients were followed up after the operation . Parameters in line with normal distribution were performed using variance analysis . For repeated measurement data accorded with spherical symmetry assumption using variance analysis, the non - corrected f critical value was used . For data that did not accord with spherical symmetry assumption, the parameters with non - normal distribution were tested using mann - whitney u or kruskal - wallis h methods . Comparisons of ranked data were performed using rank - sum test and p<0.05 was considered statistically significant . Statistical analyses were performed using spss 19.0 software (ibm corp ., ny). Parameters in line with normal distribution were performed using variance analysis . For repeated measurement data accorded with spherical symmetry assumption using variance analysis, the non - corrected f critical value was used . For data that did not accord with spherical symmetry assumption, the parameters with non - normal distribution were tested using mann - whitney u or kruskal - wallis h methods . Comparisons of ranked data were performed using rank - sum test and p<0.05 was considered statistically significant . For the pilot experimental study, the expected sample size was 23 cases in each group, which was satisfied because there were 49 and 41 cases in the sp and lp groups, respectively, who met the inclusion criteria for this study . In the sp group, there were 40 males and 9 females, with a mean age of 43.9117.54 years old, and mean bmi of 23.93.1 kg / m . In the lp group, there were 23 males and 18 females, with a mean age of 49.6315.5 years old, and a mean bmi of (24.33.8) kg / m . The sp group received intravenous infusion of propofol (1.700.40) mg / kg, fentanyl (4.601.00) g / kg, and rocuronium (0.390.06) mg / kg and the lp group received intravenous infusion of propofol (1.600.50) mg / kg, fentanyl (4.200.90) g / kg, and rocuronium (0.420.05) mg / kg . The difference in anesthetic drugs between the 2 groups was not statistically significant (p>0.05). The heart rate, mean arterial pressure, and spo2 in the baseline were not significantly different between the 2 groups . During the surgery, the hemodynamic changes at different time points showed statistically significant differences within the same group . The heart rate and mean arterial pressure at different time points were not significantly different between the 2 groups . Spo2 was maintained above 98% in all patients (table 1). In the sp group, with prolonged surgical duration, the pplat, pmean, pip, and raw were significantly elevated at t5 compared with those at t1 (p<0.05), and petco2 and compl were gradually decreased (p<0.05), while the peak inspiratory pressure (pip), mean airway pressure, peak inspiratory flow, peak expiratory flow, vd / vt and re did not display significant differences at each time point during the operation (p>0.05). In the lp group, fimax, femax, and re at time point t5 were significantly increased (p<0.05) compared to t1, and petco2, compl, and wob were significantly reduced (p<0.05). Airway pressure and vd / vt did not show significant differences at any time point during the operation (p>0.05). Pmean, pplat, and raw from time point t1 to t6 in the lp group were significantly higher than those at the corresponding time points in the sp group (p<0.05). Fimax, femax, wob, and compl from time point t1 to t6 in the lp group were significantly lower than those at the corresponding time points in the sp group (p<0.05). At time point t1, pip was significantly higher in the lp group than in the sp group (p<0.05). At t3, the petco2 in the lp group was significantly lower than that in the sp group (p<0.05). Vd / vt at t4 in the lp group was significantly higher than that in the sp group (p<0.05). Time point t5 displayed the largest difference of respiratory dynamic parameters and was selected for further analysis of the correlation between respiratory dynamic parameters and bmi (table 2). At time point t5, the ppeak, pplat, and raw in the 2 positions were both positively correlated with bmi, and compl was negatively correlated with bmi . It seems that the changes in the lp group were larger than those in the sp group, while the statistical analysis showed that only the changes in raw were significantly different (figure 2, p<0.05). When the risk of leakage of the lma and gastro - esophageal reflux were set at a condition in which the raw was higher than 25 cmh2o, the final logistic regression model demonstrated the corresponding critical value of bmi to be 34.2 in the lp group and 44.7 in the sp group (figure 3) in the sp group, bucking and pharyngalgia occurred in 4 and 2 cases, respectively, and in the lp group, bucking and pharyngalgia occurred in 5 and 2 cases, respectively . No patients in the 2 groups had gastroesophageal reflux, aspiration, myalgia, trachyphonia, or other adverse events . Lma as a supraglottic airway tool has been widely used in clinical anesthesia practice, mainly due to its advantages, including minimal airway irritation, stable hemodynamics, fast postoperative recovery, and fewer airway complications . Biedler et al . Compared leakage pressure and peak airway pressure with laryngeal tube and classical lma in surgery with different head and neck angles, and found that the leakage pressure and peak airway pressure with lma at different positions were lower than those obtained with laryngeal tube, suggesting that the lma has good airway sealing and achieves good ventilation effect . However, some researchers have found that about 10% of patients using lma have leakage with an airway pressure> 25 cmh2o, thus creating concerns about hypoventilation, gastroesophageal reflux, and aspiration in using lma when there are changes in respiratory dynamics (such as high airway resistance and attenuated chest - lung compliance). The present study aimed to determine whether surgical position and body weight lead to hypoventilation or gastroesophageal reflux by affecting respiratory dynamics . According to previous reports, positions causing hypoventilation, from severe to mild, are: deep flexibility position, head flexion lithotomy position, prone position, lateral position, and high position with gallbladder pad or kidney pad . Lithotomy position is prone to movement of the pelvis and abdominal organs towards the head, as well as decreasing space for lung activity by knee lift to elevate the sacrum, thereby creating relatively shorter lumbar vertebra height, decreasing lung compliance and increasing airway resistance . Therefore, we needed to correct tidal volume through increasing airway pressure, while the work of breathing was also increased . Therefore, in lma ventilation with a lithotomy position, a high airway pressure may be needed to ensure tidal volume, even though it may increase lma leakage and thus increase the risk of gastroesophageal reflux . We wondered whether an unstable airway, such as in use of lma, will lead to hypoventilation when chest - lung compliance is affected by lithotomy position . This study compared changes in respiratory dynamics parameters in patients undergoing general anesthesia with lma in lithotomy and supine positions . Our results showed that with these 2 positions, the inspiratory plateau pressure, inspiratory resistance, and work of breathing increased with prolonged duration of the operation, and the elevation of inspiratory plateau pressure in the lp group was significantly greater than that in the sp group . In the 2 positions, cdyn and cst decreased with prolonged duration, and the decrease in the lp group was significantly greater than in the sp group . Results at time point t7 showed a different trend from those at the other time points . Surgery ended in many patients in the 2 groups at t7, and the statistical results were unreliable due to insufficient sample size . The variation tendency of respiratory dynamics in this study was consistent with previous reports in patients undergoing general anesthesia with endotracheal catheter in trendelenburg position, head flexion lithotomy position, and prone position [46]. However, these respiratory dynamic changes presented a higher clinical risk in lma ventilation than in ventilation with endotracheal intubation . In addition, our results showed that the peak inspiratory flow and peak expiratory flow in the lp group were lower than in the sp group, which we suspect was induced by the changes in ri and re . Soto et al . Found that the airway resistance and peak airway pressure were significantly increased and the chest - lung compliance was remarkably decreased in 24 very obese patients undergoing laparoscopic bariatric surgery under general anesthesia using endotracheal intubation, but the report was focused on a very obese population . Another study involving 232 obese patients (bmi> 30 kg / m) found that, compared with endotracheal intubation, although the lma presents an increased risk of leakage, the ventilation outcome was not affected, and it may reduce the bucking at the end of anesthesia, shows faster emergence from anesthesia, and significantly reduces the incidence of postoperative hypoxemia . The present study found that the peak airway pressure, airway plateau pressure, and airway resistance in the 2 positions were positively correlated with bmi, while the dynamic and static compliances were negatively correlated with bmi, which was similar to the results in obese patients obtained by soto . However, we provided more comprehensive data on respiratory dynamics, and did not limit the study to obese subjects . The normal value of bmi is 18.524.9 kg / m, while a bmi value of 2528 kg / m is overweight, and> 30 kg / m is considered obese . The mean bmi was 23.93.0 in the supine position group and 24.02.6 kg / m in the lithotomy position group . Therefore, our results suggest the need to consider the changes in respiratory dynamics due to weight gain and the effect on ventilation, even in subjects with normal bmi . Given the specificity of lma ventilation, such as hypoventilation and risk of gastroesophageal reflux and aspiration, the results of this study have greater clinical value than the results obtained in endotracheal intubation by soto . Although we failed to show changes in respiratory dynamics that might change the ventilation effect, our results indicate that lma ventilation should be done cautiously in patients with bmi 34.2 in lithotomy position or bmi 44.7 in supine position, to avoid complications due to excessive airway resistance, as shown in figure 3 . Hypoventilation caused by surgical position and obesity has already been reported in the literature, but our study focused on the correlation between respiratory dynamics and bmi in 2 different surgical positions . We found the point at which a bmi value indicates risk of hypoventilation or gastroesophageal reflux due to exceeding airway resistance . The incidence of atelectasis at rest in obese patients was 2 times higher than in non - obese patients and is generally believed to be caused by residual gas and ineffective ventilation in blood flow due to increased closing capacity . Intrapulmonary shunt may aggravate hypoxemia, especially when the patients are in supine position, since the abdominal wall and abdominal contents may exert pressure on the diaphragm . Gaszynski conducted a study in 47 obese patients (bmi 49.547.21 kg / m) and found that overweight patients (bmi> 60 kg / m) have significantly lower preoperative lung functions and significantly higher postoperative hypoxemia . In the present study, no patients had postoperative hypoxemia, which could be because the bmi of our patients was close to normal, and patients with preoperative pulmonary dysfunctions were excluded . This is also a limitation of the present study, and further studies are needed in obese populations . In this study aspiration did not occur in either of the groups, and other complications, such as bucking and pharyngalgia, rarely occurred . The differences between the 2 groups were not statistically significant, which is consistent with previous results . However, further studies are required to validate whether this is associated with the small sample size . The airway pressure and airway resistance increased with prolonged operation time in patients undergoing general anesthesia with classical lma, and it was more significant in lithotomy position than in supine position, accompanied by decreased chest - lung compliance . The peak airway pressure and airway resistance were positively correlated with bmi, and chest - lung compliance was negatively correlated with bmi . The changes among patients in lithotomy position were even more remarkable than those in supine position . The critical value of bmi was 34.2 in lithotomy position and 44.7 in supine position, corresponding to the risk of airway resistance of 25 cmh2o . Therefore, one should be cautious while using lma in overweight patients with bmi 34.2 in lithotomy position.
We present a case of gastric outlet obstruction (goo) caused by a fibroepithelial tumour . This is the first instance of such a tumour being identified in the gastrointestinal tract . Interestingly also, our patient experienced no prodromal symptoms relating to gradual occlusion of the gastric outlet, and presented with an acute picture leading to diagnostic confusion initially . A previously well 57-year - old man presented to our emergency department with a 24hr history of epigastric fullness and discomfort, delayed non - bilious vomiting after oral intake, and anorexia . The patient took no regular medications and consumed 12 - 14 units of alcohol per week . Initial impressions were of gastritis, biliary colic, or peptic ulcer disease (pud). Blood investigations demonstrated a white cell count of 16.1 x 10 l, c - reactive protein of 247 mg l and normal amylase, liver function, and electrolytes . Endoscopy performed the following day revealed a pre - pyloric submucosal lesion with normal overlying mucosa (biopsy proven), with complete obstruction of the pyloric valve a diagnosis of goo secondary to a submucosal lesion was made . A subsequent computed tomography (ct) scan demonstrated solid and cystic components within the lesion and no associated significant lymphadenopathy (figure 1). These sequential ct image at the level of l1 demonstrates the heterogenous tumour arising from the wall of the distal stomach, and causing total occlusion of the gastric outlet . An endoscopic ultrasound examination (eus), with a view to simultaneous fine needle aspiration cytology (fna) was arranged, to further evaluate the lesion . Our patient s symptom continued unabated and despite total parenteral nutrition was failing to thrive . Therefore a laparotomy was performed in which a gastric antral mass was identified; with the transverse colon adherent to the stomach on the serosal aspect of the mass . Macroscopic examination of the specimen revealed a 9 x 8 cm section of the stomach stapled along two margins, with a 30 cm section of colon, including the caecum and appendix, attached . At the site of adhesion a 7 cm mass was felt . On opening the lumen of both the transverse colon and stomach no lesion or ulceration was seen; although there was significant bulging of the gastric mucosa overlying the mass . Upon slicing a solid white tumour was seen to arise from the wall of the stomach, approximately 3 cm from the nearest resection margin, with a well defined edge (figure 2). Here we can see a white, solid tumour, with a well circumscribed edge arising from the wall of the stomach . Microscopic analysis of sections from the stomach revealed a predominantly unremarkable mucosa, with some areas of intestinal metaplasia . Just beneath the muscularis mucosa, a nodular this consisted predominantly of epitheloid, spindle shaped cells set in a collagenous stroma containing thin walled vessels (figure 3). This haematoxylin and eosin (h&e) stained section demonstrates the epitheloid spindle shaped cells with intervening areas of stromal hyalinisation . In places the tumour also contained cleft - like spaces lined by gastric and intestinal type epithelium, seen more towards the gastric side of the tumour nodules (figure 4). This epithelium was in at least one place continuous, with the normal gastric surface mucosa which was seen to dip down into muscularis mucosa . In this h&e stained section gastric and intestinal type epithelium is seen lining numerous cleft like spaces within the lesion . Immunohistochemical analysis revealed the stromal component to be strongly smooth muscle actin (sma) positive, with patchy, weak positivity forvimentin and caldesmon . The tumour cells were negative for cluster of differentiation (cd) 117, cd34, cd99, cd68, s100, activin receptor - like kinase-1, mnf116, and human melanoma black -45 . The epithelial component showed variable positivity, in different areas, for cytokeratin (ck)-7 and ck-20 . There was no cytological atypia nor any features of malignancy in either the epithelial or spindle cell elements . Our patient experienced a protracted recovery due to a post - operative pneumonia and episode of haemorrhagic gastritis, but was eventually discharged home and remains well to this date . Considered separately, the epithelial component could not be explained by a developmental cyst or reduplication as an associated smooth muscle component was not seen; and the stromal elements did not fit with any of the recognised gastrointestinal mesenchymal tumours . The only logical way to explain the findings in this case was to regard both the stromal and epithelial components as part of the same process, and make a diagnosis of a fibroepithelial lesion of the gastrointestinal tract . Fibroepithelial neoplasias are biphasic lesions, consisting of both a stromal and epithelial components from two separate germ lines endoderm and mesoderm . They are most commonly seen in the breast as the phyllodes tumour (1). Their behaviour ranges from benign to malignant, and quantitative morphologic criteria including mitotic rate per 10 high - power fields, stromal cellularity, nuclear size, stromal overgrowth, and the largest and smallest stromal - epithelial surface area ratios - exist to stratify lesions accordingly (2). In our case gastric submucosal lesions are usually evident after endoscopic investigation, which is in turn regularly performed due to the symptom profile of these lesions . In the case of a submucosal gastric lesion, eus with fna of the lesion is the definitive investigation . This has been shown to be the most reliable in determining whether a lesion is benign or malignant, assessing the suitability of surgical versus endoscopic resection, and can also be used to survey uncertain lesions (3). The addition of fna to eus is reported at achieving an accurate tissue diagnosis of submucosal lesions in 89% of cases (4). Definitive management should be planned in accordance with the patient s symptoms and tumour in mind . Resection can be carried out successfully laparoscopically, as shown for gist s (5), or by laparotomy in difficult or uncertain cases.
The programming and reprogramming of cellular identity elicit tremendous scientific and public interest . The groundbreaking work of takahashi and yamanaka established a precedent with the generation of induced pluripotent stem cells (ipscs) by the forced expression of only four transcription factors similar to embryonic stem cells (escs), ipscs can proliferate and self - renew indefinitely under appropriate conditions and give rise to all types of cells in the body, which bestows these cells with many potential uses in regenerative medicine . Patients with degenerative diseases such as diabetes and cancer, along with aging individuals could all benefit from ipsc - based therapies . Somatic cells can be reprogrammed by nuclear transfer or by fusion with escs, suggesting that oocytes and escs contain factors that can reprogram somatic cells into stem cells . Inspired by this discovery, yamanaka and his colleagues selected 24 genes that are specifically expressed in escs, which also play important roles in the maintenance of esc identity, as candidate factors to induce pluripotency in mouse somatic cells . By transducing all 24 candidate genes together, these cells were further identified to possess esc properties . To determine which of the 24 candidates were essential, yamanaka and his colleagues tested the effects of the withdrawal of individual factors from the 24-candidate gene pool on the generation of g418-resistant colonies . Ultimately oct4, sox2, klf4 and c - myc were identified to be pivotal for the induction of pluripotency in mouse somatic cells . Furthermore, these factors were proven to be able to induce pluripotency in human somatic cells as well . Other laboratories have also been working on inducing pluripotency in somatic cells . By using lin28 and nanog together with oct4 and sox2, the thomson laboratory also independently discovered a set of four reprogramming factors that are highly enriched in escs . These first proof - of - principle studies opened the realms of ipsc research to a future in regenerative medicine . It is coherent to test whether there are novel esc - associated factors that can regulate ipsc induction . Several reports have suggested factors that are important for the maintenance of esc identity can also facilitate the induction of pluripotency . For example, nr5a2, an orphan nuclear receptor that is enriched in escs, can replace oct4 in the induction of pluripotency . Esrrb, another orphan nuclear receptor that plays a pivotal role in the maintenance of pluripotency, can replace klf4 and c - myc . Prdm14 and nfrkb were identified as novel determinants of human esc identity and can substitute for klf4 in reprogramming . Recently, by a single - cell analysis of the reprogramming process, lin28, sall4, esrrb and dppa2 were identified as a completely novel set of reprogramming factors, which are different from the factors initially identified by yamanaka et al . These factors are all important for the maintenance of esc identity . In addition to the highly expressed factors in escs, the maternal factor glis1 in oocytes for years, it was generally believed that escs are maintained by a shield of pluripotency factors . These factors function in concert with each other to prevent escs from differentiating into any lineage, thus preserving the escs at an undifferentiated state . Pluripotency factors might as well function as classical lineage specifiers that direct escs to differentiate into a specific lineage and inhibit their commitment to mutually exclusive lineages . Consistent with the notion, in escs, oct4 promotes the differentiation of mesendoderm (me) and primitive endoderm, while suppressing differentiation of the ectoderm (ect) [1517]; sox2 inhibits me differentiation but promotes neural ect differentiation . Shu et al . Provided the first proof - of - principle report showing that modulating lineage - specifying forces can restore the pluripotency of mouse somatic cells . When screening for factors that may substitute for oct4 in the induction of pluripotency, shu et al . Found that gata3, which is known to regulate me commitment and specification, can substitute for oct4 . Subsequent analysis of other lineage specifiers that mainly function in me differentiation and early embryonic patterning, which are generally not enriched in escs, found that gata6, sox7 and pax1, among others, were also able to substitute for oct4 to induce pluripotency, whereas ectodermal specifiers could not . All oct4 substitutes were also able to attenuate the upregulated expression of ect - associated genes that is triggered by the expression of sox2, klf4 and c - myc (skm), whereas knockdown of the key ectodermal marker dlx3 promoted skm - only reprogramming . These findings suggest that a novel function of oct4/gata3 is to suppress ect differentiation during reprogramming . Accordingly, the ect lineage specifiers sox1, sox3, rcor2 and gmnn can replace sox2 during reprogramming . Similarly, sox2 and its substitutes attenuate the expression of me - specific markers induced by expression of oct4, klf4 and c - myc (okm) [1820]. Strikingly, co - expression of gata6 and gmnn can substitute for oct4 and sox2 to reprogram mouse fibroblasts into ipscs in the presence of klf4 and c - myc . More recently, montserrat et al . Showed that lineage specifiers can also be used to reprogram human fibroblasts into ipscs . The authors found that gata3 can replace oct4 and the ect specifier, znf521, can replace sox2 . Lastly, they showed that gata3, together with znf521, otx2 and pax6, can substitute for both oct4 and sox2 for human ipsc induction in the presence of klf4 and c - myc . A binodal model for cell fate determination, such as gata1 and pu.1, runx2 and ppar, has been examined in various instances of pluripotent stem or progenitor cells that assume a binary cell fate decision . Proposed a new model, termed the seesaw model, in which the pluripotent state has a precarious balancing equilibrium that results from continuous mutual competition between rival lineage specification forces (figure 1). This model comprises two coupled modules the canonical pluripotency module and the lineage - antagonism module . The former module is represented by the mutual activation of oct4 and sox2, whereas mutual inhibition of the me and ect genes represents the latter module . Both the canonical pluripotency module and the lineage - antagonism module are incorporated leading into the integrated seesaw model . The novelty of the seesaw model is the proper combination of the two modules . The activation of the cross - activating pluripotency module is important for the reestablishment of the pluripotency network to achieve successful reprogramming . The self - activating pluripotency module gets activated when all of the lineage - specifying forces are counteracted at the dynamic balance point of the seesaw . In other words, no particular lineage - specifying activity is dominant in inhibiting the pluripotency module . In this case, the pluripotent state becomes achievable, eliciting the oct4 and sox2 self - activating module to coordinate with other pluripotency factors, thus collaboratively restoring the pluripotency network . Once the cross - activating pluripotency module is activated, the me and ect lineage fates are blocked by sox2 and oct4, respectively . As a result this innovative model can illustrate the aforementioned points and predicts novel strategies for cell fate conversion, including strategies for the direct conversion of somatic cells into ipscs by pluripotency factors or lineage specifiers along with the direct conversion of somatic cells into specific lineages by lineage specifiers or pluripotency factors . The direct reprogramming strategy for cell fate conversion has been widely adapted for some other cell types in addition to ipscs . The direct conversion of fibroblasts into myoblasts by overexpressing myod was reported in 1987 by davis and colleagues . Recently, increasing numbers of different cell types have been obtained by direct conversion, termed transdifferentiation . For example, a combination of three neuronal lineage - specific transcription factors, ascl1, brn2 and mytl1, is sufficient to induce neurons from fibroblasts . The cardiac - specific transcription factors gata4, tbx5 and mef2c can induce fibroblast transdifferentiation into cardiomyocytes . It is therefore assumed that the more specific and closer the endogenous regulatory network is to the factors, the more efficient the conversion will be . The seesaw model also predicts that inhibiting the mutual antagonistic lineage - specifying forces could convert one cell type into another . Furthermore, consistent with the seesaw model, reprogramming factors have been reported to directly produce lineage - committed cells . Two pivotal pluripotency factors, oct4 and sox2, were reported to regulate esc differentiation into different germ layers and to induce direct conversions between different cell types beyond ipscs . Previous studies have shown that a twofold increase in oct4 expression induces escs toward mesendodermal specification, whereas high levels of sox2 trigger the neuroectodermal commitment of escs . Recently, it was reported that oct4 and sox2 also orchestrate a germ - layer fate selection . Oct4 inhibits neuroectodermal differentiation and promotes mesendodermal differentiation, whereas sox2 promotes neuroectodermal differentiation and inhibits mesendodermal differentiation . More recently, overexpression of oct4 in fibroblasts was shown to lead to transdifferentiation into hematopoietic cells of a mesendodermal lineage, whereas overexpression of sox2 directly converted fibroblasts into neural stem cells . Additionally, decreased expression of oct4 among the four yamanaka factors can result in the direct conversion of fibroblasts into neural stem cells [2830]. These discoveries suggest that pluripotency factors, such as oct4 and sox2, can regulate not only pluripotency but also lineage specification . After the discovery of the famous yamanaka factors, a set of transcription factors consisting of oct4, sox2, klf4 and c - myc, regenerative biology has stepped into a new era . Increasing numbers of pluripotency - related factors have been identified either to replace the yamanaka factors or to boost the process . Meanwhile, direct transdifferentiation has been successfully demonstrated through a similar strategy, by using lineage - specific transcription factors for specifying each lineage fate . Model for cell fate conversion have introduced a novel scenario for cell fate conversion causing us to re - evaluate the characteristics of pluripotency factors and lineage specifiers, which are two rivals in the conventional conception of the development of cellular identity . For example, oct4 specifies me differentiation while inhibiting ect differentiation, and induces hematopoietic transdifferentiation from fibroblasts . Sox2 directs ect differentiation while prohibiting me commitment, and induces direct transdifferentiation from fibroblasts into neural stem cells . Lastly, the lineage specifiers depicted as pluripotency rivals, such as gata3 and pax6, have been identified to be able to restore pluripotency in somatic cells . Based on these discoveries, we should reconsider the definitions of pluripotency and lineage specification and present a novel perspective for understanding the determinants of cellular identity, which is one of the most important topics in modern biology.
Atopic dermatitis (ad) is a chronic inflammatory skin disease which typically affects children during the first years of life, with a prevalence around 1030% . Recent studies have shown that in 1020% of children with ad, the disease persists through adolescence . In particular, few years ago the international study isaac phase iii estimated that, among adolescents, ad is present in 510% in most european countries, 1520% in uk and south america and in 05% in asia . Peters and colleagues evaluated the course of ad in a group of german adolescents, who had been enrolled for the isaac phase ii, and were able to recognize several risk factors: family history for allergic rhinitis (or=1.6) and/or ad (or=2.7), allergic sensitization during the first years of life (or=1.8), jobs at risk for allergen exposure (hairdresser, nurse and health care professionals, cleaning staff, baker) (or=1.4). Adolescents with ad are rarely affected by animal protein food allergy (only 7.4% have a cow's milk or hen's egg allergy), but often show sensitization to inhalant allergens such as grass and birch pollens (44.4% and 55.6% respectively) and oral allergy syndrome (22%) against plant foods . The sex gender also influences the prevalence of ad: during the first 2 years of life ad more often affects males, but in the pubertal age and in adulthood the female sex has a higher prevalence; no significant differences are reported during the school age . Ziyab and colleagues performed a longitudinal study in order to evaluate a relationship between variations in ad prevalence, gender and allergic sensitization during the first 18 years of life . Puberty seems to represent a very important period, during which a change in the disease prevalence happens (16.3% of females and 8.3% of males, p<0.001); the study results indicate that this difference might be due to an increase in girls affected by intrinsic ad (5.9% females and 1.5% males, p=0.002) and to a higher number of healed males (65.4% of males and 50% of females, p=0.04). Several studies evidenced that up to 3040% of children affected by asthma, eczema and rhinitis during childhood were allergic children; the paper by ziyab et al . Indicates a higher prevalence of allergic sensitization for inhalant allergens (grass pollens, alternaria alternata, cladosporium, dog's and cat's dander) at 4, 10 and 18 years of age among male patients than in girls (p=0.024, p=0.003 and p<0.001 respectively). Mohrenschlager e coll . Performed a study on a population of 57 year - old patients and showed that girls have a higher skin ph and a lower hydration of the stratum corneum than boys, independently of being affected by ad; this might cause female patients to have a higher risk of developing eczema . Several authors highlighted that female ad patients have an important worsening of the skin condition during the pre - menstrual period, menstruations and pregnancy, suggesting that an association exists between hormonal levels and ad lesions . About 52% of patients with ad show a worsening during pregnancy, usually in the first 2 weeks of gestation; however, the mechanisms are not yet completely understood . During pregnancy a t cell switch towards a th2-type response occurs; this phenomenon is probably due to a reduction in deidroepiandrosterone (dhea) and deidro - epiandrosterone - solfato (dhea - s) levels . An increase in th2 activity might explain the eczema worsening; it is unclear whether changes in the filaggrin expression also happen . Pincus et al . Also showed the existence of an inverse association between progesterone levels during the first months of pregnancy and the risk of developing ad during adolescence in girls but not in boys . No association has been highlighted between maternal levels of estradiole during pregnancy and the risk of developing ad . Experiments on mice speculate on a possible stimulating effect of estrogens on mast cell activation . Dhea might antagonize the th2 cytokines release, but the role of testosterone and other androgens has not been clarified yet . Dhea - s levels in the peripheral blood increase in response to stress, chronic inflammation and immuno - mediated processes, but the actual role of this phenomenon has not been elucidated and no changes in the peripheral blood levels of this hormone have been detected in women affected by severe ad . In most cases, patients with ad improve or recover during childhood . However, in some cases ad persists through adulthood and is associated with the onset of allergic rhinitis and/or asthma . The risk of developing asthma in children affected by ad varies from 25% to 80% . The typical eczematous lesions appear mainly during the first two years of life with a different distribution depending on the age of the patients . During the first months of life lesions are exudative and mainly localized in the head, face (in particular forehead, cheeks, chin, with the central - face saving) and in the extensor surfaces of limbs . In older children the lesions are mainly concentrated on the flexural surfaces of the limbs, the popliteal and antecubital folds, back of hands and feet . The skin is commonly dry with lichenification and intense itch; the lips are frequently dry brittle, chapped and develop fissures . The evolution of the clinical feature is characterized by phases of remission of symptoms, which occur mostly during the summer months, alternating with periods of exacerbation, particularly during the autumn - winter . Clinical features typical of adolescence are represented by eyelid dermatitis, and the palmar and plantar juvenil dermatitis; the eczematous lesions are often localized to the neck, and in are often associated with infection by malassezia . The lesions are also localized in forehead, perioral region, neck, upper chest and shoulder girdle, flexor surfaces of the legs and backs of hands . The early onset, the severity of the framework, frequent relapses and chronic course make a disease with important psychological consequences that affect the quality of life (qol) of the children and their families . Affected children often present behavioral problems, mainly characterized by increased emotional dependency, anxiety, and sleep disturbances . The itching, which is one of the main symptoms of the disease, affects mood and sleep quality of patients and consequently of their family . The chronic course characterized by remission and exacerbation and the long - term treatments, negatively affects the quality of family life, both economically and psychologically, producing anxieties, frustrations, creating feelings of guilt and anger . Adolescents with ad exhibit greater vulnerability, anger, anxiety, insecurity, but few studies have been done about it . A recent study study of 367 american adolescents suffering from atopic diseases has highlighted the existence of a significant association between increased levels of anxiety and the presence of allergic respiratory diseases (asthma and rc) but not with the ad; in this study it seems that ad affects the qol in general terms of itching, and sleep disturbance, but it is subjectively experienced by patients as less severe than respiratory diseases . A study by brenninkmeijer et al . Considered a group of 165 patients aged between 18 and 30 years who had ad during childhood . Ad had a highly negative impact on patient qol in the 48.7% of patients, with significant correlation between disease severity and qol (r=0.518, p<0.001). Among the problems identified, patients had experienced the need to receive clearer information about the treatment of the disease (87%), greater empathy with the doctor (85%), the need to deal with other patients with ad (52%) and the need of a psychological support (68%). Furthermore, adolescents with moderate/ severe ad had shown a significant delay in development of social relationships (lower number of friends, reducing output in a group) compared to healthy ones and those with mild ad . During high school, 70% of patients reported to be ashamed of their skin, 49.1% avoided intimate situations, 43.2% abstained from sport, 90% reported intense itching, 69.2% sleep disturbance, fatigue 60.2% and 74.1% physical deterioration of the lesions with stress . A study by saunes et al . Of norwegian adolescents with ad showed a correlation between the severity of clinical feature and increased levels of psychological stress . In particular, the prevalence of psychological distress was highest in the largest age group (1719 years). Although females were more affected by psychological stress than males, the association between ad and psychological distress was higher in males than females (or=2.1 - or=1.3). Sang ho et al . Evaluated the psychological characteristics of 34 patients with ad (age 1341 years) by the following questionnaire: back depression inventory (bdi), state trait anxiety index (stai), interaction anxiousness scale (ias), private body consciousness (pbc) and the dermatology life quality index (dlqi) and found a significant increase of all parameters of the questionnaires and a significant correlation between the parameters of the questionnaires covered and the dlqi (p<0.001) the basis of therapy for the management of ad in adolescents are similar to those of younger child . It may be noted that the adherence to therapy, which often must be at least daily for prolonged periods, is certainly more difficult for the adolescents . To encourage this therapeutic compliance in adolescents, a recent study conducted in the u.s . Used short message service (sms) to the mobile phone to remember to patients the need of a daily therapy . The study showed that daily sending sms to mobile patients, led to significant improvements in patient compliance (p0.001), their self - care (p=0.002), severity (p<0.001) and qol (p=0.014). The need to create an educational and training program to educate, inform and support families with children affected by ad in the management of this disease originates the concept of the school of atopy, on the model of the first educational program initiated in germany: the berlin parental education programme based on a series of meetings between specialists and family . The schools of atopy are differently structured, depending on different countries, although in general the standard provides, in addition to a coordinator or leader, a dermatologist, a pediatrician, an allergist and a clinical psychologist, to which other figures can be added . A study by ricci et al . On a group of children with ad and their parents, who had joined this educational program for 3 years, showed a significant reduction in anxiety levels in parents mostly . The typical eczematous lesions appear mainly during the first two years of life with a different distribution depending on the age of the patients . During the first months of life lesions are exudative and mainly localized in the head, face (in particular forehead, cheeks, chin, with the central - face saving) and in the extensor surfaces of limbs . In older children the lesions are mainly concentrated on the flexural surfaces of the limbs, the popliteal and antecubital folds, back of hands and feet . The skin is commonly dry with lichenification and intense itch; the lips are frequently dry brittle, chapped and develop fissures . The evolution of the clinical feature is characterized by phases of remission of symptoms, which occur mostly during the summer months, alternating with periods of exacerbation, particularly during the autumn - winter . Clinical features typical of adolescence are represented by eyelid dermatitis, and the palmar and plantar juvenil dermatitis; the eczematous lesions are often localized to the neck, and in are often associated with infection by malassezia . The lesions are also localized in forehead, perioral region, neck, upper chest and shoulder girdle, flexor surfaces of the legs and backs of hands . The early onset, the severity of the framework, frequent relapses and chronic course make a disease with important psychological consequences that affect the quality of life (qol) of the children and their families . Affected children often present behavioral problems, mainly characterized by increased emotional dependency, anxiety, and sleep disturbances . The itching, which is one of the main symptoms of the disease, affects mood and sleep quality of patients and consequently of their family . The chronic course characterized by remission and exacerbation and the long - term treatments, negatively affects the quality of family life, both economically and psychologically, producing anxieties, frustrations, creating feelings of guilt and anger . Adolescents with ad exhibit greater vulnerability, anger, anxiety, insecurity, but few studies have been done about it . A recent study study of 367 american adolescents suffering from atopic diseases has highlighted the existence of a significant association between increased levels of anxiety and the presence of allergic respiratory diseases (asthma and rc) but not with the ad; in this study it seems that ad affects the qol in general terms of itching, and sleep disturbance, but it is subjectively experienced by patients as less severe than respiratory diseases . A study by brenninkmeijer et al . Considered a group of 165 patients aged between 18 and 30 years who had ad during childhood . Ad had a highly negative impact on patient qol in the 48.7% of patients, with significant correlation between disease severity and qol (r=0.518, p<0.001). Among the problems identified, patients had experienced the need to receive clearer information about the treatment of the disease (87%), greater empathy with the doctor (85%), the need to deal with other patients with ad (52%) and the need of a psychological support (68%). Furthermore, adolescents with moderate/ severe ad had shown a significant delay in development of social relationships (lower number of friends, reducing output in a group) compared to healthy ones and those with mild ad . During high school, 70% of patients reported to be ashamed of their skin, 49.1% avoided intimate situations, 43.2% abstained from sport, 90% reported intense itching, 69.2% sleep disturbance, fatigue 60.2% and 74.1% physical deterioration of the lesions with stress . A study by saunes et al . Of norwegian adolescents with ad showed a correlation between the severity of clinical feature and increased levels of psychological stress . In particular, the prevalence of psychological distress was highest in the largest age group (1719 years). Although females were more affected by psychological stress than males, the association between ad and psychological distress was higher in males than females (or=2.1 - or=1.3). Sang ho et al . Evaluated the psychological characteristics of 34 patients with ad (age 1341 years) by the following questionnaire: back depression inventory (bdi), state trait anxiety index (stai), interaction anxiousness scale (ias), private body consciousness (pbc) and the dermatology life quality index (dlqi) and found a significant increase of all parameters of the questionnaires and a significant correlation between the parameters of the questionnaires covered and the dlqi (p<0.001). Among the clinical parameters the basis of therapy for the management of ad in adolescents are similar to those of younger child . It may be noted that the adherence to therapy, which often must be at least daily for prolonged periods, is certainly more difficult for the adolescents . To encourage this therapeutic compliance in adolescents, a recent study conducted in the u.s . Used short message service (sms) to the mobile phone to remember to patients the need of a daily therapy . The study showed that daily sending sms to mobile patients, led to significant improvements in patient compliance (p0.001), their self - care (p=0.002), severity (p<0.001) and qol (p=0.014). The need to create an educational and training program to educate, inform and support families with children affected by ad in the management of this disease originates the concept of the school of atopy, on the model of the first educational program initiated in germany: the berlin parental education programme based on a series of meetings between specialists and family . The schools of atopy are differently structured, depending on different countries, although in general the standard provides, in addition to a coordinator or leader, a dermatologist, a pediatrician, an allergist and a clinical psychologist, to which other figures can be added . A study by ricci et al . On a group of children with ad and their parents, who had joined this educational program for 3 years, showed a significant reduction in anxiety levels in parents mostly . Ad in adolescents remains a condition in which is need to investigate several aspects: while the clinical feature is well characterized, there are also risk factors that promote the persistence of ad from the first years of life . It is also necessary to investigate better the relationship with psychological problems, the disturbing presence of ad in adolescence can become, through a vicious circle, the cause of worsening of the same.
Penile cancer is rare in developed countries, whereas in india and other asian countries the incidence is decreasing . The incidence of squamous cell carcinoma (scc) of penis varies worldwide with age, circumcision, and hygiene practices . Phimosis, lack of neonatal circumcision, human papillomavirus (hpv) infection, penile lichen sclerosis, exposure to tobacco products, and psoralen ultraviolet a (puva) are the known risk factors for development of scc of penis . Up to 50% of patients with scc of penis delay seeking medical attention by more than a year due to embarrassment, guilt, fear, ignorance, and personal neglect . The level of denial is substantial, given that the penis is observed and handled on a daily basis . A 60-year - old unmarried male, presented with a history of multiple ulcers over his right groin of 4 months duration . It started as a painless swelling over the right groin, which evolved into multiple ulcers with foul swelling discharge . . He does not give history of arthralgia, loss of weight, or loss of appetite . The patient also gave history of multiple blisters over the trunk and extremities of 1 month duration . There was no history of oral erosions or drug intake prior to the onset of lesions . On general examination, the patient was moderately built and nourished, there was no pallor, icterus, pedal edema, or generalized lymphadenopathy . Examination of the right inguinal region showed three tender excavating ulcers of sizes 6 8, 5 7, and 3 5 cm, respectively, located adjacent to each other with rolled out, everted edges, and foul smelling purulent, blood - stained discharge . Enlarged, grouped, and matted tender inguinal lymphnodes of sizes 5 7 cm were felt below the ulcer . Examination of the left inguinal region also revealed enlarged, grouped, tender inguinal nodes of size 8 6 cm present both above and below the inguinal ligament giving the appearance of groove sign of greenblatt [figure 1]. Right groin showing multiple excavating ulcers, left groin shows groove sign of greenblatt and multiple tense bullae with erosions over thighs and penile shaft genital examination revealed phimosis . A firm, indurated, non tender mass of size 3 2 cm was felt through the prepucial skin on the glans penis, located on the 11o - clock to 2 o - clock position . Multiple tense bullae filled with clear fluid and erosions were present over upper limbs, lower limbs [figure 1], and trunk . There were no oral erosions . With the above clinical findings, a differential diagnosis of lymphogranuloma venereum (lgv) or scc of penis with regional metastasis associated with bullous pemphigoid (bp) occurring as a para neoplastic phenomenon were considered . Igg antibody for chlamydia trachomatis (serovars l1, l2, l3) was negative . Vdrl for syphilis and elisa for human immunodeficiency virus (hiv) 1 and 2 were nonreactive . Wedge biopsy of the ulcer in the right groin and from the penile growth showed ulcerated hyperplastic epidermis with severe dysplasia . The dysplastic epithelial cells invade the basement membrane forming nests with keratin pearls, consistent with keratinizing scc of penis [figure 2]. Skin biopsy taken from a tense bulla over the trunk, showed a sub epidermal bulla with mild lymphocytic infiltrate in the upper dermis consistent with bp . The dysplastic epithelial cells are seen to invade the basement membrane hence, a diagnosis of keratinizing scc of penis with regional lymphnode metastasis and bp was confirmed . Scc of penis may present either as a flat growth, infiltrating, or papillary growth . The lesion is not visible in most patients since the prepuce is non - retractile . The inguinal nodes increase in size followed by erosion of the skin over the nodes . It starts as a small transient, inconspicuous lesion on the genitalia and spreads to regional lymphnodes resulting in bubo and suppurative lymphadenopathy . In our patient, scc of penis was masquerading as lgv since he presented with history of inguinal bubo, which later ulcerated . Moreover, the primary scc was not noticed by the patient and was not visible during examination since the prepuce was non- retractile . A biopsy from the groin ulcer and penile growth that was felt on palpation had confirmed the diagnosis . The occurrence of bp in this patient can be attributed as a paraneoplastic phenomenon to scc of penis . Pemphigoid has been reported in numerous patients with malignancies, like breast cancer, b - cell lymphoma, lung cancer, gastric cancer, cancers of colon or rectum, endometrial cancer, and many others . In scc, this could lead to production of antibodies directed against these antigens that results in the development of bp in these patients . To the best of our knowledge this is the first case of scc of penis with bp occurring as a paraneoplastic phenomenon . We present this case to highlight that scc of penis can masquerade as lgv and a high index of suspicion is required for an early diagnosis of scc penis.
Dental implantation is a treatment method in which fixtures are implanted in the jawbone, followed by prosthetic implantation after a resting period of approximately 36 months, at which time new bone is formed around the fixtures . Bone modeling and remodeling processes are important (1) to induce long - term stability of the implants; (2) to develop osseointegration between implant materials and the bone; (3) to allow the maturation of new bone around the implants . It has been reported that the maximum occlusal force in adults with a natural dentition is 430 n, and similar loads are likely to be applied to implants as well as normal prostheses . Therefore, to achieve long - term retention and stability of implants under such conditions, the quality of newly formed bone around implants is important . Many studies have reported that newly formed bone around implants is spongy bone . However, although the morphology of newly formed bone is reportedly like spongy bone, it is difficult to discriminate whether the bone quality is mature like cortical bone, or immature like spongy, osteoid, or cartilaginous bone; therefore, evaluation of the bone quality is required . Nakano et al . Evaluated bone density and alignment of biological apatite (bap), and boskey and pleshko used fourier - transform infrared (ftir) imaging to assess bone and cartilage quality and composition . In addition, our group has reported on the use of polarized microscopy and scanning electron microscopy (sem) to evaluate new bone and cortical bone quality, as well as microscopic raman spectroscopy to analyze phosphate peaks of bone apatite, and microcomputed tomography (micro - ct) to assess trabecular microarchitecture and bone mineral density (bmd) [810]. However, basic research on new bone formation has been sparse, and molecular and elemental characterization of bap, the basic building block of bone, has generally been overlooked . Moreover, responses to early dynamic loading in implants, with analysis of changes in new bone quality associated with mineralization, remain an important issue for further research . In this study, differences in the bone quality between newly formed bone and cortical bone formed around titanium (ti) alloy implants were investigated using x - ray photoelectron spectroscopy (xps). Six 18-week - old new zealand white rabbits (sankyo labo service co., tokyo, japan) were used in experiments . The rabbits were housed in individual metal cages at a room temperature of 23 1c and humidity of 50 1%, with ad libitum access to food and water . The experimental protocol was approved by an animal experimentation ethics committee (approval number eca 07 - 0016). All experiments were conducted according to the guidelines for the treatment of animals, nihon university, chiba, japan . Implants (diameter, 3.0 mm; length, 7.0 mm) were fabricated using ti-15%zr-4%nb-4%ta (ti-15 - 4 - 4) alloy [1115] (table 1 displays its chemical composition). Surface treatment of implants consisted of sulfuric acid - etching and grit - blasting with apatitic abrasive (particle size 250 m, himed co., ny, usa) (figure 1). Rabbits underwent general anesthesia with 2.0 mg / kg of intravenous ketalar (daiichi sankyo, tokyo, japan). Implant cavities were surgically created in the tibia 10 mm distal to the knee joint, one each bilaterally, by using a 1.0 mm and 3.0 mm diameter round bar, while irrigating the area with sterile saline . Implants were inserted into the right tibia, with each rabbit receiving one implant (8 implants were used in total). Rabbits were sacrificed by anesthesia overdose at 4 or 8 weeks after - implantation, and the tibias were resected . For xps analysis, newly formed bone in close proximity to implants and cortical bone (control) which not in close proximity to implants were analyzed using thin - cut nondecalcified histological specimens . Since both bone resorption by osteoclasts and bone formation by osteoblasts progress concurrently, differences in measurement of bone tissue results are apt to occur; therefore, it is difficult to specify the measurement area . In the present study, more than 3 areas of newly formed bone close to implants and those of cortical bone were measured, and sites within which appropriate average values were obtained were determined as the measurement areas (figure 2). An xps analysis was performed under the following conditions: x - ray source: monochromatic alk (1,486.6 ev), detection region: 20 m, and detection depth: approximately 4 - 5 nm (take - off angle: 45). Qualitative analysis was performed using wide scan measurement, and the chemical bonding conditions of detected elements were analyzed using narrow scan measurement . Hydroxyapatite powders were used as the standard specimens, followed by performance of measurements with a correction for relative sensitivity factors (rsfs). Qualitative analysis of the peak strengths on newly formed bone around implants and cortical bone at 4 and 8 weeks is shown in figure 3 . Although peaks of ca, o, p, c, mg, n, and na were detected, a marked unevenness in these peaks was observed at 4 weeks, in contrast to 8 weeks . Based on the results of wide scan measurement, narrow scan measurement of elements related to ca10(po4)6oh2 was performed . The resulting overlap of c1s, o1s, ca2p, and p2p of newly formed and cortical bone at 4 weeks is shown in figures 3 and 4, respectively; the results at 8 weeks are shown in figure 5 . The results of narrow scan measurement at 4 weeks (figure 4) indicate that, although the peak strengths of newly formed and cortical bone were almost equal at o1s, the half - width became smaller in newly formed bone . At p2p and c1s, although a shifting of the peak in newly formed bone was observed, the half - width became smaller in newly formed bone; the peak strength of newly formed bone at c1s were lower than that of cortical bone . At ca2p (ca2p 3/2), although the peak strengths of newly formed bone was lower than that of cortical bone, the half - width became smaller in newly formed bone, and a shifting of the peak was observed . The chemical bonding condition observed for each element in newly formed bone differed from that of cortical bone . As a result of narrow scan measurement at 8 weeks (figure 5), the half - width and strength of newly formed bone were almost equal to those of cortical bone at c1s, p2p, and ca2p . At o1s, the half - width became small in newly formed bone, and a shifting of the peak was observed . The chemical bonding condition observed for each element of newly formed bone was similar to that of cortical bone . Table 2 shows the results of the quantitative analysis of each element and the ca / p ratio . The results for newly formed bone were ca: 15.07 2.83 weight percent and p: 7.83 1.56 weight percent at 4 weeks; ca: 17.33 2.393 weight percent and p: 8.90 0.80 weight percent at 8 weeks . These values gradually became similar to those of cortical bone at 4 weeks (ca: 17.33 2.39 weight percent and p: 8.90 0.80 weight percent) and at 8 weeks (ca: 21.33 1.88 weight percent and p: 11.03 0.70 weight percent). The use of xps analysis in the present experiment has been employed in industry since the 1960s and is now being clinically applied to the qualitative analysis of several nanometer - sized areas on material surfaces . Changes in spectra reveal alterations in chemical bonds based on changes in interatomic transition and valence - band state density . In the present study, newly formed bone at 4 weeks showed differences in the ca / p ratio (table 2), and in peak position, peak height, and half - width on the narrow scan measurement of each element (figures 4 and 5). Many mineral elements were present in newly formed bone at 4 weeks, suggesting that the arrangement of chemical bonding and element composition in newly formed bone differed from those in cortical bone . Furthermore, the peak position, peak height, and half - width on narrow scan measurement at 8 weeks in newly formed and cortical bone showed similar traces, and the quantitative analysis of new and cortical bone at 8 weeks (table 2) showed similar results . These results suggest that newly formed bone at 8 weeks showed a similar bone quality to that of cortical bone, due to progressive bone maturation and bone metabolism of minerals . Each element showed a complex spectrum with changes in the arrangement of chemical bonding regarding the peak and element composition . The spectrum is related to changes in the chemical bonding of elements caused by interatomic transition and changes in the density or state of the valence band . Under the atomic arrangement of bap crystals in immature new bone, various minor and trace elements, including co3, na, and mg, were substituted for ca, po4, and oh . Such substitutions have been shown to affect the properties of bap crystals . For example, substitution of co3 for po4 in the apatite lattice can cause changes in chemical bonding that leads to strain and reduced crystallite size in bap [16, 17]. Such changes in chemical bonds can result in changes in binding energy in the magnitude of several electron volts (ev). In chemical bonds, trace element peaks shift to higher energy as differences in the electronegativity of bond - forming elements and the electron valence of elements increase . Peak shifts are easily influenced by the surrounding molecular environment, and peak shift and half - width variations signify the presence of compounds with different molecular weights and atomic arrangement . The peak value in a chemical bond is clear with many reports [1821]. The main combined states of each element and a corresponding peak are shown in table 3 . In the present study, differences were observed in xps spectra between new bone at 4 weeks postimplantation and cortical bone, indicating immature bone quality due to bap imperfection . In the chemical bonds of each spectrum, changes in the order of 0.1 ev affected bap crystallinity . The xps spectra of new bone at 8 weeks postimplantation resembled those of cortical bone, and quantitative analysis showed higher ca and p in newly formed bone compared to 4 weeks . This indicated that the composition of newly formed bone was closer to that of cortical bone at 8 weeks . Osseointegration around implants occurs as mineralization progresses, involving the accumulation of mineral components or bap nanocrystals . However, the present study was unable to analyze in detail the chemical bonding present in bap nanocrystals, a subject for future analysis.
Chronic lymphocytic leukemia (cll) is a cancer of elderly people since the average age of patients is over 60 years, although 10% to 15% of patients are under 50 . The neoplastic lymphocytes, mainly of lineage b, are characterized by the expression of at least one of the pan - b antigens (most often cd19) that is co - expressed with the t - cell marker cd5, the expression of cd23, weak expression of cd20 and cd79b, as well as hardly detectable expression levels of surface immunoglobulins with one of the light chains or [2, 3]. In clinical practice, two prognostic tools are used, based on rai s or binet s classifications, further extended by a recently suggested modified system . Since the course of cll is heterogeneous, these classifications are of limited use and are insufficient to predict the disease outcome at the early stages of the cancer . In the last years, some new parameters have been introduced besides the so - called standard prognostic factors such as rai / binet s classifications, lymphocyte doubling time, atypical morphology, bone marrow infiltration, increase in 2-microglobulin concentration, soluble antigen cd23 (scd23) and increased activity of thymidine kinase and lactic dehydrogenase [3, 4]. Among these parameters, the most important prognostic value is attached to the mutational status of immunoglobulin genes (igv h), the expression of tyrosine kinase zap-70 and antigen cd38, as well as cytogenetic aberrations in leukemic cells [59]. For a few years, somatic mutations of i igv h have been considered as a crucial factor determining the course of cll and the responsiveness to therapy [5, 10]. It is widely accepted that neoplastic lymphocytes pass through the germinal centers where they mutate and enter the blood in the state of anergy . Owing to these events, the mutated type of cll (in which b lymphocytes show less than 98% similarity to the igv h genes in the germ line) is characterized by a milder course, longer survival period and higher efficacy of therapy . On the other hand, a lack of mutations in igv h is related to faster progress of the disease, worse prognosis and chemoresistance [5, 10, 12, 13]. Nevertheless, it has been revealed that mutation of the igv h3 - 21 region does not confirm the correlation with favorable outcome and it is associated with an aggressive type of cll . In the assessment of cll prognosis, different sensitive techniques such as polymerase chain reaction (pcr), fluorescence in situ hybridization (fish) and dna microarray are used . Genetic aberrations are detected in more than 80% of patients with cll cases and these are mostly deletions of chromosomes 13 (13q14), 11 (11q22 - 23), 17 (17p) and 7 (7q), as well as trisomy of chromosome 12 [4, 9, 15]. Moreover, a significant prognostic value has been attached to the high expression of tyrosine kinase zap-70 (zeta - associated protein-70), a mediator of the t - cell receptor signaling pathway, and cd38 glycoprotein on the leukemic lymphocyte surface [6, 7]. Up - regulated expression of zap-70 and cd38 is related to cll cases with unmutated igv h [4, 16]. The multi - factorial analyses (igv h mutational status, cd38 and zap-70) of more than 1,000 patients with cll revealed that zap-70 expression had the strongest prognostic value, especially for the time when the therapy should be applied . In the course of cll, leukemic b - cells meet multiple interferences in the apoptotic signal transduction . In cll cells, the expression of proteins such as regulators of apoptosis, belonging mainly to the bcl-2 family, is different from that observed in normal b - cells . The prognostic significance of the bcl-2 proteins, and the polymorphisms of their genes, in cll patients have been recently reviewed in contemporary oncology . Moreover, the expression profiles of small, non - coding rna microrna (mir) can be used to distinguish between normal and leukemic b - cells, and in cll prognosis, as well as in the evaluation of disease progression [1820]. Researchers from carlo m. croce s laboratory published results indicating that the expression of 13 out of 190 analyzed mirs can be useful for differentiating aggressive from benign cll . Importantly, their expression is in close relation with the mutational status of igv h and the level of zap-70 . Mir-15a and mir-16 - 1 have been further characterized, and their genes are located on chromosome 13 (13q14.3), which is the most often deleted (in about 68% of cases) in patients with cll . It is suggested that the deletion of chromosome 13 leads to the silencing of mir-15a and mir-16 - 1 expression, which causes an increase in the synthesis of anti - apoptotic protein bcl-2 . The alterations in lipid metabolism are observed in a large number of cancers that result from e.g. Their increased hydrolysis . Researchers attention has been drawn to lipolytic enzymes, whose activity can be a valuable marker in cll prognosis . Lipoprotein lipase (lpl) is an enzyme belonging to the hydrolase class (ec 3.1.1.34) that participates in lipid metabolism . The human lpl gene, which spans about 30 kbp, was identified on chromosome 8 (8p22) and contains 10 exons (fig . 1a) it has an intron: exon ratio 9, characteristic for a mammalian gene . This enzyme hydrolyzes triglycerides (tgs) present in circulating lipoproteins, such as chylomicrons, very low density and intermediate density lipoproteins (vldl and idl, respectively) into free fatty acids (ffa). Their release supplies the cells with important energetic substrates [29, 30]. To some extent, the aforementioned lipase functions also as phospholipase a1 . A low molecular weight apolipoprotein c - ii (apo c - ii) is a main activator of lipoprotein lipase, whereas its activity is inhibited by apolipoprotein c - iii (apo c - iii). The amino - terminal domain (amino acids from 1 to 310), the carboxy - terminal domain (amino acids from 311 to 448), as well as the most significant functions of depicted fragments are shown in the picture (based on [28, 30, 32]). Lipoprotein lipase is synthesized mainly in the adipocytes and myocytes (including cardiomyocytes), but its relatively high expression is also detected in macrophages, lactating mammary glands, pancreatic islets, several areas of the nervous system, spleen, testicles, ovaries, kidneys, lungs, liver and adrenal glands [28, 29, 3235]. After biosynthesis, the enzyme is secreted into the blood vessels where it anchors by binding to heparan sulfate residues, a component of the endothelial proteoglycans . In the presence of heparin, lpl can be released to the blood, where it exists mainly as inactive monomers metabolized by the liver [26, 29]. The n - glycosylated homodimer is an active form of enzyme in which the subunits (55 kda each) interact non - covalently . Also the glycosylation of asparagine residues, asn and asn, takes place in the er, and the modification of asn is both necessary and sufficient for the activation and release of lpl from this cellular compartment . The process of folding an appropriate lpl conformation requires the participation of chaperone proteins (calnexin and calreticulin), as well as ca ions, and begins from its n - terminal domain [32, 33]. Apart from the catalytic activity, the described enzyme contributes to lipoprotein uptake by the cell that is possible thanks to its participation in the interaction with the ldl receptor - like protein (lrp) or the proteoglycans of target cells . Ldl receptor - like protein molecules can bind to either the active or inactive form of lipoprotein lipase . The binding site of lrp is situated in the c - terminal region of the lpl molecule (fig ., there is a fragment of 24 amino acids (from 415 to 438) that intermediates the binding between lpl and a lipid substrate, and is responsible for stabilization of the active homodimer . The analyses of gene expression in leukemic cells have revealed that many genes are expressed differentially when compared with the normal ones [18, 21, 22, 3638]. In the described cases, these changes are in close relationship with the presence or lack of mutations in the igv h genes . In the so - called unmutated type of cll, the overexpression of at least several dozen genes that make leukemic cells similar to myocytes or adipocytes is claimed . Lpl encoding lipoprotein lipase plays a pivotal role [21, 36, 37]. Since 2005, numerous studies on the expression of lipoprotein lipase in leukemic cells and the evaluation of this parameter as an independent prognostic marker in cll have been conducted . The patients who underwent the assessments formed a heterogeneous group with respect to the stage of the disease, age, gender, the presence of genetic aberrations and applied therapies . Simultaneously, the correlation between the level of lpl and other prognostic factors (e.g. Zap-70, cd38), including mutations of igv h genes, was evaluated [2225, 3845]. Lipoprotein lipase as a new prognostic factor in chronic lymphocytic leukemia efs event - free survival, tfs treatment - free survival, os overall survival, ldt lymphocyte doubling time, ldh lactic dehydrogenase additionally, the index lpl / adam29 (l / a index) was determined . The lpl expression was evaluated at the mrna and protein levels (in other studies lpl expression was measured only at the level of mrna). Apart from mutations in igvh, for which a statistically significant correlation was found in each study (except for). With few exceptions, patients with unmutated type of cll are characterized by significantly higher expression of the lpl gene measured at the mrna level that correlates with more complex course of the disease and worse prognosis . On the other hand, the expression of lpl at a lower level in patients with mutations in igv h genes this correlation was also confirmed at the level of intracellular protein, although this assay had a less significant differentiating value . The complex mechanisms of regulation of lpl gene expression and protein stability may underlie these observations . For that reason, estimation of mrna level seems to be a more informative analysis [22, 44]. Moreover, lpl expression on the cell surface does not correlate with its mrna level inside the cell . Lipoprotein lipase can be not only an independent factor mirroring the stage of the disease, but also a valuable prognostic marker correlating with treatment - free survival (tfs) and overall survival (os). A considerable number of published results indicate that high lpl expression is related to shorter tfs and os [22, 24, 25, 3840, 42, 44, 45]. So far, the application of a specific chemotherapy option has not caused an increase in os so the results obtained in the experiments with treated patients probably did not influence the association between lpl expression and os . Furthermore, the expression of lpl mrna does not change significantly during the course of the disease so that the level of lpl relates both to possible cancer progression in low - risk patients and the patients with more advanced disease (stage b and c according to the binet classification) [23, 38]. Moreover, the high expression of lpl in patients with binet s stage a suggests the need for applying treatment and predicts a poor prognosis even in the case of presence of favorable prognostic parameters [24, 25]. Recently published results have also supported the pivotal role for lpl as a prognostic factor in patients with cancer remission, after application of therapy . It should be emphasized that lpl expression is not a differentiating parameter between patients with mutated type of cll (with favorable prognosis) and those who have mutated vh3 - 21 segments (with poor prognosis). In both cases, for that reason, the assessment of zap-70 expression seems to be a better marker . More attention should be paid to the co - analyses of lipoprotein lipase expression and the product of adam29 (disintegrin and metalloproteinase domain 29-containing protein) gene expression . This gene is localized on chromosome 4 and codes the described enzyme that participates in the cell - cell interactions and between a cell and the extracellular matrix . It should be noted that adam29 is expressed in cll cells at different levels, with respect to mutational status of igv h, but it is not expressed in normal b lymphocytes . In patients with unmutated cll type, it seems that the lpl / adam29 (l / a) ratio is a more sensitive parameter that highly correlates with the mutations of igv h compared with the estimation of these factors separately . It was determined that, in comparison with zap-70 expression, the l / a ratio was the only one that positively correlated with event - free survival . Nevertheless, according to nckel et al ., the evaluation of each of these enzymes can be an independent prognostic factor in the course of cll . Recently published results have provided, apart from the usefulness ensured by the analysis of lpl expression, some practical advantages . Firstly, the analysis can be performed using a quite easy and widely accessible technique, quantitative real - time polymerase chain reaction (qrt - pcr) [2225, 3845]. Secondly, the comparison of lpl expression in cll cells with its level in the peripheral blood mononuclear cells from healthy donors provided the information that lpl expression is highly specific for leukemic cells . In normal blood cells, lpl mrna was absent or very low, and the presence of monocytes that express lpl did not significantly influence the final results . For that reason, the lymphocyte isolation procedure can be omitted without affecting the reliability of the results . This method can be much more simplified because the whole blood lysates can be successfully used to maintain comparative results . Taken together, the methodology of lpl identification predominates over the analysis of mutations in the igv h genes or zap-70 expression because the presence of other cells (t lymphocytes, natural killers) interferes significantly with the results among others due to high expression of kinase zap-70 in these cells [2225, 3845]. The role of lpl in cll cells remains elusive, including the fact that normal b lymphocytes do not possess this enzyme . It seems pivotal to determine whether the expression of this lipolytic enzyme is the effect of the alterations in neoplastic cells or whether it is connected with cll pathogenesis . The results reported by pallasch et al . Seem to support the second hypothesis . These researchers suggest that lpl expression in cll cells is a result of bcr (b - cell receptor) activation . The stimulation of cll cells in vitro, both with somatic mutations in the igv h genes and without them, resulted in lipoprotein lipase expression . Taking into consideration the genesis of both cell types in two cll types, this mechanism could clarify the high lpl expression in the unmutated type of this leukemia . Cll lymphocytes are more sensitive to bcr stimulation . Treatment with a lipase inhibitor, orlistat (tetrahydrolipstatin), resulted in the apoptotic death of leukemic cells, suggesting that changes in expression of lipases (including lipoprotein lipase) underlay this cancer development . Interestingly, the cytotoxic effects of orlistat on primary cll cells was enhanced by their simultaneous exposure to fludarabine, a purine analog commonly used in cll treatment . It seems that both drugs used together acted synergistically in apoptosis induction in leukemic cells . Accepting this point of view, lpl can be not only an important prognostic marker, but also a therapeutic target . The involvement of lpl in lipid metabolism suggests its role in supplying the neoplastic lymphocytes with high - energy substrates . In this context, it could be decisive for the well - being of leukemic cells, influencing their survival and proliferation . This is supported by studies that confirmed high activity of the fatty acid degradation pathway in cll cells . However, including the significant differences between lpl expression with respect to the mutated or unmutated type of cll, it can be presumed that the nature of these cells should be highly perceived . Cll cells without mutations in the igv h genes are characterized by higher sensitivity to activation through surface receptors . For that reason, it is suggested that lpl can contribute to the creation of lipid rafts that participate in b lymphocyte activation . On the other hand, in the cll cells heparin sulfate is synthesized and contributes to stabilization of the described enzyme on the cell surface, and it may be involved in tumor cell migration . The role of the microenvironment of the lymphocytes in chronic lymphocytic leukemia development and progression is undeniable . Interactions with appropriate factors and cells are indispensable in evasion of apoptosis by neoplastic b lymphocytes . Bridge between other molecules supports its participation in the interaction with dendritic and stromal cells . Supporting this, it was noted that surface lpl molecules in patients with unmutated type of cll had lower lipolytic activity although its expression level was higher . It provides a rationale that this enzymatic protein is involved in a process other than lipid metabolism . Lipoprotein lipase is a promising candidate to be an important prognostic factor in chronic lymphocytic leukemia . Owing to the feasibility of its detection, in the near future lpl is expected to become a substitute for dna sequencing in order to evaluate the presence or lack of mutations in the igv h genes, simultaneously providing the equivalent information . For the time being, lpl seems to be as good a prognostic marker as kinase zap-70, or, according to some researchers, even better [24, 39]. An open issue is to define the cut - off value and the standardization of the method used for the detection of lpl expression . The changes in the expression of genes involved in lipid metabolism are probably related to their participation in the development of chronic lymphocytic leukemia, and undoubtedly contribute to cll cell survival . Uncovering the reason for lpl expression in leukemic lymphocytes, whilst lpl is absent in normal cells, would make this knowledge applicable for the new therapeutic options . The novel findings with the lipase inhibitor orlistat provide the rationale for this approach and allow one to speculate that drugs targeting lipid metabolism might be new compounds for the therapy of this leukemia.
Chronic expanding hematoma (ceh) is a rare presentation of a hematoma characterized by a persistent increase in size for more than a month after the initial hemorrhage (1). Ceh presents as a progressively enlarging mass in patients with a history of trauma or surgery because of recurrent intracapsular bleeding from the neovasculature (2). Recently, ceh originating from the paranasal sinuses has been described using two diagnostic terms including organized hematoma or organizing hematoma which there has been no consensus on diagnostic term of this disease entity (3). Organizing hematoma of the paranasal sinuses is a diagnostic dilemma clinically and radiographically, mimicking benign, including fungal diseases, or malignant neoplastic processes and causing patients and clinicians undue worry regarding these diagnoses (3). Although the diagnostic rate of this disease entity has been increased, as characteristic imaging findings are somewhat elucidated, endoscopic examination and computed tomography (ct) imaging do not give helpful information in differentiating these lesions from malignant neoplastic processes (3). Also, because distinct pathological differences are observed between the basal and peripheral portions of organizing hematomas, preoperative biopsy taken from the periphery may usually be inconclusive (4). Recently, magnetic resonance imaging (mri) can lead to positive diagnosis by recognizing the distinct outer rims, called however, the most important limitation of mri is that patients with severe claustrophobia may not be able to tolerate mri procedures in closed gantry systems . Positron - emission tomography (pet) with f18-fluorodeoxyglucose (fdg) is an evolving diagnostic modality used for tumor detection, staging, therapeutic monitoring, and follow - up of various malignant tumors . Only five reports about fdg - pet imaging features of cehs originating from other sites of the body have been documented (5 - 9). To the best of our knowledge, fdg - pet images of organizing hematoma of the maxillary sinus have not been previously reported . In this report, we present f - fdg - pet / ct findings in organizing hematoma of the maxillary sinus (ohms) compared to concurrent ct and mri findings . He had no history of antecedent trauma, nasal surgery, or bleeding diatheses and no facial numbness or pressure . On endoscopic examination, the left lateral nasal wall was medially displaced towards the nasal septum, blocking the nasal passage completely . So, it was not possible to examine the nasal cavity endoscopically because of the narrowed passage . After shrinkage of the inferior turbinate mucosa, we observed a lobulated pinkish mass in the inferior meatus that caused medial displacement of the inferior turbinate (figure 1). Preoperative biopsy was performed from the mass of the inferior meatus, but the results demonstrated only vascular fibrous tissue with chronic inflammation . Enhanced ct of the paranasal sinuses showed a 4.3 2.8 4.3 cm sized heterogeneous enhanced soft tissue mass in the maxillary sinus, the central region of which was in particular strongly enhanced (figure 2). The expansion of the maxillary sinus and bony thinning of the posterior and medial wall of the left maxillary sinus were also noted . The mass caused obstruction of the left nasal cavity with medial displacement of the middle and inferior turbinate . Ct findings were highly suspicious for a malignant tumor of the maxillary sinus . He simultaneously underwent mri and whole - body combined f - pet / ct for evaluation of a malignant tumor of the maxillary sinus . T1-weighted mri images revealed a large, lobulated mass that was mostly isodense to the inferior turbinate diffusely interspersed with hyperintensity . T2-weighted images showed the marked heterogeneity of the lesion with a mix of hypointense (peripheral part), isointense, and hyperintense (central part) signals and a dark peripheral rim surrounding the lesion . Bright signal intensity due to mucosal inflammation of the involved maxillary sinus was distinguished from the lesion on t2-weighted images (figure 3). Integrated f - fdg - pet / ct images showed moderate uptake only at the peripheral rim of the mass with negative fdg uptake in the central portion . Other signs of abnormal uptake suggesting a malignant lesion were not observed (figure 4). Based on pet / ct and mri, the provisional diagnosis of benign tumor rather than malignancy was made . Complete resection of the mass was achieved by simple transnasal endoscopic surgery with caldwell - luc approach . Histologically, fibrous tissue, neovascularization, and extravasated red blood cells in the subepithelium with no evidence of malignancy is a combination characteristic of organizing hematoma . The patient s postoperative course was uneventful and his symptoms improved markedly . There has been no sign of recurrence 1 year after the operation . Although most hematomas resolve spontaneously, a few can persist for long periods forming slowly expanding space - occupying masses (1). This expanding process is attributable to the irritant effects of blood and the products of its breakdown, which induce repeated episodes of bleeding from the capillaries in the granulation tissue (10). Sinonasal organizing (organized) hematoma is a rare type of ceh originating from the paranasal sinuses whose clinical characteristics lead to be misdiagnosed as a malignant or locally aggressive neoplasm (3). The number of reported cases has dramatically risen since 2005 because of elucidation of the characteristic imaging findings and the specific pathologic findings (11 - 15). Because the maxillary sinus is the largest paranasal sinus that allows negative pressure, significant leakage of blood may develop in the nasal cavity into the sinus through the ostium (15). Therefore, organizing hematoma most commonly affects the maxillary sinus and is known as ohms (11). Although diagnostic criteria for correct identification of ohms are not well known (3), corrective preoperative diagnosis is important to avoid unnecessary extensive surgery because this condition is curative with a simple, conservative endoscopic approach and rarely recurs (11). The reported ct findings of organizing hematoma include an expansive, clearly demarcated and compressive mass causing bony erosion without a destructive feature (14). After administration of contrast, heterogeneous enhancement in a patchy distribution is usually found within the lesion (15, 16). Mri is superior to ct for determining the margin and extent of the lesion . In mr images, it has biphasic appearance in which the central part of the lesion presents as low intensity on t1-weighted images and high intensity on t2-weighted images, corresponding with areas of fresh hemorrhage and the peripheral part is less enhanced with a zone of fibrosis, especially called as shells of t2 hypointensity which is the most diagnostic finding on mri (3, 14, 17). However, if it is not possible to differentiate benign tumors from malignancy by ct and mri, or if the patient cannot perform mri scan because of severe claustrophobia, definite preoperative diagnosis is difficult . Cehs can be misdiagnosed as malignant tumors because of their large size and slow, progressive enlargement (5, 7). Although fdg - pet images of ceh are not widely available, fdg - pet shows moderate fdg uptake, the maximum standardized uptake value (suvmax) of 3.1 to 5.5, only at the periphery of the mass with central photon defects that is probably characteristic of ceh, suggesting chronic inflammation or granulation tissue in the fibrous wall (5 - 9). If the suvmax were set to a cut - off point of 3.0 to distinguish between benign and malignant tumors, these lesions could be misdiagnosed as malignant tumors (5). However, the use of fdg - pet imaging for tumor diagnosis is limited by the fact that fdg, a glucose analog, is taken up not only by tumor cells but also by macrophages and immature granulation tissue containing fibroblasts and inflammation (18). Ceh has abundant granulation tissue with neovascularization, macrophages and inflammation, which corresponds to organizing processes . These pathological changes are considered to have resulted in increased fdg uptake within the organizing hematoma (5 - 9). In our patient, fdg - pet images of the lesion revealed central photon defects with moderately increased fdg uptake in the peripheral rim of the space - occupying mass in the left maxillary sinus . Although the fdg uptake pattern might be seen if a malignant tumor has a tendency of central necrosis, the characteristics of fdg - pet images of ohms and ceh, including fdg uptake in the peripheral rim with central photon defect of the mass as a result of inflammation should be recognized as a potential interpretive pitfall that mimics a malignant tumor (5). In summary, we have presented fdg - pet findings of ohms that showed an increased fdg uptake in the peripheral rim of the mass with central photopenia . To our knowledge, this is the first report in literature reporting fdg - pet findings of ohms with suvmax values suggestive of a malignancy . Careful interpretation of metabolic (fdg - pet / ct) and anatomic (ct and mri) images should be performed to accurately characterize the expansile lesion of the maxillary sinus in order to increase specificity and reduce equivocal findings significantly.
Chemicals: bpa was purchased from kanto chemical co. (tokyo, japan); bpa glucuronide and bpa glucuronide / sulfate diconjugate were obtained from frontier science co. (ishikari, japan); and all other chemicals for the liver perfusion study and high performance liquid chromatography (hplc) analysis were purchased from kanto chemical co. animals: male (330400 g) and female (240280 g) sprague - dawley rats (911 weeks old) were used . Before use in experiments, all rats were housed under standard conditions and given food and water ad libitum . The animals were handled according to the laboratory animal control guidelines of rakuno gakuen university (ethics committee protocol approval number es23a06, approved jan 13, 2012). Surgical procedure for perfusion: to study perfusion, the rats were anesthetized by intraperitoneal injection of pentobarbital sodium (1.3 ml/100 g body weight). Briefly, after anesthesia, the abdomen was surgically opened, and the portal vein and common bile duct were cannulated and the caudal vena cava was incised . Oxygenated krebs - ringer buffer, described below, was infused by roller pump (mp-32n; eyela, tokyo, japan) through the liver via the portal vein at a constant rate of 30 ml / min . Once perfusion was begun, a polyethylene catheter was inserted into the vena cava . After insertion of the polyethylene catheter, each animal was euthanized by exsanguination under anesthesia . Liver perfusion: krebs - ringer buffer (nacl 115 mm, kcl 5.9 mm, mgcl2 1.2 mm, nah2po4 1.2 mm, na2so4 1.2 mm, cacl2 2.5 mm, nahco3 25 mm and glucose 10 mm) was used in all experiments . The buffer solution was aerated using 95% o2 + 5% co2, and the ph was adjusted to 7.4 . Bpa was added to the substrate buffer solution at a final concentration of 0.1 m, 1 m or 10 m . Preliminary perfusion with krebs - ringer solution was performed for 10 min, followed by 5 min inflow of the substrate buffer solution, and then reperfusion of krebs - ringer solution for 55 min . Once perfusion of the substrate buffer had begun, the excreted bile and a small amount of the perfusate in the vein were collected independently at 5-min intervals for 1 hr . High performance liquid chromatography (hplc) analysis of reaction products: the perfusate samples were independently centrifuged for 3 min at 9,000 g, and the supernatant fraction was collected . The samples were analyzed by a hplc (tosoh, tokyo, japan) system consisting of an lc-8020 pump, co-8020 oven and uv-8020 uv detector . The samples were subjected to unizon uk - c18 reversed phase column (4.6 mm i.d . 150 mm; imtakt, tokyo, japan) chromatography and eluted with a linear gradient of methanol / water at flow rate of 1 ml / min (solution a: methanol / water (24/76 v / v) and 10 mm ammonium acetate to solution b: methanol) over 15 min . The eluted samples were analyzed at 222 nm, and the results were recorded using lc-8020 integration software (tosoh). The elution peaks of bpa, bpa glucuronide and bpa glucuronide / sulfate were noted, and the concentrations were compared with the standards . Liquid chromatography - mass spectrometry conditions: liquid chromatography - electrospray ionization - time of flight mass spectrometry (lc - esi - tof ms) analysis was conducted using a lct premier mass spectrometer (waters, milford, ma, u.s.a . ). The mass spectrometer electrospray source capillary voltage was set to 2.2 kv, cone voltage to 20 v, source block temperature to 120c and desolvation temperature to 350c . The nitrogen nebulizing and desolvation gas flows were set to 50 l / hr and 650 area under the curve (auc) was used for comparison of bilious and venous excretion of bpa and its conjugates . Comparisons were made by either a student s t test or analysis of variance, and a p value of 0.05 was taken to be significant . Hplc analysis of reaction products: in the hplc samples obtained from rat liver perfused with 10 m bpa in krebs - ringer solution, two major peaks were eluted at 3.9 min and 7.2 min (fig . 1.hplc chromatograms of bile and venous perfusate derived from rat liver perfused with bpa . The liver was perfused for 5 min with krebs - ringer buffer containing bpa and then perfused with normal krebs - ringer buffer . Bile (a) and venous perfusate (b) were collected at 15 min after application of bpa to the liver . Peaks of bpa glucuronide / sulfate diconjugate (a) and bpa mono - glucuronide (b) are indicated . Panels d and e are a mass spectrum of peak a and peak b, respectively . ). In lc - ms analysis, m / z of peak a (rt=3.9 min) and peak b (rt=7.2 min) were 455.06 and 375.10, respectively . The first peak (peak a), detected mainly in the bile, was identified as bpa glucuronide / sulfate diconjugate (fig . The second peak (peak b), found in both the bile and venous perfusate, was identified as bpa mono - glucuronide (fig . The limits of detection for the bisphenol a glucuronide / sulfate diconjugate and the mono - glucuronide were 0.1 m and 0.2 m, respectively . The standard curves for the diconjugate and the mono - glucuronide resulted in a high linearity in a range of 1500 m . Hplc chromatograms of bile and venous perfusate derived from rat liver perfused with bpa . The liver was perfused for 5 min with krebs - ringer buffer containing bpa and then perfused with normal krebs - ringer buffer . Bile (a) and venous perfusate (b) were collected at 15 min after application of bpa to the liver . Peaks of bpa glucuronide / sulfate diconjugate (a) and bpa mono - glucuronide (b) are indicated . Panels d and e are a mass spectrum of peak a and peak b, respectively . Bpa conjugation and excretion in the perfused liver of male rats: on perfusion of the liver with 10 m bpa in krebs - ringer solution, 99.6% of the substrate was transferred to the liver . Bpa mono - glucuronide and glucuronide / sulfate diconjugate were subsequently excreted from the liver . The greatest glucuornide excretion was observed 1015 min after substrate application (fig . 2.bpa conjugates excreted into the bile (a) and vein (b) after liver perfusion of male sprague - dawley rats with bpa . The liver was perfused for 5 min with 10 m bpa in krebs - ringer solution and for 55 min without substrate . Bpa conjugates excreted into the bile (a) and vein (b) after liver perfusion of male sprague - dawley rats with bpa . The liver was perfused for 5 min with 10 m bpa in krebs - ringer solution and for 55 min without substrate . On application of low - doses of bpa to the perfused liver, the excretion of the conjugates diminished in a dose - dependent manner (fig . 3fig . Columns show total excretion of bpa glucuronide (hatched line) and diconjugate (empty columns) into the bile (a) and venous system (b) during 1-hr perfusion . The excretion of diconjugate was not detected following 15 mol application (0.1 m perfusion; fig . The venous excretion of glucuronide / sulfate was below quantifiable levels in all trials in this study (fig . Columns show total excretion of bpa glucuronide (hatched line) and diconjugate (empty columns) into the bile (a) and venous system (b) during 1-hr perfusion . Bpa conjugation and excretion in the liver of female rats: in female rats, liver perfusion of 10 m bpa in krebs - ringer solution resulted in up to 100% of the infused substrate being absorbed into the liver tissue, which was subsequently glucuronidated and excreted into the bile and the venous system . The greatest glucuornide excretion was observed 10 min after application of the substrate (fig . 4.bpa conjugates excreted into the bile (a) and venous system (b) after perfusion of the liver of female rats with bpa . The liver was perfused for 5 min with 10 m bpa in krebs - ringer solution and for 55 min without substrate . Bilious glucuronide excretion was estimated to be much higher than that of venous excretion . Negligible amounts of diconjugate were excreted from the liver of female rats (fig . Bpa conjugates excreted into the bile (a) and venous system (b) after perfusion of the liver of female rats with bpa . The liver was perfused for 5 min with 10 m bpa in krebs - ringer solution and for 55 min without substrate . Bilious and venous excretions of the resulting conjugates during 1-hr perfusion in male and female rats are illustrated in fig . 5fig . 5.bpa conjugates excreted into the bile (upper graph) and venous system (lower graph) during 1-hr perfusion . The livers of male (left graph) and female (right graph) rats were perfused for 5 min with 10 m bpa in krebs - ringer solution and for 55 min without substrate . Results are shown as the mean s.e .. in male rats, the amounts of bilious and venous excretions of mono - glucuronide were 49.4% and 9.9% of the infused bpa, respectively . In female rats, the amounts of bilious and venous glucuronide were 73.7% and 10.1%, respectively . The excretion via glucuronide / sulfate diconjugate production in male rats was calculated as 22.3% of the infused bpa . Total recovery of the infused substrate was 81.9% in male rats and 83.9% in female rats . Bpa conjugates excreted into the bile (upper graph) and venous system (lower graph) during 1-hr perfusion . The livers of male (left graph) and female (right graph) rats were perfused for 5 min with 10 m bpa in krebs - ringer solution and for 55 min without substrate . In sprague - dawley rats perfused with bpa, the infused compound was highly conjugated during passage through the liver, and the resultant metabolites were excreted from the liver . One - quarter of the infused bpa was eliminated as a glucuronide / sulfate diconjugate, whereas the diconjugate was virtually absent in female rats . These findings concur with a recent report, in which bpa, added to the medium of isolated hepatocytes, was metabolized into both mono - glucuronide and diconjugate; moreover, the diconjugate was almost nil in female rats . Bpa sulfo - conjugation is mediated by the sult1 family, a phenol sulfotransferase isoform . One member of the sult1 family, sult1a1, has been shown to have high - conjugation activity toward bpa . The expression level of the sult1 family is estimated to be higher in male than female rats . In contrast to the sulfotransferases, the ugt2 isoenzyme, which mediates bpa glucuronidation, exhibits gender differences in mrna expression; higher levels were shown in females compared to males . These results suggest that sex differences in the conjugation pathways of bpa are responsible for the expression level of the conjugation enzymes . In the present study, this phenomenon can be explained by the two - step conjugation of bpa, in which a carboxy radical of the substrate is glucuronidated and an addition radical is subsequently sulfated . Udp - glucuronosyltransferases are located on the endoplasmic reticulum, while sulfotransferases are found in the cytosol . It is presumable that in hepatocytes, lipophilic bpa crosses the plasma membrane and reaches the endoplasmic reticulum, where it is glucuronidated and diffuses into the cytosol . After conjugation, the resulting hydrosoluble metabolites are likely transported from inside the cell to outside via chemical transporters . We found that in rat liver, the bilious excretion of bpa mono - glucuronide is mediated by mrp2 (abcc2), a member of the atp - binding cassette (abc) family . Pharmacokinetic studies involving 4-methylumbelliferone and ethinyl estradiol demonstrate that bcrp (abcg2) mediates the transport of glucuronide and sulfate [21, 22]. While identification of the transporter exporting bpa diconjugate requires further investigation, our previous study and recent studies suggest that the abc family mediates the export of diconjugate . On application of low - dose bpa to the perfused liver in the present study, bpa diconjugate levels were negligible; however, the reaction increased in a dose - dependent manner . This indicates that bpa elimination via diconjugation occurs upon high exposure to the chemical . In animal studies of the health effects of bpa, long - term / low concentration exposure or short - term / our present results give rise to the view that, depending on the exposure level, different metabolic pathways are involved in the elimination of bpa . From bile, the excreted conjugates flow into the intestinal tract, where the metabolites are reabsorbed into the body . An in vivo study by kurebayashi et al . Showed that oral bpa exposure flows into the enterohepatic circulation before excretion to the urine and feces . In light of these findings, we suggest that the bpa conjugates pass through internal organs, such as the heart, lung, kidney and brain, before being eliminated from the body . Of concern our previous data showed that bpa glucuronide is absorbed from the maternal blood stream at the placenta . Recently, chemical transporters involved in the movement of hormone - sulfates have been identified in the placenta [18, 23]. It is highly likely that bpa diconjugate is actively transferred into the fetus at the placenta . Demonstrated the production of a bpa glucuronide / sulfate diconjugate in female mice using isolated hepatocytes . Given that greater diconjugate production in hepatocytes has been found in females, it is important that further work be done to determine the fate of the diconjugate in the pathway before excretion . In conclusion, the present study assessed bpa conjugation with mono - glucuronide and glucuronide / sulfate using a liver perfusion method . The results suggest that in the rat liver, bpa is conjugated primarily to mono - glucuronide and that diconjugate production occurs when the animal is exposed to high concentrations of the substrate . The potential public health hazards of bpa remain unknown . To elucidate the adverse effects of bpa systemically, further work focusing on the changes in bpa metabolism according to the dose is required.
Copd is a progressive disease characterized by persistent airflow limitation, chronic and progressive dyspnea, cough, and sputum production and is often complicated by exacerbations.1 copd affects approximately 24 million us adults, including 12.7 million diagnosed patients and 12 million undiagnosed, and is the third leading cause of death in the united states.2,3 the annual cost of copd in 2010 was estimated at $49.9 billion usd, with $29.5 billion attributed to direct health care costs, in which hospital care accounted for the largest share.4 treatment goals for patients with stable copd include symptom relief, prevention of disease progression, and prevention and treatment of exacerbations.1 while symptom relief is the primary target for pharmacologic therapy, prevention of exacerbations is also important because of the impact on lung function, health - related quality of life, hospitalization, and mortality . Recent copd treatment guidelines emphasize the central role of bronchodilators, including 2-agonists and anticholinergics, with drug selection depending on patient response and tolerability . Inhaled corticosteroids (ics) have a more limited role for patients unable to achieve symptom control with bronchodilators alone.1 roflumilast is a selective phosphodiesterase-4 inhibitor approved by the us food and drug administration in 2011 . It is indicated as a treatment to reduce the risk of copd exacerbations in patients with severe copd associated with chronic bronchitis and a history of exacerbations.5 no studies have compared roflumilast to other copd maintenance medications, and there is very little real - world data on utilization, clinical, and economic outcomes associated with the use of roflumilast . We hypothesized that there were no differences in the relevant clinical and economic outcomes between roflumilast and other copd maintenance medications . This was a longitudinal, retrospective cohort study using us managed - care administrative data from the lifelink pharmetrics plus health plan claims database, which includes medical and pharmaceutical claims for over 150 million unique patients from over 90 health plans across the united states . The prescription claims include national drug code, drug names, dose, strength, days supply, and quantity dispensed . It also includes demographic variables, insurance information, and plan enrollment / eligibility data . We cleaned the obtained raw data by removing the duplicates and observations with missing values for key data elements, such as age, diagnosis date, and prescription date . The data in the claims databases are de - identified and fully compliant with the health insurance portability and accountability act privacy regulations, exempting this study from an institutional review board approval requirement . Selection of patients for the study is outlined in figure 1 . To be included in the analysis, patients had to have at least one medical claim with a primary diagnosis of copd (international classification of diseases, ninth revision, clinical modification [icd-9-cm] code 491.xx, 492.xx, or 496.xx) between may 1, 2010 and september 30, 2012; have initiated therapy with roflumilast or a third copd maintenance medication between may 1, 2011 and september 30, 2012 (cohort selection period); and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date, defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . According to gold (global initiative for chronic obstructive lung disease) guidelines,1 roflumilast is recommended as the third maintenance agent for patients with severe copd . To ensure disease severity was as comparable as possible between the comparison group (patients who did not receive roflumilast) and the group of patients who received roflumilast, patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent patients were excluded if they were younger than 40 years of age, had at least one medical claim with a diagnosis code for cystic fibrosis (icd-9-cm code 277.xx) or respiratory tract cancer (icd-9-cm codes 160.xx164.xx or 231.xx) during the study period, were corticosteroid dependent (defined as 182 day supply of oral corticosteroids in the year before or after the index date), had at least one medical claim with a diagnosis of asthma (icd-9-cm codes 493.0, 493.1, or 493.9) during the 12-month preindex period (ie, baseline period), or received roflumilast in combination with theophylline . Study patients were separated into two cohorts: patients who initiated roflumilast during the cohort selection period (roflumilast group) and patients who initiated the third of three copd maintenance medications during the cohort selection period (non - roflumilast group). Patient demographics included age at the index date, sex, insurance type, and census region . Clinical characteristics included comorbid conditions, reported as a prevalence per condition and used to calculate charlson comorbidity index;6 number of copd medications and days using copd medications during the 12-month preindex period (termed as baseline period); number of copd exacerbations (defined in the following paragraph); hospitalizations; and emergency department (ed) visits during the baseline period . The primary outcome was the rate of moderate and severe copd exacerbations per patient per month (pppm) over the 3 months after the index date . Severe exacerbation was defined as a hospital admission with a primary diagnosis of copd with acute exacerbation (icd-9-cm codes 491.21, 491.22, 493.22, or 492.8) or a primary diagnosis of respiratory failure (icd-9-cm codes 518.81, 518.82, or 518.84) combined with a secondary diagnosis of copd with acute exacerbation or emphysema (491.0, 491.1, 491.21, 491.22, 491.8, 491.9, 492.0, 492.8, 493.22, or 496).7 moderate exacerbation was defined as a short course (14 days) of oral corticosteroids within 7 days of an ed visit or an office visit with a copd diagnosis (icd-9-cm codes 491.xx, 492.xx, and 496.xx); an ed visit with a primary diagnosis of copd with acute exacerbation or a primary diagnosis of respiratory failure combined with a secondary diagnosis of copd with acute exacerbation or emphysema, as defined earlier; or an ambulatory claim with a diagnosis of copd or another qualifying pulmonary condition (icd-9-cm codes 136.3, 466466.19, 480486, 487.0, 490, 491.21, 491.22, 493.02, 493.12, 493.22, 493.92, 494.1, 506.0506.3, 507507.8, 511.0511.1, 512512.8, 517.1, 518.0, 518.81, 518.82, 518.84, 770.84).8 events occurring within 15 days of each other were counted as a single exacerbation . Secondary outcomes included health care resource utilization (hcru), including the number of inpatient hospitalization admissions, ed and office visits, and total health care costs . Costs were inflated to 2012 values using the 2012 consumer price index provided by the bureau of labor statistics.9 bivariate comparisons of patient demographics and baseline characteristics between the roflumilast and non - roflumilast groups were performed using pearson chi - square tests for categorical variables and t - tests for continuous variables . Wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates (pppm), monthly hcru, and monthly costs were calculated for each patient, and difference - in - difference (did) linear regression models (appendix) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier . The did model was used to control for observed baseline differences between treatment and control groups (eg, severity of disease), as well as the effects of all time - varying determinants of the outcomes (trend change in the outcomes over time) that are common to both study groups.11 it is worth noting that the did model is based on an assumption that in the absence of treatment the difference between control and treatment groups would be constant or fixed over time . Analyses were performed using statistical analysis software version 9.2.3 (sas institute, cary, nc). For all analyses, statistical significance was defined as a p - value <0.05 . This was a longitudinal, retrospective cohort study using us managed - care administrative data from the lifelink pharmetrics plus health plan claims database, which includes medical and pharmaceutical claims for over 150 million unique patients from over 90 health plans across the united states . The prescription claims include national drug code, drug names, dose, strength, days supply, and quantity dispensed . It also includes demographic variables, insurance information, and plan enrollment / eligibility data . We cleaned the obtained raw data by removing the duplicates and observations with missing values for key data elements, such as age, diagnosis date, and prescription date . The data in the claims databases are de - identified and fully compliant with the health insurance portability and accountability act privacy regulations, exempting this study from an institutional review board approval requirement . Selection of patients for the study is outlined in figure 1 . To be included in the analysis, patients had to have at least one medical claim with a primary diagnosis of copd (international classification of diseases, ninth revision, clinical modification [icd-9-cm] code 491.xx, 492.xx, or 496.xx) between may 1, 2010 and september 30, 2012; have initiated therapy with roflumilast or a third copd maintenance medication between may 1, 2011 and september 30, 2012 (cohort selection period); and have continuous enrollment in the managed - care plan for at least 12 months before and 3 months after the index date, defined as the date of initiation of roflumilast or the first prescription of the third copd maintenance medication . According to gold (global initiative for chronic obstructive lung disease) guidelines,1 roflumilast is recommended as the third maintenance agent for patients with severe copd . To ensure disease severity was as comparable as possible between the comparison group (patients who did not receive roflumilast) and the group of patients who received roflumilast, patients who received at least three copd agents were included as the comparison group and the start of the third copd maintenance agent patients were excluded if they were younger than 40 years of age, had at least one medical claim with a diagnosis code for cystic fibrosis (icd-9-cm code 277.xx) or respiratory tract cancer (icd-9-cm codes 160.xx164.xx or 231.xx) during the study period, were corticosteroid dependent (defined as 182 day supply of oral corticosteroids in the year before or after the index date), had at least one medical claim with a diagnosis of asthma (icd-9-cm codes 493.0, 493.1, or 493.9) during the 12-month preindex period (ie, baseline period), or received roflumilast in combination with theophylline . Study patients were separated into two cohorts: patients who initiated roflumilast during the cohort selection period (roflumilast group) and patients who initiated the third of three copd maintenance medications during the cohort selection period (non - roflumilast group). Patient demographics included age at the index date, sex, insurance type, and census region . Clinical characteristics included comorbid conditions, reported as a prevalence per condition and used to calculate charlson comorbidity index;6 number of copd medications and days using copd medications during the 12-month preindex period (termed as baseline period); number of copd exacerbations (defined in the following paragraph); hospitalizations; and emergency department (ed) visits during the baseline period . The primary outcome was the rate of moderate and severe copd exacerbations per patient per month (pppm) over the 3 months after the index date . Severe exacerbation was defined as a hospital admission with a primary diagnosis of copd with acute exacerbation (icd-9-cm codes 491.21, 491.22, 493.22, or 492.8) or a primary diagnosis of respiratory failure (icd-9-cm codes 518.81, 518.82, or 518.84) combined with a secondary diagnosis of copd with acute exacerbation or emphysema (491.0, 491.1, 491.21, 491.22, 491.8, 491.9, 492.0, 492.8, 493.22, or 496).7 moderate exacerbation was defined as a short course (14 days) of oral corticosteroids within 7 days of an ed visit or an office visit with a copd diagnosis (icd-9-cm codes 491.xx, 492.xx, and 496.xx); an ed visit with a primary diagnosis of copd with acute exacerbation or a primary diagnosis of respiratory failure combined with a secondary diagnosis of copd with acute exacerbation or emphysema, as defined earlier; or an ambulatory claim with a diagnosis of copd or another qualifying pulmonary condition (icd-9-cm codes 136.3, 466466.19, 480486, 487.0, 490, 491.21, 491.22, 493.02, 493.12, 493.22, 493.92, 494.1, 506.0506.3, 507507.8, 511.0511.1, 512512.8, 517.1, 518.0, 518.81, 518.82, 518.84, 770.84).8 events occurring within 15 days of each other were counted as a single exacerbation . Secondary outcomes included health care resource utilization (hcru), including the number of inpatient hospitalization admissions, ed and office visits, and total health care costs . Costs were inflated to 2012 values using the 2012 consumer price index provided by the bureau of labor statistics.9 bivariate comparisons of patient demographics and baseline characteristics between the roflumilast and non - roflumilast groups were performed using pearson chi - square tests for categorical variables and t - tests for continuous variables . Wilcoxon rank - sum tests were used to compare the hcru and cost data between study groups because they usually have a skewed distribution.10 the differences between baseline and postindex exacerbation rates (pppm), monthly hcru, and monthly costs were calculated for each patient, and difference - in - difference (did) linear regression models (appendix) were used to adjust for covariates identified from the bivariate comparisons between roflumilast and non - roflumilast patients as described earlier . The did model was used to control for observed baseline differences between treatment and control groups (eg, severity of disease), as well as the effects of all time - varying determinants of the outcomes (trend change in the outcomes over time) that are common to both study groups.11 it is worth noting that the did model is based on an assumption that in the absence of treatment the difference between control and treatment groups would be constant or fixed over time . Analyses were performed using statistical analysis software version 9.2.3 (sas institute, cary, nc). For all analyses, statistical significance was defined as a p - value <0.05 . Of 818,457 patients identified as having copd using icd-9 codes, 804,246 met at least one of the criteria for exclusion (figure 2). The major reasons for exclusion were absence of claims for copd maintenance medications during the cohort selection period (n=501,556) and not receiving at least three maintenance copd medications during the cohort selection period (n=110,320). After these exclusions, there were 710 patients in the roflumilast group and 13,501 in the non - roflumilast group . Patients in the roflumilast group were more likely to have comorbid congestive heart failure, had a higher number of monthly exacerbations, were receiving more copd medications at baseline, had more ed visits, and were more likely to be hospitalized during the 12-month preindex period . There were no statistically significant differences in age, sex, or charlson comorbidity score between groups . In the non - roflumilast group, the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist (laba) and ics (47.2%), followed by a combination of those two classes plus a long - acting muscarinic antagonist (46.1%). In the roflumilast group, 33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist, laba, and ics, 30.4% were receiving roflumilast monotherapy (of note, these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast), and 18.9% were receiving a combination of roflumilast, laba, and ics . Figure 3 shows the change in overall rate of exacerbation pppm in the roflumilast and non - roflumilast groups from baseline . During the follow - up period, the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group (figure 3a; p<0.001 for did). While the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group, however, there was a significant difference in the rate of moderate exacerbation pppm between groups, with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations (figure 3c; p=0.001). After controlling for the key covariates (age, sex, region, insurance, baseline number of copd drugs, and charlson comorbidity index) using a did model approach, roflumilast patients still had a greater reduction in the rate of overall exacerbation (=0.0160, p=0.01) and moderate exacerbation (=0.0149, p=0.01) versus the non - roflumilast group . There was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates; however, the difference was not statistically significant (=0.0012, p=0.63). Monthly number of office visits, ed visits, and inpatient hospitalization admissions at baseline and during follow - up are shown in table 2 . During both the baseline and follow - up periods, patients in the roflumilast group had more of each type of encounter than patients in the non - roflumilast group . Both treatment groups had increased office visits, ed visits, and hospitalizations during the follow - up period compared to the baseline period . However, the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions (0.0020.152 vs 0.0050.123, p=0.024) and office visits (0.0810.938 vs 0.1220.889, p=0.01), but not for the ed visits . After controlling for key covariates using did models, roflumilast was associated with fewer hospital admissions (=0.003, p=0.57) and office visits (=0.46, p=0.26); however, the differences were not statistically significant . Table 3 shows total monthly health care costs in the roflumilast and non - roflumilast groups during the baseline and follow - up periods . During the baseline and follow - up periods, costs increased for both groups from the baseline to the follow - up period, with no significant difference in the change from baseline between groups . After controlling for the key covariates using a did model, the total cost increase was $116 more for the control of patients not treated with roflu - milast versus patients treated with roflumilast (p=0.62). Of 818,457 patients identified as having copd using icd-9 codes, 804,246 met at least one of the criteria for exclusion (figure 2). The major reasons for exclusion were absence of claims for copd maintenance medications during the cohort selection period (n=501,556) and not receiving at least three maintenance copd medications during the cohort selection period (n=110,320). After these exclusions, there were 710 patients in the roflumilast group and 13,501 in the non - roflumilast group . Patients in the roflumilast group were more likely to have comorbid congestive heart failure, had a higher number of monthly exacerbations, were receiving more copd medications at baseline, had more ed visits, and were more likely to be hospitalized during the 12-month preindex period . There were no statistically significant differences in age, sex, or charlson comorbidity score between groups . In the non - roflumilast group, the most common copd regimen exposed on index date was a combination of a long - acting 2-agonist (laba) and ics (47.2%), followed by a combination of those two classes plus a long - acting muscarinic antagonist (46.1%). In the roflumilast group, 33.5% were receiving roflumilast in combination with a long - acting muscarinic antagonist, laba, and ics, 30.4% were receiving roflumilast monotherapy (of note, these patients may have had other copd maintenance medications prior to index and discontinued to use these medications when initiating roflumilast), and 18.9% were receiving a combination of roflumilast, laba, and ics . Figure 3 shows the change in overall rate of exacerbation pppm in the roflumilast and non - roflumilast groups from baseline . During the follow - up period, the monthly exacerbation rate decreased by 11.1% in the roflumilast group and increased by 15.9% in the non - roflumilast group (figure 3a; p<0.001 for did). While the rate of severe exacerbation pppm decreased to a greater extent in the roflumilast group, however, there was a significant difference in the rate of moderate exacerbation pppm between groups, with the roflumilast group experiencing an 11.8% reduction and the non - roflumilast group having a 20.7% increase in exacerbations (figure 3c; p=0.001). After controlling for the key covariates (age, sex, region, insurance, baseline number of copd drugs, and charlson comorbidity index) using a did model approach, roflumilast patients still had a greater reduction in the rate of overall exacerbation (=0.0160, p=0.01) and moderate exacerbation (=0.0149, p=0.01) versus the non - roflumilast group . There was a numerically greater reduction in the rate of severe exacerbation in the roflumilast group after adjusting for covariates; however, the difference was not statistically significant (=0.0012, p=0.63). Monthly number of office visits, ed visits, and inpatient hospitalization admissions at baseline and during follow - up are shown in table 2 . During both the baseline and follow - up periods, patients in the roflumilast group had more of each type of encounter than patients in the non - roflumilast group . Both treatment groups had increased office visits, ed visits, and hospitalizations during the follow - up period compared to the baseline period . However, the unadjusted results showed that the change from baseline was significantly different between the roflumilast and non - roflumilast groups for monthly rate of hospital admissions (0.0020.152 vs 0.0050.123, p=0.024) and office visits (0.0810.938 vs 0.1220.889, p=0.01), but not for the ed visits . After controlling for key covariates using did models, roflumilast was associated with fewer hospital admissions (=0.003, p=0.57) and office visits (=0.46, p=0.26); however, the differences were not statistically significant . Table 3 shows total monthly health care costs in the roflumilast and non - roflumilast groups during the baseline and follow - up periods . During the baseline and follow - up periods, costs increased for both groups from the baseline to the follow - up period, with no significant difference in the change from baseline between groups . After controlling for the key covariates using a did model, the total cost increase was $116 more for the control of patients not treated with roflu - milast versus patients treated with roflumilast (p=0.62). This study found that patients initiating roflumilast had more severe copd in that they had a higher number of monthly exacerbations, received more copd medications, and used more hcru than patients initiating other copd maintenance medications (based on the observation during the baseline period). Exacerbation rates were significantly improved for patients treated with roflumilast, with an 11% decrease in the rate of exacerbations in the roflumilast group and a 16% increase in the non - roflumilast group (a relative 27% reduction). After controlling for baseline differences using a did model approach, the significant differences in exacerbation rates between groups remained . After controlling for covariates using did models, changes in hospital admissions and total costs did not differ significantly between the roflumilast and non - roflumilast groups . The cost analysis in our study focused on the overall cost (cost component shown in table s1). In order to further understand how roflumilast impacts different cost components, future studies should examine the copd- or exacerbation - related costs by component, especially when the roflumilast was used as an add - on treatment following the gold guidance . Randomized controlled trials evaluating the efficacy and safety of roflumilast in patients with copd found significant between - group differences in moderate or severe exacerbation rates (a 17% reduction, p=0.0003); however, these trials were placebo - controlled and 6 months12 or 1 year13 in duration . Significant differences in the rate of moderate, but not severe, exacerbations were initially found in two pivotal 1-year trials,12,13 which may be due to a lack of statistical power given the relatively small sample sizes of clinical trial data . Similarly, in the current study, there may not have been sufficient power to detect a significant difference in the change in severe exacerbation rates between groups . Relatively, the cohort of patients who received roflumilast was large in this retrospective cohort study; however, there may be large treatment variations from patients in real - world settings compared to the strict treatment regimens of a clinical trial . Additionally, severe exacerbations are relatively rare events, which may further reduce the power of the analysis for this outcome . As gold recommended, roflumilast can be used an add - on treatment for patients with copd in stages 3/4 (forced expiratory volume in 1 second (fev1) <50% predicted after bronchodilatation) and frequent exacerbations despite a long - acting bronchodilator treatment . It was found that the copd drugs usage before initiating roflumilast varies in our study . It may be informative to explore the subset of patients who strictly followed the gold guidance (ie, those receiving ics and a laba or other long - acting bronchodilators prior to the use of roflumilast) using the real - world data . Future studies are warranted to assess the outcomes of those patients who have used ics and long - acting bronchodilator within certain window prior to initiating roflumilast using a large sample . Previous studies examined the resource utilization and health care costs associated with exacerbations among copd patients.1417 in a us managed - care population initiating copd maintenance medications, abudagga et al found that exacerbation rates continued to be high, resulting in an estimated annual cost of $25,747 in the overall population and $29,861 in the patients with history of one or more exacerbations.14 in this study, calculated total monthly costs during the 3-month follow - up period were $2,472 and $2,851 for non - roflumilast and roflumilast group, respectively, which is higher than the abudagga study14 and likely due to the selection of patients with more severe copd . Darnell et al reported an annual 3.07 clinic visits, 0.15 ed visits, and 0.41 hospitalizations per person per year, among copd patients, calculated using a large administrative database.15 the resource utilization reported here was similar or slightly higher than data in the darnell et al study15 and again, this discrepancy may be due to the selection of patients with more severe copd . Of note, our study examined the economic burden associated with copd medications from the payer s perspective; indirect costs, such as productivity loss, were not included, and thus, cost data may be conservative estimates . Copd exacerbations result in increases in resource uti - lization, especially for severe exacerbations.9,18,19 consistent with previous studies, the current work found that reduced exacerbation rates among copd patients were associated with reduced all - cause resource utilization and costs from a managed - care payer s perspective . Therefore, it is vital for physicians and payers to be aware that preventing copd exacerbations through appropriate treatments could minimize the need for hospitalization and other medical resource use . In addition, previous research also suggested that reducing exacerbation frequency may improve the quality of life among patients with copd.20 strengths of this study include the use of a robust national commercial insurance database with a longitudinal data structure that allows for tracking of both inpatient and outpatient diagnoses and procedures as well as outpatient prescription records . The availability of prescription claims provided rich information on treatment patterns and medication use as an indicator of exacerbation frequency . The use of did models controlled for significant baseline differences in study populations, which is important as there is potential for selection bias due to the observational study design . Our study has several limitations and it is important to interpret our results in the context of these limitations . First, some clinical details such as lung function parameters are not available in the database . The cohort selection criteria is based on the use of copd maintenance treatments (ie, patients who initiated roflumilast or a third copd drug), which may identify more severe patients by the criteria itself . Second, the definition of copd exacerbations was based entirely on claims data rather than patient symptoms . The study by stein et al suggested that algorithms based on icd-9-cm codes may undercount the acute exacerbation for copd.21 the exacerbation rates among patients receiving roflumilast were similar to those reported in randomized trials,12,13,22 which validate our results . Third, the study sample for the roflumilast group was restricted to those with insurance covering roflumilast . Because data were available only for commercially insured patients, the results may not be generalizable to older patients covered by medicare . However, the mean age of patients in our study was only slightly lower than that of patients in randomized trials (63 years vs 65 years).12,13 fourth, we used a did model that mimicked an experimental study and controlled for the baseline difference between study groups . Our adjusted did regressions were based on an assumption that in the absence of treatment, the difference between control (without roflumilast) and treatment (with roflumilast) groups would be constant or fixed over time . However, in reality, it is possible that the roflumilast group was inherently more likely to experience a reduction in exacerbation rate compared to the non - roflumilast group due to having higher exacerbation rates at baseline . In this study, we observed a decreased overall exacerbation rate in the roflumilast group contrasted with an increased exacerbation rate in the non - roflumilast group . The magnitude of the difference suggests that the observed results cannot be attributed exclusively to unobserved confounders . Fifth, it is challenging to adjust for the difference in adherence in the patients without roflumilast . Patients adherence to treatments, especially to the combination therapy, may change quickly over time because patients may discontinue some drugs after the symptoms improve . It will be important for future studies to control for the adherence if a specific comparator drug is chosen . Sixth, our results may not be able to identify the seasonal effect as the follow - up duration covers only 3 months.23 however, both study groups have the same index period so the difference in the exacerbation outcomes between the two study groups may not be biased . Finally, we did not have a large sample of the roflumilast - treated group as roflumilast is only recently approved by the us food and drug administration (february 2011). This may affect the statistical power and our study may not be sufficient to determine the long - term effects of roflumilast on exacerbations, hcru, and costs . Patients with copd - initiating roflumilast treatment had greater reductions in exacerbation rates in the 3 months after the new therapy was added than patients initiating other copd maintenance medications . We also found a numerically smaller increase from baseline in the monthly rates of hospital admissions, office visits, and monthly total costs between patients treated with roflumilast versus patients not treated with roflumilast . Future studies using alternative methods such as patient survey or chart review are warranted to further evaluate the impact of roflumilast on clinical outcomes, hcru, and costs within a longer follow - up period . Monthly total health care costs and cost by component ($) notes: the result data represent average cost per patient per month . The total cost is more than the sum of inpatient, outpatient, er, and copd drug costs because there are other types of cost components not shown in this table . Abbreviations: baseline, 12-month preindex period; copd, chronic obstructive pulmonary disease; sd, standard deviation; er, emergency room.
The hypothesis that a systemic or a regional reduction of sympathetic activity for example, induced by thoracic epidural anesthesia might have positive effects on the perfusion and oxygenation (that is, increase them) of splanchnic organs like the liver and gut and that reduction of pain improves pulmonary function sounds profound . Although interest in this field of research has been increasing over the past years, detailed knowledge about the effects of increased or reduced sympathetic activity on organ perfusion and oxygenation and the mechanisms involved, as well as how these change or sympathetic activity changes immunomodulation during pathophysiological conditions, is still lacking . In recent issues of critical care, freise and colleagues and lauer and colleagues presented studies that provide interesting information concerning this subject . Sprague - dawley rats that were fitted with thoracic epidural catheters and treated with cecal ligation and puncture . Intravital microscopy was used to investigate sinusoidal diameters, loss of sinusoidal perfusion, sinusoidal blood flow, and permanent leukocyte adhesion to sinusoidal and venolar endothelium . In their experiments, cardiac output measured in an additional group of animals, which were not investigated with intravital microscopy remained constant in animals with induced sepsis with and without epidural anesthesia . From their intravital microscopy results they concluded that sinusoidal blood flow increased in the sepsis group and was normalized in the group with sepsis and thoracic epidural anesthesia however, sinusoidal vasoconstriction was not ameliorated by thoracic epidural anesthesia and nor was liver tissue injury affected . While there is broad agreement that thoracic epidural anesthesia improves postoperative pulmonary function, the underlying mechanisms for example, via reduction of abdominal pain after general abdominal surgery still remain unclear . Thoracic epidural anesthesia modulated the nitric oxide (no) pathway and exerted positive that is, lower levels of exhaled no effects on pulmonary endothelial integrity in hyperdynamic septic rats, but not in hypodynamic septic rats . In the latter, this study shows the importance of distinguishing between different phases of disease, especially during early (hyperdynamic) and late (hypodynamic) sepsis . One has to keep in mind that the authors did not describe any differences in volume management within their experimental groups and, thus, intravascular normovolemia could not be proven in either . In general, both studies add interesting results to the necessary discussion about the usefulness of epidural anesthesia during sepsis . Why is this so? Increased sympathetic activity plays an important role in the development of different pathophysiological conditions for example, during endotoxemia [4 - 7], hemorrhagic shock and even during and after routine abdominal surgical procedures . Thus, epidural anesthesia might decrease mortality during sepsis, especially as splanchnic hypoperfusion and hypoxia are said to be key factors in the development of systemic inflammatory response syndrome, sepsis and multiple organ failure . Diverse studies, however, have presented contradictory results concerning this, with some reporting decreased mortality in older animal studies and newer meta - analyses and others reporting increased mortality in an animal model . The main problem with all the published studies is that hardly any are comparable with each other . Humans and different animals (for example, pigs, rats, mice, rabbits) have been used, either systemic or regional reduction of sympathetic activity has been investigated (effects of clonidine, spinal anesthesia, epidural anesthesia), and the method of epidural anesthesia has differed, from lumbar epidural anesthesia in older studies to thoracic epidural anesthesia in recent studies, including or not the nervi accelerantes . However, to prove an effective reduction in sympathetic activity, and especially that the epidural anesthetic is working, is very difficult, especially in clinical situations . Hence, the most important considerations for future studies on the effect of epidural anesthesia on sepsis or endotoxemia are normovolaemia at any point of the experiment, a clear definition and timeline of hypodynamic and hyperdynamic circulation in sepsis, the proven spread of the epidural anesthesia, which includes or excludes the nervi accelerantes (thereby reducing or maintaining cardiac output, respectively), and the continuous, proven reduction of sympathetic activity including or excluding the adrenal glands during the different phases of the developing pathophysiological conditions . Surrogate parameters like sinusoidal width or the number of perfused sinusoids should be used with care to judge sinusoidal perfusion, as laboratory findings should be treated cautiously if not accompanied by definitive and relevant physiological changes . Although studies like those from freise and colleagues and lauer and colleagues have increased our understanding of how reduction of regional sympathetic activity can influence different organ functions during sepsis, we still largely lack understanding of the underlying mechanisms, and this will persist as long as there are no standardized, or at least fairly definitive, studies on reduced sympathetic activity during sepsis . Only with these studies
Tuberculosis persists as a common illness in developing countries, and western countries are experiencing a resurgence due to rising numbers of patients with acquired immunodeficiency syndrome1, 2). Intra - abdominal tuberculosis, including peritonitis, enterocolitis, and mesenteric lymphadenitis, is relatively rare among extra - intestinal tuberculosis, although it is of considerable clinical importance3 - 6). In korea, although the exact incidence of intra - abdominal tuberculosis is unknown, tuberculous enterocolitis has been reported to account for 4.8% of all tuberculosis, and therefore, intra - abdominal tuberculosis is clinically relevant in korea . Intra - abdominal tuberculosis can present atypical clinicopathological features, which can make a proper diagnosis difficult8 - 10). We present here an unusual case of solitary intra - abdominal tuberculous lymphadenopathy mimicking duodenal gastrointestinal stromal tumor (gist). A 22-year - old woman presented with epigastric discomfort and intermittent dark - colored stools of two months duration . She had no previous illness and denied constitutional symptoms such as fever, weight loss, or night sweating . Laboratory studies showed a hemoglobin of 13.9 g / dl, leukocytes of 7,580/mm, and an esr of 17 mm / hr . Other biochemical tests results were within normal ranges, and anti - hiv ab was negative . An esophagogastroduodenoscopic (egd) examination revealed a round mass of about 3-cm at the duodenal bulb, with normal surrounding mucosa and central ulceration (figure 1). Chest x - ray films showed inactive pulmonary tuberculosis at the left upper lobe, and abdominal computed tomography (ct) revealed a well - defined round mass that enhanced as gastric mucosa, at the posterior wall of the duodenal bulb suggestive of duodenal gist (figure 2). Endoscopic ultrasonography (eus) showed an ulcerative hypoechogenic mass at the submucosal layer of the duodenal bulb (figure 3). Exploratory laparotomy was performed to exclude the possibility of malignant gist of the duodenum . During laparotomy, a solitary mass was detected around the hepatic artery, which penetrated the duodenal bulb . The mass was excised and a histopathological analysis revealed lymphadenopathy with caseous granuloma (figure 4). Polymerase chain reaction of dna extracted from the node was positive for tuberculosis and acid - fast bacilli were found by ziehl - neelsen tissue staining . The patient was diagnosed with solitary tuberculous lymphadenopathy and discharged on anti - tuberculosis medication . Abdominal mesenteric lymphadenopathy is a relatively common manifestation of intra - abdominal tuberculosis, which occurs as a regional component of primary or secondary disease . Involved mesenteric nodes may form independent masses or they may adhere to adjacent structures . Tuberculosis usually affects multiple lymph node groups simultaneously, though isolated retroperitoneal lymphadenopathy is uncommon11, 12). Most tuberculous lymphadenopathies are visualized by abdominal ct as enlarged nodes with hypodense centers and peripherally hyperdense enhancing rims11) as conglomerate mixed density nodal masses, or as enlarged masses with homogeneous density12). In the present case, an exophytic oval mass was noted on an abdominal ct scan on the posterior wall of the duodenal bulb . Moreover, it presented as a submucosal mass with central ulceration and suspected bleeding foci on egd examination . Most cases of abdominal tuberculosis are complications of pulmonary tuberculosis, and thus in this patient with evidence of inactive pulmonary tuberculosis, a diagnosis of solitary mesenteric tuberculous lymphadenopathy with penetration into the duodenal wall appeared unlikely, and therefore, we suspected that the lesion was a duodenal gist . Gists are generally located in the stomach; only 4% of cases are reported in the duodenum . However, despite their relative infrequency small bowel gists frequently manifest as gastrointestinal bleeding, abdominal pain, or as a palpable mass13, 14), and can display unpredictable malignant behavior notwithstanding their small size . Duodenal gists can thus have a poorer prognosis than gists of the stomach15, 16). Because it is difficult to rule out duodenal gist and because of the likelihood of bleeding or malignancy laparotomy revealed a solitary mesenteric lymphadenopathy penetrating the duodenal bulb, and histopathologic analysis revealed it to be a tuberculous lymphadenopathy . Eus - guided fine - needle aspiration (fna) has recently emerged as an important modality for diagnosing gist17). Moreover, ultrasound - guided fna offers a safe method for obtaining proof of tuberculosis in patients with suspected intra - abdominal tuberculosis18, 19). It is regrettable that we did not perform fna prior to laparotomy in the present case . The preferred treatment of intra - abdominal tuberculosis is anti - tuberculous medication; surgical management is normally reserved for complications or diagnostic uncertainty . In conclusion, intra - abdominal tuberculosis can present a variety of conditions and in areas with a high prevalence the possibility of tuberculosis must be considered . Tuberculosis is curable when a diagnosis is made sufficiently early and appropriate treatment is started.
Only about 0.5% of all cancers occur among children under 15 y. in contrast to adult malignancies, where carcinomas predominate, childhood cancers are histologically very diverse . There are 12 major groups including leukemias, lymphomas, brain and spinal tumors, sympathetic nervous system tumors, retinoblastoma, kidney tumors, liver tumors, bone tumors, soft tissue sarcomas, gonadal and germ - cell tumors, epithelial tumors, and other unspecified neoplasms . Diagnostic groups are defined in the second edition of the international classification of diseases (icd) for oncology . Cancer formation is generally considered as a genetic disease involving alterations in dna structure ranging from gene mutations to gross chromosomal rearrangements . However, dysregulation of gene expression can also be acquired by epigenetic abnormalities, which are a hallmark of cancer evolution at all stages . In contrast to the rest of the genome where most cpgs are methylated, cpg islands in 5 cis - regulatory regions of genes are usually unmethylated . Methylation of these cpg islands during development or disease processes is associated with gene silencing . Inactivation of tumor suppressor genes by promoter hypermethylation provides an important mechanism for tumor initiation and progression . The genomic caretaker brca1 is necessary for faithful rejoining of broken dna ends and one mutated brca1 allele may already be sufficient to impair this process . The compromised genomic stability in brca1 germline mutation carriers may trigger the genetic changes necessary for neoplastic transformation in hereditary breast cancer patients . Sporadic breast tumors with brca1 promoter methylation are mainly estrogen- and progesterone - receptor negative and display similar pathological features as hereditary tumors with brca1 mutations . The concordance rates in monozygotic (mz) and dizygotic pairs of twins allow one to estimate the heritability of complex phenotypic traits . Mz twins arise from the same zygote, which then divides into two genetically identical embryos . Mz twins discordant for monogenic disorders are generally thought to represent rare examples of somatic mosaicism due to genetic mutations after the twinning event, which are then propagated to subsets of cells from one twin . Our results suggest that post - zygotic epimutations are another source of somatic mosaicism leading to different disease susceptibility in mz twins . The twin sisters were born in 1977 by spontaneous delivery, seven weeks before term . No intensive care or icterus treatment were necessary . Until 1982 8 mo one sister was diagnosed with precursor b - cell lymphoblastic leukemia (icd10:c910). Chemotherapy had to be discontinued because of intolerability . A first relapse of the leukemia occurred in 1984 . The second chemotherapy could be completed, but another relapse made bone marrow transplantation from her healthy twin sister necessary . Following bone marrow transplantation at age 7 y she received human growth hormone . In 2002 at the age of 25 y she was diagnosed with thyroid carcinoma (icd10:c730). When re - examined at age 34 y, the affected twin and her sister did not show any clinical manifestation of cancer . Neither family history nor clinical examination gave any hints for a dna repair syndrome or another hereditary disease . Apart from the affected twin there were no other cases of cancer in four generations . Considering her medical history, it is not unexpected that the affected twin differed from her sister in some features, including height (156 cm vs. 168 cm) and occipitofrontal circumference (52 cm vs. 51 cm). Monozygosity was confirmed by genotyping short tandem repeats . Chromosome banding analysis (at the 500 band level) of fibroblast cultures revealed normal female karyotypes in both sisters . Methylation analysis . Because mz twins are genetically identical, epigenetic differences are one plausible explanation for discordant phenotypes . To test this hypothesis, we analyzed the methylation patterns of several representative tumor suppressor genes (atm, brca1, brca2, mlh1, rad51c, and tp53). One of the studied genes, brca1, showed constitutive promoter hypermethylation in normal body cells of the affected twin, but not of the healthy twin . It can exactly (2%) quantify the methylation of individual cpg sites located in the 3050 bp 3 from the sequencing primer . Our pyrosequencing assay measures the methylation levels of five adjacent cpg sites in the brca1 5 promoter . Because the density of methylated cpgs in a cis - regulatory region rather than individual cpgs turn a gene on or off, the average methylation of all analyzed cpgs (in two independent dna samples) was used as an epigenetic marker for brca1 promoter methylation . By bisulfite pyrosequencing of exponentially growing fibroblasts, the affected twin showed an increased brca1 methylation level of 12% (fig . 1) primary skin fibroblasts were used for epimutation screening, because they constitute a homogenous cell population with intact cell cycle and dna repair checkpoints . Ten additionally analyzed two - cancer patients, who survived a childhood malignancy and then, unrelated to the first event, developed a secondary cancer, as well as 10 carefully matched one - cancer patients without a second malignancy all showed normal methylation levels (range 03%) in fibroblasts . Induction of dna damage by -irradiation (1 gy) of primary fibroblast cultures did not affect the brca1 methylation levels . In the affected twin, we found 10% methylation at 0 h, 10% at 1 h, 9% at 4 h, 11% at 12 h, and 10% at 24 h after irradiation . The control twin displayed 4%, 4%, 3%, 2%, and 4% methylation, respectively . Box plots showing the distribution of brca1 methylation values in fibroblasts of 10 one - cancer and 10 two - cancer patients . The bottom of the box indicates the 25th percentile, the top the 75th percentile . The t bars extend from the boxes to 1.5 times the height of the box . Lymphoblastoid cells of both the affected and the control twin exhibited equally low brca1 methylation levels (3% and 2%, respectively). However, because the affected twin was bone marrow - transplanted, her blood cells were also derived from the healthy twin s stem cells . The normal range of brca1 methylation in blood cells of a large number (> 100) of controls was 05% . Similar to fibroblasts, saliva dna of the affected twin showed a much higher brca1 methylation (9%) than that of her sister (2%). In addition to cells from buccal mucosa and salivary epithelium, saliva may also contain blood cells . This may explain the somewhat lower methylation level of saliva dna compared with fibroblast dna in the affected twin . To distinguish between single cpg methylation errors, which are most likely stochastic errors without pathological consequences, and true epimutations (allele methylation errors), which can be expected to interfere with gene regulation, it is necessary to study the methylation patterns of individual dna molecules . Classic bisulfite plasmid sequencing has the added advantage that it allows one to look at a larger number of cpg sites . Here we analyzed a brca1 amplicon with 13 cpg sites including the five sites targeted by the pyrosequencing assay (fig . 2). Forty - seven clones (individual dna molecules) were recovered from primary fibroblasts of each twin . Six (13%) clones of the affected twin exhibited epimutations, indicating that most (at least 70%) cpg sites on these dna molecules were aberrantly methylated, typical for epigenetically silenced alleles . In contrast, all 47 alleles of the healthy twin displayed normal hypomethylated patterns . The brca1 epimutation rate was significantly (test; p = 0.01) higher in the affected twin, compared with her sister . Both sisters showed approximately 2% stochastic (single cpg) methylation errors: 10 of 531 cpg sites in the affected twin (excluding abnormal alleles) and 14 of 609 in the healthy twin were methylated . Collectively, our results suggest a constitutively increased methylation level at the brca1 promoter in normal body cells of the affected twin . Approximately 25% cells (skin fibroblasts, cells from buccal mucosa and salivary epithelium) derived from different embryonic lineages are endowed with one hypermethylated copy of the brca1 tumor suppressor gene, representing a first hit according to knudson s two - hit hypothesis of tumor development . Methylation patterns of the brca1 promoter in fibroblasts of the affected twin and her healthy sister . Each line represents an individual allele (dna molecule) analyzed by bisulfite plasmid sequencing . Missing dots indicate cpg sites that could not be analyzed because of poor sequence quality . Six of 47 analyzed alleles (indicated by arrows) in the affected twin represent epimutations with the majority of cpgs being aberrantly methylated, whereas all 47 alleles in the healthy twin are hypomethylated . Because constitutional epimutations in different tumor suppressor genes have been linked to sequence variants in the 5 cis - regulatory region, we sequenced a 3.4 kb upstream fragment including the brca1 promoter and exon 1 . However, we did not find any genetic defect / sequence variant in the affected twin (data not shown). In addition to brca1, we performed an epimutation screening by bisulfite pyrosequencing for several other tumor suppressor genes (atm, brca2, mlh1, rad51c, and tp53) in fibroblasts of the twin pair and the 20 one- or two - cancer patients . However, apart from the brca1 epimutation in the affected twin, all analyzed samples and promoters displayed normal (<5%) methylation values (data not shown), indicating that constitutive epimutations in tumor suppressor genes are rare events in childhood - cancer patients . Customized antibody microarrays were used to compare the basal protein levels (without induction of dna damage) of brca1 and several other genomic caretakers (atm, brca2, mlh1, rad51, and tp53) in exponentially growing primary fibroblasts of the affected twin vs. her healthy sister . Triplicate measurements (technical replicates) of the protein levels were performed on protein samples from three independent cell cultures each (biological replicates). The brca1 protein expression ratio between the affected and the healthy twin (z ratio normalized with log10 transformation and z scores) was 0.75 (fig . Atm expression was slightly increased (1.25) in the affected twin, whereas actb (positive control), brca2, mlh1, rad51, and tp53 all showed comparable protein levels in both twins . The brca1 protein levels were also quantified in fibroblast cells at 1 h and 4 h after 1 gy -irradiation (fig . The amount of brca1 protein increased 1.8-fold at 1 h after irradiation, compared with untreated cells, and reached basal levels at 4 h (the z ratio of treated vs. untreated cells was 0.9). In the affected twin, brca1 protein expression was induced 2.7-fold at 1 h and also back to basal levels (z ratio of 1.1) at 4 h. figure 3 . (a) constitutive protein expression of actb (control), atm, brca1, brca2, mlh1, rad51, and tp53 in untreated fibroblasts of the affected vs. the healthy twin . Relative protein expression (z ratio normalized by log10 transformation and z scores) was measured by antibody microarrays using three biological replicates . (b) differential induction of brca1 protein in fibroblasts of the healthy twin (gray bars) and the affected twin (black bars) at 1 h and 4 h after 1 gy -irradiation . Relative protein expression in treated vs. untreated cells of each twin was measured by antibody microarrays using three biological replicates . Microarray analysis revealed a 181 kb heterozygous deletion containing the rspo3 gene on chromosome 6q22.33 and a 90 kb heterozygous deletion containing the open reading frame c5orf13 on chromosome 5q22.1 in primary fibroblasts of the affected twin . We did not see similar deletions in> 40 additionally studied childhood cancer patients and 100 normal healthy individuals (from the gutenberg heart study). We did not find evidence for a microdeletion or duplication affecting the brca1 cis - regulatory region in the affected twin . By quantitative real - time pcr (qpcr) we confirmed the presence of the heterozygous rspo3 and c5orf13 deletions in a mosaic state (fig . All qpcr experiments were performed on three independent dna samples (biological replicates) of the affected twin and a mixture of control dnas, respectively . Using rfc3 as reference for a normal diploid gene (with two copies), qpcr revealed a copy number of two for both rspo3 and c5orf13 in the controls . In contrast, the affected twin displayed copy numbers around 1.5, indicating loss of one rspo3 and one c5orf13 copy in approximately 50% of cells . Expression analysis with geneatlas microarrays using four independent rna samples (from different cell cultures of the affected and the healthy twin) demonstrated reduced (approximately 50%) rspo3 mrna levels in fibroblasts of the affected twin (fig . 4b), whereas c5orf13 levels were comparable in both twins (data not shown). (a) c5orf13 and rspo3 copy numbers in fibroblast cells of the two - cancer twin, compared with healthy controls, as determined by qpcr (using the rfc3 gene copy number as reference). Standard deviations represent three independent dna samples (biological replicates) of the affected twin and a mixture of control dnas . (b) rspo3 mrna expression ratio between the affected twin and her healthy sister, as determined by geneatlas microarrays . Standard deviations represent rna samples from four different cell cultures of the affected and the healthy twin, respectively . Traditionally, phenotypic discordances between mz twin pairs have been attributed to environmental factors; however, accumulating experimental evidence also suggests a role for genetic and, more importantly, epigenetic differences between co - twins . Microarray - based karyotype analyses revealed somatic mosaicism for copy number variations (cnvs) in a number (estimated 10%) of both phenotypically discordant (for parkinson disease, parkinsonism or lewy body dementia) and concordant mz twin pairs . Other molecular karyotype and genome sequence studies could not detect any cnv differences between mz twins that were discordant for cleft lip palate or multiple sclerosis, suggesting that post - zygotic genomic alterations are at least not a common cause of phenotypic discordance . Several recent studies clearly demonstrated the existence of genome - wide epigenetic differences between mz twins . This spontaneous epimutation rate can be modified by environmental factors, providing a link between lifestyle and phenotypic differences . In this light, stochastic and/or environmentally induced epimutations represent a major source of phenotypic variation and discordance for complex diseases between mz twins . The accumulation of epigenetic differences may also explain the phenotypic divergence of mz twins, as they age . Here we studied a mz twin pair discordant for childhood cancer and a second primary cancer . Microarray analysis and qpcr revealed mosaic heterozygous deletions on chromosome 5q22.1 (c5orf13) and 6q22.33 (rspo3) in the affected twin . It contains a conserved pest domain, which is important for targeting proteins for degradation by the ubiquitin / proteasome . R - spondin family members are modulators of the wnt/-catenin signaling pathway which plays a crucial role in cell growth, development and disease pathogenesis . Rspo3 is frequently upregulated in human breast cancers and correlated with several tumor parameters, suggesting that it can function as a breast cancer oncogene . Although c5orf13 and rspo3 haploinsufficiency may contribute to the discordant cancer phenotype, they are unlikely to be the primary cause . We propose that epigenetic changes, specifically hypermethylation of one brca1 allele constituted the first hit leading to inactivation of an important tumor suppressor gene and tumor initiation . Germline mutations in brca1 are associated with hereditary breast cancer, ovarian cancer and some other malignancies; however, so far they are generally not considered as a risk factor for childhood cancer . Our index patient developed precursor b - cell lymphoblastic leukemia at the age of 4 y and 8 mo . Leukemia, expression of the bcr - abl fusion protein leads to posttranscriptional downregulation of brca1 . One study reported partial hypermethylation and reduced expression of brca1 in primary and therapy - related acute myeloid leukemia, whereas others did not find abnormal brca1 methylation in leukemia samples . In the mz twin pair studied here, only one sister exhibited a brca1 epimutation in a mosaic state in multiple tissues / cell types, whereas the unaffected twin did not . This epimutation most likely originated by a methylation error during early embryogenesis after the twinning event . It affects a substantial subset of cells derived from different embryonic cell lineages, resulting in somatic mosaicism . Genome - wide demethylation waves in the early embryo erase most germline methylation patterns, followed by de novo methylation and establishment of somatic methylation patterns . Most constitutive epimutations may occur during this vulnerable time window, where epigenetic genome reprogramming occurs, and represent stochastic and/or environmentally induced errors in the establishment or maintenance of an epigenetic state . Similar to our case, discordant methylation patterns at the kcnq1ot1 locus (imprinting control region 2 on chromosome 11p15) have been described in a number of mz twin pairs discordant for beckwith - wiedemann syndrome, which is characterized by tumor predisposition and multiple congenital abnormalities . Constitutive h19 epimutations (imprinting control region 1 on chromosome 11p12) were found in several patients with sporadic wilms tumor without features of beckwith - wiedemann syndrome . Somatic gene mutations and mosaicism (with or without involvement of the germline) are an important etiological mechanism for monogenic disorders with high de novo mutations rates such as duchenne muscular dystrophy, neurofibromatosis type 1 and retinoblastoma . In contrast, constitutive epimutations, characterized by soma - wide methylation abnormalities in functionally relevant cis - regulatory regions of disease genes, are an understudied phenomenon . Constitutive epimutations in the dna mismatch repair gene mlh1 have been identified in a small number of mutation - negative cases of hereditary non - polyposis colorectal cancer (hnpcc). In rare familial cases with dominant transmission of mosaic mlh1 methylation, the constitutional epimutation was linked to a single nucleotide variant in the 5 utr of mlh1 . Similarly, constitutional epimutations in the dna mismatch repair gene msh2, another rare cause of hnpcc, were linked to 3 end deletions of epcam, a gene directly upstream of msh2 . Constitutive epimutations in the dapk1 gene that predispose to b cell chronic lymphocytic leukemia are caused by a point mutation upstream of the dapk1 promoter . Neither sequencing nor microarray analysis of the 5 cis - regulatory region of brca1 revealed any detectable genetic differences between the discordant mz twin pair studied here . Although we cannot exclude a causative genetic defect elsewhere, our findings are consistent with the view that the affected twin exhibits a true brca1 epimutation . Our study revealed the existence of a constitutive brca1 epimutation only in the affected sibling of a mz twin pair . Compared with her healthy sister, the two - cancer twin showed reduced basal brca1 protein levels (in untreated fibroblasts) and a higher induction of protein expression by dna damage . Upregulation of brca1 in irradiated cells of both the affected and the healthy twin was not mediated by demethylation of the brca1 promoter . In the affected twin the percentage of aberrantly methylated alleles remained constant (at about 10%) after dna damage and in the healthy twin the brca1 promoter was already completely demethylated in untreated cells . It is well known that the methylation of cpgs in 5 promoters that are usually protected from methylation in somatic tissues can suppress gene expression during development, differentiation and disease processes . However, other mechanisms, i.e., gene body methylation, may be more important for regulating the cell - context specific efficiency of transcription . The basal atm protein levels were also increased in the affected twin, although both twins showed equally low promoter methylation levels (around 1%) of the atm gene . We speculate that atm is constitutively upregulated by a compensation mechanism to counterbalance the reduced brca1 levels . Brca1 is regulated by an atm - dependent mechanism and, on the other hand, essential for the recruitment of previously activated atm to the sites of dna damage . Although we cannot exclude the formal possibility of a chance coincidence or a late (i.e., therapy - related) somatic event, it is plausible to assume that epigenetic silencing of one copy of the brca1 tumor suppressor gene in 25% body cells and differences in the dna damage response contributed to the discordant cancer phenotype in the affected twin . Preliminary evidence suggests that brca1 promoter hypermethylation can also be found in blood cells of a subset of breast cancer patients . Collectively, these results suggest that constitutive brca1 epimutations occur in normal tissues and may be associated with an increased cancer risk . Our pyrosequencing assay may prove useful for high - throughput screening of larger patient populations . The dna methylation analysis performed in this study was limited to a number of key tumor suppressor genes . Considering the enormous epigenetic variability due to stochastic methylation errors or extrinsic variations during post - zygotic genome reprogramming, we cannot rule out the possibility that epimutations in the affected twin in genes other than those studied are responsible for the discordant phenotype . Nevertheless, 10% soma - wide promoter methylation in an essential tumor suppressor gene such as brca1 can be expected to contribute to a tumor susceptibility phenotype . The study was approved by the ethics committee of the medical association of rhineland - palatinate [(nr . 837.440.03(4102)]. With the help of the german childhood cancer registry, we recruited a discordant mz twin pair . One twin suffered from childhood leukemia and later on thyroid carcinoma, whereas her twin sister was completely healthy (without malignancy). In addition, we recruited 10 two - cancer patients as well as 10 carefully matched one - cancer patients (same sex, same primary cancer, equal age at first diagnosis) who did not develop a second malignancy . Primary fibroblasts from skin biopsies were cultured in minimal essential medium with earle s salts (invitrogen, karlsruhe, germany), supplemented with 10% fetal bovine serum, vitamins and antibiotics . The cell cultures of the twin sisters were always passaged in parallel and harvested in a subconfluent stage . To induce dna damage, subconfluent cultures were -irradiated with a dose of 1 gy using a gammacell 2000 (cs) irradiator . Cells were harvested at 1 h, 4 h, 12 h, and 24 h after irradiation . Lymphoblastoid cell lines were established by epstein - barr virus transformation of peripheral blood lymphocytes . Dna sequence analysis . For sanger sequencing of the 5 utr and exon 1 of the brca1 gene, a 3.4 kb fragment on chromosome 17 (41.277.11241.280.530 bps, ensembl release 64) was divided into 11 amplicons (table s1). For amplicons 15 and 8, the pcr reaction mixture (25 l) consisted of 2.5 l 10x pcr buffer, 20 mm mgcl2, 0.5 l 10 mm dntp mix, 1 l (10 pmol) of each forward and reverse primer, 0.2 l (1 u) faststart taq dna polymerase (roche diagnostics, mannheim, germany), 18.8 l pcr - grade water, and 1 l (~100 ng) template dna . For amplicons 6, 7, 8, 10, and 11, the pcr mixture (25 l) contained 25 mm ammonium sulfate, 750 mm tris - hcl, 0.1% tween 20, 240 m dntps, 2.4 mm magnesium sulfate, 2.4x pcrx enhancer solution (invitrogen), 0.4 m of each primer, 1 unit of platinum taq (invitrogen), and 1 l (~100 ng) template dna . All pcr amplifications were performed with an initial denaturation step at 95c for 5 min, 40 cycles of 95c for 30 sec, primer - specific annealing temperature (table s1) for 30 sec, 72c for 45 sec, and a final extension step at 72c for 7 min . Sequencing of the resulting pcr products was done on an abi 3130 x l automated sequencer . Genomic dna from fibroblasts and ebv - transformed lymphoblasts was extracted using the qiagen mini dna kit (qiagen, hilden, germany). Saliva dna was extracted using the oragene dna extraction kit (dna genotek, kanata, ontario, canada). Bisulfite conversion of genomic dna was performed with the epitect bisulfite kit (qiagen). Bisulfite pyrosequencing was performed on a pyromarktmq96 md pyrosequencing system with the pyromark gold q96 cdt reagent kit (qiagen). An assay quantifying the methylation levels of five representative cpg sites in the brca1 promoter was designed using the pyromark assay design 2.0 software (qiagen). A 232 bp amplicon (hsa17: 41,277,29241,277,523 bps; ensembl release 60) was pcr amplified from bisulfite - treated dna using forward primer 5-atttagagtagagggtgaagg-3 and biotinylated reverse primer 5-tctatccctcccatcctctaatt-3. The reaction mixture consisted of 2.5 l 10x pcr buffer, 20 mm mgcl2, 0.5 l 10 mm dntp mix, 1 l (10 pmol) of each primer, 0.2 l (1 u) faststart taq dna polymerase (roche diagnostics, mannheim, germany), 18.8 l pcr - grade water, and 1 l (~100 ng) template dna . Pcr amplifications were performed with an initial denaturation step at 95c for 5 min, 35 cycles of 95c for 30 sec, 55c for 30 sec, and 72c for 45 sec, and a final extension step at 72c for 5 min . Sequencing was performed with primer 5-ttgagaaattttatagtttgtttt-3. The pyro q - cpg software (qiagen) was used for data analysis . For classical bisulfite plasmid sequencing brca1 pcr products were cloned into pcr2.1-topo vector using t4-dna ligase, the ta cloning kit and one shot top10 chemically competent escherichia coli (invitrogen). Plasmid dna of individual clones was isolated with the zr plasmid miniprep classic kit (zymo research, freiburg, germany). Insert - containing clones were sequenced using dye terminator cycle sequencing with m13 primers on an abi 3130xl automated sequencer (life technologies, frankfurt, germany). Cells were resuspended two times in 500 l of 10 mm hepes, 1.5 mm mgcl2, 10 mm kcl, ph 7.9 and incubated on ice for 10 min . The pellet was resuspended in 100 l of 20 mm hepes, 0.42 m nacl, 1.5 mm mgcl2, 0.2 mm edta, 25% glycerol, ph 7.9 and homogenized . After incubation on ice for 30 min and centrifugation for 30 min at maximum speed at 4c, the supernatant containing the nuclear protein extract was separated from the cytoplasmic pellet and stored at -80c . The protein concentration was measured according to bradford, using roti quant (roth, karlsruhe, germany). Customized antibody microarrays for quantification of brca1 (antibody #sc-1553, santa cruz biotechnology, heidelberg, germany) and several other dna repair - associated proteins (atm, brca2, mlh1, rad51, and tp53) were prepared by spotting triplicate drops, each containing approximately 0.5 pg antibody onto nitrocellulose - coated slides (oncyte, nitrocellulose 16 multi - pad slides, grace bio - labs, bend, or, usa), using a sciflexarrayer 3 non - contact spotter (scienion, berlin, germany). Antibodies against -actin (actb) (#a5441, sigma - aldrich, munich, germany) served as positive, spotting buffer as negative control . Nuclear proteins were labeled with an amine reactive fluorine dye, which forms a covalent amide bond between the primary amines of proteins . Two micrograms of protein and 0.12 l fluorescent dye (dylight 649 nhs ester, pierce, rockford, usa) were incubated for 1 h at room temperature in the dark . Then, excess fluorescent dye was inactivated by adding 100 mm glycine to the reaction . Prior to use, antibody microarrays were covered with 16-pad fast frame hybridization chambers (whatman, maidstone, uk). Unspecific binding sites were blocked for 1 h at 4c with 120 l pbs containing 4% non - fat dry milk per subarray, followed by three washes with 120 l pbs each for 10 min . Labeled protein samples were incubated on subarrays overnight at 4c . Afterwards, the slides were washed two times for 15 min with pbs, 5% tween 20 and two times for 15 min with hplc - grade water . Finally, the slides were dried in a speedvac and scanned with a high - resolution confocal affymetrix array scanner 428 tm (affymetrix, high wycombe, uk). Slide images were analyzed using the tm4 spotfinder (version 3.1.1) software (dana faber cancer institute, boston, usa). Background subtraction was performed according to the formula: spot intensity = mean intensity sp - (sum bkg sum top25bkg)/(number of pixelsp - number of pixel top25bkg), where sp represents any spot, bkg the corresponding background and top25bkg the top 25% of background pixel . Three biological replicates (using protein samples from different cell cultures of the affected and the healthy twin) were performed for each experiment . Molecular karyotype analysis . High - resolution screening for microdeletions and duplications was performed with the affymetrix genechip genome wide human snp array 6.0 and the genechip genome wide snp sty assay kit 5.0/6.0, following the protocol developed by the manufacturer . Copy numbers were determined using the affymetrix genotyping console 4.0 and chromosome analysis suite 1.0.1 . Quantitative real - time pcr with the universal probe library set 04683633001 (roche diagnostics) was used to validate copy number changes of c5orf13 (upl probe #24) and rspo3 (#23). Pcr was performed on an abi 7500 fast real - time pcr system (life technologies) with one cycle of 95c for 10 min and 45 cycles of 95c for 30 sec and 60c for 60 sec . Copy number calculation was performed with the -ct method, using the rfc3 (upl probe #32) gene copy number as reference . Genome - wide expression analyses were performed with rna samples from four fibroblast cell cultures of each twin using the geneatlas personal microarray system (affymetrix, santa clara, ca, usa). Data were analyzed with the affymetrix expression console program.
Prostatic stromal sarcoma (pss) is quite rare, comprising only 0.1 - 0.2% of all malignant prostate tumors (1). In 1998, gaudin et al . (2) classified sarcoma - related proliferative lesions of the specialized prostatic stroma, including prostatic phyllodes tumors, into two subtypes: prostatic stromal proliferation of uncertain malignant potential and pss (2, 3). Herein, we present a case of pss in a young man . To the best of our knowledge, only three reports of pss with findings from computed tomography (ct) have been reported in the english literature (1, 4, 5). However, none of those reports described findings on magnetic resonance imaging (mri). This represents the first report in the radiological literature on the mri appearance of pss . A 26-year - old man presented with a history of dysuria and hematuria for about one month . Serum levels of prostate - specific antigen (psa) were not elevated (0.44 ng / ml). Mri was performed using a 1.5-t signa excite scanner (ge medical systems, milwaukee, wi) and a phased - array torso coil . T2-weighted fast spin - echo (fse) imaging in the transverse and coronal planes, t2-weighted echo - planar imaging (epi) in the transverse plane, t1-weighted fse imaging in the transverse plane, diffusion - weighted imaging (dwi) (using b - factors of 0 and 800 s / mm) in the transverse plane, dynamic contrast - enhanced mri (dce - mri) in the transverse plane, and contrast - enhanced t1-weighted fse imaging in the transverse plane were performed . Data acquisition for dce - mri began simultaneously with the initiation of intravenous injection of gadopentetate dimeglumine (magnevist; bayer schering pharma, osaka, japan) at 0.1 mmol / kg body weight within 10 s through a peripheral intravenous cannula . Multiphase dce images (6 phases) were obtained every 30 s for 150 s without breath - holding . Mri revealed an uneven multinodular mass located mainly within the central zone of the prostate and extending to the right peripheral gland at the bottom level of the central gland mass . 1b). In dce - mri, the central gland portion of the mass showed a weak gradual enhancement containing cystic areas, whereas the right peripheral gland showed a moderate gradual enhancement (fig . Dwi showed the prostatic mass as an area of marked high signal intensity (fig . The resected specimen predominantly comprised elongated ducts and cellular stroma that contained areas of necrosis with calcification (fig . Although epithelial components did not show malignant changes, stromal cells showed ovoid, spindle - shaped and hyperchromatic nuclei in cellular areas, and spindle - shaped nuclei in myxomatous areas . Epithelial cells showed positive immunostaining for pan - cytokeratin (ae1/ae3, cam5.2). In stromal cells, positive immunostaining was detected for vimentin, cd34, and progesterone receptor; whereas, negative results were obtained for pan - cytokeratin, cd31, s-100, hhf-35, desmin, smooth muscle actin, myod1, c - kit, and estrogen receptors . Although the patient was treated by chemotherapy, he showed progressive disease with bone, lung, liver, and mediastinal lymph node metastases and died seven months after admission . Primary prostate sarcomas are rare malignant tumors of the prostate gland (1, 2). Leiomyosarcoma is the most common histological subtype of prostate sarcoma seen in adults (6). Prostate stromal sarcoma is rarer with fewer than 30 documented cases (1 - 5, 7 - 16). Most patients with prostatic sarcoma, including pss, present with symptoms of urethral obstruction (1 - 3, 5, 6), as in the present case . The mass effect of the tumor, mainly located in the central gland, probably caused the early onset of symptoms (17). In previous reports on pss, age at diagnosis has ranged from 19 to 86 years (mean, 48 years) (1 - 5, 7 - 16). Psa levels in patients with pss including the present case have been relatively low (0.1 - 4.5 ng / ml) in comparison with prostatic adenocarcinoma (1, 3 - 5). Pss is often large, with most tumors having a diameter> 4 cm (1, 3 - 5). In immunohistochemical studies, pss are typically positive for vimentin and cd 34, and negative for estrogen receptor and hhf-35 (1, 2). On mri, a prostatic adenocarcinoma typically demonstrates an unidentified area with signal isointensity relative to background prostatic structures on t1-weighted imaging; an area in the peripheral zone with homogeneous low signal intensity with mass effect, or an area in the transition zone with homogeneous low signal intensity, ill - defined margins, and lack of a capsule, with or without a lenticular shape and invasion of anterior fibromuscular stroma on t2-weighted imaging; an area with focal early enhancement on dce - mri; and an area with focal high signal intensity relative to background prostatic structures on dwi (18 - 21). In the present case, the main tumor in the central gland showed a multinodular shape with heterogeneous high signal intensity and a low signal intensity pseudocapsule on t2-weighted imaging . Pss may differ from a typical adenocarcinoma with respect to shape, vascularity on dce - mri, and signal intensity on t2-weighted imaging, thus reflecting tissue construction and cellular structure . Conversely, these pss findings resemble those of leiomyosarcoma and rhabdomyosarcoma, which are a more common form of sarcoma involving the prostate (22, 23). Furthermore, dwi clearly demonstrated both central gland and peripheral zone parts of the tumor as showing marked high signal intensity, suggesting the usefulness of this modality for determining the local extent of the lesion . Mri features of pss have not previously been reported in the english literature . However, two reports of mri findings for stromal sarcoma of uterine endometrium by koyama et al . (25) demonstrated that stromal sarcoma was relatively large and showed heterogeneous signal intensity on t2-weighted imaging, heterogeneous contrast enhancement of the tumor, marginal nodules, multiple nodule formation, hemorrhage, and necrosis in the tumor . Mri findings including t2-weighted imaging, dce - mri, and dwi in the present case seemed to reflect the pathological features of pss, which include aggressive tumor growth, greater cellularity, and necrosis with mitotic activity . In the present case, the tumor was mainly located in the central gland, and appeared as a signal hypointense mass on t1-weighted imaging, and as a heterogeneously signal hyperintense multinodular mass on t2-weighted imaging . Pss did not resemble prostatic adenocarcinoma on mri, which may thus play a role in differentiating pss from prostatic adenocarcinoma . When an adult male shows mri findings as mentioned above, although these imaging findings are not pathognomonic, pss should be included in the differential diagnoses of a prostatic adenocarcinoma.
Although, eus is already available in many countries, there is no official data regarding clinical practice in latin america (la). The main objective of this study was to evaluate the current practice of eus in la and methods of existing training . Other objectives were to obtain information about cost, fee and reimbursement in eus procedures . A web survey form containing four pages with 34 questions (appendix) was developed in three languages (english, spanish and portuguese). Questions 1 - 27 were obligatory to the completion of the questionnaire and were related to clinical practice and training . Questions 28 - 34 were optional and related to costs, fees and reimbursement for eus . A message containing a link to the online questionnaire was sent directly via e - mail, in august 2012, to 268 captulo latino americano de ultrasonido endoscpico (cleus) members organization, which represents physicians interested in eus in la). The message was also forwarded to other physicians who perform eus (non - cleus members) through e - mail by the cleus members who had received the link . The questionnaire was developed together with an it company (trajettoria information technology ltda, so paulo, sp) and was available through the site http://www.cleus-encuesta.com until january 2013 . To ensure confidentiality of the survey and the respondents, an account with login and electronic messages were sent twice a month, before 31 january 2013 requesting the participation of the members who had nt responded and those with incomplete questionnaires . The variables in some multiple choice questions were evaluated based on a score of frequency of responses, expressing a ranking of importance among the variables . The survey was sent to 268 cleus members and 13 new registrations were made, possibly, by referral of cleus members . Of these 70 questionnaires, 57 (81%) participants answered all mandatory questions . Of the 57 endosonographers, 41 (72%) also responded partially or all the optional questions related to cost, fees and reimbursement . Among the la countries, latin american countries represented in the survey characteristics of respondents (n = 70) respondents have an average of 6 years of independent practice in eus (mode: 1 year, median: 3.5 years). Regarding the place of practice, 37 (53%) respondents are employees of private hospitals . A total of 22 endosonographers performed 100 or fewer procedures in their entire career, while 17 performed between 1001 and 5000 exams . The median number of procedures performed in 2011 was: 100 upper eus, 20 lower eus, 30 upper fine - needle aspirations (fna) and 0 lower fna (tabs . Current practice in eus in latin american median of eus procedures performed in entire career the indications for anatomical segments in order of frequency are: pancreatic - biliary - ampulary, gastroduodenal, esophageal, anorectal and mediastinal . The most common indications for pancreatic - biliary - ampulary segment are evaluation of solid and cystic tumors of the pancreas (tab . Score of indications for eus (n = 67) the most frequent complication related to echoendoscopic procedure is bleeding which needed treatment or hospitalization (26 occurrences from 63 respondents). When asked about sedation, nearly 82% of respondents use propofol in most or all procedures . Regarding the type of equipment, the linear echoendoscope is the most used, followed by electronic radial echoendoscope . Other probes (for example, rigid rectal probe) are also used less frequently . 7 and 8 shows the combination of different endoscopes, needles and different brands available on the market . Equipment used by latin american endosonographers score of preferred needles (%) a total of 88% of participants check the result of anatomopathology . The related average number of positive punctures to solid lesions is 80% (n = 54, average = 79.61, = 18.53) and to cystic lesions is 70% (n = 54, average = 70.56, = 23.89). About 48% of respondents have more than 6 months of dedicated hands - on eus training . Almost 90% of respondents consider that formal training in eus is needed to acquire competence . Training methods used to learn eus (n = 70) the minimum average training should be at least 6 months for 72.6% of respondents (tab . 10). Most of the endosonographers consider that the minimum number of supervised procedures should be greater than 50 pancreatic - biliary - ampulary, 20 anorectal, 20 fna and nine therapeutic procedures (tab . Opinion of latin america endosonographers about training numbers of eus performed during training versus opinion on number of eus necessary for training the questions relating to reimbursement by health maintenance organization show that 13 of 28 respondents did not know how much was reimbursed in diagnostic procedures and 13 of 25 respondents did not know about fna procedures . Reimbursement by the government institution is unknown among 18 of 26 respondents for diagnostic procedures and 18 of 25 for procedures with fna . The average costs for eus procedures in private practice ranges from $200 to $4000 among the respondents (n = 37). The costs with the needle ranges from $190 to $1200 (n = 39 respondents, average = $534.77 dollars, = 275.21). Doctors fee also varied considerably, ranging from $170 to $1500 (n = 35 respondents). Respondents have an average of 6 years of independent practice in eus (mode: 1 year, median: 3.5 years). Regarding the place of practice, 37 (53%) respondents are employees of private hospitals . A total of 22 endosonographers performed 100 or fewer procedures in their entire career, while 17 performed between 1001 and 5000 exams . The median number of procedures performed in 2011 was: 100 upper eus, 20 lower eus, 30 upper fine - needle aspirations (fna) and 0 lower fna (tabs . Current practice in eus in latin american median of eus procedures performed in entire career the indications for anatomical segments in order of frequency are: pancreatic - biliary - ampulary, gastroduodenal, esophageal, anorectal and mediastinal . The most common indications for pancreatic - biliary - ampulary segment are evaluation of solid and cystic tumors of the pancreas (tab score of indications for eus (n = 67) the most frequent complication related to echoendoscopic procedure is bleeding which needed treatment or hospitalization (26 occurrences from 63 respondents). When asked about sedation, nearly 82% of respondents use propofol in most or all procedures . Other probes (for example, rigid rectal probe) are also used less frequently . 7 and 8 shows the combination of different endoscopes, needles and different brands available on the market . Equipment used by latin american endosonographers score of preferred needles (%) a total of 88% of participants check the result of anatomopathology . The related average number of positive punctures to solid lesions is 80% (n = 54, average = 79.61, = 18.53) and to cystic lesions is 70% (n = 54, average = 70.56, = 23.89). About 48% of respondents have more than 6 months of dedicated hands - on eus training . Almost 90% of respondents consider that formal training in eus is needed to acquire competence . Training methods used to learn eus (n = 70) the minimum average training should be at least 6 months for 72.6% of respondents (tab . Most of the endosonographers consider that the minimum number of supervised procedures should be greater than 50 pancreatic - biliary - ampulary, 20 anorectal, 20 fna and nine therapeutic procedures (tab . Opinion of latin america endosonographers about training numbers of eus performed during training versus opinion on number of eus necessary for training the questions relating to reimbursement by health maintenance organization show that 13 of 28 respondents did not know how much was reimbursed in diagnostic procedures and 13 of 25 respondents did not know about fna procedures . Reimbursement by the government institution is unknown among 18 of 26 respondents for diagnostic procedures and 18 of 25 for procedures with fna . The average costs for eus procedures in private practice ranges from $200 to $4000 among the respondents (n = 37). The costs with the needle ranges from $190 to $1200 (n = 39 respondents, average = $534.77 dollars, = 275.21). Doctors fee also varied considerably, ranging from $170 to $1500 (n = 35 respondents). Respondents have an average of 6 years of independent practice in eus (mode: 1 year, median: 3.5 years). Regarding the place of practice, 37 (53%) respondents are employees of private hospitals . A total of 22 endosonographers performed 100 or fewer procedures in their entire career, while 17 performed between 1001 and 5000 exams . The median number of procedures performed in 2011 was: 100 upper eus, 20 lower eus, 30 upper fine - needle aspirations (fna) and 0 lower fna (tabs . Current practice in eus in latin american median of eus procedures performed in entire career the indications for anatomical segments in order of frequency are: pancreatic - biliary - ampulary, gastroduodenal, esophageal, anorectal and mediastinal . The most common indications for pancreatic - biliary - ampulary segment are evaluation of solid and cystic tumors of the pancreas (tab score of indications for eus (n = 67) the most frequent complication related to echoendoscopic procedure is bleeding which needed treatment or hospitalization (26 occurrences from 63 respondents). When asked about sedation, nearly 82% of respondents use propofol in most or all procedures . Other probes (for example, rigid rectal probe) are also used less frequently . 7 and 8 shows the combination of different endoscopes, needles and different brands available on the market . Equipment used by latin american endosonographers score of preferred needles (%) a total of 88% of participants check the result of anatomopathology . The related average number of positive punctures to solid lesions is 80% (n = 54, average = 79.61, = 18.53) and to cystic lesions is 70% (n = 54, average = 70.56, = 23.89). About 48% of respondents have more than 6 months of dedicated hands - on eus training . Almost 90% of respondents consider that formal training in eus is needed to acquire competence . Training methods used to learn eus (n = 70) the minimum average training should be at least 6 months for 72.6% of respondents (tab . Most of the endosonographers consider that the minimum number of supervised procedures should be greater than 50 pancreatic - biliary - ampulary, 20 anorectal, 20 fna and nine therapeutic procedures (tab . Opinion of latin america endosonographers about training numbers of eus performed during training versus opinion on number of eus necessary for training the questions relating to reimbursement by health maintenance organization show that 13 of 28 respondents did not know how much was reimbursed in diagnostic procedures and 13 of 25 respondents did not know about fna procedures . Reimbursement by the government institution is unknown among 18 of 26 respondents for diagnostic procedures and 18 of 25 for procedures with fna . The average costs for eus procedures in private practice ranges from $200 to $4000 among the respondents (n = 37). The costs with the needle ranges from $190 to $1200 (n = 39 respondents, average = $534.77 dollars, = 275.21). Doctors fee also varied considerably, ranging from $170 to $1500 (n = 35 respondents). This is the first survey to assess eus practice in la with participation of 17 different countries and one latin territory . Furthermore, it is the first survey that jointly assesses issues relating to practice, training, costs and reimbursement . Furthermore, it is a new method among cleus members, using electronic mail to enable a large number of respondents in different countries . Other north american studies also used electronic mail as a method to reach a larger number of respondents.1234 although the initial number of electronic mails was considerable and the reason why only 25% of participants responded this research can possibly be explained by the fact that not all participants of cleus perform eus and many of them are surgeons and gastroenterologists interested in eus . The largest number of brazilians endosonographers can be possibly explained because it is the most populous country and the human development index is relatively better than many la neighbors.567 according to the annual report of the united nations, brazil (85) is behind four countries in south america, chile (40), argentina (45), uruguay (51) and peru (77), ahead of ecuador (89) and colombia (91). In 1999, in the united states, the median age was 39 years, 57% were in academic practice and 84% performed endoscopic retrograde cholangiopancreatography (ercp).8 in another international study comparing the practice of international and united states respondents, the mean age of participants was 40.5 years and 89.5% were men . The majority (63.4%) were in some type of academic practice.2 in an asian study conducted in 2006, the respondents were mostly young (median age 40 years), male (97%), practicing in public hospitals (50.7%).9 in this survey, endosonographers tend to be male, young, interventional endoscopists (perform ercp) and in most cases were affiliated to a private institution . When compared with other studies, there is a correlation as regards endosonographers being young and mostly perform ercp . In 2004, an international survey proposes to determine the practice patterns of international and united states endosonographers participating in a biennial international eus symposium . This study showed that in the united states, endosonographers are likely interventional gastroenterologists who also perform therapeutic ercp and are more likely to perform eus - guided interventional procedures . International endosonographers were less likely to perform ercp and other interventional eus procedures.2 in this study, it was not possible to perform this comparison between different countries . In the 1999 united states survey, although the median total number of procedures ever performed was 200, 41% of respondents performed half or more of their total eus procedures during the year prior to the survey.8 in the 2004 international survey, approximately 90% of international as well as the united states endosonographers had been performing eus for more than 1 year and more than a third had been performing eus for at least 5 years.2 in the 2006 asian survey, the respondents had median experience of 5 years in eus practices and performed a median of 500 procedures in their career.9 in this survey, most endosonographers have been performing eus for at least 1 year . The vast majority of procedures are diagnostic upper eus compared to eus / fna and lower eus . Lower eus was performed much less frequently than upper eus, probably about the least frequent indications for the procedure and/or rectal and anal sphincter evaluation by radiologists using magnetic resonance . This is consistent with research conducted in 1999;8 however, a 2006 study assessed the level of knowledge of gastroenterologists american society of gastroenterology (asge) members about the indications of eus in four segments: esophagus, gastroduodenum, hepatopancreatobiliary and colorectum . A research showed that gastroenterologists who perform eus have greater knowledge of indications of eus . However, the levels of knowledge of colorectal applications of eus are the poorest among the four studied organ systems . This refers to the fact that educational initiatives should focus on applications of eus in this category.4 regarding indications for upper eus, pancreatobiliary segment was among the most studied and the indications in this segment are the study of cystic and solid lesions of the pancreas . In another study,8 esophagogastric and pancreatico - biliary were the most common, but there is no detail about the lesions studied in each segment . In this study, linear echoendoscope was the most used, but when compared with radial echoendoscope (electronic and mechanic), the difference is not significant . Most participants also perform ercp . In 2004, das et al.2 compared north american endosonographers with other international endosonographers and showed that north americans are more likely to use linear echoendoscope and perform ercp more frequently . This possibly explained why north american endosonographers were the most of participants also perform ercps were propense to perform eus interventional procedures are more likely to use liready likely to perform eus - fna and other interventional procedures . In this study, most endosonographers perform ercp and use the linear echoendoscope, but it is not possible to conclude that there is a propensity for eua - fna and other interventional procedures . Perhaps, this reflects the fact that the indication for pancreatic segment is the most common and most studies in eus - fna performed in the pancreatic masses setting have been conducted using 22-g needles.10 in this study, the complication most often reported in eus is bleeding; followed by infection and perforation . In the literature, the incidence of bleeding reported in large prospective series range between 0% and 0.5%.10 in relation to learning methods, the majority of respondents had a formal training of 6 months or more in eus after graduating in fellowship programs in gastroenterology or gastrointestinal endoscopy . This can be a reflection that new strategies are being put in place and newly trained endosonographers have a well - established training program than older endosonographers who did their training mainly in france, usa, japan and germany . In la, few training centers came into being in recent years, for instance, centro franco - brasileiro de ecoendoscopia in brazil and clinica alemana in chile . About 24% of endosonographers also learned eus observing other more experienced physicians . This may be related to the fact that even younger endosonographers spend a period during their training programs in other centers outside la . Even in europe, the combination of two methods of learning eus is practice; consisting of dedicated fellowship program and informal training (short repeated exposures to hands - on experiences).10 in this study, 44% of endosonographers train others in eus . This number does not differ much from what was seen in the study published in 1999 with the united states endosonographers, where 40% trained other doctors . Nearly 90% of participants agree that formal training is necessary to acquire competence, 80% think that training strategies must depend upon local endoscopy societies and 73% of respondents consider a time period longer than 6 months to acquire competence . In addition, during the training program, a minimum number of procedures for each anatomical segments, fna and therapeutic procedure are indicated by the respondents . Thus, as ercp, learning in eus is considered technically more demanding and there is a variation in individual learning curve that should be evaluated by criteria goals . Currently, there is no consensus on the number of procedures required to acquire competence . The asge guidelines published in 2006 states the minimum number of procedures: 75 cases of esophagus, stomach and rectum tumors; 40 cases of submucosal abnormalities; 75 pancreatobilliary cases; 25 eus - fna (non - pancreatic) and 25 eus - fna (pancreatic). The competence recommended generally in all aspects of eus would be a minimum of 150 supervised cases, of which, 75 should be pancreatobilliary and 50 eus - fna . Trainees may start performing eus - fna after 50 procedures or more.1112 a study in the asia - pacific region also considered that there should be a median number of 100 supervised procedures with a minimum of 6 months training . Most respondents (90%) also believe that formal internship is necessary to acquire competence.9 as a result, new training strategies become imperative and each country should implement more appropriate training orientations to ensure quality in training and clinical practice of eus in la . Insufficient statistical data did not allow us to reach a conclusion regarding costs, fees and reimbursement . This is because, 58 of the participants answered the questions related to these topics, about half of the respondents had no knowledge as regards refund of eus procedures and there are different payment systems in different countries in la . The questionnaires were sent to all cleus members and there was an increase in the sample through the e - mails sent to non - members . Therefore, there are limitations in the methodology of this research, as we do not know accurately the number of doctors who perform eus in la and it is unlikely that all endosonographers have responded this survey . Furthermore, it is likely that some respondents did not participate in the survey because they do not perform a large number of procedures . Even sending reminders over the months in which the research was online, some doctors may not have received the questionnaire due to the limitations of the tool used for dissemination (incorrect addresses, the presence of anti - spam, among others). This study provides an overview on the status of eus in la and from our standpoint, the greatest contribution of this study is the perception of the need to standardize eus training strategies in la.
Dental restorations require finishing and polishing procedures to fulfill the requirements of form and function and to promote aesthetics, longevity, and periodontal health [1, 2]. Different methods can be used for finishing and polishing, and the surface smoothness obtained is dependent on the composition of the composite, the presence of bubbles, and the instruments and procedures used . There is controversy regarding the best time to perform finishing and polishing of composite restorations . Although composite manufacturers recommend performing the procedure immediately after the restoration, it should be delayed to avoid the negative effects of heat generation, such as smearing of the resin matrix and the creation of local hot spots before the final polymerization of the composite . However, delayed finishing and polishing resulted in lower microhardness than immediate polished composite specimens . A silorane - based low - shrinkage composite has been introduced as an alternative to methacrylates and has low filler content by volume with a combination of fine quartz particles and yttrium fluoride . When compared to methacrylate - based resins, the silorane - based composite has exhibited lower water sorption and solubility, lower adhesion potential of oral streptococci, and similar adhesion potential of candida albicans [810]. Cusp deflection caused by polymerization contraction was significantly lower on teeth restored with an experimental silorane - based composite compared to teeth restored with a methacrylate - based composite . Furthermore, no leakage was found when mesio - occluso - distal cavities were restored with silorane - based composites . Silorane has shown lower compressive strength and microhardness and greater flexural strength and fracture toughness . However, silorane has shown lower degrees of conversion and polymerization depth than methacrylate - based composites . Changes in matrix composition, the introduction of new monomers, filler content optimization, and variations in particle size, type, and morphology can all increase the surface roughness of composites and can result in plaque accumulation, gingival inflammation, and surface staining . Few previous studies have compared the use of different finishing and polishing systems on a silorane - based composite [15, 17] and, to date, no studies have verified the effect of immediate or delayed polishing on this restorative material . The objective of the present study was to evaluate the effect of immediate or delayed finishing / polishing using different systems on the surface roughness, hardness, and microleakage of a silorane - based composite . The null hypothesis tested was that the time to perform finishing / polishing and the use of different systems do not affect the surface roughness, microhardness, and microleakage of a silorane - based composite resin . The main factors evaluated in this in vitro study were finishing / polishing systems at four levels control (light - cured in contact with polyester strip), aluminum oxide discs (sof - lex, 3 m espe), diamond - impregnated silicone tips (astropol, ivoclar vivadent), and aluminum oxide - impregnated silicone tips (enhance, dentsply)and the time to perform finishing / polishing at two levels (immediately and after 7 days). The specimens were made of silorane - based composite (filtek p90, 3 m espe) following a randomized complete block design . The dependent variables were mean surface roughness (ra, m) (n = 20), vickers microhardness (n = 10), and microleakage at the enamel and dentin margins, evaluated by dye penetration scores (n = 10). The surfaces of the specimens were analyzed using scanning electron microscopy (sem). A two - part teflon mold (diameter = 10 mm and high = 2 mm) was filled with the composite in a single increment, using a 70 spatula (ss white / duflex, rio de janeiro, rj, brazil). A polyester strip and a glass slide were positioned on the strip, and a load of 500 g was applied for 30 seconds, followed by light - curing (40 seconds, 600 mw / cm) using a qht light - cure unit (demetron lc, kerr corporation, middleton, wi, usa) positioned in direct contact with the polyester strip . A marking was made on the outer edge of each specimen to standardize the direction of rotating device application . Half of the specimens of each group randomly divided received finishing / polishing immediately after preparation . The other half was stored in dark container on gauze soaked in distilled water at 37c for 7 days . The surface finish was provided by the polymerization of the composite in contact with a polyester strip . It is the sequential application of medium, fine, and superfine grain discs, mounted on the handpiece . Each disc was applied to the specimen for 10 seconds under constant cooling with a water jet . It is the application of disc - shaped enhance tips, mounted on the handpiece for 30 seconds under constant cooling with a water jet . This is the sequential application of astropol hp (gray), astropol p (green), and astropol f (pink) discs, mounted on the handpiece . Each disc was applied to the specimen for 10 seconds under constant cooling with a water jet . The specimens were washed for 10 seconds with compressed air / water and cleaned (ultrasonic bath for 30 seconds in distilled water), dried with paper towels, and stored dry . A single operator performed all laboratory procedures in an air - conditioned environment at temperature of 21 2c . The same specimens were used sequentially for measurements of surface roughness and microhardness . A profilometer (mitutoyo sj-301 surftest, aurora, il, usa) calibrated with a standard of known roughness the arithmetic mean of the absolute distance of the roughness profile (ra, m) was recorded within a measuring length of 4 mm and with a cut - off of 0.8 mm . Four readings were taken for each specimen, one parallel, one perpendicular, and two diagonal in relation to the direction of the finishing / polishing instrument application . Vickers microhardness was conducted on a mvk - h1 microhardness tester (hardness testing machine, mitutoyo, kanagawa, ken, japan) under 50 g load, over 45 seconds . Four indentations were made on each specimen, one in each quadrant, equidistant from the center . The readings were recorded immediately after removal of the penetrator to minimize the effect of elastic recovery . The mean of the four indentations was used to determine the vickers hardness number of each specimen . For illustrative purposes, two specimens representative of each experimental condition were prepared for surface characterization using sem (quanta 200f, fei, hillsboro, or, usa). The specimens were metallized with carbon, and the surface was examined with up to 3,000x magnification and an accelerating voltage of 10,000 v. extracted bovine teeth were scaled to remove organic and inorganic debris and were stored in 0.05% thymol solution . Eighty teeth were visually selected based on the absence of cracks, stains, and excessive incisal wear . After thorough washing in running water, cylindrical cavities (2.0 mm in diameter and 1.5 mm in depth) were prepared using 2294 diamond bur (kg sorensen, cotia, sp, brazil) in a high - speed handpiece under constant air / water cooling . Two cavities were prepared on the buccal surface of each tooth to 3 mm of the enamel - cement junction, one on the coronal portion and the other on the root surface . The cavities dimensions were checked using a millimeter probe (hu - friedy, chicago, il, usa). The coronal restoration operative steps were (a) application of 37% phosphoric acid (condac, fgm, joinville, sc, brazil) for 30 seconds on the enamel, washing with air / water spray for 60 s, and drying with paper towels; (b) active priming (silorane adhesive system, 3 m espe) application for 15 seconds, applying a gentle air stream until the primer was spread into a uniform film, and light - curing for 10 seconds; and (c) adhesive application (silorane adhesive system, 3 m espe) into the cavity, applying a gentle stream of air until the adhesive was spread into a uniform film, and light - curing (10 seconds). The composite was then inserted into the cavity in a single increment and was light - cured (40 seconds, 600 mw / cm) in contact with a polyester strip . Root cavities were restored following the operative steps described above, except for the application of phosphoric acid . The restorations were subjected to the same finishing / polishing procedures as described for the roughness and microhardness tests . The other half were stored in a dark container on a gauze pad soaked in distilled water at 37c and received finishing / polishing seven days after preparation . A single operator performed all laboratory procedures in an air - conditioned environment at a temperature of 21 2c ., so paulo, sp, brazil) 3 mm in diameter was applied to the restoration and adjacent tooth structure . The apical foramen was covered with plastified wax (wilson, polidental, cotia, sp, brazil), and a double layer of nail polish (impala, mundial sa, so paulo, sp, brazil) was applied over the entire tooth surface . After the nail polish dried, the tape discs were removed to expose the restoration and the 1 mm adjacent tooth structure . The specimens were immersed in 0.5% basic fuchsin for 24 hours at room temperature, washed in running water for 2 minutes, and dried with absorbent paper . The teeth were sectioned on the enamel - dentin junction to separate the crowns of the roots . Then they were sectioned longitudinally in a buccolingual direction through the center of the restorations using a double - faced diamond disc (kg sorensen, cotia, sp, brazil). A trained examiner (kappa = 0.86) examined both crown and root hemisections to determine the scores of dye penetration in the restoration margins . The cervical and incisal or apical margins of the coronal and root restorations were examined separately using a stereomicroscope (stemi dv4, carl zeiss, gttingen, ls, germany) with 32x magnification and were classified according to the following scores: (0) no evidence of dye penetration at the tooth / restoration interface; (1) dye penetration up to half the length of the wall; (2) dye penetration along all the length of the wall; and (3) dye penetration along all the length of the wall and in the axial direction . Kolmogorov - smirnov and shapiro - wilk tests verified the normal distribution of surface roughness (p = 0.094) and microhardness data (p = 0.200). Levene's test was used to verify the equality of variances for the surface roughness (p = 0.547) and microhardness (p = 0.916) data . The effects of the finishing and polishing systems, the time of application, and the interactions were analyzed using two - way analysis of variance (anova) and scheffe post hoc test . Kruskal - wallis test compared median microleakage scores to determine the effects of finishing / polishing systems and time of application on the enamel and dentin margins . Two - way anova found a significant effect of finishing / polishing system on surface roughness (p <0.0001). There was no significant effect of the application time (p = 0.2851) or the interaction between polishing system and application time (p = 0.0669). The control group produced significantly less surface roughness than the other types of polishing, which did not differ from each other (table 2). Two - way anova showed a significant effect of finishing / polishing system on microhardness (p <0.0001). There was no significant effect of the application time (p = 0.500) or of the interaction (p = 0.313). The control group had significantly lower microhardness than the astropol and sof - lex groups but did not differ from the enhance group . Enhance did not differ from astropol but produced significantly lower microhardness than sof - lex . Sof - lex produced significantly higher microhardness than control and enhance but did not differ from astropol (table 3). For the enamel margins, delayed polishing produced significantly higher microleakage than immediate polishing (p = 0.009), but there was no significant effect of finishing / polishing system (p = 0.309). Medians, minimum and maximum values, and mean ranks of microleakage observed for immediate and delayed finishing / polishing in enamel and dentin are presented in table 4 . For the dentin margins, there was nosignificant effect of the application time (p = 0.313), but there was a significant effect of finishing / polishing type on microleakage (p = 0.033). Table 5 shows median, minimum and maximum values, and mean ranks of microleakage for the finishing / polishing systems studied . Sem analysis showed that when subjected to immediate or delayed finishing / polishing procedures, the composite surface showed more irregularities than the control . The control group exhibited a smoother texture and unscratched surface (figures 1(a) and 1(b)). Scratches and porosity resulting from the displacement of particles were observed on the surfaces treated with astropol (figures 1(c) and 1(d)), enhance (figures 1(e) and 1(f)), and sof - lex discs (figures 1(g) and 1(h)). In agreement with the profilometric findings, rougher surfaces were observed in the groups that underwent finishing and polishing, with no differences between them and between times of application . The polyester strip provides a smoother surface, but its use is limited by the complex occlusal anatomy and need for functional adjustments in almost all restorations, requiring the use of tools to provide a smooth final surface . Some of these factors are intrinsic to the material and are related to its composition, such as filler type, shape, size, and distribution, the type of resin matrix, the degree of final cure achieved, and the bond efficiency at the filler / matrix interface . Extrinsic factors are associated with the type of polishing system used, such as the flexibility of the material in which the abrasives are incorporated, the hardness of the abrasives, the geometry of the instruments, and the way they are used [3, 17, 18]. In the present study, the surface roughness obtained for the silorane - based composite light - cured in contact with a polyester strip (0.193 0.063 m) was similar to the mean found in a previous study (0.220 0.292). These values are close to the critical value of 0.2 m proposed as the roughness threshold required for the accumulation of biofilm . Silorane - based composite presented comparable or less roughness than microhybrid and nanoparticle composites for restorations in posterior teeth when subjected to standardized polishing with 1000 grade abrasive sandpaper [2, 3, 13, 19]. The relatively low quartz and yttrium fluoride particle contents (76% p / p or 55% p / v) and the mean particle size of 0.5 m (0.12 m) in silorane - based composite may have contributed to this surface roughness value [13, 14, 20]. The images obtained by sem confirmed these findings, showing a smooth surface texture without scratches in the control group . However, in this study, after the application of the finishing and polishing systems, the resulting mean roughness in all groups was approximately more than twice the value of 0.2 m for both immediate and delayed polishing . The mean values measured were higher than those shown in other studies [3, 8, 10, 18, 21]. Some studies employed 1000 to 4000 grade sandpaper in a mechanical polisher with water lubrication and controlled rotation before the application of the polishing systems under testing conditions [3, 8, 10, 18]. This procedure may have favored to obtain a lower mean ra because it promotes more uniform removal of the surface layer of the composite . In the present study, no statistically significant difference was observed between the systems studied regarding surface roughness . This result is in agreement with another study that compared different finishing and polishing systems for silorane - based composites . No significant difference was observed for the application times of finishing and polishing procedures, demonstrating that immediate or delayed polishing did not affect this property . This behavior has been previously described for microparticulate and hybrid methacrylate - based composites [4, 5, 22]. Images obtained by sem showed scratches and porosities resulting from particle displacement on the surfaces submitted to finishing and polishing instruments at both application times . To ensure the effectiveness of the finishing system the present study showed similar performance between aluminum oxide discs, diamond - impregnated silicone tips, and silicone discs covered with aluminum oxide . Different effects could be expected because astropol hp contain diamond particles in its composition, while sof - lex discs and enhance tips use aluminum oxide as abrasive particles . Diamond is harder than aluminum, causing deeper grooves on the surface of the composite, which results in more roughness . Nonetheless, instruments impregnated with aluminum oxide were able to wear quartz and yttrium fluoride particles and the silorane matrix uniformly [17, 18, 21]. Hardness can be defined as the resistance of solid structures to permanent indentation or penetration . Changes in hardness may reflect the cure state of a material and the presence of either a continuous reaction or the maturity of the restorative material [4, 5, 15, 22]. In general, increasing the particle size increases the resistance and surface hardness of the composite . Moreover, the type, morphology, distribution, and volume fraction of filler particles and the concentration of diluent monomers affect the hardness of the composite [15, 23]. The vickers hardness number (vhn) reported for silorane - based resin (measured after polishing with abrasive sandpaper) has ranged from 59.26 to 80.8 [10, 18, 20, 24]. In this study, the mean vhn measured immediately after the use of different finishing and polishing systems ranged from 55.77 to 60.91 . This lower range may be related to the fact that no prior polishing with abrasive sandpaper was performed to mimic the clinical conditions in which finishing and polishing systems are responsible for the removal of the surface layer rich in organic matrix . In this experiment, the use of different finishing and polishing systems resulted in a significant difference in the microhardness of silorane - based resin when it was applied either immediately or after 7 days . The use of sof - lex resulted in significantly higher microhardness compared to enhance, which was not different from astropol, which yielded intermediate microhardness values . We did not find previous studies that have evaluated the microhardness of silorane - based resin in response to different finishing and polishing systems . It is likely that the use of the abrasive disc sequence more effectively removed the organic matrix rich layer compared to other systems, exposing a harder surface . One might expect to find greater hardness for a silorane - based composite polished after 7 days because its polymerization reaction is characterized by continuous cationic ring opening initiated at the time of light - curing . However, no significant differences were observed in the hardness of the silorane - based resin after various storage times (1 to 30 days), probably due to the presence of siloxane, which has reduced water solubility and sorption . The dye penetration test was performed to determine the marginal sealing capacities of the restorations under the conditions evaluated . Overall, median microleakage scores were low (0.00 to 0.50) for the enamel and dentin margins . Silorane - based composite restorations of class ii and class v cavities with enamel and dentin margins had similarly low microleakage results [13, 19, 2529]. This result can be attributed to the ring opening chemical of the silorane system, which provides the system with less polymerization shrinkage, and to the enamel and dentin adhesion promoted by the self - etching silorane adhesive system . The effects of the finishing and polishing systems on microleakage were observed only for the dentin substrate . The use of the sof - lex discs promoted greater microleakage compared to the control and enhance and did not differ from astropol . It seems that the use increasing grit sequential tips or discs produced some kind of damage to the tooth - restoration union, notably on the dentin margins, which have less mineral content and more moisture compared to the enamel . It suggests that after 7 days of storage in a humid environment at 37c, there was a loss of bond quality of the self - etching adhesive system, even when enamel etching had previously been performed with 37% phosphoric acid . Immediate polishing did not make the silorane - based composite more susceptible to the supposed adverse effects of heat generation, probably due to the stability of its aromatic rings . Considering the limitations of this in vitro study, it can be concluded that immediate polishing does not adversely influence the surface roughness, microhardness, and microleakage of silorane - based composite resin . The sequential aluminum oxide discs system produced a beneficial effect on microhardness but negatively influenced microleakage on the dentin margins . Considering the three outcomes studied as a whole, no finishing and polishing system performance was superior to the others.
The increase in incidentally found small renal tumors has served as an impetus to develop less invasive parenchymal - sparing techniques for tumor resection . Recent studies have shown that renal parenchymal - sparing procedures yield comparable outcomes with regard to tumor control compared with outcomes of radical nephrectomy for small tumors . To reduce the morbidity of partial nephrectomy, several investigators have reported laparoscopic partial nephrectomy (lpn) in select patients with small lesions . Several hemostatic modalities have been used during lpn with variable success, including argon beam coagulation, neodymium: yag laser, harmonic scalpel, hand assistance, bipolar electrical current, unipolar spray electrical current, ultrasound scissors, microwave tissue coagulation, cable - ties, and radiofrequency ablation . Based on the success of holmium: yag laser prostatectomy, we designed a study to evaluate the feasibility and efficacy of the holmium laser in performing lpn in a porcine model . In an animal care and use committee - approved study, laparoscopic transperitoneal lower pole partial nephrectomy was performed using the holmium: yag laser in 5 female farm pigs (average weight, 45 to 50 kg). A pilot study in a single animal four separate lpn were performed using the holmium laser at settings of 0.5 joules at 55 pulses / sec, 0.8 joules at 40 pulses / second, or 0.2 joules at 60 pulses / second . All settings resulted in successful partial nephrectomy with good hemostasis (estimated blood loss, <50 cc). The setting of 0.2 joules at 60 pulses / second provided an almost continuous delivery of energy, which produced smooth cutting of the kidney and was therefore chosen for the current study . The holmium laser was used in all 5 animals to perform laparoscopic transection of the lower pole of the left kidney; 2 weeks after the procedure, a right lower pole laparoscopic partial nephrectomy was performed, and the animals were immediately sacrificed . For the purposes of analysis, the initial left lpn kidneys are referred to as the survival kidneys, and the right lpn kidneys are the acute kidneys . General endotracheal anesthesia was induced using an initial dose of terazol 4 mg / kg and ketamine 2.2 mg / kg with isoflurane 2% used for maintenance . Three ports were placed: a 12-mm trocar just lateral to the rectus muscle at the level of the umbilicus, a 12-mm trocar in the lower lateral quadrant at the midclavicular line, and a 12-mm trocar approximately halfway between the umbilicus and the xiphoid at the midclavicular line . The lower pole of the kidney was mobilized, and the proximal ureter was displaced medially, away from the lower pole of the kidney . A 2-mm port was then inserted under laparoscopic guidance at the level of the lower pole of the kidney to accommodate and stabilize the end - fire holmium: yag laser fiber (trimedyne corporation, irvine, ca) (1000 m [n=6] or 550 m [n=4]). With the laser set at 0.2 joules / pulse and 60 pulses per second, an incision was initiated 1 cm below the level of the hilum with direct laser contact on the parenchyma . Then a groove was cut into the cortex of the kidney from medial to lateral (figure 1), which was deepened until the lower pole was completely excised . The laser was defocused as necessary to coagulate small areas of bleeding . By withdrawing the laser from contact with the cut surface, a larger surface area was encompassed by laser energy (defocused), permitting coagulation of any bleeding surface . Fibrin glue (1 cc) was then applied to the cut edge of the kidney to seal the collecting system . In the animals undergoing left lpn (survival kidney), the specimen was removed intact using an organ entrapment sack via an extension of a trocar site . Serum creatinine was obtained on postoperative days 0, 1, 4, and 7 . At 14 days, the pigs were anesthetized and a laparoscopic right lower pole partial nephrectomy was performed . The kidneys were then harvested and an in situ left retrograde pyelogram was performed (n=4). General endotracheal anesthesia was induced using an initial dose of terazol 4 mg / kg and ketamine 2.2 mg / kg with isoflurane 2% used for maintenance . Three ports were placed: a 12-mm trocar just lateral to the rectus muscle at the level of the umbilicus, a 12-mm trocar in the lower lateral quadrant at the midclavicular line, and a 12-mm trocar approximately halfway between the umbilicus and the xiphoid at the midclavicular line . The lower pole of the kidney was mobilized, and the proximal ureter was displaced medially, away from the lower pole of the kidney . A 2-mm port was then inserted under laparoscopic guidance at the level of the lower pole of the kidney to accommodate and stabilize the end - fire holmium: yag laser fiber (trimedyne corporation, irvine, ca) (1000 m [n=6] or 550 m [n=4]). With the laser set at 0.2 joules / pulse and 60 pulses per second, an incision was initiated 1 cm below the level of the hilum with direct laser contact on the parenchyma . Then a groove was cut into the cortex of the kidney from medial to lateral (figure 1), which was deepened until the lower pole was completely excised . The laser was defocused as necessary to coagulate small areas of bleeding . By withdrawing the laser from contact with the cut surface, a larger surface area was encompassed by laser energy (defocused), permitting coagulation of any bleeding surface . Fibrin glue (1 cc) was then applied to the cut edge of the kidney to seal the collecting system . In the animals undergoing left lpn (survival kidney), the specimen was removed intact using an organ entrapment sack via an extension of a trocar site . Serum creatinine was obtained on postoperative days 0, 1, 4, and 7 . At 14 days, the pigs were anesthetized and a laparoscopic right lower pole partial nephrectomy was performed . The kidneys were then harvested and an in situ left retrograde pyelogram was performed (n=4). Gross inspection and histopathologic evaluation using hematoxylin and eosin staining were performed . All lower pole partial nephrectomies were successfully completed, and hemostasis was adequate in all cases . No statistically significant differences were noted in specimen length (as a percentage of renal length) or weight between the acute and survival groups . The differences in the operative time can be accounted for by the additional time required to apply fibrin glue, remove the specimen, and close the trocar sites in the survival animals . No difference in the cutting or hemostatic ability existed between the 550 and 1000 m fibers . Left retrograde pyelograms performed on 4 survival animals demonstrated extravasated contrast in 2 animals that was contained by small bowel that adhesed to the cut surface of the kidney . The acute amputation sites had a slightly irregular surface contour with minimal amounts of adhesed blood and fibrin . Figure 2 shows a representative cut surface of the kidney after an acute and 2-week survival lpn . Microscopic sections from the acute amputation sites showed mild histologic changes including increased cytoplasmic eosinophilia of the renal tubules with blurring of the cytoplasmic borders and elongation of the nuclei . Sections from the 2-week specimens showed a 1.5- to 2.0-mm zone of organized fibrosis with admixed lymphohistiocytic inflammation . With the increase in incidentally detected renal lesions, the indications for partial nephrectomy are expanding . However, the procedure is more technically challenging than radical nephrectomy, both with the open and laparoscopic approaches . In contradistinction to open partial nephrectomy where hilar occlusion limits blood loss, the laparoscopic approach precludes safe hilar occlusion due to the risk of warm renal ischemia . Furthermore, routine suturing of transected vessels during lpn is technically challenging and time - consuming . These concerns have prompted a search for techniques that provide safe, reliable hemostasis during lpn . Winfield and colleagues performed the first lpn in 1992 in a patient with a lower - pole stone - bearing caliceal diverticulum . An electrosurgical blade and laparoscopic argon beam coagulator were used to achieve hemostasis, which was further facilitated by closure of gerota's fascia . Winfield and associates subsequently reported on lpn in 6 patients, among whom 2 required open conversion . The argon beam coagulator was also used to achieve hemostasis in this group of patients . Janetschek and colleagues used a neodymium: yag laser (n=1), argon beam coagulator (n=1), and bipolar coagulation forceps (n=5) along with oxidized cellulose and gelatin resorcinol formaldehyde glue to perform 7 laparoscopic wedge resections for small solid masses (1 cm to 2 cm) without significant complications . Hoznek and coworkers performed retroperitoneal lpn in 13 patients using rotating tip coagulating scissors, bipolar coagulator, harmonic scalpel, and occasional hilar occlusion to incise the kidney and collagen mesh with gelatin resorcinol formaldehyde glue to achieve hemostasis . Recently, yoshimura and coworkers utilized a microwave tissue coagulator to perform laparoscopic partial nephrectomy in 6 patients with small renal tumors (<25-mm). Also, wolf et al performed 10 lpn using hand assistance in 8 with comparable convalescence to open partial nephrectomy . In these lpn as can be discerned from the various methods used to achieve hemostasis in these series, an ideal surgical technique has not yet been developed . Since 1994, the availability of high - powered laser systems has expanded the applications of laser technology from stone fragmentation to tissue ablation and incision . The holmium: yag laser has a wavelength that is strongly absorbed by water (2100 nm) and has a 0.5-mm depth of penetration . The ability to cut and ablate with the holmium laser is the result of rapid heating of water in tissue . Johnson and colleagues found that the area of coagulative necrosis produced with the holmium laser was less than that associated with the neodymium: yag laser, although both lasers achieved a similar degree of hemostasis in renal tissue . In a porcine model, the holmium laser effectively transected renal parenchyma while maintaining hemostasis without the need for hilar dissection or vascular occlusion . Both the 1000-m and 550-m laser fibers resulted in successful lpn, but the 1000-m fiber was easier to use due to greater stiffness that increased manual precision . First, the 2-mm port was occasionally vented to facilitate removal of smoke that accumulated during laser activation . Second, when the laser was defocused to coagulate bleeding sites, visibility was occasionally obscured by blood spray, requiring that the camera be maintained at a distance to avoid the spray . This is a drawback of using the laser in a dry co2 pneumoperitoneum as opposed to submersed in saline as is the case with wet lastly, minimizing traction on the cut surfaces of the kidney reduced mechanical trauma to the kidney and subsequent blood loss . Instead in addition, the loss of renal function resulting from a unilateral partial nephrectomy is not adequately quantified by serial serum creatinine measurements and would be better assessed with differential renal function from a renogram . Although the holmium laser provided adequate hemostasis, sealing of the collecting system was inconsistent, (2 of 4 animals had extravasation on retrograde pyelogram). The reliability of fibrin glue and other products to consistently seal the peripheral collecting system requires further investigation . Of note, none of the pigs developed clinical symptoms from these minor leaks . In clinical use, a perinephric drain would be placed and perinephric fat would be positioned over the cut surface . Finally, a size differential exists between the pig kidney and the adult human kidney . The holmium laser at lower power settings may not adequately coagulate larger vessels in human kidneys . Our pilot study animal showed that hemostasis was easily attained with higher energy levels (0.5 and 0.8 joules), and these settings may prove more appropriate for human use . In fact, we have successfully performed this technique clinically in 3 patients and found the setting of 0.8 joules was able to achieve adequate hemostasis in adult kidneys (in submission). Further clinical evaluation will be necessary to determine this technique's utility for performing lpn . The holmium: yag laser is an effective modality for achieving partial nephrectomy in a porcine model . The procedure is quick, safe, and relatively simple . With further study, this technique may be added to the armamentarium of the laparoscopic renal surgeon.
This article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by any of the authors . Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study . This article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made.
Neurodegeneration is known as the progressive loss of structure or function of neurons, including death of neurons . Many neurodegenerative diseases including alzheimer's disease, parkinson's disease, and huntington's disease occur as a result of neurodegenerative processes . Regenerative therapy could be a promising approach as the regeneration of lost or altered cellular functions, which could significantly reverse functional decline to an extent that raises the patient's survival rate and physiological function . As a representative of the neurodegenerative disorders, alzheimer's disease (ad) is pathologically characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions . Neurofibrillary tangles, and senile plaques involve the basal forebrain cholinergic system, amygdala, hippocampus, and cortical areas . Neural loss results in gross atrophy of the affected regions, including degeneration in the temporal lobe and parietal lobe, and parts of the frontal cortex and cingulate gyrus . The national institute of neurological and communicative disorders and stroke (nincds) established the most commonly used criteria for ad . According to these criteria, the presence of cognitive impairments (learning & memory, language, perceptual skills, orientation), and a suspected dementia syndrome confirmed by neuropsychological testing are contributed to the clinical diagnosis of ad . The observable symptoms are often mistakenly thought to be age - related concerns, or manifestations of stress . In the early stage, the most common symptom is unable to acquire new memories, observed as difficulty in recalling recently observed events . When ad is suspected, the diagnosis is usually confirmed with behavioral assessments and cognitive tests, often followed by a brain scan if available . Nowadays, specific treatments for particular symptoms of ad are available . However, specific disease - modifying treatments aimed at preventing or reversing the basic pathophysiologic processes of ad still remain under investigation . In this literature, the recent research advances in stem cell treatments of ad are reviewed from an experiment to a clinical research . Currently, there is no proven cure for ad . In terms of drug therapy, no drug treatment can reverse, stop, or even slow this inexorable neurodegenerative process . Also, non - drug treatments, including gene therapy and behavioral interventions, can only bring temporary symptomatic relief and not result in halting the progression of these diseases . In recent decades, neurogenesis has been proved to exist in restricted regions of the adult brain in many kinds of species including humans . This continuing neurogenesis in the subventricular zone, olfactory bulb, and hippocampal dentate gyrus is supported by the identification of neural stem cells (nscs), suggesting that the adult central nervous system (cns) may be amenable to the cell intervention . A variety of animal models have been developed to examine the etiology of ad and to determine the responses of endogenous and/or transplanted stem cells to the pathological microenvironment of the brain . Under many conditions, adult neurogenesis is impaired, and the dysfunctional neurogenesis, both decreased and increased, has been reported for ad transgenic models . Increasing research evidences in recent decades may promise a bright future for stem cell based neuroreplacement therapies for ad . The adult - born neurons in the diseased brain seem to be a good candidate for those lost or apoptotic neurons . Because stem cells can be genetically modified in vitro and have high migratory capacity after transplantation into the brain, they can be an efficient way to delivery neurotrophic factors or enhance gene expression that can modify the course of the disease . Since neural progenitor / stem cells have been proven to exist in the adult cns and to be involved in the neurogenesis process, the activation of endogenous neural progenitor / stem cells populations that can migrate to the injured regions, proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain . This activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors, such as brain derived neuronal factor (bdnf) and vascular endothelial growth factor . A recent research has demonstrated that the self - repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke . Currently, it is widely accepted that neurogenesis in the hippocampus are involved in learning and memory formation . The hippocampus - dependent learning tasks can significantly increase the proliferation of endogenous neuronal progenitors, survival of new neurons and, the task performance by animals correlates positively with the amount of adult born neurons . Alterations in the microenvironment where the endogenous nscs reside play an important role in nsc activation . In ad brain, for example, immune responses include the activation of microglia and astrocytes around the senile plaques area and t - lymphocyte infiltration into the damaged brain . These cells produce cytokines and other molecules that promote or inhibit the neurogenic function of the nscs . Therefore, it is possible for damaged cells to be replaced from endogenous nsc pools . Studies have also suggested that the capacity of endogenous nscs to compensate for lost cells is limited . In spite of the activated proliferation, obviously, most of the new migrated neurons in and near the injured area die before differentiating into functional neurons, possibly because of a lack of factors and stimulation to support their survival and differentiation; thus, only 0.2% of the dead neurons are replaced . However, it has not been determined whether these effects depend directly on the promotion of neuronal regeneration by nscs, or whether accompanying events, such as enhanced glial regeneration and other types of trophic support, are more important . Moreover, a key issue in the field of neuronal regeneration is that newly generated neurons need to make the appropriate connections, although the details of this process are still largely unknown . Further studies are needed to clarify how newly generated neurons are associated with neurological improvement and to elucidate the comprehensive mechanism regulating the endogenous regeneration system . The success of isolation and easy gene - engineered modification of embryonic stem cells (escs) and nscs in vitro profoundly provides researchers a promising tool to replace the injured neurons in the ad brain . Therefore, the cell transplantation strategy has given rise to hopes for clinical application of these in vitro produced neuronal cells in the cell replacement procedures for ad . Since chronic inflammation is a characteristic property of ad brain, transplantation of neuronal precursor cells (npcs) has been proven to particularly inhibit ongoing inflammatory reactivity . Researchers have tested that the intrahippocampal transplantation of npcs is effective in attenuating inflammatory responses and plays a neuroprotection role in beta - amyloid 42 (a-42) peptide - injected rat hippocampus, indicating effects of npcs transplantation in ad models are consistent with cellular actions to attenuate inflammatory reactivity . The progressive degeneration of cholinergic neurons occurs in the forebrain cholinergic projection system especially in the nucleus basalis of meynert . Moghadam et al used escs - derived npcs to treat ibotenic acid - induced ad models in order to investigate the production of cholinergic neurons derived from engrafted cells . After transplantation, not only a significant behavioral improvement in memory deficits was observed, but also the majority (about 70%) of the npcs retained neuronal phenotype and about 40% of them had a cholinergic cell phenotype with no tumor formation . Similarly, in the a-induced ad model, the transplantation of escs - derived npcs into the injured hippocampus can also improve the memory dysfunction of the ad models . Furthermore, the transplanted cells demonstrate characteristics of proper synapse formation between host and grafted neural cells . Recently, another group reported the in vivo functional integration of the human escs - derived nscs after cell transplantation . At 6 months after transplantation, human axons identified with the human - specific middle - weight neurofilament protein antibody were found inside the stratum radiatum . Alongside these projections, small patches of human synaptophysin immunoreactivity were detected, suggesting the formation of presynaptic terminals . Hippocampal grafts placed in the dentate gyrus were projected to both the ipsilateral and contralateral pyramidal cell layers, while axons of donor neurons placed in the motor cortex extended via the external and internal capsules into the cervical spinal cord and via the corpus callosum into the contralateral cortex . Their data indicate that neurons derived from human pluripotent stem cells (pscs) are endowed with a remarkable potential to establish orthotopic long - range projections in the adult mammalian brain . Meanwhile, in another mouse ad model, the transplantation of nscs was reported to improve cognition function mediated by the neurotrophic factor bdnf . The differentiation of transplanted nscs in response to local cues in the brain suggests that environmental cues have the ability to direct the fate of stem cells to become the specific, terminally differentiated cells that are required to restore functions . Alternatively, stem cells are differentiated prior to transplantation and then directed to the correct areas through surgery . However, care should be taken with the characterization of these cells in regard to their multipotentiality and genetic stability with increasing passages in culture, as transformed cells may contribute to the formation of tumors . Efforts to investigate the pathophysiology of human ad are hampered by the lack of genuine in - vitro models . Stem cells generated by induced direct reprogramming of adult somatic cells using are termed as ipscs, offering paradigm shifting opportunities by providing specific / personalized models for studying ad, and personalized renewable source of cells for practical autologous cell therapies and regenerative medicine applications, that avoid immune rejection . The possibility of using ipscs as a tool for development of such ad patient specific model systems, however, remains at best challenging . In 2006, takahashi et al discovered that four transcription factors could reprogram mouse fibroblasts to a pluripotent state . Ever since then ipscs have created excitement among researchers, as ipcs are more available, easier to make, and less ethically conflicted than escs . This has been widely regarded as a milestone advance in stem cell research, as it may allow researchers to obtain pscs without the controversial use of embryos . Because ipscs are developed from an adult somatic cell, it is believed that ipscs can avoid immunogenic responses . Ipscs are a specific cell type compromised by disease, even lost in patients, that can be recreated in culture . Furthermore, ipscs have the potential to provide an unlimited source for any desired cell type . Ultimately, aside from being an exciting research tool to probe embryogenesis and disease pathogenesis, ipscs are so - called disease modeling for drug screening, identifying novel drugs to treat diseases and patient - tailored cell therapy . Most (9095%) of ad patients are sporadic populations (sad) while 510% patients are diagnosed as having early - onset ad, half of whom are familiar ad (fad). Fad is caused by a mutation in at least one of three genes: presenilin 1/2 and amyloid precursor protein (app). Obviously, ipscs technology contributes to capture the genomes of ad patients and to generate live cellular models of both the fad and sad . These models allow us to identify the earliest events of ad and to investigate aspects of ad pathogenesis that are not replicated in animal models . Yagi's group recently pioneered to generate ipscs from fibroblasts of fad patients with mutations in presenilin 1 (a246e) and presenilin 2 (n141i), and characterized the differentiation of these cells into neurons . Their remarkable data showed that fad - ipsc - differentiated neurons increased a42 secretion, recapitulating the molecular pathogenesis of mutant presenilins . Furthermore, a42 secreted from these neurons sharply responded to -secretase inhibitors and modulators, indicating the potential for identification and validation of candidate drugs . This finding significantly demonstrates that the fad - ipsc - derived neuron is a valid model of ad and provides an innovative strategy for the study of age - related neurodegenerative diseases . Marchetto et al in their study of rett syndrome using ipscs, reported the in vitro differentiation of ipscs into neurons that contained glutamatergic synapses and were capable of generating spontaneous synaptic activity . The spontaneous synaptic activity observed in the differentiated neurons hinted that ipsc technology can be used to study not only human neurons but also patient - specific neural networks . Previous research has reported the marked differences in differentiation propensity between psc lines, even between ipsc lines generated from the same individual . And furthermore, differentiation variability has become an important issue . This issue becomes more complex if this novel method is to investigate a disease with unclear developmental changes . Thus, it is unclear if ipscs and ipsc - derived npcs from ad patients would generate neurons differently than control cells, such as embryonic stem cells and neural stem cells . Despite some successes in animal models, ipscs technology is not yet ready for human trials . Current ipscs protocols cannot efficiently eliminate undifferentiated cells and, tend to be oncogenic and form teratomas, just like escs . Additionally, most patient - specific ipscs have been generated using integrating vectors, which may not get silenced efficiently or can disrupt endogenous genes that are a potential impediment in human ipscs therapy . Many mouse ipscs harbor epigenetic abnormalities are reported in recent studies, which may develop genetic mutation on prolonged culture and continue to retain epigenetic memory of their donor cells . In terms of the ipscs technique, (1)ethical hurdles: undoubtedly, it turns out that ipscs are not entirely problem - free . Researchers working with ipscs still must countenance certain ethical concerns, and they may also face newly discovered scientific hurdles . (2)scientific hurdles: whether ipscs are truly equivalent to escs? Some studies have raised the possibility of significant differences . Lanza group compared differentiated cells derived from a series of ipscs lines and escs lines, and found thatboth cells differentiated to form blood cells, vascular cells or retinal cells . However, the ipscs did so at a significantly lower rate and had a higher rate of cell death . Another study raised a question about whether ipscs can serve as tools for modeling disease . Researchers compared escs with ipscs that carried the mutation for the mental impairment disorder fragile x syndrome . Some scientists hope that the retroviruses which these problems arise from can be used to generate ipscs . Since neural progenitor / stem cells have been proven to exist in the adult cns and to be involved in the neurogenesis process, the activation of endogenous neural progenitor / stem cells populations that can migrate to the injured regions, proliferate and functionally integrate into the existing circuit represents a significant strategy to promote neural regeneration in the diseased brain . This activation within the brain is to protect the remaining tissues and prevent secondary neuron loss through the production of neurotrophic and neuroprotective factors, such as brain derived neuronal factor (bdnf) and vascular endothelial growth factor . A recent research has demonstrated that the self - repair in the adult brain can be augmented by the infusion of growth factors to activate endogenous neural precursor cells that contribute to new tissue formation and functional recovery after stroke . Currently, it is widely accepted that neurogenesis in the hippocampus are involved in learning and memory formation . The hippocampus - dependent learning tasks can significantly increase the proliferation of endogenous neuronal progenitors, survival of new neurons and, the task performance by animals correlates positively with the amount of adult born neurons . Alterations in the microenvironment where the endogenous nscs reside play an important role in nsc activation . In ad brain, for example, immune responses include the activation of microglia and astrocytes around the senile plaques area and t - lymphocyte infiltration into the damaged brain . These cells produce cytokines and other molecules that promote or inhibit the neurogenic function of the nscs . Therefore, it is possible for damaged cells to be replaced from endogenous nsc pools . Studies have also suggested that the capacity of endogenous nscs to compensate for lost cells is limited . In spite of the activated proliferation, obviously, most of the new migrated neurons in and near the injured area die before differentiating into functional neurons, possibly because of a lack of factors and stimulation to support their survival and differentiation; thus, only 0.2% of the dead neurons are replaced . However, it has not been determined whether these effects depend directly on the promotion of neuronal regeneration by nscs, or whether accompanying events, such as enhanced glial regeneration and other types of trophic support, are more important . Moreover, a key issue in the field of neuronal regeneration is that newly generated neurons need to make the appropriate connections, although the details of this process are still largely unknown . Further studies are needed to clarify how newly generated neurons are associated with neurological improvement and to elucidate the comprehensive mechanism regulating the endogenous regeneration system . The success of isolation and easy gene - engineered modification of embryonic stem cells (escs) and nscs in vitro profoundly provides researchers a promising tool to replace the injured neurons in the ad brain . Therefore, the cell transplantation strategy has given rise to hopes for clinical application of these in vitro produced neuronal cells in the cell replacement procedures for ad . Since chronic inflammation is a characteristic property of ad brain, transplantation of neuronal precursor cells (npcs) has been proven to particularly inhibit ongoing inflammatory reactivity . Researchers have tested that the intrahippocampal transplantation of npcs is effective in attenuating inflammatory responses and plays a neuroprotection role in beta - amyloid 42 (a-42) peptide - injected rat hippocampus, indicating effects of npcs transplantation in ad models are consistent with cellular actions to attenuate inflammatory reactivity . The progressive degeneration of cholinergic neurons occurs in the forebrain cholinergic projection system especially in the nucleus basalis of meynert . Moghadam et al used escs - derived npcs to treat ibotenic acid - induced ad models in order to investigate the production of cholinergic neurons derived from engrafted cells . After transplantation, not only a significant behavioral improvement in memory deficits was observed, but also the majority (about 70%) of the npcs retained neuronal phenotype and about 40% of them had a cholinergic cell phenotype with no tumor formation . Similarly, in the a-induced ad model, the transplantation of escs - derived npcs into the injured hippocampus can also improve the memory dysfunction of the ad models . Furthermore, the transplanted cells demonstrate characteristics of proper synapse formation between host and grafted neural cells . Recently, another group reported the in vivo functional integration of the human escs - derived nscs after cell transplantation . At 6 months after transplantation, human axons identified with the human - specific middle - weight neurofilament protein antibody were found inside the stratum radiatum . Alongside these projections, small patches of human synaptophysin immunoreactivity were detected, suggesting the formation of presynaptic terminals . Hippocampal grafts placed in the dentate gyrus were projected to both the ipsilateral and contralateral pyramidal cell layers, while axons of donor neurons placed in the motor cortex extended via the external and internal capsules into the cervical spinal cord and via the corpus callosum into the contralateral cortex . Their data indicate that neurons derived from human pluripotent stem cells (pscs) are endowed with a remarkable potential to establish orthotopic long - range projections in the adult mammalian brain . Meanwhile, in another mouse ad model, the transplantation of nscs was reported to improve cognition function mediated by the neurotrophic factor bdnf . The differentiation of transplanted nscs in response to local cues in the brain suggests that environmental cues have the ability to direct the fate of stem cells to become the specific, terminally differentiated cells that are required to restore functions . Alternatively, stem cells are differentiated prior to transplantation and then directed to the correct areas through surgery . However, care should be taken with the characterization of these cells in regard to their multipotentiality and genetic stability with increasing passages in culture, as transformed cells may contribute to the formation of tumors . Efforts to investigate the pathophysiology of human ad are hampered by the lack of genuine in - vitro models . Stem cells generated by induced direct reprogramming of adult somatic cells using are termed as ipscs, offering paradigm shifting opportunities by providing specific / personalized models for studying ad, and personalized renewable source of cells for practical autologous cell therapies and regenerative medicine applications, that avoid immune rejection . The possibility of using ipscs as a tool for development of such ad patient specific model systems, however, remains at best challenging . In 2006, takahashi et al discovered that four transcription factors could reprogram mouse fibroblasts to a pluripotent state . Ever since then ipscs have created excitement among researchers, as ipcs are more available, easier to make, and less ethically conflicted than escs . This has been widely regarded as a milestone advance in stem cell research, as it may allow researchers to obtain pscs without the controversial use of embryos . Because ipscs are developed from an adult somatic cell, it is believed that ipscs can avoid immunogenic responses . Ipscs are a specific cell type compromised by disease, even lost in patients, that can be recreated in culture . Furthermore, ipscs have the potential to provide an unlimited source for any desired cell type . Ultimately, aside from being an exciting research tool to probe embryogenesis and disease pathogenesis, ipscs are so - called disease modeling for drug screening, identifying novel drugs to treat diseases and patient - tailored cell therapy . Most (9095%) of ad patients are sporadic populations (sad) while 510% patients are diagnosed as having early - onset ad, half of whom are familiar ad (fad). Fad is caused by a mutation in at least one of three genes: presenilin 1/2 and amyloid precursor protein (app). Obviously, ipscs technology contributes to capture the genomes of ad patients and to generate live cellular models of both the fad and sad . These models allow us to identify the earliest events of ad and to investigate aspects of ad pathogenesis that are not replicated in animal models . Yagi's group recently pioneered to generate ipscs from fibroblasts of fad patients with mutations in presenilin 1 (a246e) and presenilin 2 (n141i), and characterized the differentiation of these cells into neurons . Their remarkable data showed that fad - ipsc - differentiated neurons increased a42 secretion, recapitulating the molecular pathogenesis of mutant presenilins . Furthermore, a42 secreted from these neurons sharply responded to -secretase inhibitors and modulators, indicating the potential for identification and validation of candidate drugs . This finding significantly demonstrates that the fad - ipsc - derived neuron is a valid model of ad and provides an innovative strategy for the study of age - related neurodegenerative diseases . Marchetto et al in their study of rett syndrome using ipscs, reported the in vitro differentiation of ipscs into neurons that contained glutamatergic synapses and were capable of generating spontaneous synaptic activity . The spontaneous synaptic activity observed in the differentiated neurons hinted that ipsc technology can be used to study not only human neurons but also patient - specific neural networks . Previous research has reported the marked differences in differentiation propensity between psc lines, even between ipsc lines generated from the same individual . And this issue becomes more complex if this novel method is to investigate a disease with unclear developmental changes . Thus, it is unclear if ipscs and ipsc - derived npcs from ad patients would generate neurons differently than control cells, such as embryonic stem cells and neural stem cells . Despite some successes in animal models, ipscs technology is not yet ready for human trials . Current ipscs protocols cannot efficiently eliminate undifferentiated cells and, tend to be oncogenic and form teratomas, just like escs . Additionally, most patient - specific ipscs have been generated using integrating vectors, which may not get silenced efficiently or can disrupt endogenous genes that are a potential impediment in human ipscs therapy . Many mouse ipscs harbor epigenetic abnormalities are reported in recent studies, which may develop genetic mutation on prolonged culture and continue to retain epigenetic memory of their donor cells . In terms of the ipscs technique, (1)ethical hurdles: undoubtedly, it turns out that ipscs are not entirely problem - free . Researchers working with ipscs still must countenance certain ethical concerns, and they may also face newly discovered scientific hurdles . Lanza group compared differentiated cells derived from a series of ipscs lines and escs lines, and found thatboth cells differentiated to form blood cells, vascular cells or retinal cells . However, the ipscs did so at a significantly lower rate and had a higher rate of cell death . Another study raised a question about whether ipscs can serve as tools for modeling disease . Researchers compared escs with ipscs that carried the mutation for the mental impairment disorder fragile x syndrome . Some scientists hope that the retroviruses which these problems arise from can be used to generate ipscs . Although stem cell - based replacement strategies carried out in animal models have shown promising results, there are still many hurdles to overcome before these approaches can be translated into the ad patients . One major challenge is the development of a safe method to deliver stem cells to the injury region . In addition, the stage of differentiation of those cells needs careful consideration: fully differentiated cells are associated with a smaller efficiency due to poor viability, while undifferentiated cells present a higher risk of undirected differentiation and uncontrolled proliferation . Adult neurogenesis is important for cellular therapy and physiopathology of the cns, as for development and pharmacology of the adult brain . Hence, the role, contribution and significance of newborn neurons in the adult brain remain to be fully elucidated and understood . One of the main limitations in determining and understanding the role of newborn neurons in the adult brain is the use of bromodeoxyuridine (brdu) for studying neurogenesis . The microenvironment in which stem cells are placed also needs consideration, as local soluble factors are likely to affect differentiation events in the tissue . Environmental factors need to be included: pro - inflammatory cytokines are associated with a negative effect on neural differentiation, while anti - inflammatory cytokines may have the opposite effect . The inflammatory status of the brain and the possible activation of inflammatory responses therefore need to be considered with cell replacement strategies . In all, developing our understanding of the processes controlling the activation, migration and differentiation of stem cells will be a critical step towards the usage of stem cells for new regenerative therapies . It has been a century since the first description of ad . In the past decades, tremendous advances in understanding of the molecular pathogenesis of ad have been springing out . However, no proven effective treatment is able to delay the onset or slow the progression of ad . Now, many new therapies directly targeting the mechanisms underlying ad are now in the pipeline . A combination of psychosocial, behavioral, and pharmacologic strategies aims at slowing the process of ad and preserving quality of life for as long as possible . Until medical research discovers definitive disease modifying treatments for patients with ad, we must continue to maximize all available resources to provide the best possible individualized patient - centered and family care . Despite a better understanding of the pathology of cognitive impairments and clinical features of ad, which have aided diagnosis and management of the disease, further work is required to improve screening and decrease the burden of care on healthcare systems and families . Furthermore, more research is needed to elucidate the mechanisms of the disease to enable new drug targets to be developed based on biological models, which may provide novel treatments for the prevention and management of ad . The situation for neuronal replacement aiming at functional restoration in ad is extremely complex because the stem cells have to be pre - differentiated in vitro to many different types of neuroblasts for subsequent implantation in a large number of brain areas . However, to give long - lasting symptomatic benefit, a cholinergic cell replacement approach will require intact target cells and host neurons that the new cholinergic neurons can act on . Stem cell - based cell replacement strategies are very far from clinical application in ad . But the neuroreplacement strategy will undoubtedly become more feasible as we advance our understanding of the pathogenesis of ad and foster creativity in research aiming to elucidate the physiological role of nscs in the adult brain . Also, the nscs indeed exist in the complexity and intricacy of the architecture of the human brain, the mechanisms and therapeutic potential of nscs just need to be further explored.
Vascular dementia sometimes can precede or accompany alzheimer's disease, and in these cases the development of alzheimer's disease becomes more dramatic . The dementia of ischaemic type and alzheimer's disease is synergistic or additive in the earliest stages of alzheimer's disease, although the interactive mechanisms are not known [1, 2]. Both types of dementia are neurodegenerative diseases and for both the dysfunction and degeneration of cholinergic projective systems in the cortex and the hippocampus from the forebrain nuclei are critical [38]. A number of studies on animal models demonstrated a possible trigger role of cholinergic projective neurons in brain ischaemia . The early activation of cholinergic projective neurons was found to occur simultaneously with glutamatergic activation in the cortex and the hippocampus [912]. Correlations between the development of cholinergic dysfunctions and the destruction of pyramidal neurons in the hippocampus [6, 13, 14], and also damage to the cognitive functions of animals [48, 10], led to the presumption that a dysfunction in cholinergic afferents plays a major role in the development of ischaemic pathologies [9, 12, 14, 15]. Modern electrophysiology accumulated numerous data that interneurons of the cortex and the hippocampus actively participate in the modulation of neuronal activity including the hippocampal pyramidal neurons [1618]. It was revealed that the cholinergic effects on the interneurons of the cortex and the hippocampus was substantially mediated through nicotinic receptors (nachrs) [1621]. On the other hand the role of the cholinergic interneurons in behavioural, and neurodegenerative mechanisms is still unknown . Our investigations on the light and heavy synaptosomal fractions of the cortex and the hippocampus allowed the study of the major cholinergic projection systems of the cortex and the hippocampus and their minor intrinsic systems of cholinergic interneurons . According to immunochemical data, both the cortex and the hippocampus the first major sources are the neuronal projections from the forebrain nuclei basalis magnocellularis into the cortex (precursor of the meynert nucleus in primates and humans) and projections from the forebrain medial septal nuclei and vertical limb nuclei of the diagonal band of broca into the hippocampus . The synaptosomes are presynaptic parts of synapses with their junction complexes; these shall be termed presynapses in the present study . We previously showed that for both the cortex and the hippocampus the cholinergic presynapses from different sources are isolated in different synaptosomal fractions during preparation in the sucrose density gradient . The presynapses of cholinergic projections from the forebrain nuclei are accumulated mainly in the light synaptosomal fractions whereas the presynapses of cholinergic interneurons are accumulated mainly in the heavy synaptosomal fractions [2931]. It is probable that the heavy synaptosomal fraction of the hippocampus may also accumulate a small part of the cholinergic projective presynapses (lateral projection pathway into the hippocampus). Our studies on the cortical synaptosomal fractions of cats allowed suggest the involvement of the cholinergic interneurons in cognitive functions . In the studies on the cortical and hippocampal synaptosomal fractions of rat we revealed that during the first three hours of chronic brain ischaemia the cholinergic projective neurons were reactive, as were the interneurons of the cortex and the hippocampus as well . At present, the molecular, genetic and neurochemical mechanisms of cognitive functions are widely investigated in different behavioural models and widely discussed as well . Some among these data induce to revise generally conception that memory formation involves an irreversible passage via labile phases, such as working and short - term memory to the stable form of long - term memory . Thus it was shown, that several drugs inhibited short - term memory without altering long - term memory and that working, short - term, and long - term memory were differentially regulated in the various brain regions by the various neurotransmitter systems, including cholinergic one [3437]. The authors concluded that different types of memory had the separate mechanisms, various neurotransmitter systems, and regions . The basis of our investigation is importance of the cholinergic systems in human and animal cognition, and also the existence of general mechanisms in the development of dementias of different aetiologies . In the present study, the cholinergic synaptic organization of different forms of learning and memory in rats with normal and chronic ischaemic brains a marker of cholinergic neurons, enzyme of acetylcholine synthesis choline acetyltransferase (chat; ec 2.3.1.6) was used for estimation of the cholinergic systems . Chat activity and also protein contents (total synaptic parameters) were measured in subfractions of the synaptic membranes and the synaptoplasm isolated from light and heavy synaptosomal fractions of the cortex and the hippocampus . Thus, the participation of projective and intrinsic cholinergic systems of the cortex and hippocampus in mechanisms of learning and memory under normal and ischaemic conditions was researched . In addition, the regulation of learning performance under prolonged action of selective agonist of 42 subtype of nachr metanicotine (rjr) and selective antagonist of non7 subtypes of nachr mecamlamine was studied . Outbred white adult male rats (220270 g) were supplied from the animal's nursery light mountains (russia) and then kept in the vivarium of our institute of general pathology and pathophysiology . The rats were housed in a temperature - controlled room (2024c) with free access to food and water and kept on a 12 h light / dark cycle according to the national institutes of health animal care and the principles of laboratory animal care guidelines and the study was approved by the ethical committees of the institutes . Chronic rat ischaemia was induced by permanent occlusion of the common carotid arteries (two - vessel occlusion, 2vo) by ligation . The bilateral common carotid arteries were tied with silk threads whilst the rats were under an appropriate level of pentobarbital anaesthesia . The common carotid arteries were separated from the cervical sympathetic and vagal nerves through a ventral cervical incision . The sham - operated animals (control groups) underwent a similar surgery but vessel ligation was excluded . Behaviour was studied in spatial contextual (noncued) or spatial cued models of learning and memory in the morris water maze following standard procedures . The experimental apparatus consisted of a circular water pool (diameter, 120 cm; height, 60 cm) filled with milk - clouded water at 24c to a depth of 40 cm . A plexiglas hidden platform (10 10 cm) was submerged 2 cm below the water surface and was placed at the midpoint of one quadrant . The rats were trained during three daily sessions in the contextual (sham - operated/2vo rats) or the cued learning models . In both learning models the rats were given four daily attempts to find the hidden platform in a 60 s time interval and the estimated swim time for platform achievement (latency time) was recorded . Rats which failed to find the platform within 60 s were considered unable to solve the task and were softly guided there by the investigator with 60 s scored . The other rats remained on the platform for 30 s and were returned to their home cage during the intertrial interval (60 s). In the contextual model the location of the hidden platform remained the same throughout the training period . The pool was located in a test room containing no prominent visual marks . At the start of all trials, the rats were placed in the pool at one of four starting positions . In the cued model a prominent visual mark (cue) was placed on the maze wall over the hidden platform to help the animal locate the platform . In this model the rats had the same starting position but the hidden platform with its cue was moved to four different positions during the session . The following forms of cognitive functions were observed and investigated: the inherited abilities (the first noncasual attempts at decision making in the task, that is, 1s1 trial in the cued model and 1s2 trial in the contextual model); working memory in the first session (1s24 and 1s3 - 4 averaged out over the following trials, resp . ); learning in the second and the third sessions (2s24 and 3s24 averaged trials, resp . ); and long - term memory on the days after the first and the second sessions of training (2s1 and 3s1 trials, resp . ). All behavioural experiments were carried out by investigators who had no knowledge of the experimental groups . Metanicotine (rjr 2304, tocris), a selective agonist of 42 subtype of nachr and mecamilamine (sigma), a selective antagonist of non-7 subtypes of nachr, were used . The preparations were subchronically administered (i.p .) Three times daily in doses of 26 and 3.9 nmoles / kg, respectively . Both the sham - operated and the ischaemic rats received the first injection of the preparations immediately after the end of narcosis (1.53 hours after surgical intervention). Some of animals were decapitated for biochemical analysis 3 - 4 days after the third session of training . It means, the rats which trained from 6 day after the surgery were decapitated at 11 or 12 days after the surgery and so on . The biochemical group included the control/2vo animals trained in the contextual model (contextual biochemical subgroup) or the cued model (cued biochemical subgroup). Briefly, the brain, cortex and hippocampus were removed, separated and homogenized . From each sample the light and heavy synaptosomal fractions were isolated, with further separation of the subfractions of the synaptic membranes and the synaptoplasm, following preparative and the disruptive procedures and the discontinued gradients of sucrose density as described previously [30, 39]. The fractions of the synaptosomes were obtained from the rough mitochondrial fraction by centrifugation using a bucket rotor (84,000 g 120 min, 24c) in layers between 1.01.2 m sucrose densities (the light synaptosomes) and between 1.21.4 m sucrose densities (the heavy synaptosomes). The synaptosomes were disrupted by combined shock procedures: the synaptosome pellets were suspended in hypo - osmotic solution containing 6 mm tris - ncl buffer, ph 8.1 (100 mg tissue / ml) and they were then exposed by freeze - thawing . The synaptoplasm subfractions were obtained as supernatants by centrifugation from the disrupted synaptosomal fractions (14,000 g 30 min, 24c). The pellets were suspended in the hypo - osmotic solution and stratified on discontinued gradients again . The synaptic membrane subfractions were obtained by centrifugation using the bucket rotor (130,000 g 120 min, 24c) in layers between 0.61.2 m sucrose densities . The clean synaptic membrane subfractions were free from glial, mitochondrial and synaptic vesicle contamination . The activity of chat in subfractions of synaptic membranes and synaptoplasm of the cortex and the hippocampus was determined by the radiometric method of fonnum and the protein contents were determined by the method of lowry et al . . Accordingly, the membrane - bound mchat activity and m - protein contents were determined in the synaptic membrane subfractions, and the water - soluble cchat activity and c - protein contents were estimated in synaptoplasm subfractions . Moreover, this is the reason why changes in mchat activity could be exposed and changes in cchat activity could be masked in the small presynapses, whereas changes in cchat activity, but not in mchat activity, could be exposed in the large presynapses . Therefore, estimations of mchat and cchat activities (as well as m- and c - protein contents) could give additional information on the characteristics of changes caused by ischaemia . The reactive mixture contained a final concentration of 0.2 mm acetyl coasa (fluka) and [1-c]-acetyl coasa (amersham pharmacia bioscience) with spa 5 mci / mmol, 300 mm nacl, 3 mm mgcl2, 0.2 mm physostigmine salicylate (sigma), 10 mm choline chloride (serva), 0.5% triton x-100 (serva), 0.5 mg / ml albumin from bull serum (koch - light), 10 mm sodium phosphate buffer/1 mm edta - na2, ph 7.8 and the subfraction samples (near 3.5 mg of protein) at a common volume of 0.050.1 ml . The reactive mixture was incubated in a water shaker at 37c for 3060 min . The reaction was stopped by adding 2 ml of ice - cold stop solution (0.2 mm acetylcholine in 10 mm sodium phosphate buffer/1 mm edta - na2, ph 7.8) and by placing the mixture in an ice bath . Then, a 1 ml solution of sodium tetraphenylborate (sigma) in butyl acetate (15 mg / ml) was added and quickly subjected to intensive mixing in a shaker (500 turns / min, 4 min, room temperature). The organic phase was separated from the inorganic phase by centrifugation (1000 g 15 min, 24c). The organic phase with acetylcholine (0.50.7 ml) was placed into scintillation liquid for organic solutions and the radioactively synthesized acetylcholine (dpm) was quantified with a beta counter . Reactive solution (biuret reagent) was prepared at the day of experiment by mixing 0.5 ml of 1% cupric sulfate with 0.5 ml of 2% sodium potassium tartrate, followed by the addition of 50 ml of 2% sodium carbonate in 0.1 n naoh . Bovine serum albumin (bsa) powder was dissolved in distilled water and diluted to a concentration of 1000 g / ml . A series of dilutions of the basic bsa solution (50, 100, 200, 400 and 500 g / ml) was made by mixed thoroughly of the aliquots of basic bsa solution and water with repeated pipeting . Samples were within the bsa standard range (120 g in assay volume). Reaction was started by intensive mixed of 0.02/0.04 ml of bsa or subfractions samples with 1 ml of the reactive solution . The mixture was then allowed to incubate at room temperature for 1015 min prior to the addition of 0.1 ml per tube of 1.0 n folin & ciocalteu's reagent . Color was allowed to develop for 2 hours in dark at room temperature and the absorbance of the reduced folin reagent measured at 750 nm and blanked on the water only control . After then the reaction was found to be stable for up to an hour at room temperature and kept in refrigerator at 58c for up to 1 - 2 days . The behavioural results were expressed in terms of time taken to swim to the hidden platform (s) and the biochemical results were expressed in terms of chat activity (nmoles acetylcholine / min) or protein content (mg) in 1 g wet weight of cortex and hippocampus tissue, respectively . The data were calculated using the nonparametric fisher's exact test and the r - criterion of the pearson's correlative test in microsoft excel with a glance of adjusting formula for small number of observations . The period of 610 days of chronic brain ischaemia led to a strong decline in training efficiency in the morris water maze . Learning and long - term memory were impaired in both the contextual and cued models (figure 1). Learning in 2s24 and 3s24 were impaired in a similar manner in both of the behavioural models, whereas impairment of the long - term memory had the specificity in each model . In the contextual model (figure 1, top), impairment of long - term memory developed gradually and only 3s1 was significantly impaired . In the cued model (figure 2, bottom), long - memory 2s1 was impaired and 3s1 was the same as the control . It can be noted that although the investigated cognitive functions were impaired, they were still performed in ischaemic rats . From all of the investigated animals (n = 27), only two rats could not solve the tasks in our experimental conditions (in the contextual model). As a rule, the prolongation of solving tasks and/or the delay in learning (successful solving of the task only occurred in the third session) was observed . The period of 1114 days of chronic ischaemia resulted in significant changes in chat activity and protein content in the investigated subfractions of the synaptosomes, both of the cortex and the hippocampus (figure 2-all rats). In the cortical light synaptosomal fraction, mchat activity and m - protein content were increased, and these changes were positively correlated amongst themselves (r = + 0.770, n = 18, total control and 2vo groups data, p <.001; in the control group r = + 0.788, n = 9, p <.02). The cchat activity did not significantly vary or correlate with mchat activity but it was positively correlated with increasing c - protein content (r = + 0.669, n = 9, p <.05; in the control group r = + 0.305, n = 9, this indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic projective neurons in the cortex . But mchat activation was accompanied by a reinforcement in the positive correlation between its values and the m - protein content (r = + 0.694, n = 9, p <.05; in the control group r = + 0.291, n = 9, p>.05). Also, a reinforcement of the positive correlation between the values of cchat activity and c - protein content was observed (r = + 0.835, n = 9, p <.01; in the control group r = + 0.633, n = 9, p>.05). At the same time, the correlation between the activity of mchat and the activity of cchat became weaker than in the control (r = + 0.579, n = 9, p>.05; in the control group r = + 0.754, n = 9, p <.02). This indicated a reorganization of the synaptic pool in more than one synaptic population of the cholinergic interneurons in the cortex . In the hippocampal light synaptosomal fraction, however, significant correlations between the values of mchat and cchat activity, and between the values of chat activity and protein content, were absent . This indicated a reorganization of the synaptic pool in more than one synaptic population in the hippocampus, in both the cholinergic systems and some noncholinergic systems . In the hippocampal heavy synaptosomal fraction, m - protein content decreased and c - protein content increased . Changes in the m - protein content did not correlate with chat activity and thus reflected reorganization of noncholinergic presynapses in the hippocampus . The activities of mchat and cchat did not differ from the controls although at the same time a positive correlation arose between their values (r = + 0.802, n = 8, p <.02; in the control group r = 0.300, n = 9, p>.05). Also, the positive correlations between values of c - protein content and mchat activity (r = + 0.927, n = 8, p <.01; in the control group r = + 0.265, n = 9, p>.05) and cchat activity (r = + 0.844, n = 8, p <.01; in the control group r = + 0.091, n = 9, this indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons in the hippocampus . It seems that the unchanged values of chat activity reflected parallel processes of activation and inactivation of chat in different synaptic populations of this fraction . Also, the biochemical data of the contextual biochemical subgroup were compared with the cued biochemical subgroup . Analysis of the biochemical parameters in the control biochemical subgroups did not reveal significant changes between the subgroups (table 1). Only the values of mchat activity were lower in the cortical heavy and in the hippocampal light synaptosomal subfractions in the cued biochemical subgroup of rats as compared with the contextual one . Analysis of the biochemical parameters in the 2vo biochemical subgroups confirmed our observations about the reorganization of the synaptic pool . In the cortical light synaptosomal fraction in the contextual biochemical subgroup (figure 2-contextual), independent correlations were detected between the values of mchat activity and m - protein content (r = + 0.765, n = 10, total control and 2vo contextual biochemical subgroups data, p <.01; in the control contextual biochemical subgroup r = + 0.774, n = 5, p>.05) and between the values of cchat activity and c - protein content (r = + 0.987, n = 5, p <.01; in the control contextual biochemical subgroup r = + 0.441, n = 5, p>.05). In the cued subgroup (figure 2-cued), a correlation was only detected between mchat activity and m - protein content (r = + 0.783, n = 8, total control and 2vo cued biochemical subgroups data, p <.05; in the control cued biochemical subgroup r = + 0.622, n = 4, p>.05) and a correlation was revealed between mchat and cchat activities (r = + 0.995, n = 4, p <.01; in the control biochemical cued biochemical subgroups r = 0.404, n = 4, p>.05). In the cortical heavy synaptosomal fraction in the contextual biochemical subgroup, independent correlations were detected between mchat activity and m - protein content (r = + 0.981, n = 5, p <.01) and between the values of cchat activity and c - protein content (r = + 0.966, n = 5, p <.01), whereas there was no correlation between the activities of mchat and cchat among themselves (r = + 0.652, n = 5, p>.05). In the cued biochemical subgroup, an increase in mchat activity was detected (a tendency) whereas a decrease in m - protein content was revealed . The decrease in m - protein content allows to suppose the changes in noncholinergic presynapses . In the hippocampal light synaptosomal fraction in the contextual biochemical subgroup, an independent decrease in mchat activity and an increase in cchat activity only, and in the cued biochemical subgroup an increase in cchat activity only, and a decrease in m - protein content and an increase in c - protein content were detected . In the hippocampal heavy synaptosomal fraction in the contextual biochemical subgroup, positive correlations were detected between the increased values of c - protein content and mchat activity (r = + 0.922, n = 5, p <.01) and cchat activitiy (r = + 0.919, n = 5, p <.05), and between mchat and cchat activities (r = + 0.910, n = 5, p <.05; in the control contextual biochemical subgroup r = 0.266, n = 5, p>.05). However, in the cued biochemical subgroup, a decrease in mchat activity and m - protein content was revealed and these changes did not correlate among themselves . This indicated a reorganization of the presynapses of the cholinergic interneurons / lateral pathway projective neurons and noncholinergic neurons in the hippocampus . Differentiation of the rats into biochemical subgroups, tested in the contextual and cued models, permit to compare the behavioural performance and chat activity in these rats . Under the normal conditions, each form had individual cholinergic composition (table 2: sham - contextual, figure 3-i: sham). The inherited ability 1s2 cholinergic composition included large presynapses of projective cortical neurons (positive correlation with cchat activity) and presynapses of hippocampal interneurons / lateral pathway projective neurons (negative correlation with mchat and cchat activities). The same cholinergic structures associated with the long - term memory 3s1, but with inverse symbols of r - criterions . The long - term memory 2s1 had composition other than 3s1 which involved small presynapses of the projective hippocampal neurons (negative correlation with mchat activity) and some populations of hippocampal interneurons (positive correlations with mchat and cchat activities). The composition of the working memory 1s3 - 4 involved small presynapses (positive correlation with mchat activity) and large presynapses (negative correlation with cchat activity) of the projective hippocampal neurons . The composition of learning 2s24 and 3s24 was identical and involved small presynapses of the projective cortical neurons (positive correlation with mchat activity in both forms of learning) and the hippocampal interneurons / lateral pathway projective neurons (positive correlations with mchat activity). The similar analysis in the cued biochemical subgroup revealed other individual cholinergic compositions of learning and memory (table 2: sham - cued, figure 3-ii: sham). According to our data, working memory 1s24 composition involved small presynapses of both cortical cholinergic systems (negative correlation with mchat activity) and of the hippocampal interneurons / lateral pathway projective neurons (positive correlation with mchat activity). The composition of learning 2s24 and 3s24 was identical and comprised large presynapses of cortical interneurons and projective hippocampal neurons (positive correlations with cchat activity in both cases). The long - term memory composition in 2s1 involved large presynapses of the cortical interneurons and small presynapses of projective hippocampal neurons (in both cases, there were positive correlations with cchat or mchat activities). And the long - term memory composition in 3s1 involved large presynapses of the cortical projective neurons (positive correlations with cchat activity). Chronic brain ischaemia had considerable effects on the cholinergic organization of the investigated cognitive functions . In the contextual model (table 2: 2vo - contextual, figure 3-i: 2vo), inherited abilities 1s2, learning 2s24 and long - term memory 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the cortical cholinergic populations . The composition of working memory 1s3 - 4 only kept negative connections with the large presynapses of projective hippocampal neurons . The positive connections of learning 3s24 with the small presynapses of the hippocampal interneurons / lateral pathway projective neurons inversed to negative ones . The long - term memory composition 2s1 consisted of only new, positive connections with large presynapses of the projective hippocampal neurons . In the cued model (table 2: 2vo - cued, figure 3-ii: 2vo), long - term memory 2s1 and 3s1 completely lost correlations with the cholinergic populations and all forms of cognition lost correlations with the hippocampal cholinergic influences . The working memory 1s24 lost its negative connections with the small presynapses of cortical projective neurons and its negative connections with the small presynapses of cortical interneurons inversed to positive ones . The composition of learning 2s24 included a reversal to negative connections with the large presynapses of cortical interneurons, new negative connections with presynapses of cortical projective neurons and new negative connections with the small presynapses of cortical interneurons . The learning composition 3s24 kept its connections with the large presynapses of cortical interneurons and added new negative connections with presynapses of the cortical projective neurons . So, under 2vo conditions as in the contextual and in the cued biochemical subgroup quantity of the cholinergic connections with the cognitive functions significantly reduced and some new links arose . Each form of cognition as resulting had 2vo cholinergic synaptic composition organized differently from normal ones . We attempted to analyze the dependence of impairment of the investigated cognitive functions in 2vo conditions from the reorganization of key cholinergic systems . It seems, in the contextual biochemical subgroup only preservation of the inherited abilities 1s2 from damage could be explaned by preservation of the key cholinergic populations, revealed in the normal conditions (figure 4, middle row). But long - term memory 3s1 had the same cholinergic composition with inverse symbols of r - criterions . In this case it would be expected that 3s1 would also be protected; however, this did not take place . Moreover, according to the cholinergic organization under normal conditions, working memory 1s3 - 4 would be considerably impaired, whereas long - term memory 2s1 and learning 2s24 and 3s24 would be considerably improved; however, these did not occur either . On the other hand, the new cholinergic composition in 2vo conditions had accordance between reinforcement of the negative influence of the hippocampal interneurons / lateral pathway projective neurons on learning 3s24 and impairment of this function (figure 4, bottom row). But reinforcement of new negative and positive influences of projective hippocampal neurons was not reflected in the performance of either working memory 1s3 - 4 or long - term memory 2s1 . In the cued biochemical subgroup, only the preservation of long - term memory 3s1 could be explained by the resistance to ischaemia of the key synaptic population revealed in normal conditions (figure 5, middle row). At the same time, working memory 1s24 would be considerably impaired, learning 2s24 and 3s24 would be equally improved or otherwise unchanged and long - term memory 2s1 would be unchanged, but these were not observed . On the other hand, the absence of 1s24 impairment could also be explained by the new, weakly expressed positive cholinergic influence (tendency) of the large presynapses of cortical interneurons (figure 4, bottom row). Then the distinctions in learning 2s24 and 3s24 performance in 2vo conditions would be explained by their new cholinergic compositions if we suppose more considerable influence of the large presynapses of the cortical interneurons on these functions in comparison with the influence of the other new key synaptic populations . The impairment of 2s1 also would be explained by the reduction of the link with this key synaptic population under the normal conditions . So, it seems that performance of the cognitive functions as in the contextual and in the cued model under 2vo conditions, as a rule, did not depend on their cholinergic organization, revealed in normal conditions . Contrary, new cholinergic organization, revealed in 2vo conditions showed more significant correlations with changes in behavioral performance . Whereas, the contextual and cued learning 2s24 and 3s24 revealed cholinergic synaptic compositions identical for normal conditions and different ones, revealed in 2vo conditions, it was investigated prolonged action on the learning performance of the selective agonist of 42 subtype of nachr rjr and the selective antagonist of non-7 subtypes of nachr mecamilamine . In the contextual model under normal conditions, both the agonist rjr and the antagonist mecamilamine did not influence on learning as 2s24 and 3s24 performance . It seems, this fact indicate that non-7 subtypes of nachr did not participate in the regulation of ones (figure 6, contextual learning). Under the 2vo conditions, effects of rjr on both learning performance evidently, the 42 subtype did not participate in the regulation of the contextual learning, as before, while some non-7 and non-42 subtypes participated with negative influences on learning 3s24 . In the cued model under normal conditions, the agonist rjr impaired learning 2s24 and did not affect learning 3s24 (figure 6, cued learning). The negative effect of the agonist on learning in 2s24 was significantly greater than that of the antagonist (p <.05). The difference between the agonistic and antagonistic actions on learning in 3s24 at were also significant (p <.05). It follows that the 42 subtype (negative influence) and some non-7 and non-42 subtypes of nachr (positive but weak influence) participated in the regulation of learning in 2s24 . At the same time, the 42 subtype did not participate in the regulation of learning 3s24, while some non-7 and non-42 subtypes participated with negative influences . Under the 2vo conditions, rjr did not correct the impaired functions 2s24 and 3s24, and mecamilamine did not correct learning 2s24 but resulted in normal learning 3s24 performance . It seems, non-7 subtypes of were removed from the receptor composition of learning 2s24 . At the same time, the importance of some non-7 and non-42 subtypes of nachr was reinforced or new connections arose in the receptor organization of learning 3s24 (negative influence). So, in the contextual learning 2s24 and 3s24, nachr were absent in normal and were acquired in 2vo receptor composition (3s24). Then, the cued learning 2s24 and 3s24, with identical cholinergic synaptic compositions in the norm, had differences in receptor compositions . Moreover, the cued learning 2s24 and 3s24 had also differences in 2vo receptor compositions via another means . This research showed that the chronic 2vo brain ischaemia model was an efficient model of neurodegenerative disorders, which was the first purpose of our investigation . The period of 610 days of 2vo ischaemia provoked typical attributes of vascular dementias such as impairment of learning and long - term memory in both spatial - contextual and spatial - cued models of behaviour in the morris water maze . The inherited abilities and working memory remained intact, and damage to the cued long - term memory was transient in this ischaemic period . A considerable reorganization of the synaptic pool of all investigated cholinergic systems in the cortex and the hippocampus was revealed in these same 2vo rats 1114 days after the surgery . A decrease in mchat or cchat activity in one synaptosomal fraction and an increase in another were obtained as result of the 2vo influence, but not of the training, whereas the biochemical parameters did not reveal similar changes between the control contextual and cued biochemical subgroups except one . It is possible that decrease in mchat activity in the hippocampal heavy synaptosomal fraction in the cued biochemical subgroup was result of the training (see table 1 and figure 2-cued). The decrease in mchat and cchat activity reflected cholinergic hypofunction or a degeneration of the cholinergic presynapses . Neurodegeneration was observed in different brain ischaemia models starting from the second day up to half a year of ischaemia [45, 46]. Dysfunction of chat in the projective fibres in the hippocampus (representing 8090% of the total activity of this enzyme, and see table 1) was described at 714 days of ischaemia [6, 13, 14, 47, 48]. It was shown in vitro that the activity of mchat was selectively suppressed when the exchange of acetylcholine was damaged by inhibition of the vesicular acetylcholine transporter or the high affinity transport of choline . The function of the vesicular acetylcholine transporter depends on the proton gradient, which in turn is disturbed due to falling atp levels (inhibition of the proton atpase) or acidosis . We did not reveal a decrease in protein content correlated with chat activity in our research . But we did suppose that the correlation between chat activity and protein content could be masked because of the complex opposing processes that took place in some of the synaptosomal fractions . At the same time, according to data in the literature, sprouting and destruction with the swelling of neurons and their terminals predominates in late brain ischaemia or postischaemic reoxygenation (in days and months) [3, 48, 53, 54]. In our research, activation of chat was also observed in the majority of the synaptic subfractions, and it could have reflected cholinergic hyperfunction or synaptogenesis (sprouting). It is known that synaptic hyperfunction is accompanied with an enhanced structuring of proteins from the synaptoplasm . Under these conditions, the m - protein content will increase and the c - protein content will decrease . Therefore, the correlated increase between mchat activity and m - protein content will be reflected as cholinergic hyperfunction and synaptogenesis (cortical light synaptosomes in the biochemical total group and both subgroups), whereas the correlated increase between cchat / mchat activity and the c - protein contect will only reflect synaptogenesis (cortical light synaptosomes in the biochemical total group and the contextual subgroup, hippocampal heavy synaptosomes in the total group and the cued subgroup). Selective activation of mchat in vitro was shown under conditions of impaired ionic balance such as an accumulation of [ca]i and [zn]i [55, 56]. It was revealed that [zn]i precedes [ca]i accumulation and [ca]i in turn results in the functional hyperactivation and swelling of synapses [53, 57, 58]. According to data in vitro activation of cchat reflect cholinergic hyperfunction under normal ionic and metabolic conditions [49, 59, 60]. Therefore, we suppose that the activation of cchat reflected synaptogenesis, in agreement with other researchers . Thus, chronic brain ischaemia for 1114 days resulted in a complex reorganization of the cortical and hippocampal synaptic pool which involved synaptogenesis or hyperfunctions in the unbalanced ionic conditions of one of the cholinergic synaptic populations and degeneration or dysfunction of the others . We supposed that all of the cholinergic processes revealed under 2vo conditions were present in both cholinergic subgroups of the rats but with different intensities (see figure 2-all rats, contextual and cued). We also supposed that the variety of cholinergic reactions was revealed by the phenotypical variety of the outbred rats and it was useful for understanding some of the principles of the organization of different forms of cognition . The second purpose of our research was a comparative analysis of the behavioural and biochemical parameters for identification of the cholinergic composition of the investigated cognitive functions under normal and 2vo conditions . In the first place it is necessary to note that the results of this research confirmed and expanded the knowledge about cholinergic mechanisms of cognitive functions under normal brain conditions . Our data showed the active involvement of cholinergic projective systems and also regional ones of the cortex and the hippocampus in cognitive processes . Cholinergic synaptic connections with the investigated cognitive functions revealed under normal conditions indicate that each form of cognition has an individual cholinergic synaptic and probably receptor compositions . This conclusion is conformed to the results of investigations, obtained in the morris water maze and some other behavioural models [3437]. Then, the data showed the participation of the cholinergic systems not only in mechanisms of learning and working memory, which was repeatedly observed in previous studies [35, 45, 61], but also in mechanisms of the inherited abilities and long - term memory . Our inherited abilities in the contextual task in the morris water maze was firstly detected by r g morris and u frey as a distinct type of memory and termed as it seems the problem of future discussions . Our data, concerning individual cholinergic organisation of function support contextual inherited abilities as a distinct form of cognition . Our data concerning the same cholinergic structures associated with inherited abilities 1s2 and long - term memory 3s1 also testify to possible tight interaction between these two forms of cognition . The involvement of cholinergic projective systems in mechanisms of the long - term memory is usually denied [6467], and was only discussed in a few studies [36, 68, 69]. Our data confirmed that synaptic populations of cholinergic projective neurons and of the interneurons of the cortex and the hippocampus can have positive and negative connections with cognitive functions . A negative dependence of cognitive functions on cholinergic cortical efficiency was also revealed earlier in cats using a similar methodology for researching the cholinergic synaptic organization of cognitive functions . Therefore, our data demonstrate that the cholinergic mechanisms of learning and memory are more complex than currently perceived . It is evident that this can complicate the detection of cholinergic effects on some cognitive functions by means of nonselective influences on cholinergic efficiency . For example, according to our data in the contextual model, the nonselective pharmacological cholinergic means as well as use of different methods of degeneration of the cholinergic projective systems would certainly have revealed the participation of the cholinergic projective systems in learning 2s24 and 3s24, but it would probably have concealed a cholinergic participation in the mechanisms of the inherited abilities, the long - term and the working memory . Such results would correspond to the data in the literature [62, 63, 68, 70]. From the numerous data in the literature, preservation of the cholinergic projective systems is critical for the success of cognitive processes, and it was thought that cholinergic dysfunction or degeneration results in the impairment of memory in neurodegenerative diseases of different aetiologies . Therefore, we analyzed the connections between reorganization of the cholinergic synaptic pool and impairment of learning and memory under 2vo conditions . The comparative analysis showed that the connections between the functional and cholinergic parameters revealed under normal conditions were practically lost in the ischemic rats . In our research, only impairment of cued long - term memory 2s1 was really dependent on degeneration of the key synaptic population of the cholinergic cortical interneurons, and also, probably, the intact cued memory 3s1 by the unchanged key synaptic population of the cholinergic cortical projective neurons . At the same time, our data also showed different cholinergic compositions of the cognitive functions under 2vo and under normal conditions in as the spatial contextual and the spatial cued models . Under 2vo conditions, most connections of the investigated functions with cholinergic synaptic populations revealed under normal conditions disappeared and new connections with other cholinergic synaptic populations arose . The quantity of cholinergic synaptic populations, involving in mechanisms of the investigated cognitive functions, was considerable reduced . Furthermore, cholinergic connections in general disappeared from the mechanisms of the following forms of cognition: inherited abilities 1s2, learning 2s24 and long - term memory 3s1 in the contextual model, and long - term memory 2s1 and 3s1 in the cued model . Moreover, brain region specializations of both the contextual and the cued functions were changed . Cortical cholinergic influences had been completely removed from the contextual functions and hippocampal ones from the cued functions . All considerable differences between cholinergic organisation of the cognitive functions in the normal and 2vo conditions stated above are clear demonstrated in figure 3 . It is important that a consistency between the performances of cognitive functions and their new key cholinergic synaptic populations was found in the majority of the remaining cholinergic - dependent functions under 2vo conditions (from four to six functions). Thus, according to our data, we suggest that the normal cholinergic synaptic connections in learning and memory were progressively reduced and changed during chronic ischaemia . It is known that anticholinesterase drugs are only effective in the early stages of alzheimer's disease (early and mild alzheimer's disease). It can be noted that the new cholinergic connections with the cognitive functions were not necessarily a consequence of degeneration or dysfunctions in the key cholinergic synaptic populations (it was evident for contextual learning in 3s24 and long - term memory in 2s1, that is, these new links could arise by other, indirect reasons). The dependence on the inclusion of cholinergic links in the realization of cognitive functions from the functional background of neuronal environments was recently revealed . This corresponds with the theory by d. a. sakharov about the nonsynaptic transfer of chemical information [71, 72]. According to this theory, any change in any functional system results in a change in all systems as a result of the change in neuroactive compounds of intercellular environments (the matrix). The changes in the matrix determine the activation of one or another neuronal ensemble which finally determines the behavioural act . From all of these viewpoints, it seems that the main value for cognitive functions is its receptor composition and its change in neurodegenerative pathology . Our data concerning the different consequences on the learning performance under normal and 2vo conditions by the action of rjr and mecamilamine on the same subtypes of nachr testify to this version . It seems that the reasons for changes in the cholinergic organization of cognitive functions in an ischaemic pathology can be any neurodegenerative or, on the contrary, reparative process (sprouting) of cholinergic and noncholinergic synaptic populations . In spite of the brain reparative potentials, the cholinergic and the whole neurochemical organization of cognitive functions under the chronic actions of pathological factors will be formed as optimally as possible under the new conditions . A new organization of cognitive functions will be constructed on neuronal elements which are stable against pathological influences . This new organization can provide an optimum realization of some cognitive functions but not of others . In any case, the study of new key neurochemical links in the organization of cognitive functions may be promising . The plasticity of neurochemical links in the individual organization of certain types of cognition can be used in the future for alternative corrections of vascular and other degenerative dementia.
In general, the third molar extraction is considered a procedure of minor proportion into the field of maxillofacial surgery . However, this procedure requires a specific surgical indication, planning, technical approach, and follow - up . Transoperative accidents related to the extraction of third molars often involve fracture of the adjacent bone and teeth, laceration of soft tissue, haemorrhage, neural lesions, and dental displacement into the maxillary sinuses, extracranial fossae, and cervicofacial spaces . Specifically in the medical literature, mandibular third molars were reported displaced into the infratemporal fossa; the pterygomandibular fossa; and the lateral pharyngeal, submandibular and sublingual spaces . In this context, the sublingual area is a triangular virtual space, located in the floor of the mouth, above the mylohyoid muscle, under the free portion of the tongue . The lateral limit of the sublingual space is the muscle complex hyoglossus - styloglossus, while the anterior limit is the genioglossus muscle . Important morphologic structures are observed in the sublingual space, such as the duct of the submandibular salivary gland, branches of the lingual artery, and the lingual and hypoglossal neural bundles . Thus, third molar extractions should be preferentially performed by maxillofacial surgeons, who are highly familiarized with the surgical morphology of head and neck . In addition, preoperative imaging must be properly used to avoid potential accidents in the routine of dental surgery . Based on that, the present study reports a case of a transoperative complication, in which a mandibular third molar was accidentally displaced into the sublingual space . In december, 2012, an 18-year - old female patient underwent extraction of the mandibular left third molar (tooth #38) for orthodontic reasons . Accidentally, the tooth #38 was displaced from the dental socket to an unknown adjacent site . The surgery was interrupted after the patient complains about the prolonged time taken to find the tooth . Anti - inflammatory (ibuprofen 600 mg) on every 12 hours, and analgesic (metamizole sodium 500 mg) on every 6 hours, were prescribed covering a period of 3 days after the surgery . The patient was referred to a radiology clinic for a proper diagnosis by means of multislice computed tomography, using a toshiba aquilion 64-channels (toshiba corporation, tokyo, japan) device . Three - dimensional reconstructions and axial and coronal slices enabled to detect the third molar in the medial surface of the left mandibular ramus in an inverted position (figures 1 and 2). In addition, a detailed examination revealed that the third molar was displaced above the mylohyoid muscle, laterally to the tongue: into the sublingual space (figure 3). Superior (a) and inferior (b) views of the mandible, presented in three - dimensional reconstruction by means of multislice computed tomography, illustrating the position of the tooth #38 (indicated by the arrows). Axial (a) and coronal (b) views of the mandible, presented in two - dimensional slices for bone analysis by means of multislice computed tomography, revealing a small bone fragment of the left lingual cortical plate (indicated by the arrow), broken during the surgery . Coronal (a) and sagittal (b) views of the mandible, presented in two - dimensional slices for soft tissue analysis by means of multislice computed tomography, illustrating the relation between the tooth #38 (indicated by the arrows) and the morphologic limits of the sublingual space . Despite advised about the importance of a second surgical intervention, the patient became apprehensive and uneasy in the following days . Additionally, functional and psychological conditions were worsened due to the presence of trismus, hampering daily activities . Based on that, the patient was convinced to undergo a second surgery only after 21 days from the accident . The surgical costs were afforded by the first dentist . During the preoperative examination the patient physical status was classified as asa i, enabling the surgery . Based on that, the patient was medicated with 8 mg of orally administered corticosteroid dexamethasone 1 hour prior to the surgery, aiming optimal postoperative outcomes . The second surgical procedure was performed under mandibular nerve block, using lidocaine 2% with adrenaline 1:100.000 (nova dfl, rio de janeiro, brazil). The third molar was accessed and successfully removed through an envelope incision, with a mucoperiosteal flap detachment from the retromolar trigone to the medial surface of the mandibular left first molar (tooth #36). In the following week the patient received the same medication prescribed in the first surgical attempt . Despite the close relation between the tooth #38 and the submandibular salivary gland and the lingual nerve, the patient did not report postoperative sequels within a follow - up period of 45 days . Displacement represents one of the most important accidents during the extraction of third molar due to the common need for major secondary surgical steps [2 - 6]. Additionally, accidentally displaced third molars may cause iatrogenic / traumatic neural injuries and even temporomandibular joint pain and dysfunction . The current literature reports the displacement of both maxillary and mandibular third molars . Specifically, gomz - oliveira et al . Described a case in which a maxillary third molar was displaced into the infratemporal fossa . Despite the close relation with the internal maxillary artery and the venous pterygoid plexus, the third molar was removed through a secondary surgical intervention, performed 2 weeks after the initial attempt, under local anesthesia . Similarly, hoekema et al . Reported the accidental dental displacement into the same anatomic site . However, the authors state that asymptomatic patients could be treated with periodic clinical and radiographic follow - ups prior to additional invasive approaches . Yet in extractions of mandibular third molars, most of the accidents involve the displacement of dental roots . It is justified due to the tilted position in which mandibular third molars are often observed, which makes necessary the crown resection prior to the dental extraction . Huang et al . Reported a case in which a third molar root was displaced into the pterygomandibular space . In this situation, the patient was successfully reoperated 5 months after the initial attempt, under general anesthesia and intraoral access . Specifically, the authors observed that 2 out of the 6 cases required a secondary intraoral surgical intervention for root retrieval . Both of the patients presented postoperative impairment of the inferior alveolar and lingual nerves . The other 4 patients were asymptomatic, excluding the need for a new surgery . Differently, in the present report an entire third molar was displaced into the sublingual space, indicating that the tooth was in a favourable position for extraction, in which the crown resection was not necessary . Besides, wrong surgical techniques are closely related to the transoperative displacement of third molars, potentially justifying the accident reported in our study . The same was observed in the reports of pippi and perfetti, and olusanya et al ., in which general practitioners performed unsuccessful extractions, highlighting the relevance of not performing surgical interventions without having a proper expertise on the field . Based on that, we recommend that third molar extractions should be preferentially managed by maxillofacial surgeons for optimal surgical outcomes . Considering the current literature, most of the third molars displacements into the sublingual space allows for a second surgical intervention under local anesthesia, making feasible a faster postoperative recovery, as observed in our study . An optimal postoperative recovery also depends on the technique addressed during the second approach for third molar removal . In some situations invasive techniques, such as double mucoperiosteal flaps, are necessary; while in other cases high - tech performances, such as endoscopic - assisted retrieval, are feasible . In the present case a single large mucoperiosteal surgical flap was necessary to reach the displaced third molar . Despite a close relation between the third molar and the submandibular salivary gland and the lingual nerve, independent from the situation, the level of surgical difficulty must be examined in forehand . Specifically, cases involving tilted and multiradicular third molars, and in close relation with neurovascular bundles, must detailed studied for an optimal surgical intervention . In this context, the radiographic surgical planning is the most adequate approach to avoid third molar accidental displacement . Cone - beam and multislice computed tomography play an important role, allowing for detailed analysis through three - dimensional reconstructions and slice navigation . In combination, the knowledge concerning the anatomy of head and neck is essential for an adequate and planned intervention . In parallel to the surgical care, accidents involving iatrogenic performances must be promptly reported to the patient, or legal responsible, informing the patient s health status and treatment choices . Thus, the bioethical principle of autonomy, which comprehends the patient s rights of being informed, and making decisions, is respected . Above all, more important is the preoperative description of technical steps and potential risks, and further record of the patient s decision within a signed informed consent form . Despite the best approach for clinical solutions involving iatrogenic performances, the dialogue not always avoids juridical complaints . In this context, the combination of preoperative imaging data, surgical plan, technical knowledge, informed consent, and the correct registration of the patient s files plays a valuable part as the main tool to support the professional against legal and ethic complaints . Despite common in the routine of maxillofacial surgeons, the third molar extraction is subject to transoperative complications . Based on that, professionals must be aware to the fact of keeping patients informed prior to the technical procedure, and supported after the treatment . Additionally, the present case report highlights the importance of technically planning and performing oral surgeries; requesting and interpreting complementary exams; and registering the patient s consent; in face of legal and ethic potential complaints.
Parent involvement is reduced, and students may live away from families in dormitories and may have added responsibilities . These factors appear to be stress generating, particularly at the start of the course . There is increased attention to the health and well - being of students at institutions of higher learning as they represent the future of families, communities, and countries . Of the students in institutes of higher education, medical students appear to have more emotional challenges, physical and psychosocial hazards, and mood disorders as they progress and think of their future and professional goals . A systematic review of 40 studies concluded that the overall psychological distress and prevalence rates of depression and anxiety in medical students are higher than nonmedical students or age - matched peers from the general population . In the arab world, studies on medical students have reported similar high levels of perceived psychological stress and depression related to internal and external variables, which accord with results reported in the international literature . The excessive amount of stress in medical training may lead to negative consequences such as diminished attention and concentration, increased incidence of errors, negligence, absenteeism, self - medication, and cheating during examinations . Medical education has been reported throughout the world as one of the most stressful academic curricula, which negatively affects the physical and mental health of medical students . Fear of examinations, high parental expectation, peer pressure, lack of leisure time, financial problems, relationship disharmony, and aspirations of higher studies are some of the many factors known to contribute to the development of stress in undergraduate medical students . This study aimed at assessing the perceived level of stress in medical students in the college of medicine, king saud bin abdulaziz university for health sciences (ksau - hs), king fahad medical city . These were graduate students with bachelor's degrees in allied health professions such as pharmacy, laboratory, radiology, physical therapy, and rehabilitation . Their educational background was different from their colleagues in the main university who were from secondary schools . The objectives of this study was to determine the prevalence and level of perceived stress among medical students, and to identify risk factors associated with perceived stress . This cross - sectional study was conducted among all applied medical sciences or science college bachelor program students using an anonymous self - administered questionnaire . Batches 9 and 10 had male students only (15 and 18 students respectively) while batch 11 had both males and females (14 males and 23 females). Batches 9, 10, and 11 were labeled as such by the ksau - hs according to the year of admission . The study tool was kessler10 (k10) psychological distress instrument, which is widely used in population - based epidemiological studies to measure current (1-month) distress . Its aim was to measure the level of stress and severity associated with psychological symptoms in population surveys . It consisted of ten questions in the form of how often in the past month did you feel and offers specific symptoms, such as tired for no good reason, nervous, and sad or depressed . The five possible responses for each question ranged from never to all of the time and were scored from 1 to 5, respectively . A total score of <20 denotes no stress, a score of 2024 represents mild stress, 2529 represents moderate stress, and 3050 represented severe stress . Questions related to basic demographics of the study participants such as age, gender, and grade point average were added to k10 tool . The study was approved by the scientific research committee of the college of medicine, kfmc and ethically approved by the institution review board of kfmc . All collected data were entered and analyzed using spss version 22 (ibm spss statistics for windows, version 21.0 . Quantitative variables were expressed as means and its related standard deviations while qualitative variables were presented in the form of frequency and percentages . Logistic regression analysis was done for severely stressed students versus others as a dependent variable with each of age, gender, bachelor of allied health sciences versus others, and living with parents versus others . Factor analysis techniques with varimax rotation with kaiser normalization and scree plot criteria were used to discover hidden factors for stress . Of 94 students, 80 (85.1%) completed the questionnaire giving a response rate of 85.1% . The mean age for males (26.3 0.3 years) was slightly higher than that of females (25.3 1.4 years). The majority (46.3%) of the students were from batch 11 and 75% held a bsc from applied medical sciences . About 51.2% of the students had been employed before enrolling in the college; 51.2% lived with their parents [table 1]. Only 5.9% were not satisfied with their work in the course; 15% rated the quality of education in the college as unsatisfactory while 47.5% rated it as satisfactory . One student described his physical health as poor while 10% students suffered from chronic diseases . Besides, 35% of the students had considered leaving the university at least once [table 2]. Mean stress score was higher among females (26.9 8.2) than males (25.6 10.4) with no statistical significance . According to kessler psychological distress scale (k 10), 30% students could be considered well, 18.8% as having a mild disorder, and 17.5% as having moderate disorder . The prevalence of severe stress was 33.8% among the participants, 40.7% of them belonged to the junior batch in the college (batch 11), and 59.3% did not live with their parents . Table 3 shows that the feeling of nervousness, hopelessness, restlessness, and depression were the most important factors affecting students stress scores . Since there were more of those who were likely to have severe disorder in the junior batch, a separate analysis was done for this batch and it showed that the only significant factor affecting stress was gender . One male (7.1%) was likely to have severe stress compared to 10 (43.5%) females (p = 0.02). Logistic regression analysis done for students with severe stress versus others and as dependent variables with each of age, gender, bachelor of allied health sciences versus others, and living with parents versus others showed that gender was the only significant predictor of stress in this study group (p = 0.04). Females were 11.8 times more at risk of developing stress than males [table 4]. Demographic characteristics of study participants students perceptions of their health status and education environment perception of stress factors by students during the past 4 weeks logistic regression model to predict stress score from some important variables factor analysis techniques with varimax rotation with kaiser normalization and scree plot criteria revealed three hidden factors for this group, namely, depression, nervousness, and age [figure 1]. The response rate of this study was more than 80%, which is comparable or even better than response rates reported in similar studies . The results of this study revealed that the mean stress score was 26.03 9.7 (range from 10 to 50). This overall stress prevalence of just over 52% is lower than the 67% prevalence reported in a previous ksa study . Overall stress among medical students using k10 instrument ranged from 38% to 62% in many countries, which is higher than that of the general population . The highly individualized and competitive environment in medical schools generates stress which tends to continue even after graduation . The learning and recreational environment of medical education have to be more friendly to medical students to reduce their current levels of stress . In this study, females showed higher stress levels than males but the difference did not reach statistical significance . It is to be noted that females were only in batch 11, the junior batch . The association of gender with stress levels in medical students has been addressed in many studies . Some studies have claimed that the gender differences in mean scores of stress were rare and not significant . Others found that males were more stressed than females, which as suggested by the authors might be because male students needed to have high scores and complete their courses within the shortest possible time to start their careers . The majority of studies, including the present study, found that females were much more stressed than males . The inconsistency of gender association with stress in medical students may be related to differences in social and educational environment as well as subjectivity in measuring self - reported stress . Women are more likely to perceive challenging and threatening events as stressful than men as suggested by some authors . The stress level was higher in the new batch of students compared to those at the senior levels . This is in agreement with studies which found that stress appears much more profound in the first academic year and that the level of stress decreased as the years progressed . There have been similar results in which stress was found to be higher in first - year medical students (28%) than in second - year medical students (16%). This difference was explained by the fact that the second - year medical students gradually adapted to the new living environment and the medical course thus reducing their stress and burnout . Important causes of stress in new medical students included unsuitable teaching methods, an unsatisfactory study environment in college, fear of failure in examinations, and social problems . All of these results in perceived anxiety and depression, negative lifestyle practices, and the state of physical and mental health worsened from the start of their college studies . This is contradictory to the findings of studies reporting that the level of stress increased progressively during their education and training . Variations and inconsistencies may be due to differences in curricula, educational, and social environments . In this study, no significant differences in stress levels were detected according to the state of physical health as shown in other studies . This is at variance with studies that reported that stress was significantly associated with students self - reported physical problems . The subjectivity in reporting episodes of illness and the perception of the nature of the illness and behavior may partly explain the inconsistency . This study revealed no significant effect on the stress level of medical students who lived with their families, with friends, or on their own . This contradicts another study which found that medical students living with their families tended to have less stress and burnout levels than those living alone . Because of the cultural norm of close family ties, family members are generally supportive however, they can also be a source of stress, especially in the case of students who have family problems, or who are married and/or with children, who may be struggling to balance family life and medical school training . The evaluation of the teaching and learning environment and the intention to discontinue studies were not significantly associated with stress levels . One would expect students who rated the educational environment poorly or were thinking of leaving the college to show higher stress levels . Medical students are trained to take care of the health of individual patients and populations . Unfortunately, their training is so physically and mentally demanding that it may have undesirable effects on their own physical, social, psychological, and mental health . As this study revealed worrying stress levels in medical students, much work is needed to reduce the prevalence of stress and promote good physical, psychological, and mental health . Some medical colleges implemented successful intervention strategies and programs to prevent or reduce stress among medical students . Those interventions and programs reduced stress, depression, and anxiety, increased spirituality and empathy, and made better use of positive coping skills among medical students . These interventions resulted in reducing the negative effects of stress on the health and academic performance of medical students . Much work is required to make the medical education environment more acceptable and engender less stress . It is recommended that universities start conducting stress coping strategies courses and workshops for medical students . Provision of free professional services to help cope with stress and psychological support services are also highly recommended . This was a cross - sectional study, so cause - effect relationships could not be established . This was a cross - sectional study, so cause - effect relationships could not be established.
Metabolic syndrome (ms) is a clustering of cardiovascular risk factors represented by high blood pressure, overweight / obesity, hypertriglyceridemia, low high - density lipoprotein - cholesterol (hdl - c), and glucose intolerance.1 the diagnosis of ms in adults, and recently in children and adolescents, is established when three or more of the five individual elements exist together in the same subject . Ms comprises a major risk for chronic diseases and in association with rising childhood obesity, and a sedentary lifestyle, is rapidly increasing in prevalence.3 it is reported that up to 47.2% of the iranian population had sedentary lifestyle.4 it is estimated that 21.9% of iranian adults living in central iran have ms.5 coronary heart disease is the leading cause of death in industrialised countries and is rapidly becoming a primary cause of death worldwide.6 in adults, ms is associated with a significantly elevated risk of coronary heart disease.7 physical activity helps to promote a healthful body composition, maintain muscle mass and thus preserve the resting metabolic rate.89 physical activity and fitness are associated with a lower incidence of morbidity and mortality from a number of chronic diseases, including cardiovascular diseases (cvds), diabetes and obesity.1011 there is a substantial body of evidence associating physical inactivity or low cardio respiratory fitness with the development of ms in adults.1213 in contrast, physical activity has favourable effects on all components of the ms and on the resulting cardiovascular risk.14 walking is the most common physical activity among adults,615 and an accessible form of moderate physical activity particularly relevant for the obese, which are less likely to perform vigorous physical activity . The quantity and the frequency of physical activity necessary to produce beneficial effects has not been defined as yet, but brisk walking is considered particularly appropriate, as it can be practiced by a large number of individuals, without any additional cost, and has a low rate of injury.16 in one study on hypertensive patients, walking and running produce similar reductions in mortality.17 in this study, walking with intensity, which is usual in daily activity, was evaluated in relation to ms in a sample of iranian population . The programme began in 2000 - 2001 and its third phase was done in 2007.1819 it is a quasi - experimental trial that includes a reference area and several levels of evaluation including process, impact and outcome evaluations . This was a cross - sectional study of the isfahan healthy heart program (ihhp). Ihhp is a comprehensive integrated community based programme for cvd prevention and control among adults via reducing cvd risk factors and improvement of cardiovascular healthy behaviours . The ihhp evaluation included four annual independent sample surveys in four specific sub - groups (adults, adolescents, health professionals and individuals at high risk for non - communicable disease) in both intervention and reference areas . In each community, ms was defined as subjects who had three or more of the following criteria as defined by the national cholesterol education program:1 (1) central obesity as the waist circumference (wc)> 102 cm in men and> 88 cm in women; (2) fasting plasma triglycerides 150 mg / dl; (3) low hdl - c with fasting hdl - c <40 mg / dl in men and <50 mg / dl in women; (4) hypertension with systolic blood pressure 130 mmhg and/or diastolic blood pressure 85 mmhg and/or anti - hypertensive agents and (5) hyperglycaemia with fasting plasma glucose 100 mg / dl and/or hypoglycaemic medications . A total of 2196 persons who had no components of ms were considered as the reference group . Walking time was estimated by participants . Walking time is composed of two components, leisure walking time and transfer walking time; in this study, the sum of these two components was considered as walking time . The weight measured by a seca scale, and wc was measured at the part of the trunk located midway between the lower costal margin (bottom of the lower rib) and the iliac crest (top of the pelvic bone). Body mass index (bmi) was calculated as weight / height (kg / m). Blood pressure was measured twice on the right arm, in sitting position and after 15 minutes rest . The first and fifth korokov's sounds were considered as systolic and diastolic blood pressure, respectively . To measure blood sugar and lipid profile (cholesterol, triglyceride, hdl and low - density lipoprotein (ldl)), approximately 10 ml of blood sample the demographic and baseline data of two groups were compared by chi - square and t - test . The prevalence for each component of ms was calculated by chi - square and analysis of variance (anova) tests were used to compare the means . The relations between walking - duration quartiles and ms were analysed by logistic regression test . To test for linear trend and determine p - value for trend across quartile of walking, we assigned the median walking time to individual's variable as continuous variable in logistic regression for> =3 component vs. 0 component . The results are adjusted by age, sex, socioeconomic status (ses), life style and food items (fat and oil, sweet and sweet drink, rice and bread, fried food). The demographic and baseline data of two groups were compared by chi - square and t - test . Walking time was divided in to the 4 quartiles . The prevalence for each component of ms was calculated by chi - square and analysis of variance (anova) tests were used to compare the means . The relations between walking - duration quartiles and ms were analysed by logistic regression test . To test for linear trend and determine p - value for trend across quartile of walking, we assigned the median walking time to individual's variable as continuous variable in logistic regression for> =3 component vs. 0 component . The results are adjusted by age, sex, socioeconomic status (ses), life style and food items (fat and oil, sweet and sweet drink, rice and bread, fried food). The study population was 4151 persons with a mean age of 40.21 16.26 years (49.4% female and 50.6% male). Ms is more prevalent in women than in men (62.7% vs. 31.9%) (p <0.001). It is also more prevalent in people who are in lower economic status (65% vs. 27.5%) (p <0.001). Ms prevalence is higher in persons with higher stress score and in people who had lower daily physical activity (p <0.001). Basic characteristics of the study population in [table 2], mean and prevalence of ms components, among quartile of walking time are shown . Wc, fasting blood glucose, ldl and total cholesterol all are negatively associated with increasing walking time (p <0.001). The results of logistic regression test for relation between ms and walking duration are shown in [table 3]. Mean and prevalence of the components of metabolic syndrome in study participants among quartile of walking time crude and adjusted odds ratios (95% ci) for metabolic syndrome vs. no elevated component of ms among quartiles of walking time in all models, the odds ratio of ms decreases with increasing walking time (p <0.05). In model 1, logistic regression without adjustment the odds ratio for existing ms in persons with usual walking time between 60 and 300 minutes / day is 0.71, which means that this level of daily walking time decreases the probability of occurrence of ms by about 29% . In model 6, adjusting for age, sex, ses, life style, food items and bmi the odd ratio changes only 1%, this means that these factors had a trivial effect on the relation between daily walking time and ms . Regular physical activity is an important protective factor against several diseases, such as obesity, hypertension, type ii diabetes318 and ms.19 in a cross - sectional school - based study on 417 adolescents (243 girls) aged 15 - 18 years from the azorean islands, portugal, daily step counts and physical activity levels were negatively associated with having one or more metabolic risk factors.22 in another study on 456 adolescents in brazil, the inactive adolescents and the adolescents with low cardio respiratory fitness had higher prevalence of ms; there was no difference with respect to gender.23 in the woolf study on 207 adult women (20 - 70 years), significant inverse correlations were found between activity (steps per day) and bmi, insulin level, crp concentration, leptin level, wc and body fat, glucose levels, crp concentration, wc and body fat.24 in our study, lower physical activity was associated with higher prevalence of ms (p <0.001). In the woolf study, with increasing age (from 30 to70 years) in women, the incidence of ms components increases . The relation between physical activity and ms components is also more prominent in younger women.24 kim et al ., from japan, reported that the risk for ms among physically inactive men was significantly higher than that for physically active men after adjustment for age, sedentary time, low intensity activity, smoking, calorie intake and bmi . In contrast, the risk for ms in women was not significantly different between physically active and physically inactive women after adjustment for age, sedentary time, low intensity activity, smoking, calorie intake, bmi and menopausal status.25 in our study, the age and sex had a small effect (> 4%) on risk of having ms for patients with low physical activity . Regarding the mentioned studies, the age and sex in different region of world may have different impact on relation between physical activity and ms . This may be related to daily level of physical activity, daily calorie intake, daily stresses and other measures of life style . In our study, it was a negative association between ms and self - reported walking time in adults aged 19 - 55 years, regarding high prevalence of sedentary life in the iranian population (47.2%),4 replacing daily activity with activity that increase the usual daily walking time would decrease the incidence of ms in this population . The main limitation of our study was estimation of walking time by participants, hence this variable depends on patient cooperation . Also this study was cross - sectional and these kinds of studies are weak for evaluation of relations between variables . In individual adults aged 19 - 55 years, daily estimated walking time is negatively associated with ms . This indicates that a mild physical activity such as regular daily walking is an effective way of preventing metabolic syndrome in the adult population.
The cadherin superfamily consists of classic cadherins and nonclassic cadherins, including desmosomal cadherins and the recently discovered protocadherins . Major cadherins in the skin include the adherens junction cadherin e - cadherin and the desmosomal cadherin desmoglein 1; these are expressed by virtually all epidermal keratinocytes and melanocytes and play important roles in maintaining proper cohesive arrangement of the epidermal cells . A cadherin less well studied in the context of the skin is t - cadherin . T - cadherin is a unique cadherin superfamily member: it possesses the five extracellular cadherin domain repeat structures typical of the classical cadherins but lacks transmembrane and cytoplasmic domains and is instead anchored to the cell membrane via a glycosylphosphatidylinositol moiety . T - cadherin is strongly expressed on epidermal basal layer keratinocytes of human and murine skin . Very little information exists regarding expression of t - cadherin in other structures of the skin . Its presence on the hair follicle, sebaceous glands, and eccrine glands has been reported; however, details on cellular distribution and pattern of expression are imprecise . In order to gain insight into the potential functions of t - cadherin in the skin, we performed an extensive examination of t - cadherin expression and its distribution in the epidermis and adnexal structures of normal skin . Ten normal skin specimens obtained from the samples of wide surgical resection of cutaneous tumors were used . Immunohistochemical studies were performed on 4-m - thick sections cut from formalin - fixed paraffin - embedded tissue using a universal immunoperoxidase polymer detection system (n - histofine simple stain max po; nichirei biosciencies, tokyo, japan). Sections were deparaffinized in xylol and hydrated through grades of ethanol, and then heated in a citric acid buffer (ph 6.0) at 95c for 60 min for antigen retrieval . The primary antibodies used were goat anti - t - cadherin (1:300; cat . Af3264, r&d systems europe ltd ., abingdon, uk), monoclonal mouse anti - cd34 (undiluted [rtu - end], novocastra), and rabbit anti - cytokeratin 15 (ck15) mab epr1614y (1:300; cat . Immunoreactivity was visualized by using the histofine simple stain max po reagent and diaminobenzidine tetrahydrochloride as the substrate . Sections were counterstained with mayer's hematoxylin, dehydrated with grades of ethanol, cleared with xylene, and mounted with tissue tec coverslipping film . For negative controls, immunohistochemical reactions on any given tissue sample and using any given antibody were run in duplicate . Stained sections were viewed using a nikon eclipse 50i microscope and images captured using a colorview iiiu camera (olympus). T - cadherin was strongly expressed in the basal keratinocyte layer, with a typically global distribution over the cell membrane and pronounced expression at intercellular junctions and on the basal and apical sides of the epidermal basal cells (fig . There was a weak cell body and/or nuclear expression of t - cadherin on occasional suprabasal keratinocytes (fig ., there was no t - cadherin expression in the upper spinous layer of the epidermis . T - cadherin was present on endothelial cells in all types of dermal blood vessels . Subendothelial smooth muscle cells and pericytes also showed immunoreactivity for t - cadherin (fig . Moderately to strongly positive staining for t - cadherin was observed in the basal cell layer surrounding maturing sebaceous cells . 2). In the eccrine glands, moderately to strongly positive cytoplasmic and membranous staining for t - cadherin was found in the majority of the single layer of cells within the secretory portion . T - cadherin was also strongly expressed on the outer myoepithelial cells surrounding the coiled gland (fig . The eccrine ducts consisting of a double layer of cells exhibited a strong membranous staining on the inner layer of cuticular cells, although infrequently, a few cells with cytoplasmic staining were seen (fig . In contrast to the pattern of staining in eccrine glands, both the coiled secretory gland and excretory ducts of apocrine glands were consistently negative for t - cadherin, whereas the myoepithelial cell layer around the periphery of the glands demonstrated moderately strong staining for t - cadherin (fig . 4). In order to define the distribution for t - cadherin in terminal hair follicles, we used vertical consecutive sections of the hair follicles and compared the localization of t - cadherin with that of two usually used stem cell markers, cd34 and ck15 . In terminal hair follicles, the basal cell layer of the epidermis and follicular infundibulum was strongly positive for t - cadherin and ck15 but negative for cd34 . Cd34 was strongly expressed in the endothelial cell and perifollicular spindle - shaped cells (fig . A strong staining for t - cadherin was observed in the outer root sheath (ors) layers within the isthmus of the follicle: the distribution was typically global with staining intensity being more prominent on the apical surface and at intercellular contacts of cells (fig . The area of the ors around the site where the arrector pili muscle attaches to the hair follicle (otherwise known as the bulge) demonstrated intense staining for t - cadherin (fig . Strong ck15 immunoreactivity was found in the ors of the isthmus and with increasing staining intensity at the site of attachment of the arrector pili muscle at the bulge level (fig . Cd34 staining was not observed in the ors within the level of the isthmus (fig . Cd34 was expressed in the most external layer of the ors below the level of the isthmus, but was not found in the lower part of the suprabulbar ors region (fig . 6). Within the thin ors layers surrounding the bulb of the follicle, positive t - cadherin was expressed in most cells, whereas ck15 expression was more focal (fig . 7). In the surrounding fibrous root sheath of the hair bulb and in the dermal papilla, isolated cells with immunoreactivity for t - cadherin were observed . T - cadherin was strongly expressed in the basal keratinocyte layer, with a typically global distribution over the cell membrane and pronounced expression at intercellular junctions and on the basal and apical sides of the epidermal basal cells (fig . There was a weak cell body and/or nuclear expression of t - cadherin on occasional suprabasal keratinocytes (fig ., there was no t - cadherin expression in the upper spinous layer of the epidermis . T - cadherin was present on endothelial cells in all types of dermal blood vessels . Subendothelial smooth muscle cells and pericytes also showed immunoreactivity for t - cadherin (fig . Within sebaceous glands, moderately to strongly positive staining for t - cadherin was observed in the basal cell layer surrounding maturing sebaceous cells . 2). In the eccrine glands, moderately to strongly positive cytoplasmic and membranous staining for t - cadherin was found in the majority of the single layer of cells within the secretory portion . T - cadherin was also strongly expressed on the outer myoepithelial cells surrounding the coiled gland (fig . The eccrine ducts consisting of a double layer of cells exhibited a strong membranous staining on the inner layer of cuticular cells, although infrequently, a few cells with cytoplasmic staining were seen (fig . In contrast to the pattern of staining in eccrine glands, both the coiled secretory gland and excretory ducts of apocrine glands were consistently negative for t - cadherin, whereas the myoepithelial cell layer around the periphery of the glands demonstrated moderately strong staining for t - cadherin (fig . In order to define the distribution for t - cadherin in terminal hair follicles, we used vertical consecutive sections of the hair follicles and compared the localization of t - cadherin with that of two usually used stem cell markers, cd34 and ck15 . In terminal hair follicles, the basal cell layer of the epidermis and follicular infundibulum was strongly positive for t - cadherin and ck15 but negative for cd34 . Cd34 was strongly expressed in the endothelial cell and perifollicular spindle - shaped cells (fig . A strong staining for t - cadherin was observed in the outer root sheath (ors) layers within the isthmus of the follicle: the distribution was typically global with staining intensity being more prominent on the apical surface and at intercellular contacts of cells (fig . The area of the ors around the site where the arrector pili muscle attaches to the hair follicle (otherwise known as the bulge) demonstrated intense staining for t - cadherin (fig . 5b). In contrast, the inner root sheath of the hair follicles was t - cadherin negative . Strong ck15 immunoreactivity was found in the ors of the isthmus and with increasing staining intensity at the site of attachment of the arrector pili muscle at the bulge level (fig . Cd34 staining was not observed in the ors within the level of the isthmus (fig . Cd34 was expressed in the most external layer of the ors below the level of the isthmus, but was not found in the lower part of the suprabulbar ors region (fig . 6). Within the thin ors layers surrounding the bulb of the follicle, positive t - cadherin was expressed in most cells, whereas ck15 expression was more focal (fig . 7). In the surrounding fibrous root sheath of the hair bulb and in the dermal papilla, isolated cells with immunoreactivity for t - cadherin were observed . Expression of t - cadherin on keratinocytes and specific localization on the basal cell layer of the epidermis in normal skin was first reported by zhou and colleagues more than a decade ago . This finding was confirmed in a number of subsequent studies addressing expression of t - cadherin in a variety of keratinocytic disorders including psoriasis, basal cell carcinoma (bcc), actinic keratosis, and squamous cell carcinoma (scc). In this study, we demonstrate that t - cadherin exhibits a precisely confined pattern of expression not only in the normal epidermis but also in adnexal structures of the skin, including the sebaceous, eccrine and apocrine glands and the hair follicle . The function of t - cadherin in skin (patho)biology remains unclear . In vitro investigations exploiting ectopic modification of t - cadherin expression levels in human keratinocyte (hacat) and scc (a431, hsc-1) cell lines have demonstrated functions for t - cadherin as a negative regulator of proliferation, migration, and invasion and a positive regulator of cell - matrix adhesion . In keeping with these putative functions, immunohistochemical studies of human cutaneous specimens have reported inverse relationships between t - cadherin expression levels and progression of several hyperproliferative keratinocytic disorders including psoriasis vulgaris, bowen's disease, and scc . On the other hand, and as recapitulated in this study, in the healthy epidermis, notably, cells that move away from the basal layer compartment toward the skin surface, withdraw from cell cycle, and commit to terminal differentiation no longer express t - cadherin . T - cadherin is also expressed in actinic keratosis, an in situ scc characterized by exaggerated keratinocyte proliferation . Furthermore, t - cadherin is prominently upregulated in bcc regardless of grade of proliferation and invasiveness (superficial, nodular, and infiltrative), with expression being particularly strong in the palisading peripheral cells of invading tumor nests . Noteworthy too is that in well - differentiated scc, t - cadherin remains strongly expressed on cells at the periphery of tumor nests invading the dermis, whereas in moderately - to - poorly differentiated scc (i.e., transformed cells acquire dedifferentiated status), t - cadherin expression is reduced or lost . Studies using a431 scc keratinocyte cell line and a three - dimensional spheroid cell culture model to mimic multicellular tumor organization demonstrated that spheroid structure was remarkably more compact following ectopic upregulation of t - cadherin but loose following silencing of t - cadherin . These in vitro findings, together with the collective immunohistochemical observations of the specific expression of t - cadherin in the basal keratinocyte layer of the epidermis and its maintained expression in the periphery of bcc and well - differentiated scc, might be interpreted to indicate an important role for t - cadherin in maintenance of organized structure . Support for such an interpretation is provided by our findings on the pattern of t - cadherin expression in skin adnexal structures, which, to date, has received rare attention . In sebaceous glands, the basal cell layer exhibited strong expression of t - cadherin, whereas sebocytes in the inner compartment of the gland showed no expression . The expression of t - cadherin in the basal layer of the sebaceous gland is not surprising since the physiological processes of the glands are in many ways similar to those of the continual renewal of the interfollicular epidermis . Although epidermal terminal differentiation differs from sebocyte maturation, in both the epidermis and sebaceous glands undifferentiated cells of the proliferative basal cell layer are required to maintain the continual renewal of the epidermis as well as the continuous sebum production and regeneration of sebaceous glands . As for the interfollicular epidermis, basal cells of the sebaceous gland typically lack expression of t - cadherin once they have left the basal compartment, withdrawn from the cell cycle, and commit to terminal sebocyte differentiation . In normal eccrine glands, we observed positive cytoplasmic staining for t - cadherin in the majority of the inner layer of cells within the secretory coils, confirming the finding of takeuchi et al . . However, we additionally found that outer layer of myoepithelial cells in the secretory portion and epithelial cells lining the excretory ducts strongly expressed t - cadherin . Pulse and pulse - chase studies using the sensitive nucleotide analog ethynyldeoxyuridine demonstrated proliferation of both luminal and myoepithelial cells within mature coiled secretory sweat glands, supporting that both cell populations are capable of regenerative self - renewal . In contrast to eccrine glands, the inner layer of secretory cells in apocrine sweat glands was consistently negative for t - cadherin, whereas myoepithelial cells within the periphery of the secretory components exhibited positive staining . As for eccrine glands, apocrine gland myoepithelial cells are likely also capable of self - renewal . Expression of t - cadherin in the hair follicle, especially in the ors, has been reported by takeuchi et al . . Our study clearly demonstrates that expression of t - cadherin in terminal hair follicles is strictly confined to the most external layer of the ors . It is perhaps not surprising that t - cadherin is expressed throughout the ors, as the ors is contiguous with, and biochemically similar to, the basal layer of the epidermis . Epithelial hair follicle stem cells localize to the outermost ors layer of the distal hair follicle epithelium at the proximal end of the isthmus in the region known as the bulge, and in a cluster of cells below the bulge known as the hair germ . Using consecutive sections of terminal hair follicles and k15 and cd34 antibodies as stem cell markers, we found that both ck15-positive cells in the bulge region and cd34-positive cells in the suprabulbar ors localize with t - cadherin positivity . Like the basal keratinocyte layer, the bulge and suprabulbular regions of the hair follicle are regarded as important stem cell reservoirs for hair follicle renewal and also for regeneration of sebaceous glands and of the epidermis in case of injury . The presence of t - cadherin throughout the bulge and suprabulbular regions might not endow t - cadherin with usefulness in specific hair follicle stem cell identification, but nevertheless invokes potential participation in regenerative processes . Accumulating evidence from different fields suggests that t - cadherin acts as a signaling receptor participating in the control of tissue architecture, recognition of the environment, regulation of cell motility, guidance of moving structures, and guarding integrity of functionally connected tissue layers . Herein, we have shown that t - cadherin is expressed in cell layers considered to house regenerative stem cell populations, namely the basal keratinocyte layer of the epidermis, the basal layer of the sebaceous gland, the myoepithelial layer of the eccrine gland, the myoepithelial layer of the apocrine glands, and the hair follicle ors . A function for t - cadherin as a guardian of the structural integrity of the epidermis and skin samples, previously collected for medical diagnostic, were obtained from the archival histopathology collection of the laboratory for histologic diagnostic, basel.
The oral bioavailability of many drugs is limited by their unfavourable physicochemical characteristics or absorption in well - defined part of the gastrointestinal tract (git) referred as absorption window . Prolonged gastric retention improves bioavailability, reduces drug waste, and improves the solubility for drugs that are less soluble in a high ph environment . Various approaches have been investigated to increase the retention of oral dosage form in the stomach, including floating systems, swelling and expanding systems, bioadhesive systems, modified shape systems, high density systems, and other delayed gastric emptying devices . Atenolol is a beta (1)-adrenergic antagonist or more commonly known as a beta - blocker used in the treatment of hypertension and angina pectoris . Atenolol undergoes little or no hepatic first pass metabolism and its elimination half - life is 6 to 7 hours . It is incompletely absorbed from the gastrointestinal tract and has an oral bioavailability of only 50%, while the remaining is excreted unchanged in faeces . Therefore, it is selected as a suitable drug for the design of a gastroretentive floating drug delivery system (gfdds) with a view to improve its oral bioavailability . Hydroxy propyl methyl cellulose (hpmc) is hydrophilic cellulose ether widely used as release retarding material . Hcso belongs to usp - nf type 1 consisting of triglycerides of hydroxy stearic acid widely used as a tablet lubricant . In the present study the objective of the present study was to develop a gastroretentive floating drug delivery system (gfdds) of atenolol and to examine the effects of both hydrophilic and hydrophobic retardant on in vitro drug release . In the present study, atenolol floating tablets were prepared by using hydrophilic polymer, hpmc k4 m, hpmc k15 m, and hcso as a hydrophobic retardant, alone and in combination to study the release kinetics and find out the effects of both the retardants and their combinations . Hpmc k4 m and hpmc k15 m were supplied by colorcon pvt . Ltd ., goa . Compatibility studies were carried out to know the possible interactions between atenolol and excipients used in the formulation . Physical mixtures of drug and excipients in the ratio 1: 1 were prepared to study the compatibility . Floating tablets containing atenolol were prepared by direct compression technique using varying concentrations of retardants (hpmc and hcso) with sodium bicarbonate . All the powders were accurately weighed and passed through 40 mesh sieve . Then, except magnesium stearate all other ingredients were mixed thoroughly for 15 minutes . After sufficient mixing of drug as well as other components, magnesium stearate was added, as post lubricant, and the blend was further mixed for additional 2 - 3 minutes . The final blend was compressed into tablets having average weight of 300 mg using a single punch tablet machine (royal artist, india) fitted with an 10 mm round flat punches . The preformulation studies including bulk density, tapped density, hausner's ratio, and angle of repose were performed of the powder . Twenty tablets were accurately weighed and placed in the friabilator (roche's friabilator) and operated for 100 revolutions . The% friability was then calculated by (1)% friability=(initial weightfinal weight)initial weight100 . Twenty tablets were selected randomly from the lot and weighed individually to check for weight variation . Ten tablets were finely powdered; quantities of the powder equivalent to 50 mg of atenolol were accurately weighed and transferred to a 100 ml of volumetric flask . The flask was filled with 0.1 n hcl (ph 1.2 buffers) solution and mixed thoroughly . Dilute 1 ml of the resulting solution to 100 ml with 0.1 n hcl . The absorbance of the resulting solution was measured at 226 nm using a shimadzu uv - visible spectrophotometer . The linearity equation obtained from calibration curve was used for estimation of atenolol in the tablet formulations . The tablets were placed in a 250 ml beaker, containing 200 ml of 0.1 n hcl . The time required for the tablet to rise to the surface and float was determined as floating lag time (flt) and the time period up to which the tablet remained buoyant is determined as total floating time (tft). The tablets were weighed individually (designated as w0) and placed separately in petri dish containing 5 ml of 0.1 n hcl and incubated at 37c 1c . At regular 2 h time intervals until 12 h, the tablets were removed from petri dish, and the excess surface liquid was removed carefully using the tissue paper . The swollen floating tablets were then reweighed (wt) and% swelling index (si) was calculated using the following formula: (2)si (%) = (wtw0w0)100 . The in vitro dissolution of all the batches were carried out in 0.1 n hcl as the dissolution medium using usp type ii apparatus (tdt-08l, electrolab) apparatus at 50 rpm . The temperature was maintained at 37 0.5c . The absorbances of the samples at different time intervals were carried out using uv visible spectrophotometer (uv 1800, shimadzu) at max of 226 nm . The mechanism of atenolol release from the floating tablets was studied by fitting the dissolution data of optimized formulation in following models: zero order: m = m0 k0 t; first order: log c = log c0 kt/2.303; higuchi square root law: q = kt; korsemeyer's model: mt / m = kt; zero order: m = m0 k0 t; first order: log c = log c0 kt/2.303; higuchi square root law: q = kt; korsemeyer's model: mt / m = kt; where m, c, and q are the amount of drug released at time t, m0, and c0 are total amount of drug, and k0, k, and k are corresponding rate constant . In case of korsemeyer's model mt / m is the fractional drug release at time t, k is a constant incorporating the properties of the macromolecular polymeric systems and the drug, n is a kinetic constant, which is used to characterize the transport mechanism . The value of n for a cylinder is <0.5 for fickian release, 0.5 <n <1.0 for anomalous transport (nonfickian diffusion), 1.0 for case - ii transport,> 1.0 for super case - ii transport type release . The optimized formulation of atenolol were packed in amber color bottle and aluminum foil laminated on the upper part of the bottle and these packed formulation was stored in stability chamber maintained at 40c 2c and 75% 5% rh for 3 months . The samples were withdrawn periodically and evaluated for their drug content, in vitro buoyancy studies and for in vitro drug release . The peaks obtained in the spectra of each formulation correlates with the peaks of drug spectrum . It does not show any well - defined interaction between atenolol and excipients the spectra for pure drug, drug - excipients mixture and optimized formulation are shown in figures 2, 3, 4, 5, and 6 . Results of the precompression parameters performed on the blend for batch f1 to f18 are tabulated in table 2 . The bulk density and the tapped density for all the formulations varied from 0.384 to 0.486 g / ml and 0.4809 to 0.5667 g / ml, respectively . Angle of repose of all the formulations was found to be less than 30, which indicates a good flow property of the powders . The formulated tablets were subjected for post compressional evaluation such as thickness, hardness, weight variation, friability, drug content, in vitro buoyancy studies, swelling studies, in vitro dissolution studies, and stability studies . Tablet thickness (n = 3) was almost uniform in all the formulations and values for tablets ranged from 3.2 to 3.88 mm . The hardness of all formulations was in the range of 8 to 12 kg / cm, indicating satisfactory mechanical strength . All the tablets passed weight variation test as the% weight variation was within the acceptable limits of 5% of the weight as per indian pharmacopoeia . All the values are below 1% indicating that the tablets of all formulations are having good compactness and showing enough resistance to the mechanical shock and abrasion . The percent drug content of the tablets was found to be in between 97 to 103% . Table 3 shows the results of physicochemical characters of atenolol tablets . In vitro buoyancy of the tablets from each formulation f1 to f18 was evaluated and the results are mentioned in table 4, where the highest and lowest floating lag time (flt) were observed with the formulation hydrogenated cottonseed oil and hpmc, respectively . The swelling index of the tablets from each formulation f1 to f18 was evaluated and the results are mentioned in table 5 and plot of% swelling index versus time (hrs) is depicted in figure 7, where the highest and lowest swelling was observed with the formulation f5 and f12 after 12 hrs, respectively . The swelling index was increased with concentration of hpmc since this polymer gradually absorbs buffer due to hydrophilic nature . The in vitro drug release profiles for the formulations f1f18 were depicted in tables 6 and 7 . The plot of cumulative percentage drug release versus time (hrs) for formulations f1f4, f5f8, f9f12 and f15f17 were plotted and depicted in figures 8, 9, 10, and 11, respectively . And f2 showed release of 87.17% and 78.22% at end of 4th hr, respectively . While f3 and f4 showed release of 66.81% and 44.58% at the end of 4th hr, respectively, indicating sustained effect due to higher concentration of hcso . Batches formulated with hpmc k4 m showed decrease in% drug release with increase in polymer concentration . Formulation f6 showed release of 97.13% at end of 12th hr, while f7 and f8 exhibited higher retardation . Its viscous nature also affects the diffusion coefficient of the drug . As a result reduction in drug release batches formulated with hpmc k15 m and hpmc k4 m showed similar release pattern till 4th hr . After 4th hr, release was retarded with formulations containing hpmc k15 m to higher extent because of its high viscosity as compared to hpmc k4 m . Thus, all hpmc k15 m formulations exhibited sustained effect for more than 12 hrs . It was observed that the type of polymer / retardant influences the drug release pattern . It was observed that as the concentration of polymer / retardant increased in formulations, the% drug release was decreased . Formulations f13 and formulation f15 with hcso, hpmc k4 m, and hpmc k15 m in ratio 2: 1: 1 showed release of 57.2% at end of 4th hr . While f16 with hcso, hpmc k4 m, and hpmc k15 m in ratio 1: 2: 1 showed release of 51.13%, but complete drug release occurred at 11th hr . Formulation f17 was considered as optimized formulation because it showed 51.08% drug release at end of 4th hr and successful sustained effect up to 12 hrs . Formulation f18 with higher amount of hydrophilic polymers, hpmc k4 m and hpmc k15 m showed release more than 12 hrs . Thus, the optimum combination of hydrophilic - hydrophobic matrix forming material required in formulation to get buoyancy and release of drug over 12 hrs . The data obtained from in vitro dissolution studies were fitted to zero - order, first - order, higuchi, and korsemeyer - peppas equations . The dissolution data obtained were plotted as time versus cumulative percent drug released as zero order, time versus log cumulative percent drug remaining as first order release kinetics, square root of time versus cumulative percent drug released as higuchi equation, and log time versus log cumulative percent drug released as per korsemeyer - peppas equation . The best fit with the highest determination r coefficients was shown by both peppas and zero order model followed by higuchi model which indicate the drug release via diffusion mechanism . Zero - order rate equation, which describes the system where release rate is independent of the concentration of the dissolved species . The korsemeyer - peppas equation is used to analyze the release of pharmaceutical polymeric dosage forms, when the release mechanism is not well known or when more than one type of release phenomena could be involved . The values of n with regression coefficient of all the formulations are shown in table 8 . The value of n obtained was in the range of 0.519 to 0.765, indicating nonfickian diffusion in case of tablets formulated with hpmc k4 m only . While tablets of hydrogenated cotton seed oil and hpmc k15 m followed fickian diffusion, matrix tablet of hpmc and hydrogenated cottonseed oil followed nonfickian diffusion . From the results, it was confirmed that all the formulations are following zero order models followed by higuchi model which indicate the drug release via diffusion mechanism . Formulation f17 gave 99.08% drug release at 12th hr fulfilling the aim of study and, hence, was selected as optimized batch . The results of stability studies did not show any significant change in the physical appearance, drug content, in vitro buoyancy studies, and in vitro dissolution studies of above four formulations as shown in table 9 . The results of the present research work demonstrates that the combination of both hydrophilic and hydrophobic polymers successfully employed for formulating the sustained release matrix tablets of atenolol . It is observed that optimum concentration of each of the polymer in combination was able to produce desired formulation which releases complete drug in 12 hours . The mechanism of drug release has observed the combined effect of diffusion and erosion for sustained drug release . So, the combination of both hydrophilic and hydrophobic retardant was suitable to produce the matrix tablet rather than using a single type of polymer . The present study also revealed that hcso can be used as a matrix - forming agent for the preparation of floating tablets.
While it is often a symptom of psychiatric or medical illness, for many older persons, it is not possible to identify the underlying cause . Studies during the last 15 years have consistently shown that fatigue in older adults is a strong predictor of functional limitations, disability, and mortality . Thus, fatigue might represent an early stage of frailty, defined as a physiologic state of increased vulnerability to stressors that results from decreased reserves in multiple physiologic and biologic systems . Cellular and organismal damages caused by an accruing burden of oxidative stress could be an underlying factor to fatigue . Increased oxidative stress can be caused by defective mitochondria as well as by chronic low - grade inflammation . Both these conditions are known to be associated with increasing age, and evidence suggests that oxidative stress and inflammation are associated with frailty . It is generally accepted that oxidative stress and inflammation are important contributors to the pathology of aging - related diseases such as cardiovascular disease, dementia, and cancer . Such conditions often predispose to frailty . Leukocyte telomere length (ltl) ostensibly undergoes an accelerated shortening due to inflammation and increased oxidative stress [6, 9]. This might explain the association of shortened ltl with aging - related diseases and some of their risk factors, including coronary heart disease, dementia, and insulin resistance [10, 11]. Potentially, shortened ltl and the consequent cellular aging could thus be an underlying mechanism to fatigue . Found no apparent association between ltl and frailty in a study that applied a composite frailty index score but had not examined directly the potential connection between ltl and fatigue . To examine whether a link in fact exists between ltl and fatigue, we have exploited the same - sex twin design that has previously made it possible to show relationsships of ltl with mortality, and physical ability . In the present study the longitudinal study of aging danish twins (lsadt) began in 1995 with an assessment of same - sex twin pairs born in denmark before 1920 . Since 1995 twins from the lsadt have participated in a comprehensive face - to - face interview including detailed questions about health, lifestyle, and physical functioning . This interview has been repeated every second year for a total of six examinations . At the second examination, blood samples were collected from 283 twin pairs . Of these, a total of 274 pairs (548 individuals) had their ltl measured . For this study, the population was restricted to the 439 individuals who were nondisabled, that is, who were able to rise from a chair or bed, walk indoors, get outdoors, walk outdoors in nice weather, walk outdoors in bad weather, and climb stairs without help . These restrictions were applied as previous studies have shown fatigue to be related to onset of disability, functional limitations, and even cardiovascular disease among nondisabled and nondiseased . We also repeat the analyses in a restricted sample of persons below 80 years, as these persons are more likely to be without conditions, which we have not been able to adjust for . For further information on the participants, see table 1 . Ltl was measured using southern blots of the terminal restriction fragments after digestion with hphi and mnli restriction enzymes; for 377 of the nondisabled twins the terminal restriction fragments were also generated by hinfi and rsai digestion . The hphi / mnli products include predominantly true telomeric repeats, whereas the hinfi / rsai product includes a proximal telomere segment that is not strictly ttaggg . The hphi / mnli products were on average 1.1 kb shorter than the hinfi / rsai products, and the two ltl measures correlated strongly (r = 0.88, p <0.001). Samples from the cotwins in each twin pair were resolved in adjacent lanes of the same gel . Running dna digests from the two cotwins in adjacent lanes on the same gel diminished the methodological variation within twin - pairs . In between - pair analysis the gel effect was included as covariate . Each sample was measured in duplicate (performed on different gels) and the mean of the two samples was used . We present data from the hphi / mnli digest, while results obtained with hinfi / rsai digest can be found in the corresponding supplementary (s) material (see tables in the supplementary material available online at http://dx.doi.org/10.1155/2014/403253). Fatigue was measured by the mob - t scale based on questions about six mobility items ((a) rising from a chair or bed, (b) walking indoors, (c) getting outdoors, (d) walking outdoors in nice weather, (e) walking outdoors in bad weather, and (f) climbing stairs). Every activity was described relative to whether the respondents did or did not feel fatigued afterwards . The mobility - tiredness scale (mob - t) counts the number of activities managed without fatigue . Was tested by the rasch model for item analysis, which showed that the scale had satisfactory homogeneity [19, 20]. The data have recently been reanalyzed using a new test based on the rasch model on the assumption of equal item discrimination with satisfactory results . Reliability tests showed agreement from 94.3% to 98.3% and kappa values from 0.72 to 0.88 for the included items on intrarater and interrater tests . As this measure of fatigue focuses on fatigue in performing certain activities it is likely that it reflects the physiologic and biologic consequences of doing these activities . Covariates of lifestyle, mental health, and aging - related somatic diseases were included as potential confounding factors since previous studies have shown that they might be associated with fatigue and ltl [10, 2329]. Physical activity was scored on a five - point scale deducted from self - reported level and frequency of physical activity . The answers were scored as follows: 0: no physical activity, 1: seldom low intensity physical activity, 2: frequent low intensity physical activity, 3: seldom high and frequent low intensity physical activity, and 4: frequent high intensity physical activity . Cognitive performance was scored by a cognitive composite score . This was evaluated by a battery of five brief cognitive tests, which were selected to be sensitive to age - related changes . The tasks included a verbal fluency test, forward and backward digit span, and immediate and delayed recall of a 12-item list . Depression symptomatology was assessed using an adaptation of the depression section of the cambridge mental disorders of the elderly examination (camdex). Mcgue and christensen factor analyzed the lsadt depression items identifying two factor scales (9-item affective and 8-item somatic scales). Because these scales were highly correlated (r> 0.50), a 17-item total scale score has been defined . Cardiovascular disease included angina pectoris, heart failure, hypertension, irregular heart rhythm, and myocardial infarction . For diabetes and cancer any positive history is regarded as being a positive outcome and no distinction is done between type and duration . Rheumatologic disease covers the presence of either of the following conditions: rheumatic arthritis, osteoarthritis, and/or gout . Possible association was further studied using an intrapair twin design in which difference in ltl was regressed on the difference in fatigue . This design enables us to control for age, sex, zygosity, and gel effect, as well as for early common environment and genetic factors . If an association is attenuated among mz and dz twins it suggests early environmental confounding . A p value of 0.05 was chosen as significance level for all tests and the statistical analyses were done using stata 11.1 (stata corp, college station, tx, usa). A total of 439 twins representing 257 twin pairs were studied . 145 (33%) were men, and 294 (67%) were women; 235 (53.5%) came from a dizygotic twin pair (dz) and 204 (46.5%) from a monozygotic twin pair (mz). The mean ages of the participants were 78.1 years for men (range 7388) and 78.5 years for women (range 7394). Women were found to have longer ltl than men (age adjusted: 4.65 kb and 4.46 kb, resp . ). Fatigue was inversely correlated with age (r = 0.25, p <0.001), that is, the higher the age the more fatigued (lower score). The association between the examined covariates and, respectively, ltl and fatigue is presented in table 2 . Only smoking and depression symptomatology were significantly associated with ltl, while bmi, physical activity, cognitive function, depression symptomatology, and rheumatic disease were significantly associated with fatigue . The regression model revealed a positive association between ltl and fatigue when adjusting for effects of sex and age . For every unit increase in fatigue score, ltl increased by 0.038 kb after adjustment for sex and age (p = 0.023; table 2), which is equivalent to two years of age - related ltl attrition . There was no effect of zygosity nor was there an interaction effect of sex and age (data not shown) on the association . Hence, the association between fatigue and ltl was similar for each gender and zygosity . The examined lifestyle factors had a minimal effect on the association between ltl and fatigue (table 3, model 3), diminishing the association coefficient from 0.038 to 0.032 . Including mental health in the model (table 3, model 4) however, inclusion of these covariates was not found to give a significantly better fit by a likelihood ratio test . Adjusting for somatic diseases separately or jointly did not influence the association between ltl and fatigue (table 3, model 5). Including all covariates, that is, sex, age, gel effect, zygosity, somatic diseases, mental health, and lifestyle resulted in slight attenuation of the association between ltl and fatigue (table 3, model 6) (p = 0.166). However, likelihood ratio test indicated that the optimal model of the ltl - fatigue association includes only sex and age (model 2). The analyses were repeated in a subsample of the study population restricted to individuals below 80 years . The intrapair analysis confirmed that for every one unit change in fatigue score, ltl differed by 0.039 kb (p = 0.054) within the pair (table 4). This effect was not significantly different between mz and dz twin pairs; however, the effect only reached significance in mz twin pairs . The association was attenuated when including lifestyle factors from coef = 0.039 to coef = 0.030 due to a small confounding effect of smoking and physical activity not seen in the regression analysis . Also inclusion of depression symptoms provided a better fit of the data but hardly influenced the association (coef = 0.036, p = 0.097) (data not shown). There was no noteworthy difference on the demographics of two populations (table s1). There were similar associations between the examined covariates and, respectively, ltl and fatigue (table s2). The association with fatigue was of lesser magnitude and no longer reached significance (table s3, model 2), neither for the younger subpopulation . Likelihood ratio test showed that the inclusion of lifestyle factors (especially smoking) as well as depression symptomatology had a better fit of data . Including these factors completely attenuated the association (coef = 0.005, p = 0.846). The intrapair analyses were in agreement with findings from the hphi / mnli digest, however not significant, and could mostly be attributed to physical activity and smoking, as well as depression symptomatology . In this study we observed an association between fatigue and ltl in nondisabled older twins . Previous studies have found fatigue to be related to onset of several adverse health outcomes, also when adjusted for multiple covariates . The predictive value of fatigue has been especially strong in nondisabled older persons and in persons without cardiovascular disease, indicating that fatigue may be an early marker of the aging process, before the onset of diseases and disability . We have earlier shown that ltl was related to physical ability, measured by a self - reported strength scale, in a study population of 7093-year - old participants in the lsadt study . The present study shows that ltl is associated with fatigue before the onset of disablement, thus suggesting that shortening of ltl is an early detectable event in this process . We have chosen to include four major aging - related diseases (cvd, diabetes, rheumatic disease, and cancer) as well as cognitive function as potential confounders since these have previously been suggested to be associated with ltl dynamics . However, we found no influence of either diseases or cognitive function on the ltl - fatigue association . Depression is a condition closely correlated with fatigue and it is likely that this finding is merely an over adjustment since the depression symptomatology score contains questions on feeling of lack of energy, which was highly correlated with fatigue (r = 0.323, p <0.001). The coexistence of fatigue and depression has previously been reported and found to be associated with an increased risk of death in older adults, as has the coexistence of depression and other diseases predisposing to fatigue . A common inflammatory and oxidative stress pathway has been suggested, necessitating a clarification of this coexistence in future studies . Of the included lifestyle factors, we found that only smoking had a slight effect on the association between ltl and fatigue . We have previously found that physical activity level was associated with ltl . In this subpopulation we suggest that this is simply due to lack of power and therefore chose to include physical activity as a possible confounder . However, physical activity seemed only to have an effect on the association when ltl was measured with the hinfi / rsai digest . Smoking is therefore the only of the analyzed covariates that we suggest as a confounder to be included in the model besides sex and age . A study by woo et al . Found no association between frailty and ltl . The discrepancy is likely due to the focus on fatigue in the present study, while woo and colleagues used a composite frailty index . Reported a cv of 11.1% in their qpcr based method, while the cv of the trf assay for this cohort was only 3.4% . Moreover, although our cohort was of limited size, we gained statistical power by exploiting the twin design . The low coefficient of variation is a strength to our study, as well as the twin design . We have included lifestyle factors and major aging - related diseases as covariates; in future studies inclusion of medications could be of value since these could contribute to fatigue . Various tools to evaluate fatigue exist . Further different performance based measures are often used (e.g., an oxygen uptake measure or accelerometers). The used fatigue measure is reliable [1, 22] and has the advantage that it is a self - reported measure of fatigue in performing certain activities and is therefore likely to reflect the physiologic and biologic consequences of doing these activities . Our study suggests a possible common physiological pathway between ltl attrition and fatigue; however, to establish a better insight into the dynamics in fatigue and cellular aging, future studies should examine changes over time of ltl and fatigue score in the same individuals . Overall, our findings indicate that ltl is associated with fatigue and that this cannot be explained by common lifestyle factors, aging - related diseases, or cognitive function . These findings largely agree with previous studies showing that fatigue is a strong indicator of adverse health outcomes in nondisabled older adults and in middle - aged healthy men . The findings are suggestive of oxidative stress and inflammation being underlying factors of fatigue and should spur the search for more biomarkers leading to a better understanding of the physiology behind fatigue.
The metals with densities over 5 gr / cm are called heavy metals (1). In recent years, the presence of these metals such as arsenic(as), cadmium (cd), mercury (hg), lead (pb), nickel (ni), and chrome (cr) in drinking water have become an international environmental and health concern (2 - 4). The entry of the heavy metals in water resources can be due to the natural processes such as wastewater municipal, industrial, and agricultural sewage (5). Many of these elements have a dual role in the human body (6). The heavy metals could be dangerous for human health at higher values than the standard (7, 8). These elements have biological accumulation, toxicity, and environmental sustainability properties (9). The epidemiological studies show that there is a significant relationship between tooth decay, kidney disorders, neurological disorders, and cancers with heavy metals (10 - 12). Cadmium can enter into the drinking water by the penetration of industrial wastewater containing cadmium into water distribution network and also penetration via polyethylene tubes and containers (water bottles) (13, 14). The international agency for research on cancer (icrp) has classified cadmium as a group a carcinogen (15). Cadmium s biological half - life in bone and kidney is 38 and 10 years, respectively (16). Chronic exposure to cadmium causes kidney failure and itai - itai disease (osteoporosis and severe pain) (17, 18). The standard limit of who and epa for cd in bottled drinking water is 3 and 5 g / lrespectively (19, 20). In the last 30 years, the use of bottled water has been growing in many nations (21, 22). The packed water is divided into two groups of mineral water and bottled water (23, 24). In many studies, the concentration of heavy metals in bottled water and their carcinogenic and non - carcinogenic risks in different age groups have been measured (25 - 31). Due to the health hazards of cadmium and high water consumption in bandar abbas, especially the bottled water (due to hot and humid weather), in this study, we have tried to evaluate the non - carcinogenic risk in adult men and women, and children . The coastal city of bandar abbas (center of hormozgan province) is located at the south of iran (54227e and 271153 n) and at a height of 9 meters above the sea level (25). The climate of this city is hot and humid and its population is growing by the day because of economic and industrial development . The sample collection was made from 8 marks of popular packaged water in bandar abbas at 13 different places . Per month 9 number of 1.5 liter 216 samples of water were collected in summer and 216 samples were collected in winter (totaling 432 samples of bottled water). The samples were transferred to the chemistry laboratory of health faculty of medial science, university of hormozganin 4 - 6c in order to measure the heavy metals concentration (26). Areas of collecting samples of bottled water in the surface of bandar abbas in the south of iran 1 ml nitric acid was added to water samples per each liter of sample water in a laboratory to get to ph<2 (to save up the cd up to 28 days in the water samples). For condensation, water samples were passed through whatsman glass micro - fiber filter (gf / c) (27). Cd concentration was measured by atomic absorption spectrophotometer in model dr2800 through the dithizone method (28). The difference of mean concentration of cdin different marks of the bottled water, the difference of cdconcentration in summer and winter, the difference of r and hqin different age groups was analyzed byt- test and anova test by software spss16 . The calculation of the chronic daily intake (cdi) was performed through the equation which was presented by the united states environmental protection agency (usepa); in this equation, cdi is chronic daily intake (mg / kg - d), c is the concentration of cd in drinking water (g / l), di is mean daily intake water(l / d), and bw is a body weight (kg). Because of, not exist data about the mean daily water consumption and the weights of different age groups population in bandar abbas; we used date suggested by who and epa . Hence, di for adult men (17 - 65 years old), adult women (17 - 65 years old) and children (4 - 14 years old) 2.723, 2.129, 1.8 l / d and bw 76, 64 and 22.3 kg, are respectively (29, 30). Since the safety factor (sf) for the carcinogenic risk from oral exposure to cadmium has not been estimated by epa (integrated risk information system), the carcinogenic risk of cadmium was not calculated (31). Hazard quotient (hq) for the calculation of non - carcinogenic risk of cd in water was calculated using equation 2; in equation 3, rfd is dose of polluter s reference (mg / kg - d) which was considered 0.0005 mg / kg - d forcd(3). A population is located in a safe area when hazard quotient is less than 1: hq<1 (32). The coastal city of bandar abbas (center of hormozgan province) is located at the south of iran (54227e and 271153 n) and at a height of 9 meters above the sea level (25). The climate of this city is hot and humid and its population is growing by the day because of economic and industrial development . The sample collection was made from 8 marks of popular packaged water in bandar abbas at 13 different places . Per month 9 number of 1.5 liter 216 samples of water were collected in summer and 216 samples were collected in winter (totaling 432 samples of bottled water). The samples were transferred to the chemistry laboratory of health faculty of medial science, university of hormozganin 4 - 6c in order to measure the heavy metals concentration (26). Areas of collecting samples of bottled water in the surface of bandar abbas in the south of iran 1 ml nitric acid was added to water samples per each liter of sample water in a laboratory to get to ph<2 (to save up the cd up to 28 days in the water samples). For condensation, water samples were passed through whatsman glass micro - fiber filter (gf / c) (27). Cd concentration was measured by atomic absorption spectrophotometer in model dr2800 through the dithizone method (28). The difference of mean concentration of cdin different marks of the bottled water, the difference of cdconcentration in summer and winter, the difference of r and hqin different age groups was analyzed byt- test and anova test by software spss16 . The calculation of the chronic daily intake (cdi) was performed through the equation which was presented by the united states environmental protection agency (usepa); in this equation, cdi is chronic daily intake (mg / kg - d), c is the concentration of cd in drinking water (g / l), di is mean daily intake water(l / d), and bw is a body weight (kg). Because of, not exist data about the mean daily water consumption and the weights of different age groups population in bandar abbas; we used date suggested by who and epa . Hence, di for adult men (17 - 65 years old), adult women (17 - 65 years old) and children (4 - 14 years old) 2.723, 2.129, 1.8 l / d and bw 76, 64 and 22.3 kg, are respectively (29, 30). Since the safety factor (sf) for the carcinogenic risk from oral exposure to cadmium has not been estimated by epa (integrated risk information system), the carcinogenic risk of cadmium was not calculated (31). Hazard quotient (hq) for the calculation of non - carcinogenic risk of cd in water was calculated using equation 2; in equation 3, rfd is dose of polluter s reference (mg / kg - d) which was considered 0.0005 mg / kg - d forcd(3). A population is located in a safe area when hazard quotient is less than 1: hq<1 (32). The (mse) mean and range of cadmium (cd) concentration is 1.730.19 g / l and 0 - 7.1 g / l, respectively . The mean concentrations of cadmium in marks bw1, bw2, bw3, bw4, bw5, bw6, bw7 and bw8 are 1.330.17, 0.680.09, 0.770.13, 0.950.17, 1.320.17, 0.670.09, 4.80.43, and 3.370.32 the orders of the bottled water marks with respect to the mean cadmium concentration are bw7>bw8>bw1>bw5>bw4>bw3>bw2>bw6 . The most and the least mean cadmium concentration goes to the marks bw7 (3.370.32 g / l) and bw6 (0.670.09 g / l), respectively (table1). The range of cadmium concentrations in brands bw1, bw2, bw3, bw4, bw5, bw6, bw7 and bw8 are 0 - 2.9, 0 - 2.7, 0 - 2.1, 0 - 2.3, 0 - 4.1, 0 - 1.7, 1.9 - 6.1 and 1.5 - 7.1 g / l, respectively . Mean (mse), standard deviation (sd), the range of cadmium concentration (g / l), in 8 marks of the bottled water in bandar abbas in summer and winter 2013 (n=432) the mean cdi for age groups of adult men, adult women and children is 17e-7, 18e-5 and 39e-7 mg / kg - d, respectively . (70e-7, children) and bw4 (3e-7, adult women), respectively (table 2). The cdi order for different age groups is as the following manner: children> adult women> adult men . The cdi for children is 2.22 times the cdi for adult men and women (p value<0.05). Cdi, r and hq in adult men, adult women and children due to cadmium in the bottled water of bandar abbas the mean concentration of cadmium in pip s study (0.20.04 g / l), is much lower than that of our study (33). The cadmium concentration in yazd s water distribution network in the study of salmani et al ., (3.350.79 g / l), is more than that of ours (34). The maximum cadmium concentration in the bottled water in the research done by miranzadeh et al ., (4.751.25 g / l), is lower than the maximum cadmium concentration in our study (7.10.83 g / l, bw8) (35). The mean concentration of cadmium in all the brands off bottled water is lower than the epa s maximum allowable concentration (5 g / l). But, in the mark of bw8 and bw7, the mean concentrations of cadmium are more than who guidelines (3 g / l) (19, 36). Concentration ranges in marks bw5, bw7 and bw8, in some samples are more than the standard limit (figure 2). Comparison of mean and range of cadmium concentration in bottled water with standard of who and epa as it is seen in figure . 3, 33.2% of all the samples (i.e. 144 sample out of 432), have concentrations more than the standard of who and 22.4% (i.e. 97 samples) have concentrations more than the epa standard . The percentage of relative frequency distribution of cadmium concentration in the bottled water of bandar abbas (n=432) there was a significant difference (p value <0.05) between the mean concentrations of cadmium during summer and winter in bw5 and bw6 marks . This difference in cadmium concentration could be due to different water sources, variation in the type of processing, possible contamination in the water source or the cadmium leakage from the polymer the bottle into the water (14). Hq for adult men, adult women and children is 0.034, 0.032 and 0.078, respectively . Hq in the study of muhammed et al ., (0.033 in jijal - dubair area) is almost equal to that of our study (0.034) (27). On the contrary to our study,, due to the absence of cadmium in water source (zero concentration) (37). The order of the non - carcinogenic risk is children> adult men> adult women . Hq in children is 2.29 times that of adult men and women (p value<0.05). The mean cadmium hq of the bottled water in age groups of adult men, adult women and children is less than 1 (figure 4). Comparison of hq in age groups of adult men, adult women and children with the safe limit g / l) is less than the standard limits of who and epa, some samples have cadmium concentrations more than the standard limit . Hq between the adult men and women is almost equal and there is no significant difference between these two . However, the hq in children age group is more than that of the adult men and women . As hq is less than 1 in all the age groups, so it could be inferred that the whole bandar abbas population is secure (hq <1).
Human immunocompetence and survival depend on the egress of newly produced t cells from the thymus . Approximately 1% of the thymocyte population emigrates from the thymus each day to populate the peripheral t - cell compartment . Following the arrival of early thymic progenitors, the thymus supports all steps of t - cell development, from the differentiation of progenitors into t - cell receptor (tcr)-expressing cd4 and cd8 double - positive (dp) cells in the cortex to the maturation of dp into cd4 or cd8 single - positive (sp) cells that localize to medullary regions . Sp thymocytes initially possess an immature phenotype but over time acquire markers associated with maturation (figure 1). Egress is thought to occur via blood vessels and lymphatics, but the route that predominates remains unknown . An initial insight into the mechanism of thymic egress came with the finding that transgenic expression in thymocytes of pertussis toxin, an inhibitor of gi signaling, strongly inhibited egress . A role for gi2 in thymocyte emigration was later suggested though may instead reflect an accelerated transition of gi2-deficient cells from the dp to sp stage of development . Important further advances regarding the mechanism of thymic egress emerged from a combination of pharmacological and genetic studies that established an essential role for sphingosine-1-phosphate (s1p) and one of its g - protein - coupled receptors . A thymocyte is depicted undergoing maturation from the cortical cd4 and cd8 double - positive (dp) stage to the medullary cd4 single - positive (sp) stage . The sp cell is initially in an immature state, lacking krppel - like factor-2 (klf2) and sphingosine-1-phosphate receptor-1 (s1p1) and having a characteristic surface phenotype . After a period of a few days, if the cell survives negative selection, it upregulates klf2 and then s1p1 (green 7-transmembrane structure) and undergoes other changes in surface marker expression . Acquisition of s1p1 allows the cell to respond to s1p (green dot) that is present at approximately 1,000 nm in the vessel lumen (supplied by red blood cells) and locally supplied by radiation - resistant cells, perhaps endothelial cells or pericytes . Thymic pericytes are unusual in being neural crest cell (ncc)-derived . The final egress step triggered by s1p, likely the reverse transmigration step, requires coronin-1a- and mdia1-mediated reshaping of the actin cytoskeleton and thus the cell . Cells that have just exited the thymus are referred to as recent thymic emigrants (rtes). T cells that are genetically deficient in s1p1 or klf2 can reach the mature sp stage but then fail to egress and accumulate in the medulla . Treatment with fty720 or with s1p lyase inhibitors that promote increases in intrathymic s1p causes s1p1 down - modulation from the cell surface and likely degradation (black crosses) and interrupts egress, again causing accumulation of mature sp cells in the medulla . The active phosphoryl metabolite of fty720 acts as an agonist for four of five s1p receptors, and of these, s1p1 is strongly upregulated in maturing sp thymocytes . Remarkably, genetic deletion of s1p1 in thymocytes phenocopied the pertussis transgenic mouse, indicating that s1p1 is the primary gi - coupled receptor mediating egress . Deficiency in the two kinases that generate s1p led to a similar thymic egress defect . A second small molecule, 2-acetyl-4-tetrahydroxybutyl - imidazole (thi), known to reduce thymic egress when fed to mice, was found to inhibit s1p lysase, causing 1,000-fold increases in thymic s1p and down - modulation of thymocyte s1p1 . Similar findings were made when s1p lyase was knocked down using a short hairpin rna (shrna) and through analysis of mice lacking the s1p lyase gene . The latter mice showed severe growth abnormalities and poor viability, perhaps reflecting roles of the lyase in sphingolipid metabolism, and thymic function was strongly affected . Introduction of a low - expressing s1p lyase transgene rescued non - lymphoid pathologies while failing to fully restore thymic egress . These studies highlight the importance of appropriately compartmentalized s1p distribution to support normal thymic egress (figure 1). A study aimed at characterizing the altered t - cell compartment in mice lacking krppel - like factor-2 (klf2), a zinc - finger transcription factor, revealed a block in thymic egress associated with reduced s1p1 mrna in mature thymocytes . However, sufficient s1p1 may remain in klf2-deficient cells to permit low amounts of thymic egress and further work will be needed to fully define the factors controlling s1p1 expression . Insight into factors that act upstream of klf2 has come from work on the foxo family of transcription factors . Deficiency in foxo1 led to a slight accumulation of mature sp thymocytes, particularly of the cd8 lineage, though the block was less severe than for klf2 deficiency . Other foxos appear likely to help drive klf2 expression the inhibition of foxo transcription factors by phosphoinositide 3-kinase (pi3k) signaling might explain the thymic egress block seen in transgenic mice overexpressing pi3k in thymocytes . The factors determining when expressions of foxo, klf2, and thus s1p1 are turned on remain undefined, but recent work suggests the involvement of a timer mechanism . In transgenic mice expressing green fluorescent protein (gfp) from the rag2 locus, the amounts of gfp in the cell can be used as a molecular timer to determine how much a thymocyte has aged since its rag2-expressing dp stage of development . By means of this approach, a medullary dwell time for sp thymocytes of 4 to 5 days was calculated, with egress occurring in a conveyor belt fashion with cells that enter the medulla first exiting the thymus first . To exit the thymus, t cells must reverse - transmigrate across an endothelial barrier . Several recent observations suggest that molecules required for reorganization of the thymocyte actin cytoskeleton are central to this process . One study followed up on the description of a spontaneous mutant mouse line with a thymic egress defect and peripheral t - cell deficiency to identify a role for the arp2/3 regulatory protein coronin-1a in thymic egress . Notably, although dp cell migration was also affected by the mutation when tested in vitro, the in vivo thymic defect revealed itself only as an accumulation of mature sp cells, suggesting an especially stringent need for appropriate control of actin branching during the transmigration step . Since formins support polymerization of unbranched actin, this work further highlights the necessity of fine actin cytoskeleton control to coordinate the cell shape changes required for egress . In addition to molecules involved in cytoskeletal reorganization, cell adhesion molecules may play a role in thymocyte egress . Recent data suggest that p - selectin glycoprotein ligand-1 (psgl-1) may be involved . P - selectin marks a subset of thymic endothelial cells, and thymocytes express psgl-1 . However, the accumulation of thymocytes at the immature sp stage in psgl-1-deficient mice is unusual for a thymic egress defect (figure 1). Further study of how psgl-1 influences sp thymocyte behavior is needed before it can be concluded to function at the egress step . In addition to endothelial cells, the vascular barrier involves a layer of pericytes and two layers of basement membrane, the latter forming the so - called perivascular space (pvs). The significance of the pvs in egress remains unclear, though a reconstitution study has correlated the appearance of sp thymocytes in the pvs with the time when cells begin to egress from the thymus . Two recent studies made the striking observation that the pericytes surrounding thymic blood vessels are neural crest cell - derived . The significance of this specialized origin for thymic pericytes is unknown but may relate to the barrier properties of thymic vessels or to their unique requirement to support thymocyte egress . The active phosphoryl metabolite of fty720 acts as an agonist for four of five s1p receptors, and of these, s1p1 is strongly upregulated in maturing sp thymocytes . Remarkably, genetic deletion of s1p1 in thymocytes phenocopied the pertussis transgenic mouse, indicating that s1p1 is the primary gi - coupled receptor mediating egress . Deficiency in the two kinases that generate s1p led to a similar thymic egress defect . A second small molecule, 2-acetyl-4-tetrahydroxybutyl - imidazole (thi), known to reduce thymic egress when fed to mice, was found to inhibit s1p lysase, causing 1,000-fold increases in thymic s1p and down - modulation of thymocyte s1p1 . Similar findings were made when s1p lyase was knocked down using a short hairpin rna (shrna) and through analysis of mice lacking the s1p lyase gene . The latter mice showed severe growth abnormalities and poor viability, perhaps reflecting roles of the lyase in sphingolipid metabolism, and thymic function was strongly affected . Introduction of a low - expressing s1p lyase transgene rescued non - lymphoid pathologies while failing to fully restore thymic egress . These studies highlight the importance of appropriately compartmentalized s1p distribution to support normal thymic egress (figure 1). A study aimed at characterizing the altered t - cell compartment in mice lacking krppel - like factor-2 (klf2), a zinc - finger transcription factor, revealed a block in thymic egress associated with reduced s1p1 mrna in mature thymocytes . However, sufficient s1p1 may remain in klf2-deficient cells to permit low amounts of thymic egress and further work will be needed to fully define the factors controlling s1p1 expression . Insight into factors that act upstream of klf2 has come from work on the foxo family of transcription factors . Deficiency in foxo1 led to a slight accumulation of mature sp thymocytes, particularly of the cd8 lineage, though the block was less severe than for klf2 deficiency . Other foxos appear likely to help drive klf2 expression . The inhibition of foxo transcription factors by phosphoinositide 3-kinase (pi3k) signaling might explain the thymic egress block seen in transgenic mice overexpressing pi3k in thymocytes . The factors determining when expressions of foxo, klf2, and thus s1p1 are turned on remain undefined, but recent work suggests the involvement of a timer mechanism . In transgenic mice expressing green fluorescent protein (gfp) from the rag2 locus, the amounts of gfp in the cell can be used as a molecular timer to determine how much a thymocyte has aged since its rag2-expressing dp stage of development . By means of this approach, a medullary dwell time for sp thymocytes of 4 to 5 days conveyor belt fashion with cells that enter the medulla first exiting the thymus first . To exit the thymus several recent observations suggest that molecules required for reorganization of the thymocyte actin cytoskeleton are central to this process . One study followed up on the description of a spontaneous mutant mouse line with a thymic egress defect and peripheral t - cell deficiency to identify a role for the arp2/3 regulatory protein coronin-1a in thymic egress . Notably, although dp cell migration was also affected by the mutation when tested in vitro, the in vivo thymic defect revealed itself only as an accumulation of mature sp cells, suggesting an especially stringent need for appropriate control of actin branching during the transmigration step . Since formins support polymerization of unbranched actin, this work further highlights the necessity of fine actin cytoskeleton control to coordinate the cell shape changes required for egress . In addition to molecules involved in cytoskeletal reorganization, cell adhesion molecules may play a role in thymocyte egress . Recent data suggest that p - selectin glycoprotein ligand-1 (psgl-1) may be involved . P - selectin marks a subset of thymic endothelial cells, and thymocytes express psgl-1 . However, the accumulation of thymocytes at the immature sp stage in psgl-1-deficient mice is unusual for a thymic egress defect (figure 1). Further study of how psgl-1 influences sp thymocyte behavior is needed before it can be concluded to function at the egress step . In addition to endothelial cells, the vascular barrier involves a layer of pericytes and two layers of basement membrane, the latter forming the so - called perivascular space (pvs). The significance of the pvs in egress remains unclear, though a reconstitution study has correlated the appearance of sp thymocytes in the pvs with the time when cells begin to egress from the thymus . Two recent studies made the striking observation that the pericytes surrounding thymic blood vessels are neural crest cell - derived . The significance of this specialized origin for thymic pericytes is unknown but may relate to the barrier properties of thymic vessels or to their unique requirement to support thymocyte egress . Our current understanding of t - cell egress has profited enormously from the discovery of the egress inhibitory drug, fty720, and the potential of this molecule for treatment of human autoimmune disorders is under ongoing investigation . The immunosuppressive potential of s1p lyase inhibition is also under exploration with a small molecule inhibitor, lx2931, entering clinical trial . An important question in this area will be to define the impact of prolonged inhibition of thymic egress on thymic function . The importance of normal thymic egress in humans has also been highlighted by the identification of a tb natural killer (nk) severe combined immune deficiency (scid) patient who lacked functional alleles of the coronin-1a gene . Unusual for tbnkscid but consistent with a thymic egress defect, the patient possessed normal thymic mass . Given that the coronin-1a gene resides in a region of the human genome subject to copy number variation, it seems likely that further cases of scid resulting from deficiency in this gene will be identified . Work over the last several years has led to an increasingly detailed model for the essential final step in thymocyte maturation, acquisition of egress competence, but many questions remain . Chief among these are identifying the precise location and cellular dynamics of thymocyte egress, understanding how klf2 induction is triggered over time, and establishing whether alterations in thymic egress occur as part of the change in thymic function that takes place in certain infectious and autoimmune diseases and during aging . Given the translational advances that have already been made, we can be sure that further research on thymic egress will continue to impact human medicine.
Schistosomes are parasitic helminths, most important in terms of socio - economic and public health in tropical and subtropical areas . Chronic disease may also lead to cancer (oh and weiderpass, 2014). In addition, there are often systemic symptoms, such as retarded growth, slowing of cognitive development and the effect of continuing low - level blood loss (bergquist et al ., 2004). Current treatment is based on the anti - helminthic drug praziquantel (pzq) (trainor - moss and mutapi, 2015). Unfortunately, following its massive use, there are reports of induction of resistance and reduced susceptibility to praziquantel in field isolates (melman et al ., 2009, artemisinin derivatives (araujo et al ., 1991, del villar et al ., 2012,, 2016; wikipedia, 2016) and artemisinin analogs - synthetic peroxides e.g. Ozonides and trioxolanes (keiser et al ., 2012; xiao et al ., 2012) have been proposed as alternative or complementary drugs against schistosomes . Artemisinins are widely used against malaria wherein their mechanism of action is generally accepted to involve activation by heme or non - heme iron to generate cytotoxic carbon radicals that alkylate vital intraparastic molecules (klonis et al ., 2013). However, based on a consideration of the extensive literature on c - radicals and their evident inability to act as alkylating agents, coupled with the low propensity of artemisinins to react with iron in the first place, the thesis is not without contradictions (haynes et al ., 2013). Alternatively, artemisinins may accept electrons from reduced flavin cofactors within flavin disulfide reductases such as glutathione reductase (gr), thioredoxin reductase (trxr) and others that maintain redox homeostasis in the malaria parasite (haynes et al . As in the case of plasmodia, action of artemisinins may be due either to heme initiated formation of free radicals (arajo et al ., 2008, muangphrom et al ., 2016) or to abrogation of redox homeostasis by the artemisinins interacting with reduced flavin cofactors of flavin disulfide reductases . Notably within s. mansoni, the multifunctional disulfide reductase thioredoxin glutathione reductase (tgr) functionally replaces trxr and gr of plasmodia and therefore tgr is a potentially important drug target (alger and williams, 2002, kuntz et al ., 2007) and is likely to be so for artemisinins . Be that as it may, whilst praziquantel affects adult schistosomes, artemisinins affect both larval and mature stages of the parasites (shuhua et al ., 2000, fenwick et al ., 2006). However, because of the complexity of the parasite, most artemisinins would have to be administered by repetitive injections, although artesunate and artemether that are water soluble and lipid soluble, respectively, could possibly be used orally (barradell and fitton, 1995, bunnag et al ., 1996). In this work, we have conducted a preliminary assessment of the in vivo activity of the clinically used artemisinin derivative artemether compared with the newer aminoartemisinins artemiside and artemisone, both of which are shown to be potently active against the apicomplexan parasite plasmodium falciparum (pf) in vitro and in vivo . Artemisone has improved pharmacokinetics and anti - plasmodial activity, and reduced toxicity compared to the artemisinin derivatives clinically used against malaria (haynes et al . We also include the newer n - sulfonyl aza - artemisinin derivative ckw03, that previously has also been shown to be potently active against pf and like other n - sulfonyl azaartemisinin derivatives, has notable thermal stability (haynes et al ., 2007). Finally, we have examined the effect of controlled artemisone release from biodegradable gels on s. mansoni in mice, with the aim of improving treatment by exposing the parasites for a longer period of time to a drug concentration sufficient to eliminate the pathogen, but at dose levels non - toxic to the host . Artemisone was prepared as previously described (haynes et al ., 2006) or artemisone in polyricinoleic acid (ra)-sebacic acid (sa) gel was produced as previously described: poly (ester anhydride) was prepared by the insertion of ra into poly sa (shikanov et al ., 2004) with modifications . Briefly, 70 g of ra and 30 g of poly sa (psa) were mixed by raising the temperature to 160 c for 24 h under nitrogen atmosphere to yield ra - sa carboxylic acid terminated oligoesters . These oligoesters were activated by acetic anhydride and polymerized for 4 h at 140 c under vacuum (1015 mbar) to yield poly(ra - sa)70:30 pasty injectable polymer . Different amounts of artemisone powder were mixed in the pasty polymer and used for injection . Block polymer pcl - mpeg was synthesized according to a previously published procedure (bubel et al ., 2013). Blockcopolymers of pcl - mpeg were fabricated by different ratios of pcl: mpeg . To create a homogeneous mixtures of pcl - b - mpeg and artemisone, different ratios of both compounds after all particles were dissolved, the solvent was completely evaporated . Using a heat press at 65 c, the mixture was pressed into a polytetrafluoroethylene matrix (8 4x2 mm) and then cooled down to room temperature under a second press at about 20 c . The polymers were sterilized by brief (5 s) washing in 70% ethanol and exposure to uv for 45 min . Artemisone suspension for gavage was prepared by suspending artemisone (32 mg) in 7% tween 80 (1 ml) and 3% alcohol . A liberian strain was used in the swiss tropical institute (table 1). The life cycle of s. mansoni was maintained in nmri and icr mice and biomphalaria glabrata snails . Seven to 8 weeks post - infection, shistosome eggs were extracted from the granuloma - containing livers . Pba strain (mra-311, cdc, atlanta, ga, usa) was maintained in vivo by serial transfer of parasitized erythrocytes from infected to naive mice . Parasites that stably and constitutively express luciferase were cultivated at 5% hematocrit in rpmi 1640 medium, 0.5% albumax ii (invitrogen, carlsbad, california, united states), 0.25% sodium bicarbonate, and 0.1 mg / ml gentamicin . Parasites were incubated at 37 c in an atmosphere of 5% oxygen, 5% carbon dioxide, and 90% nitrogen . Parasites were cultured in media containing 4 nm wr99210 to select for stable luciferase expression . Parasite viability assays were performed either by measuring their luciferase activity (see bioassay below). Mice were maintained in environmentally controlled conditions (temperature, 23 c; 50% humidity, free access to a standard diet and water, a 12/12-h automatically timed light / dark cycle . Female nmri mice were purchased from charles river (sulzfeld, germany) and harlan laboratories (blackthorn, united kingdom). These mice were used for the schistosome infections in tel - aviv university (tel aviv university ethical committee number 01 - 13 - 076). The mice were infected a few days later and treated as indicated at various intervals following infection . Male c57bl/6 mice were purchased from harlan laboratories (jerusalem, israel) and used for plasmodial infections in the hebrew university . The animal study protocol was approved by the hebrew university institutional animal care and use committee (protocol no . Treatment of mice was conducted by intraperitoneal or subcutaneous injections, and gavage or subcutaneous insertions of a solid polymer or a gel containing artemisone . Gavage was conducted by oral administration of 0.4 ml containing the test drugs at 400450 mg / kg in tween 7% and 3% ethanol in ddw . Nmri female mice, 5 week old, 22 g at infection with 80 cercariae, were treated at day 21 post infection (pi) by gavage of 400 mg / kg drug suspension (table 1). Icr male mice, 78 weeks old, 33 g at infection, were treated by gavage of 450 mg / kg at days 18 and 25, and at day 21, 29 and 35 pi . Mice treated by gavage once on day 42, or twice on day 42 and 49 post infection were dissected on day 61 post infection and schistosomes counted (fig . 2).similar icr mice were infected and treated 20 days pi by artemisone (2mg / mouse, 60 mg / kg) in solid polymers that were inserted subcutaneously (sc) into the abdomen of mice anesthetized by ketamine / xylazine injection . Artemisone was injected sc and ip at days 18 and 25 pi by in dmso to similar mice, 4mg/40microl / mouse (120 mg / kg). Mice were dissected at day 49 post infection and schistosomes counted (data not shown). Artemisone in gel (0.1 or 0.2 ml) was injected sc in the abdomen to infected icr male mice, when 78 weeks old, 33 g, at various days post infection with schistosomes . Male c57bl/6 mice (78 weeks old, 25 g) were used for plasmodial infections . The animals were infected by ip injection of 50,000 erythrocytes parasitized by pba and were treated by sc injections of artemisone in gel . Plasmodial parasitemias in mice were estimated by microscopic determination of the percent of parasitized erythrocytes in 5000 erythrocytes in giemsa stained blood smears obtained from the mouse tail vein . Schistosoma numbers were determined by counting worms from dissected and squashed livers, and mesenteric veins of dissected intestines of the mice, mostly 4951 days pi unless otherwise stated . Granuloma density in the liver was assessed by press - flattening the livers between two glass plates and observation under a dissecting microscope . P. falciparum bioassay for in vitro release of artemisone from gels was conducted as follows: artemisone released from the gel samples was quantified in a bioassay based on two - day cultures of drug sensitive p. falciparum that stably expresses a luciferase gene (see above). Gel samples containing two mg artemisone were sterilized by uv exposure and transferred to 1 ml rpmi 1640 medium in 24 well, nunc disposable sterile plates that were incubated at 37 c . Once a day the polymers were washed twice in 2 ml medium, 1 ml fresh medium was added and the plates were returned to the incubator . The collected supernatants in different dilutions were examined for p. falciparum growth inhibition in triplicates, in nunc flat bottom 96 well plates (nunc microwell 96-well optical - bottom plates with polymer base). Luciferase activity was measured in parasitized erythrocytes after removing 100 l of the medium, following addition of 100 l bright - gloh luciferase reagent (promega) in a fluoroskan fl luminometer (thermo). Percent inhibition of the luciferase counts reflecting anti - plasmodial activity by supernatants from experimental gels compared to control (blank) gels, is presented . The mice were dissected at day 49 pi and schistosomes counted; n = 5 in experimental groups and 10 in the control group (table 1). The effect was significant (p <0.01) in all experimental groups . 1 depicts the results of treatment 18 and 25 (a), or 21, 29 and 35 (b) days pi . Differences were significant between the treated and the control groups (p <0.05) but more pronounced in mice that were treated thrice, compared to the ones that were treated twice (97.1% vs. 70.4% reduction, respectively). 2 depicts the results of late treatment, 42, or 42 and 49 days pi . Artemisone administered by gavage at a late stage significantly reduced the number of adult worms only after an additional treatment (68.5% inhibition, p <0.05). However, the method of treatment by gavage is not optimal because of the large amounts of drug needed for treatment . Mice were treated by artemisone (2mg / mouse, 60 mg / kg) in a solid polymer that was inserted s.c . One treatment of this relatively low concentration of artemisone significantly reduced schistosome load (79.4%, p <0.01). This surgical procedure would not be recommended for repeated treatments; therefore we shifted to drug injections, which correlate better with improved compliance . Infected mice were treated by artemisone in gel (0.2 ml) that was administered s.c . This procedure was applied in day 62 in the late treatment groups for enabling expression of drug effect (table 2). The effect on schistosome recovery varied with the timing and number of drug injections . One treatment at an early stage, 21 days pi, did not significantly reduce schistosome number but profoundly reduced induction of granulomas in the liver of the treated mice, in contrast with their abundance in the control mice . A relatively late single treatment on day 34 pi reduced also the schistosome number by 82% . Two treatments, at an early stage (on days 7 and 14 or 21 and 28) pi also reduced schistosome number by about 73 and 82%, respectively . The effect was further increased to 96% reduction due to an additional treatment at day 35 . There was no significant effect when using increased amounts of artemisone in quantities above 4 mg in identical gels, probably due to artemisone crystallization in the gel (data not shown). Identical doses of artemisone and praziquantel were tested in days 7 and 14, and days 42 and 49 pi . While the praziquantel did not affect parasite development at the early stage pi in comparison with the significant activity of the artemisone, it was highly active, similarly to artemisone after administration at the late stage . Infected mice were treated at day 18 and 25 days pi by i.p ., and s.c . Treatment (18.6 3.4 schistosomes in the control group vs. 14.8 4.2 in the treated group). Gels containing artemisone were incubated in medium and their dilutions were examined in p. falciparum cultures . Supernatants from media incubated with blank gels had no effect on p. falciparum development . In contrast, considerable amounts of artemisone were released in vitro, spanning at least 7 days (table 3). For example, a dilution of 15,625 of supernatant collected on day 3 killed most of the parasites (meaning that the amount of released artemisone was above 16 g on that day according to the assay of free artemisone (concentrations of 0.110 ng / ml were examined and the ed50 was about one ng / ml). The ed50 of free artemisone was estimated by identical methods and was about 1 ng / ml . Mice were treated 3 days before infection with pba by injections of artemisone in gel, 2mg/0.2ml / mouse (80 n = 3 in the experimental group and 5 in the control group . All control mice treated with empty gel died within eight days pi of cerebral malaria while the experimental mice treated by artemisone in the gel died 1618 days pi, of anemic malaria (fig . Current treatment of schistosomiasis is based on the anti - helminthic drug praziquantel (pzq). However, following its massive use, there are already reports of pzq reduced sensitivity (references are cited in the introduction). Artemisinin and its current clinical derivatives collectively referred to as artemisinins, have been proposed as alternative or complementary drugs . Current reports reflect a significant but not total reduction of worm load after repeated (at least four doses) of intra - gastric treatment with high amounts of an artemisinin (saeed et al ., 2016). Pzq is effective mainly against mature schistosomes (as could be also seen in our experiments) while artemisinins are also effective against early developmental stages (when compared to pzq at the second to the fifth week pi (utzinger et al ., 2007). Therefore, improved results are expected to derive from a combination therapy using pzq and an artemisinin . Experiments in mice reveal an additive effect of artemisinin and pzq in s. mansoni infection (botros et al ., 2010) and in hamsters infected with s. japonicum (utzinger et al ., 2001). However, treatment of infected human patients with the combinations in endemic areas did not improve the performance of pzq probably because certain worm stages were not exposed simultaneously to the two drugs (pratic et al . Artemisone proved superior to other artemisinin derivatives, especially in terms of its greatly enhanced efficacy against p. falciparum, its metabolic profile that precludes formation of dihydroartemisinin, its relative stability and reduced toxicity (schmuck et al ., 2003, haynes et al ., 2006, haynes et al ., 2008, obaldia et al ., 2009, waknine - grinberg et al ., 2010). Given thes advantages)and considering the similarity of mechanism of action in plasmodia and schistosomes (discussed in the introduction), coupled with our current preliminary results (table 1), artemisone was selected for further experiments in mice infected with s. mansoni . A similar effect on female and male reduction that was detected in this experiment hints to an identical mechanism of action in both genders . However, it is possible that an injury induced in the female may affect the male and vice versa, due to the obligatory relationship between the genders (lu et al ., 2016). Such a sequential effect would be eventually reflected in an equal mortality rate of both genders . In general, sustained release may limit the amount of the drug in question to a non - toxic level whilst maintaining its efficacy during an extended period . This is highly relevant for artemisinins that at certain levels are neurotoxic (wesche et al ., 1994, schmuck and haynes, 2000, schmuck et al ., 2002, campos et al ., 2008). Sustained release also allows for extended treatment of the changing stages of schistosomes . As a control for the sustained mode of the treatment we used subcutaneous (sc), intraperitoneal (ip) injections and gavage . Two methods of sustained release of the drug were applied, namely release from a solid polymer that necessitates insertion using a relatively straightforward surgical procedure, and injectable gel . Artemisone administered by gavage at a relatively early stage, namely on day 21 or on days 18 and 25 pi, significantly reduced the number of adult worms and was most pronounced in mice that were treated thrice (97.1%). Late gavage at day 42 pi had no effect, but an additional treatment one week later induced 68.5% adult worm elimination . However, the method of treatment by gavage is not optimal because of the large amounts of drug needed for treatment probably due to low degree of absorption . Consequently, we used artemisone incorporated into polymers . One treatment 20 days post - infection (pi) using a relatively low concentration of artemisone (60 mg / kg) released from a solid polymer significantly reduced the schistosoma load (79.4%). This result confirms the curative potential of artemisone at a relatively early stage of infection . However, insertion of a solid polymer that is associated with a surgical procedure might not be recommended for repeated treatments . Therefore, we evaluated the effect of injections of artemisone within a soft biodegradable gel that would be more convenient for repetitive treatments . The applicability and safety of this polymer has been described by shikanov and domb (2006). The effect on the number of recovered schistosomes was dependent on the number of treatments and the timing of the injection pi . One injection, 34 days pi, was more effective than 21 days pi (82.1% vs. 39.1% inhibition respectively). However, even the single treatment at 21 days pi prevented induction of granulomas in the liver of the treated mice, in contrast with their abundant formation in the control mice . This is a critical finding because granulomas are the main cause and indication of schistosoma pathology (hams et al ., 2013). Control repeated sc injections of 4mg/40 l dmso had no effect on the maturation of the schistosomes . The half - life of artemisone is about 3 h (haynes et al ., 2006, nagelschmitz et al ., 2008) so the amount retained in the plasma of injected mice would not be detectable three days after injection of this dose of artemisone . In contrast, we show that artemisone is released from the gels in vitro during at least seven days, and retain in vivo efficacy for at least three days by using an in vivo bioassay of mice infected with drug sensitive plasmodium berghei anka . The effect of the released drug also was examined in mice in a prophylactic manner, in which the artemisone containing gel was injected three days before plasmodial infection . While all mice that were injected with empty gels died of cerebral malaria within seven days pi, the mice treated with the prophylactic gel survived during a much longer period (6.2 vs. 17.3 days, respectively). Overall, we demonstrate that a sustained release procedure for artemisone induced both elimination of schistosomes and reduction of hepatic pathology greatly in excess of that achieved by per os delivery . The results justify further consideration of treatment of schistosomiasis with artemisinins, as well as using these in with combination with a partner drug in sustained release systems . The main advantage of artemisinins over praziquantel is their efficacy against early developmental stages of the parasite, which in many cases are the cause of acute schistosomiasis syndromes.
Ot-2 t cells (510/well) were incubated for 30 min at 37c in 96-well u - bottom plates with dcs (10/well) or lps - activated b cells (210 per well) that had been pulsed with antigen . Conjugates were enumerated by flow cytometry after the cell mixture was stained at 4c for cd4, cd11c, and cd19 and repeatedly washed . To visualize t - dc interactions (fig . 1), ova323-pulsed dcs were injected subcutaneously at 210 per mouse 24 hours prior to intravenous transfer of sap and sap t cells (310 each). Imaging was conducted 12 to 24 hours later . To examine activation phenotypes, 10 dcs and 210 gfp - expressing t cells per mouse 2), 310 ot-2 t cells of each genotype were co - transferred into mice together with 510 wild - type b cells . Immunization was done 12 hours prior to cell transfer, and imaging was conducted 24 to 36 hours thereafter . To visualize t - b interactions under non - competitive conditions and to assay t and b cell expansion (fig . 3), 610 gfp - expressing ot-2 t cells were co - transferred together with 310 cfp - expressing b cells 24 hours prior to immunization . Imaging and cytometric analyses were conducted 6072 and 96 hours later, respectively . To visualize gc recruitment and retention of t cells (fig . 4), 310 cfp - expressing sap and 310 gfp - expressing sap ot-2 t cells were co - transferred with 310 non - fluorescent md4 b cells . Dye - labelled nave b cells (2410) were given 1 day before imaging to provide follicle / gc landmarks . Longer than 2 hours, the animal s hydration was maintained by lactated ringer s solution given via a catheter . The typical x - y - z dimension was 0.51.10.51.13 m, and the time resolution was 3045s . For experiments involving co - transfer of two types of dye - labelled t cells, the cells were always reciprocally labelled to control for potential dye - induced behavioural differences . The mann - whitney rank sum test was used to calculate p values for highly skewed distributions . For gaussian - like distributions, ot-2 t cells (510/well) were incubated for 30 min at 37c in 96-well u - bottom plates with dcs (10/well) or lps - activated b cells (210 per well) that had been pulsed with antigen . Conjugates were enumerated by flow cytometry after the cell mixture was stained at 4c for cd4, cd11c, and cd19 and repeatedly washed . 1), ova323-pulsed dcs were injected subcutaneously at 210 per mouse 24 hours prior to intravenous transfer of sap and sap t cells (310 each). Imaging was conducted 12 to 24 hours later . To examine activation phenotypes, 10 dcs and 210 gfp - expressing t cells per mouse 2), 310 ot-2 t cells of each genotype were co - transferred into mice together with 510 wild - type b cells . Immunization was done 12 hours prior to cell transfer, and imaging was conducted 24 to 36 hours thereafter . To visualize t - b interactions under non - competitive conditions and to assay t and b cell expansion (fig . 3), 610 gfp - expressing ot-2 t cells were co - transferred together with 310 cfp - expressing b cells 24 hours prior to immunization . Imaging and cytometric analyses were conducted 6072 and 96 hours later, respectively . To visualize gc recruitment and retention of t cells (fig . 4), 310 cfp - expressing sap and 310 gfp - expressing sap ot-2 t cells were co - transferred with 310 non - fluorescent md4 b cells . Dye - labelled nave b cells (2410) were given 1 day before imaging to provide follicle / gc landmarks . Imaging sessions longer than 2 hours, the animal s hydration was maintained by lactated ringer s solution given via a catheter . The typical x - y - z dimension was 0.51.10.51.13 m, and the time resolution was 3045s . For experiments involving co - transfer of two types of dye - labelled t cells, the cells were always reciprocally labelled to control for potential dye - induced behavioural differences . The mann - whitney rank sum test was used to calculate p values for highly skewed distributions . For gaussian - like distributions,
Periodontitis is characterized by the presence of inflammation and progressive destruction of supporting structures of the teeth, including the periodontal ligament, alveolar bone and gingival tissues . The use of rat models has been validated in the evaluation of pathogenesis of periodontal diseases and regarding the influence of risk factors, such as nicotine, alcohol and diabetes on disease progression . The induction of periodontal disease in rats can be achieved in many ways . In numerous studies, the use of cotton ligatures is reported, involving the cervical portion of the maxillary second molar or the mandibular first molar . Previous studies have validated the use of morphometric analysis to evaluate alveolar bone loss in ligature - induced periodontitis in rats . According to klausen (1991), this is the most appropriate methodology to measure periodontal bone loss in dissected rat maxillas . Alveolar bone loss located on molar buccal and lingual faces can be quantified by measuring the linear distance of the cementoenamel junction (cej) from the alveolar bone crest (abc) in molar roots . In addition, measurements of the area of bone loss using the alveolar bone tissue, the cej and the proximal region of roots as references has also been validated, as has measuring the area of the exposed root . The purpose of this study was to evaluate morphometrically the alveolar loss bone caused by experimental induction of periodontitis in rats, comparing different locations (lingual mandible, palatal maxilla and buccal maxilla) and methods (area and distance). Fourteen 4-month - old adult female wistar rats weighing 300 g on average were used in the study . All rats were housed under similar conditions and received solid diet (guabi nutrilabor, mogiana alimentos, campinas, sp, brazil) and water ad libitum . The institutional animal research committee of so jos dos campos dental school, so paulo state university, so jos dos campos, sp, brazil, approved the research protocol . General anesthesia was induced by intramuscular administration with a solution of 13 mg / kg of 2% xylazine hydrochloride (rompum, bayer, so paulo, sp, brazil) and 33 mg / kg of ketamine hydrochloride (francotar, virbac, roseira, sp, brazil). To induce periodontitis, cotton ligatures (coats - corrente, so paulo, sp, brazil) were placed around the cervix of the maxillary second molar and mandibular first molar, both on the right side, leaving the left side molars unligated to serve as controls . After 4 weeks, the rats were sacrificed by decapitation, the mandibles and maxillas were removed and separated into right and left sides, and the specimens were fixed in 10% formalin solution . The procedure included immersion in sodium hypochlorite for 4 h and mechanical scavenging of the remaining tissue . Next, the specimens were stained with methylene blue dye (1 g/100 ml) for 1 min to demarcate the cej . The specimens were examined under stereomicroscopy . To obtain sufficient reproducibility of the alignment of the image, the buccal cusp tip of the first and second molars should be superimposed on the corresponding lingual / palatal cusp tip . The image was digitalized at 25x magnification for the buccal and palatal sides of the maxilla and for the lingual side of the mandible . Measurements were made on the maxillary second molar and mandibular first molar three times using image tool v.3.0 image - analysis software (uthscsa, san antonio, tx, usa), and the mean values were used in statistical analysis . All measurements were made in a blinded fashion to the group to which the rat belonged . The area method was defined by delimitation of the area of bone loss, using the alveolar bone crest (abc), the cej and the proximal region of roots as reference . The distance method applied by crawford, taubman and smith (1978) on digitalized images was used to perform linear measurements from the cej to the abc, on half of each root following the axis . Three measurements were obtained for the mandibular first molar (lingual) and four for the maxillary second molar, two each for the buccal and palatal sides . Both methods can be observed in figure 1 for the buccal maxilla, in figure 2 for the palatal maxilla, and in figure 3 for the lingual mandible . The percentage of periodontal bone loss (pbl) was measured by two methods (area and distance) in teeth with (lig) and without ligature (unlig) for the palatal and buccal maxilla, and the lingual mandible . Pbl was determined using the following equation: pbl= (lig unlig) x100/lig . Photograph of alveolar bone loss evaluated morphometrically by measuring the linear distance between the cementoenamel junction and alveolar bone crest at two buccal sites on the maxillary second molar (a). The area method was defined by delimitation of the area of bone loss using the alveolar bone crest, the cementoenamel junction and the proximal region of roots of the maxillary second molar on the buccal side as references (b). Original magnification: x 25 photograph of alveolar bone loss evaluated morphometrically by measuring the linear distance between the cementoenamel junction and alveolar bone crest at two palatal sites on the maxillary second molar (a). The area method was defined by delimitation of the area of bone loss using the alveolar bone crest, the cementoenamel junction and the proximal region of roots of the maxillary second molar on the palatal side as references (b). Original magnification: x 25 photograph of alveolar bone loss evaluated morphometrically by measuring the linear distance between the cementoenamel junction and alveolar bone crest at three lingual sites on the mandibular first molar (a). The area method was defined by delimitation of the area of bone loss using the alveolar bone crest, the cementoenamel junction and the proximal region of roots of the mandibular first molar on the lingual side as references (b). Original magnification: x 25 prior to the statistical analyses, the intraexaminer reproducibility was checked by evaluating two sets of measurements of all specimens with a 1-week interval . Paired t test statistical analysis showed no differences between the mean values obtained for the area method (p=0.4179) and the distance method (p=0.3273). Additionally, pearson's correlation coefficient was obtained between the two sets of measurements and revealed a very high correlation in both the area (99%: r=0.99986, p=0.000) and distance methods (99%: r=0.9986, p=0.000). Data were expressed as mean values and standard deviation of percentage (%) of periodontal bone loss (mm or mm). The t test for independent variables (=0.05) was used for comparisons between the area and distance methods . One - way anova (=0.05) and the tukey's test for subsequent multiple comparisons (=0.05) were used to determine significant differences in periodontal bone loss between the locations analyzed . Fourteen 4-month - old adult female wistar rats weighing 300 g on average were used in the study . All rats were housed under similar conditions and received solid diet (guabi nutrilabor, mogiana alimentos, campinas, sp, brazil) and water ad libitum . The institutional animal research committee of so jos dos campos dental school, so paulo state university, so jos dos campos, sp, brazil, approved the research protocol . General anesthesia was induced by intramuscular administration with a solution of 13 mg / kg of 2% xylazine hydrochloride (rompum, bayer, so paulo, sp, brazil) and 33 mg / kg of ketamine hydrochloride (francotar, virbac, roseira, sp, brazil). To induce periodontitis, cotton ligatures (coats - corrente, so paulo, sp, brazil) were placed around the cervix of the maxillary second molar and mandibular first molar, both on the right side, leaving the left side molars unligated to serve as controls . After 4 weeks, the rats were sacrificed by decapitation, the mandibles and maxillas were removed and separated into right and left sides, and the specimens were fixed in 10% formalin solution . The procedure included immersion in sodium hypochlorite for 4 h and mechanical scavenging of the remaining tissue . Next, the specimens were stained with methylene blue dye (1 g/100 ml) for 1 min to demarcate the cej . The specimens were examined under stereomicroscopy . To obtain sufficient reproducibility of the alignment of the image, the buccal cusp tip of the first and second molars should be superimposed on the corresponding lingual / palatal cusp tip . The image was digitalized at 25x magnification for the buccal and palatal sides of the maxilla and for the lingual side of the mandible . Measurements were made on the maxillary second molar and mandibular first molar three times using image tool v.3.0 image - analysis software (uthscsa, san antonio, tx, usa), and the mean values were used in statistical analysis . All measurements were made in a blinded fashion to the group to which the rat belonged . The area method was defined by delimitation of the area of bone loss, using the alveolar bone crest (abc), the cej and the proximal region of roots as reference . The distance method applied by crawford, taubman and smith (1978) on digitalized images was used to perform linear measurements from the cej to the abc, on half of each root following the axis . Three measurements were obtained for the mandibular first molar (lingual) and four for the maxillary second molar, two each for the buccal and palatal sides . Both methods can be observed in figure 1 for the buccal maxilla, in figure 2 for the palatal maxilla, and in figure 3 for the lingual mandible . The percentage of periodontal bone loss (pbl) was measured by two methods (area and distance) in teeth with (lig) and without ligature (unlig) for the palatal and buccal maxilla, and the lingual mandible . Pbl was determined using the following equation: pbl= (lig unlig) x100/lig . Photograph of alveolar bone loss evaluated morphometrically by measuring the linear distance between the cementoenamel junction and alveolar bone crest at two buccal sites on the maxillary second molar (a). The area method was defined by delimitation of the area of bone loss using the alveolar bone crest, the cementoenamel junction and the proximal region of roots of the maxillary second molar on the buccal side as references (b). Original magnification: x 25 photograph of alveolar bone loss evaluated morphometrically by measuring the linear distance between the cementoenamel junction and alveolar bone crest at two palatal sites on the maxillary second molar (a). The area method was defined by delimitation of the area of bone loss using the alveolar bone crest, the cementoenamel junction and the proximal region of roots of the maxillary second molar on the palatal side as references (b). Original magnification: x 25 photograph of alveolar bone loss evaluated morphometrically by measuring the linear distance between the cementoenamel junction and alveolar bone crest at three lingual sites on the mandibular first molar (a). The area method was defined by delimitation of the area of bone loss using the alveolar bone crest, the cementoenamel junction and the proximal region of roots of the mandibular first molar on the lingual side as references (b). Prior to the statistical analyses, the intraexaminer reproducibility was checked by evaluating two sets of measurements of all specimens with a 1-week interval . Paired t test statistical analysis showed no differences between the mean values obtained for the area method (p=0.4179) and the distance method (p=0.3273). Additionally, pearson's correlation coefficient was obtained between the two sets of measurements and revealed a very high correlation in both the area (99%: r=0.99986, p=0.000) and distance methods (99%: r=0.9986, p=0.000). Data were expressed as mean values and standard deviation of percentage (%) of periodontal bone loss (mm or mm). The t test for independent variables (=0.05) was used for comparisons between the area and distance methods . One - way anova (=0.05) and the tukey's test for subsequent multiple comparisons (=0.05) were used to determine significant differences in periodontal bone loss between the locations analyzed . Morphometric analysis showed no significant differences among pbl values (p>0.05) for the area and distance methods for three locations (buccal maxilla: p=0.6981; palatal maxilla: p=0.0816 and lingual mandible: p=0.3789). Evaluating the percentage of pbl derived from the cotton ligature insertion in different locations, there was showed significant differences among pbl values (p<0.05). Analysis revealed a greater percentage of pbl in the maxilla, bone loss was more accentuated on the buccal side than the palatal side (p<0.05). On the lingual side of the mandible, there was a lower percentage in pbl values (table 1). Mean and standard deviation of alveolar bone loss (%) at the evaluation location independent sample t test (p<0,05). Lower case letters should be considered in the area method column and capital letters should be considered in the distance method column (anova and tukey, p <0.05). The most commonly used teeth for periodontitis induction in rats are the maxillary second molar and the mandibular first molar, both of which were evaluated in the present study . (2004) verified that the presence of a cotton ligature caused bone loss, which is detectable by morphometric analysis on day 15 after its placement . Subsequent observation verified the stagnation of bone loss progression up to day 60 . Based on that study, it can be concluded that the ligature must remain in place for a minimum of 15 days and the experimental period can range from 15 to 60 days . Regarding the period of ligature placement, several studies in rats used 30 days for periodontitis induction in the maxilla and mandible . This period was very similar to that used in the present work of four weeks . Alveolar bone loss in rats can be caused by physiological remodeling or periodontitis induction . The present study only identified the effects of ligature on the alveolar bone because the bone loss values obtained in unligated teeth (physiological bone remodeling) were subtracted from the bone loss values obtained in the presence of ligature (periodontitis induction effects). The aim of this methodology was to define the amount of bone loss caused by periodontitis and to determine which location was more sensitive to the evaluation methods . The results revealed greater bone loss in the maxilla on the buccal side than on the palatal side, and in the lingual side of the mandible . The present study showed that both the area and the distance morphometric methods could be used, as there was no significant difference between each other regarding bone loss caused by periodontitis . (2004) compared both methods on the buccal maxilla, their results suggested that the distance method was preferable to the area method . Moreover, the linear method has long been studied and has recently been more commonly used for morphometric analyses of alveolar bones in dissected skulls . The present study showed that both area and distance measurements can be used to evaluate bone loss caused by ligature - induced periodontitis in rats, and suggests applying the morphometric methodology to the maxilla on the buccal side.
The protocols for flim sample preparation do not differ from those for confocal or wide - field intensity - based fluorescence microscopy . The data acquisition is followed by the main task of data analysis, i.e. Extracting the fluorescence lifetimes from the raw data . Prepare a stock solution (10 ml) by dissolving approximately 1 mg / ml of the dye in an appropriate solvent (e.g. Methanol for bodipy - c12) using an accurate balance and a pipette . The cells (a model cancer cell line, hela in our case) to be stained are grown on in a multiwell plate with a coverslide underside for microscopy, in an incubator at 37 c with a 5% co2 atmosphere until ~ 80% confluent . Add 10 - 20 l of the stock solution to the living cells growing in a multi - well plate (smartslide 50 micro - incubation system, wafergen) in 4 ml of opti - mem medium (gibco) per well for a 6-well plate . Return the multi - well plate to an incubator at 37 c with a 5% co2 atmosphere for 10 - 45 mins for staining . Remove the multiwell plate from the incubator and wash the cells 3 - 4 times with 4 ml optically clear cell culture medium (e.g. Opti - mem) to remove excess dye . Transfer the multiwell plate to the microscope stage and connect to a temperature controller / 5% co2 gas inlet as required, in preparation for imaging . Place the sample on the microscope stage and obtain a transmission and fluorescence image to identify fluorescent cells . A schematic diagram of the experimental set - up is shown in fig . 1.verify that the fluorescence emanates from the locations expected (e.g. Cell membrane, cytoplasm). Obtain a fluorescence emission spectrum, and verify that it is that of the dye or protein expected, in this case the spectrum of the molecular rotor . As a negative control, although this step is not essential specifically for flim, it is good practice in general and does help to verify that the sample is what you think it is . Switch to flim mode - this is easily accomplished by moving a mirror out of the fluorescence detection beam path (" external detector " button on " beam path setting " panel on the leica tcs sp2 acquisition control software). An appropriate fluorescence emission filter to block any exciting light from reaching the detector must be in the fluorescence detection beampath . Figure 1 . Experimental arrangement for time - domain flim using a confocal laser scanning microscope . Scan the sample and check, on the computer controlling the flim acquisition, that the detector count rate (black bar labeled cfd on acquisition control software of the becker & hickl spc 830 board) is no more than about 1% of the laser repetition rate (green bar labeled sync acquisition control software). If it is, reduce the laser excitation intensity, e.g. By placing a neutral density filter in the laser beam path, to avoid collecting pile - up distorted fluorescence decay curves . Acquire a flim image, typically for 3 - 5 min, stop scanning and save the raw data (a 3d data " cube " consisting of spatial coordinates x and y, and time). Open the raw data in the fluorescence decay analysis software package, for example tri-2 or commercial software, to display the fluorescence intensity image . This is simply the integrated fluorescence decay, i.e. The area under the fluorescence decay curve, in each pixel . Select a typical pixel by placing the cursor on it, and inspect the fluorescence decay in that pixel . The counts of adjacent pixels (e.g. 3x3 or 5x5) are added into the central pixel, so that a higher peak count is obtained there . Alternatively, the measurement could be repeated for a longer acquisition time (step 5). For 30 - 50min, an approximately 10 times higher peak count (and total counts) is obtained, but this is far too long an acquisition time for most biological samples because of the danger of introducing artifacts due to sample movement, microscope drift, phototoxicity and photobleaching . Select a global pixel threshold value (above which the decay in a pixel is fitted) and apply a single exponential decay fit to the image . The result yields a fluorescence lifetime for each pixel above the threshold, which is then encoded in color . Each pixel is colored with the result of the fit, and a flim map is obtained . Check the reduced chi - squared values for various pixels - around 1 (and up to 1.3) indicates a good fit . The fluorescence lifetime histogram plots how often certain fluorescence lifetimes occur versus the fluorescence lifetime itself . Adjust the colour range such that the fluorescence lifetime distribution fits into the colour range . If a monoexponential fit does not yield a chi - squared value of around 1 (and up to 1.3), and there is a systematic deviation of the residuals from zero, a more sophisticated model is required . For example, try fitting a double exponential model to the fluorescence decays, to account for two different environments the probe may be in . The fit will also yield the pre - exponential factors or amplitudes which give an indication of the relative amount of dye in one environment or the other . Alternatively, a stretched exponential function may be appropriate to account for a distribution of fluorescence lifetimes . The results for the fluorescence lifetimes, pre - exponential factors, and the lifetime ratio and the pre - exponential factor ratio for each pixel can then be encoded in color . Each pixel is coloured according to its value, and contrast due to fluorescence lifetimes, pre - exponential factors and their ratios is obtained . Again, check the reduced chi - squared values (which can also be encoded in colour and displayed as an image) - around 1 (and up to 1.3) indicates a good fit . Inspect the residuals, which should be randomly distributed around zero . Fluorescence lifetime histograms should accompany all images for easy visualisation of average fluorescence lifetime values, and the fluorescence lifetime distribution . Fluorescence decays measured for the fluorescent molecular rotor at increasing viscosity in methanol / glycerol mixtures are shown in fig . The fluorescence decays are monoexponential, and the fluorescence lifetime varies markedly as a function of viscosity . It increases from around 300 ps in methanol (viscosity 0.6 cp) to 3.4 ns in 95% glycerol (viscosity 950 cp). Fluorescence decay profiles for bodipy - c12 in methanol / glycerol mixtures of varying viscosity . The logarithmic calibration plot of fluorescence lifetime versus viscosity for the fluorescent molecular rotor is shown in fig . 3 . It is a straight line as demanded by the frster hoffman equation where k0 is the radiative rate constant, and z and x are constants, with 0<x<1 . Taking the logarithm on both sides yields where x is the gradient of the straight line . Figure 3 . A plot of log fluorescence lifetime vs log viscosity for bodipy - c12 yields a straight line in accordance with the frster - hoffmann equation . Following incubation of living cells with the fluorescent molecular rotor a punctate dye distribution flim images of hela cells incubated with a meso - substituted bodipy dye are shown in fig . The fluorescence decays in every pixel of the image can be adequately fitted using a single exponential decay model . (a) fluorescence intensity and (b) flim images of hela cells stained with bodipy - c12 . This shorter liftime corresponds to a lower viscosity in the puncta, probably lipid droplets, according to the frster - hoffmann equation . By plotting the lifetimes extracted from every pixel, we obtain a fluorescence lifetime histogram of the whole image as shown in fig . Histograms of fluorescence lifetimes from flim images of hela cells stained with meso - substituted bodipy molecular rotors . Prepare a stock solution (10 ml) by dissolving approximately 1 mg / ml of the dye in an appropriate solvent (e.g. Methanol for bodipy - c12) using an accurate balance and a pipette . The cells (a model cancer cell line, hela in our case) to be stained are grown on in a multiwell plate with a coverslide underside for microscopy, in an incubator at 37 c with a 5% co2 atmosphere until ~ 80% confluent . Add 10 - 20 l of the stock solution to the living cells growing in a multi - well plate (smartslide 50 micro - incubation system, wafergen) in 4 ml of opti - mem medium (gibco) per well for a 6-well plate . Return the multi - well plate to an incubator at 37 c with a 5% co2 atmosphere for 10 - 45 mins for staining . Remove the multiwell plate from the incubator and wash the cells 3 - 4 times with 4 ml optically clear cell culture medium (e.g. Opti - mem) to remove excess dye . Transfer the multiwell plate to the microscope stage and connect to a temperature controller / 5% co2 gas inlet as required, in preparation for imaging . Place the sample on the microscope stage and obtain a transmission and fluorescence image to identify fluorescent cells . A schematic diagram of the experimental set - up is shown in fig . 1.verify that the fluorescence emanates from the locations expected (e.g. Cell membrane, cytoplasm). Obtain a fluorescence emission spectrum, and verify that it is that of the dye or protein expected, in this case the spectrum of the molecular rotor . As a negative control, image a non - stained sample and verify that it does not fluoresce . Although this step is not essential specifically for flim, it is good practice in general and does help to verify that the sample is what you think it is . Switch to flim mode - this is easily accomplished by moving a mirror out of the fluorescence detection beam path (" external detector " button on " beam path setting " panel on the leica tcs sp2 acquisition control software). An appropriate fluorescence emission filter to block any exciting light from reaching scan the sample and check, on the computer controlling the flim acquisition, that the detector count rate (black bar labeled cfd on acquisition control software of the becker & hickl spc 830 board) is no more than about 1% of the laser repetition rate (green bar labeled sync acquisition control software). If it is, reduce the laser excitation intensity, e.g. By placing a neutral density filter in the laser beam path, to avoid collecting pile - up distorted fluorescence decay curves . Acquire a flim image, typically for 3 - 5 min, stop scanning and save the raw data (a 3d data " cube " consisting of spatial coordinates x and y, and time). Open the raw data in the fluorescence decay analysis software package, for example tri-2 or commercial software, to display the fluorescence intensity image . This is simply the integrated fluorescence decay, i.e. The area under the fluorescence decay curve, in each pixel . Select a typical pixel by placing the cursor on it, and inspect the fluorescence decay in that pixel . The counts of adjacent pixels (e.g. 3x3 or 5x5) are added into the central pixel, so that a higher peak count is obtained there . Alternatively, the measurement could be repeated for a longer acquisition time (step 5). For 30 - 50min, an approximately 10 times higher peak count (and total counts) is obtained, but this is far too long an acquisition time for most biological samples because of the danger of introducing artifacts due to sample movement, microscope drift, phototoxicity and photobleaching . Select a global pixel threshold value (above which the decay in a pixel is fitted) and apply a single exponential decay fit to the image . The result yields a fluorescence lifetime for each pixel above the threshold, which is then encoded in color . Each pixel is colored with the result of the fit, and a flim map is obtained . Check the reduced chi - squared values for various pixels - around 1 (and up to 1.3) indicates a good fit . The fluorescence lifetime histogram plots how often certain fluorescence lifetimes occur versus the fluorescence lifetime itself . Adjust the colour range such that the fluorescence lifetime distribution fits into the colour range . If a monoexponential fit does not yield a chi - squared value of around 1 (and up to 1.3), and there is a systematic deviation of the residuals from zero, a more sophisticated model is required . For example, try fitting a double exponential model to the fluorescence decays, to account for two different environments the probe may be in . The fit will also yield the pre - exponential factors or amplitudes which give an indication of the relative amount of dye in one environment or the other . Alternatively, a stretched exponential function may be appropriate to account for a distribution of fluorescence lifetimes . The results for the fluorescence lifetimes, pre - exponential factors, and the lifetime ratio and the pre - exponential factor ratio for each pixel can then be encoded in color . Each pixel is coloured according to its value, and contrast due to fluorescence lifetimes, pre - exponential factors and their ratios is obtained . Again, check the reduced chi - squared values (which can also be encoded in colour and displayed as an image) - around 1 (and up to 1.3) indicates a good fit . Fluorescence lifetime histograms should accompany all images for easy visualisation of average fluorescence lifetime values, and the fluorescence lifetime distribution . Fluorescence decays measured for the fluorescent molecular rotor at increasing viscosity in methanol / glycerol mixtures are shown in fig . The fluorescence decays are monoexponential, and the fluorescence lifetime varies markedly as a function of viscosity . It increases from around 300 ps in methanol (viscosity 0.6 cp) to 3.4 ns in 95% glycerol (viscosity 950 cp). Fluorescence decay profiles for bodipy - c12 in methanol / glycerol mixtures of varying viscosity . The logarithmic calibration plot of fluorescence lifetime versus viscosity for the fluorescent molecular rotor is shown in fig . 3 . It is a straight line as demanded by the frster hoffman equation where k0 is the radiative rate constant, and z and x are constants, with 0<x<1 . Taking the logarithm on both sides yields where x is the gradient of the straight line . Figure 3 . A plot of log fluorescence lifetime vs log viscosity for bodipy - c12 yields a straight line in accordance with the frster - hoffmann equation . Following incubation of living cells with the fluorescent molecular rotor a punctate dye distribution is observed in the fluorescence images . Flim images of hela cells incubated with a meso - substituted bodipy dye are shown in fig . The fluorescence decays in every pixel of the image can be adequately fitted using a single exponential decay model . (a) fluorescence intensity and (b) flim images of hela cells stained with bodipy - c12 . This shorter liftime corresponds to a lower viscosity in the puncta, probably lipid droplets, according to the frster - hoffmann equation . By plotting the lifetimes extracted from every pixel, we obtain a fluorescence lifetime histogram of the whole image as shown in fig . Histograms of fluorescence lifetimes from flim images of hela cells stained with meso - substituted bodipy molecular rotors . It can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, because it can separate them from fluorophore concentration effects . The fluorescence lifetime can readily be converted into a viscosity using a calibration graph, as shown in fig . Instrumental artifacts include scattered light which will show up as a peak on top of the beginning of the fluorescence decay and may be confused with a short decay time, or a small peak after the irf which may be caused by reflections inside the microscope . These scattered light artifacts can be identified as such because they can be distinguished with spectral discrimination they are always at the same wavelength as the exciting light . Remembering that in air, light travels 30 cm in 1 nanosecond helps to pinpoint the origin of reflections . Filter or glass fluorescence could also cause an artifact, especially at low sample fluorescence, but this can easily be identified by taking a measurement without the sample: if a decay is obtained under these circumstances, it is due to the instrument and has nothing to do with the sample! On the other hand, note that sample autofluorescence may also contribute to a fluorescence decay . In time - correlated single photon counting (tcspc), time - to - amplitude converter (tac) non - linearities may cause poor fits, but can be identified by blocking the excitation and shining ambient light, e.g. From the transmitted light source onto the sample and measuring the timing . A constant background should be obtained in each pixel of the image . Regions where deviations from a constant background occur, will never yield a good fit and should be avoided for the measurement if they cannot be eliminated by adjusting the parameters for the tcspc card . One infamous artifact in tcspc is photon pile - up which is caused by too high a photon detection rate . This leads to only the first photon being timed, ignoring any subsequent photons because the electronics are busy timing and processing the first photon . Pile - up leads to a shortening of the fluorescence lifetime, and the best way to avoid this is to keep the photon count rate at around 1% of the laser repetition rate . There are various implementations of flim, and, depending on the application, each has its advantages and drawbacks . The ideal fluorescence microscope would acquire the entire multidimensional fluorescence emission contour of intensity, position, lifetime, wavelength and polarization in a single measurement, with single photon sensitivity, maximum spatial resolution and minimum acquisition time . There is presently no technology with this unique combination of features, and to build one remains a challenge for instrumentation developers . The application of new physical techniques to important problems in cell biology is often the path to unexpected discoveries, and there is a long way to go before we are close to saturating the capabilities of fluorescence imaging for cell biology . Indeed, imaging fluorescence parameters such as lifetime, spectrum and polarization, as well as imaging more rapidly in 3d at higher spatial resolution, are certain to reveal new aspects in cell biology . There are various implementations of flim, and, depending on the application, each has its advantages and drawbacks . The ideal fluorescence microscope would acquire the entire multidimensional fluorescence emission contour of intensity, position, lifetime, wavelength and polarization in a single measurement, with single photon sensitivity, maximum spatial resolution and minimum acquisition time . There is presently no technology with this unique combination of features, and to build one remains a challenge for instrumentation developers . The application of new physical techniques to important problems in cell biology is often the path to unexpected discoveries, and there is a long way to go before we are close to saturating the capabilities of fluorescence imaging for cell biology . Indeed, imaging fluorescence parameters such as lifetime, spectrum and polarization, as well as imaging more rapidly in 3d at higher spatial resolution, are certain to reveal new aspects in cell biology.
Naturally produced biopolymers in living organisms play crucial roles in materials discovery and development . They have inspired scientists to synthesize novel biomaterials through mimicking mother nature, or they can further serve as templates and building blocks to prepare new generations of biocompatible, bioregenerative, or biodegradable materials for biomedical applications . For instance, dna has been used to rationally design plasmonic nanostructures, to build nanoscaffolds for incorporating multiple - affinity ligands, and to self - assemble into numerous prescribed 3d shapes . Cellular membranes have also been widely imitated by phospholipids and polysaccharides to form liposome or micelles for drug and imaging agent delivery . Leukocyte membranes have also been used to coat silicon nanoparticles (nps) to yield hybrid nps that achieve cell - like functions, including avoiding clearance by the immune system . Multimodal imaging combines different modalities together to provide complementary information and achieve synergistic advantages over any single modality alone . It has emerged as a very promising strategy for preclinical research and clinical applications . One major challenge of multimodal imaging is to develop an efficient platform to load various components with individual contrast properties together while maintaining compact size, good biocompatibility and targeting capability . Exogenous inorganic nps - based reporters have attracted considerable interests, such as iron oxide nps for magnetic resonance imaging (mri) and quantum dots for fluorescence imaging . Compared with inorganic nps, organic nps generally exhibit good biocompatibilities, biodistribution and clearance, although most of them only appear to possess optical imaging properties . Some biomolecules based nps such as liposomes have been widely used for loading contrast agents and drugs . Therefore, such biomolecules need complicated and time - consuming processes to prebuild various contrast properties or require chemical modifications to integrate different reporting moieties into one entity, which we term as a passive platform . For example, organic ligands are generally incorporated into a nanoplatform before chelating to radioactive or magnetic metal ions for positron emission tomography (pet) and magnetic resonance imaging (mri). Melanin, an amorphous, irregular functional biopolymer and a ubiquitous natural pigment that presents in many organisms including human skin, is a typical biomarker for disease imaging including melanoma detection and parkinson diseases diagnosis . In this study, we report the successful transferring of this biomarker into an imaging nanoplatform . By mimicking natural melanin, water - soluble melanin nanoparticle (mnp) has been synthesized and used as the active platform for multimodal imaging of tumors . We demonstrate that mnp cannot only offer its native optical properties for photoacoustic imaging (pai), but also actively chelate to metal ions (cu, fe) for pet and mri with a high loading capacity and stability utilizing its intrinsic chelating function . Furthermore, ultrasmall size mnps (4.5 nm) can be easily prepared and surface - modified . Overall, these unique properties significantly simplify the process of preparation of multimodal imaging probes and make mnp a highly promising nanomaterial for biomedical applications . Figure 1 schematically illustrates the procedure to prepare ultrasmall water - soluble mnp with multimodal imaging properties . To change the intrinsic poor water - solubility of melanin, pristine melanin granule was first dissolved in a 0.1 n naoh and then neutralized under the assistance of sonication to decrease interchain aggregation . Ultrasmall mnp in high water monodispersity and homogeneity with a size of 4.5 0.5 nm, which was termed as plain water - soluble mnp (pws - mnp), were successfully obtained (figure 2a, b and figure s1a, supporting information). Pws - mnp exhibited excellent water - solubility of 40 mg / ml and stability, which can be attributed to the highly negative potential of approximately 22.2 mv on the np surface that efficiently blocks the np aggregation through electrostatic repulsion (figure s1b). Furthermore, pws - mnp can be stored as lyophilized powder for over six months and effectively redissolved in water allowing long - term usage (figure 2a). The ft - ir spectra of pristine melanin granule and pws - mnp were similar to each other, indicating no significant change of molecular structure (figure s2a). The h nmr spectrum of pws - mnp in d2o showed no obvious signal belonging to the hydrogen atom on the arylene groups, suggesting most of the conjugated backbones were buried in the np (figure s2b). The molecular weight of a pws - mnp was calculated from the nanoparticle size and its density (1.3 g / cm), which is about 40 kda . The melanin granules were first dissolved in 0.1 n naoh aqueous solution, and then neutralized under sonication to obtain melanin nanoparticles in high water monodispersity and homogeneity . After peg surface - modification, rgd was further attached to the mnp for tumor targeting . Then fe and/or cu were chelated to the obtained mnps for pai / mri / pet multimodal imaging . Characterization of physical properties of mnps . (a) from left to right: pictures of (1) pristine melanin granule in h2o, (2) melanin neutralized without sonication in h2o, (3) freeze - dried pws - mnp, (4) freeze - dried pws - mnp redissolved in pbs (ph = 7.4), (5) freeze - dried peg - mnp, (6) freeze - dried peg - mnp redissolved in pbs (ph = 7.4). (b) tem of pws - mnp (left) and peg - mnp (right), scale bar = 20 nm . (c) the plot of the relationship between the number of metal ions attached on one mnp with feed ratio (wions: wmnp). (d) stability study of metal ion - chelated mnps in pbs (ph = 7.4). In vitro and in vivo study of pai of mnps . (a) the photoacoustic signal produced by peg - mnps at concentrations of 0.625, 1.25, 2.5, 5.0, 10, and 20 m, and it was observed to be linearly dependent on its concentration (r = 0.995). Mice were injected subcutaneously (region enveloped by blue dotted line) with peg - mnp at concentrations of 0, 5, 10 (from left to right in top row), and 20, 40, 80 (from left to right in bottom row) m . One vertical slice in the photoacoustic image (red) was overlaid on the corresponding slice in the ultrasound image (gray). The linear regression is calculated on the five most concentrated inclusions (r = 0.998). (d) the overlaying of ultrasonic (gray) and photoacoustic (red) imagings of u87 mg tumor (region enveloped by yellow dotted line) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice (n = 3) and their subtraction imagings . (e) quantitative analysis of enhanced pa signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h, compared with at 0 h. to retain the water - solubility of pws - mnps for further biomodification and metal ion - chelating, polyethylene glycol (peg) chains were introduced to the mnp . Nh2-peg5000-nh2 was used because the amine groups can react with dihydroxyindole / indolequinone groups in melanin . The number of peg chains per mnp was determined to be 19 (figure s3a and s3b). The diameter of the peg - functionalized mnp (peg - mnp) became large and reached 7.0 nm (figure 2b and figure s1a). Moreover, the surface potential of peg - mnp decreased to 6.1 mv (figure s1b) because of introduction of peg and positive nh2 groups on the mnp surface . The similar absorption spectrum of peg - mnp to pws - mnp demonstrated that the peg - modification did not influence the absorption properties of melanin (figure s3c). Lastly, for demonstrating that mnp can be used as a platform for biomodification, peg - mnp was further modified with biomolecules such as cyclic arg - gly - asp - d - phe - cys [c(rgdfc)] peptide (abbreviated as rgd), which can target tumor v3 integrin . The number of rgd attached to the mnp was calculated to be about 8 per mnp and the size of rgd - functionalized peg - mnp (rgd - peg - mnp) increased a little to 9.6 nm (figure s4). In vitro and in vivo study of mri of fe - chelated mnps . (a) t1 relaxation rates (1/t1, s) as a function of fe - rgd - peg - mnp (mm) in agarose gel (1.0 t, 25 c). (b) mri detection of fe - rgd - peg - mnps in living mice . Mice were injected subcutaneously (region enveloped by red dotted line) with fe - rgd - peg - mnps at concentrations of 0, 1.25, 2.5 (from left to right in upper layer), and 5, 10, 20 (from left to right in bottom layer) m . (c) quantitative analysis of enhanced mr signal of u87 mg tumor after tail - vein injection with rgd - peg - mnp at 4 h, compared with at 0 h. (d) mri images of u87 mg tumors (region enveloped by yellow dotted line) before and after tail - vein injection of 250 l of 200 m rgd - peg - mnp in living mice (n = 3) (tr: 700 ms, te: 5.2 ms). Top row shows black and white images, and bottom row shows the pseudocolored images . To investigate the possibility of mnp as a platform for pet and mri, its chelating properties to cu (cu for pet) and fe (for mri) were studied . After adding metal ions (0.2 ml of 10 mm fecl3 or cucl2) into mnp aqueous solutions (1 ml of 20 m for pws - mnp and peg - mnp), the precipitation of pws - mnp quickly appeared, while peg - mnp maintained good water - solubility (figure s5). The fe or cu - chelated mnp (fe - peg - mnp, fe - rgd - peg - mnp, cu - peg - mnp and cu - rgd - peg - mnp) exhibited high loading capacities . The maximum quantities of one mnp to chelate to cu and fe are about 100 and 90 ions, respectively, no matter whether rgd is attached to the mnp or not (figure 2c). After fe - chelating, the mnp sizes increased to 8.9 nm and 10.7 nm for fe - peg - mnp and fe - rgd - peg - mnp respectively and their zeta - potential remained in the neutral region (figure s4 and table s1). The optical stabilities of peg - mnp and rgd - peg - mnp under increasing durations of light exposure were further tested . Compared with those reported dyes for pai, which exhibit significant reduced absorption (> 30%) under light exposure, peg - mnp and rgd - peg - mnp showed intriguing photostability (only 3% reduced absorption) (figure s6), indicating their high capability for pai . Further stability assay of fe or cu - chelated mnps in pbs solution showed that only about 3% cu and 7% fe were released from those mnps at the first 2 h, and there was no further release at longer incubation time points, indicating the high stability of the chelating platform (figure 2d). The first 2 h released metal ions may derive from those that were absorbed on the mnps through weak electrostatic interaction . Furthermore, the high viability of nih3t3 and u87 mg cells (about 90110% as compared to the nontoxic control) after 24 h of incubation with peg - functionalized mnps was found, indicating high biocompatibility and low cytotoxic effect of peg - functionalized mnps (figure s7). To investigate the possibility of mnps to be used as a photoacoustic agent, we first studied the detection sensitivity of peg - mnp in aqueous solution at increasing concentrations from 0.625 to 20 m . The peg - mnp with 0.625 m was detected, and the photoacoustic signals increased linearly with the increase of peg - mnp concentrations (r = 0.995) (figure 3a). The detection sensitivity of mnp in living body was further tested by subcutaneous injection of peg - mnp on the lower back of mice (n = 3) at increasing concentrations of 5 to 80 m (figure 3b). A linear correlation (r = 0.998) between the mnp concentration and the corresponding photoacoustic signal 2.5 m of peg - mnp was found to give the equivalent photoacuoustic signal strength as the tissue background . To further investigate the in vivo pai properties, one group of u87 mg tumor mice were tail - vein injected with 250 l of rgd - peg - mnp at a concentration of 200 m . Mice showed obvious increase of photoacoustic signal in tumors after injection with rgd - peg - mnp at 4 h than that of prescan (figure 3d). The increased photoacoustic signal of rgd - peg - mnp (figure 3e) could be attributed to the enhanced permeability and retention (epr) effect and the tumor targeting ability of rgd - peg - mnp to v3 integrin . Furthermore, instead of vevo lazr pai system, using inveon research workplace (nexus 128) was able to obtain 3d pa imaging, which provided the more clearly enhanced blood vessel signals in tumor after mnp injection (figure s8). In vitro and in vivo study of pet of cu - labeled mnps . (a) uptake of cu - rgd - peg - mnp with and without blocking in u87 mg cells at 37 c for 1, 2 and 4 h incubation . All results, expressed as percentage of cellular uptake, are mean of triplicate measurements sd . (b) representative decay - corrected coronal (top) and transaxial (bottom) small animal pet images (left three images) and the overlaying of ct (gray) and pet (color) images (right three images) of u87 mg tumors (region enveloped by yellow dotted line) acquired at 2, 4, and 24 h after tail vein injection of cu - rgd - peg - mnp . (c) biodistribution of cu - rgd - peg - mnp in mice (n = 3) at 2, 4, and 24 h after injection . The radioactive signal from each organ was calculated using a region of interest drawn over the whole organ region . To study whether fe (t1 contrast agent) retains mr signal - enhancing property after loading into mnps, t1-weighted mri images of various concentrations of fe - rgd - peg - mnp in agarose gel was investigated (figure s9a). With the increase of np concentration, mr signal was significantly enhanced, suggesting fe - rgd - peg - mnp generate a high magnetic field gradient on their surface . R1 value of fe - rgd - peg - mnp (the slope of the fitted curve in figure 4a, using gd as standard) was calculated to be 1.2 mm s. the magnetic sensitivity in living mice was then tested by subcutaneous injection of fe - rgd - peg - mnp on the lower back of mice (n = 3) at increasing concentrations of 1.25 to 20 m . It was extrapolated that 1.25 m of fe - rgd - peg - mnp produced the equivalent mri signal intensity as the tissue background (figure 4b). To demonstrate the use of mnp as the platform for mri of tumors, t1-weighted images were obtained from mice bearing u87 mg tumors (n = 3). U87 mg tumors displayed increased signals after 4 h mnp injection (figure 4d). The relative mr signal intensity of tumor at 4 h increased 30% compared with at 0 h, demonstrating that mnp can be used as a platform for mri (figure 4c). Furthermore, further optimizing the mr imaging conditions can provide more clearly enhanced mri signal and mnp accumulation in tumor (figure s9b). To investigate the pet imaging properties of mnp, cu was selected as a pet radiolabel for mnp because it can be readily chelated by melanin and the intermediate half - life of cu (12.7 h) makes it suitable for radiolabeling of biomolecules and imaging . Simple mixing of rgd - peg - mnp and peg - mnp with cu allowed successfully labeling the nps in the yield of 80% . The resulting mnps, cu - rgd - peg - mnp and cu - peg - mnp, displayed excellent stability in pbs solution (figure s10). Similar to cu - chelated mnps, only 3% cu released from the mnps after 24 h of incubation . Thus, cu - labeled mnps were easily and reliably produced and exhibited reasonable stability in vitro . In vivo multimodality imaging of tumor (region enveloped by yellow dotted line) bearing mice with pai and mri / pet respectively . (b) the overlaying of ultrasonic (gray) and photoacoustic (red) imaging of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp (200 l of 10 m) in living mice and their subtraction imaging . (c) the overlaying of representative decay - corrected coronal (top) and transaxial (bottom) small animal ct (gray) and pet (color) images of u87 mg tumors acquired at 2, 4, and 24 h after tail vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp (250 l of 200 m). (d) mri images of u87 mg tumor before and after tail - vein injection of cu - fe - rgd - peg - mnp and fe - rgd - peg - mnp (250 l of 200 m) in living mouse . Top row shows black and white images, and bottom row shows the pseudocolored images . Uptake of cu - rgd - peg - mnp by u87 mg cells with or without blocking agent rgd at 1, 2, and 4 h are shown in figure 5a . Cu - rgd - peg - mnp exhibited higher uptakes than blocking group at all the incubation time, with a value of 3.32 0.37%, 5.32 0.43% and 7.18 0.33% for cu - rgd - peg - mnp at 1, 2, and 4 h. in comparison, for cu - rgd - peg - mnp blocking group, much lower uptake of cu - rgd - peg - mnp was observed with a value of 2.00 0.15%, 3.16 0.20% and 3.48 0.29% at 1, 2, and 4 h, respectively, indicating the successful biomodification of mnps with rgd peptide and the specific targeting ability of rgd contribute to the uptake of cu - rgd - peg - mnp by u87 mg cells . The in vivo pet of mnps was performed in u87mg - tumor - bearing mice . Cu - rgd - peg - mnp showed tumor accumulation and clear tumor contrast after 2 h postinjection (figure 5b). Quantification analysis revealed that the tumor uptake values of cu - rgd - peg - mnp gradually increased with time to 24 h, and they were 4.75 0.63, 5.87 0.87, and 5.93 0.89% id / g at 2, 4, and 24 h, respectively (figure 5c). In addition to the tumor, moderate activity accumulation was observed in the liver (e.g., 15.78 2.55% id / g at 24 h for all mnps), and relative lower activity accumulation was also found in the kidneys (e.g., 5.34 0.62% id / g at 24 h for all mnps). These data indicated the mnp was cleared mainly through hepatobiliary system . To further investigate the possible targeting property of mnps, the cu radiolabeled rgd - peg - mnp and control cyclic arg - ala - asp - d - phe - cys [c(radfc)] peptide (abbreviated as rad, with nontargeting property for tumor v3 integrin) functionalized peg - mnp (cu - rad - peg - mnp) for u87 mg tumor pet imaging the obvious stronger pet signal of cu - rgd - peg - mnp can be found in tumor at 4 h than that of cu - rad - peg - mnp (p <0.05), indicating the good and specific targeting property of rgd - peg - mnp . To investigate the possibility of using mnp platform for multimodality imaging, mnp were mixed with fe and cu in sequence to form the multifunctional probes fe - rgd - peg - mnp and cu - fe - rgd - peg - mnp (the amount of fe per mnp is 56) for pet / pai / mri . Pet of mice bearing u87 mg tumors were then obtained first at 2, 4, and 24 h postinjection of cu - fe - rgd - peg - mnp . After 48 h, t1-weighted mri and pai of mice bearing u87 mg tumors were then obtained respectively at 4 h after another injection of large dose of fe - rgd - peg - mnp . In figure 6, cu - fe - rgd - peg - mnp showed very similar pet, mri and pai properties on u87 mg tumor, compared with the corresponding single modality imaging from cu - rgd - peg - mnp, fe - rgd - peg - mnp, and rgd - peg - mnp respectively . These results showed that using mnp as the active platform to load cu and fe together can efficiently combine its native photoacoustic properties with radioactive and magnetic properties together for multimodality imaging . To change the lack of contrast properties of biomolecule - based nanoplatform for multimodality imaging, recently porphyrin were successfully introduced into phospholipid to provide the platform with desirable optical properties, while it still requires complicated and time - consuming chemical modifications and other reporting molecules to achieve multimodality imaging ability . We herein developed the functional biomarker, melanin, as a novel nanoplatform with its native optical property and multifunctions, which can simply and actively collecting optical, magnetic and radioactive properties together for multimodality imaging . Melanin, the oxidation products of tyrosine, plays an important role in living organism . Accompanied with the development of molecular imaging probes in the past decade, melanin has been used as an effective molecular target as well as endogenous contrast agent for pai because of its strong light absorption properties . Besides, melanin has intrinsic strong chelating properties to many metal ions including cu and fe, which can be used to nuclear imaging and mri . Consequently melanotic melanomas show hyperintensity on t1-weighted mri images . Considering the attractive properties of the biomarker melanin, we and others have engineered cancer cells to biologically produce melanin for multimodality imaging (pai / mri / pet) of cancer . However, this method requires genetic modification of cells, which is time - consuming and may have limited clinical value . Thus, water - soluble mnps are more appropriate to behave as a natural active platform to simplify the preparation procedure for multimodal applications . Considering only trace amount of cu ions utilized for pet and its final decay to zn ions, which is necessary for life process, and the abundant amount of fe ions in living body, cu and fe ions used in our system are expected to be metabolized in living subjects . In comparison, although gd ion has higher t1 mri effect than fe ion, its potential toxicity is still a problem . Concomitantly, the traditional nanoparticle - based platform needs complicated functionalization of ligand to chelate gd for mri and cu for pet . More interestingly, the new mnp system can serve as an active nanoplatform and easily bind with metal ions without the traditional needs of surface modification and introducing chelating groups, which significantly simplifies the preparation process and reduces the heterogeneity of the resulting multimodal nps . Furthermore, the mnp is an organic and biodegradable material and showed relatively good tumor imaging properties . All of these properties make mnps highly promising for potential clinical translation . Despite its important functions, developing melanin for molecular imaging therefore, preparing mnps is desired while still a challenge for well - dispersing in water, especially for those with size around 10 nm that can provide not only appropriate blood circulation time, but also high surface - to - volume ratio to chelate enough metal ions for efficient bioimaging . Although the formation mechanism of polymeric melanin is not clear, its molecular structure is generally considered to be composed of dihydroxyindole / indolequinone segments with hydrophobic conjugated main chain having strong interaction and hydrophilic hydroxyl groups on the benzene rings . Therefore, to realize melanin water - soluble at neutral environments, decreasing the interchain aggregation of conjugated main chains and lowering the formed melanin particle size to expose more hydrophilic hydroxy groups on the surface of melanin is a promising way . In our work, we first realized the synthesis of ultrasmall mnps in water with high monodispersity and homogeneity under the assistance of sonication . Another problem that should be resolved is the metal ion - initiated cross - linking and the formation of mnp precipitation . Recent reports showed that fe is a strong cross - linker for catechol groups, which is one of the components in melanin molecular structure . In our work, peg encapsulation is found cannot only enhance the biocompatibility and the water - solubility, but also efficiently prevent the formation of metal ion - initiated precipitation . Investigation on the in vivo subcutaneous phantom showed that the signals between mri and pai had good linear relationship (figure s12). In addition, the slope of enhanced pa signal with different concentrations was higher than that of mri signal, indicating the pai imaging of mnp was more sensitive than the mri imaging . Moreover, because of their easy conjugation with targeting groups, the mnps modified with rgd exhibited the tumor targeting ability to the v3 integrin overexpressed on the surface of tumor vasculatures and the u87 mg tumor cells (from pet data), which afforded rgd - conjugated mnps higher accumulation capability in tumor than those rad - modified mnps through enhanced permeability and retention (epr) effect . This further indicated that mnps can conveniently function as a good nanoplatform for targeted imaging . Though it is difficult to compare the imaging effects of our platform with other type of nanoplatforms (considering the lack of the triplemodality analogues), we demonstrated the unique ability of mnp nanoplatform to combine pet, photoacoustic and mr imaging modalities together to get complementary information for tumor imaging . For example, pet efficiently provides the in vivo pharmacokinetics and biodistribution of the nanoprobes and can also provide physiological information on disease with whole body imaging capability, but it cannot image tissues at high spatial resolution; pai provides functional and molecular information on the tumor with high spatial resolution . It is a cheap and convenient way to image tumor in real time but suffers from limited tissue penetration ability, which can be compensated by pet . Mri provides high spatial resolution image and anatomical information on disease but generally lacks of molecular imaging capability . Combination of pet / pai / mri thus allows us to image diseases at different depths with molecular and anatomical information . Recent developments showed that multimodality imaging is promising not only for accurate tumor imaging but also for guiding tumor resection . For example, pet / mri, which combines the exquisite anatomical information by mri with the extreme sensitivity of pet, can be used for whole - body imaging and deep tumor localization . Mri / fluorescence imaging (fi) is appropriate for guiding superficial tumor resection . In comparison, combining pet / mri / pai together is anticipated to help for guiding both superficial and deep tumor surgery . To our knowledge, our triple - modality mnp system can be first used for pet and mri to obtain the detailed information on tumor for surgical planning in presurgery . Pai can then be used to localize the superficial and relatively deep tumors in surgery for helping the tumor resection . Overall, a reliable method for preparation of water - soluble mnp has been developed in our work, which lays down a foundation for its future biomedical applications . It can be easily envisioned that mnp can serve as a nanoplatform not only for molecular imaging but also for theranostics . Considering the abundant functionalities of melanin, such as binding drugs, mnp - based platform used for drug delivery and therapy are now being investigated . In conclusion, we report mnp as the natural biomarker - transferred active platform for multimodality imaging . Mnp is of particular interest because such an endogenous agent with native photoacoustic signals and strong chelating properties with metal ions can act as an active platform to simplify the assembling of different imaging moieties . Mnp can be easily modified with biomolecules for targeted tumor multimodality imaging, and it showed good in vivo tumor imaging properties . We expect this work will stimulate further studies of multifunctional endogenous material as nanoplatforms for potential imaging and therapeutic applications . The following reagents were acquired and used as received: melanin (sigma - aldrich), sodium hydroxide (sigma - aldrich), hydrochloric acid (37 wt%, sigma - aldrich), nh4oh solution (28 wt%, sigma - aldrich), amine - peg5000-amine (nh2-peg5000-nh2, 5 kda, laysan bio), dimethylthiazolyl - diphenyltetrazolium (mtt; biotium), phosphate buffered saline (pbs, gibco), and agarose (invitrogen). Tyrosine - derived synthetic melanin (20 mg) was first dissolved in 10 ml of 0.1 n naoh aqueous solution under vigorous stirring . After dissolving, hcl aqueous solution (0.1 n) was swiftly dropped into the obtained basic melanin solution to adjust the ph to 7.0 under sonication with output power = 10 w for 1 min . The neutralized solution was further centrifuged with a centrifugal - filter (amicon centrifugal filter device, mwco = 30 kda) and washed with deionized water and repeated several times to remove the produced nacl . The aqueous solvent was removed by freeze - drying to obtain 15 mg black solid of pws - mnp . Nh4oh solution (28 wt%) was added to 5 ml of pws - mnp aqueous solution (1 mg / ml of water) to adjust the ph of the solution to 9 . This mixed solution was added dropwise into nh2-peg5000-nh2 (5, 10, 25, and 50 mg) aqueous solution with ph = 9 . After vigorous stirring for 12 h, peg - modified mnp was retrieved by centrifugation with a centrifugal - filter (amicon centrifugal filter device, mwco = 30 kda) and washed with deionized water several times by redispersion / centrifugation processes to remove the unreacted nh2-peg5000-nh2 . The aqueous solvent was removed by freeze - drying and the obtained peg - mnp was weighed to preliminary calculate the quantity of the peg attached on mnps . Because of the existence of one nh2 group per peg chain on the surface of mnp, we then accurately determined the nh2 group on mnp with fluorescamine by spectrofluorometer to calculate the amount of peg on the nanoparticles (using ethamine as the standard). The cross - linker solution was prepared freshly . The 4-(n - maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo - n - hydroxysuccinimide ester sodium salt (sulfo - smcc) (1.2 mg) the water - soluble peg - mnp [1 mg in 1 ml of pbs (ph = 7.2)] was incubated with the above cross - linker solution for 2 h at room temperature . The resultant thiol - active mnp ran through a pd-10 column prewashed with pbs (ph = 7.2, 10 mm) to remove the excessive sulfo - smcc and byproducts . The purified mnp was concentrated to the final volume of 0.5 ml with a centrifugal - filter (mwco = 30 kda). The crgdfc stock solution (120 l of 5 mm in the degassed water) was added to the above mnp solution with stirring . The uncoupled rgd peptide was removed through a pd-10 column and collected to analyze its quantity through hplc . The resultant product, rgd - peg - mnp, were concentrated by a centrifugal - filter (mwco = 30 kda) and stored at 4 c for one month without losing targeting activity . The final rgd - peg - mnp were reconstituted in pbs and filtered through a 0.22 m filter for cell and animal experiments . The mnp (1 mg in 1 ml h2o) was labeled with fe or cu by addition of 20 l of fresh fecl3 (10 mg / ml) in pbs (ph = 7.4) or 20 l of cucl2 (10 mg / ml) in buffer solution of ph = 5.5 followed by a 1 h incubation at 40 c (the chelating mechanisms of mnps were shown in figure s13). The products were washed out by pbs and passed through a 0.22-m millipore filter into a sterile vial for in vitro and animal experiments . The fe and cu concentrations of mnps were measured by inductively coupled plasma - mass spectrometry (icp - ms) analysis . The stability of metal ion - chelated mnps were studied by incubating those mnps in pbs (ph = 7.4) at 37 c . Those mnps were placed in dialysis tube (mwco 10k) with magnetic stirring, dialysis against 10 ml pbs . At a certain time, dialysate was removed for icp - ms analysis and replaced with fresh pbs solution . For icp - ms analysis, 100 l of the detected sample was first heated to evaporate the water solvent and then digested with 0.5 ml of concentrated nitric acid (70% w / w) under heating . After the solvent was evaporated, the residue was then dissolved in 7 ml of dilute nitric acid (2% w / w) for final icp - ms analysis . Ft - ir spectra were measured in a transmission mode on a bio - rad ft - ir spectrophotometer (model fts135) under ambient conditions . Samples of pristine melanin granules and functionalized mnps were ground with kbr and then compressed into pellets . Transmission electron microscopy (tem) images were recorded on a jeol 2010 transmission electron microscope at an accelerating voltage of 100 kv . The tem specimens were made by placing a drop of the nanoparticle aqueous solution on a carbon - coated copper grid . The hydrodynamic sizes of the mnps were determined by dynamic light scattering (dls) using a 90 plus particle size analyzer (malvern, zetasizer nano zs90). Zeta potentials were measured using a zeta potential analyzer (malvern, zetasizer nano zs90). The h nmr spectra were recorded at 20 c on a 400 mhz nmr spectrometer (bruker), using d2o as solvent . The mnps with or without fe were further radiolabeled with cu by addition of 11.5 mci of cucl2 in 0.1 n naoac (ph 5.5) buffer followed by 1 h incubation at 40 c . The radiolabeled mnps were then purified by a pd-10 column (ge healthcare, piscataway, nj, usa). The product was washed out by pbs and passed through a 0.22-m millipore filter into a sterile vial for in vitro and animal experiments . The investigation of the radiolabeling stability of mnps is similar to the metal ion - chelated mnps except that the detector icp - ms was replaced by perkinelmer 1470 automatic gamma - counter for counting radioactivity . In vitro cytotoxicity of mnps was determined in nih-3t3 and u87 mg cells by the mtt assay . Nih-3t3 and u87 mg cells were incubated on 96-well plate in dmem medium containing 10% fbs and 1% penicillin / streptomycin at 37 c in 5% co2 humidified atmosphere for 24 h and 0.5 10 cells were seeded per well . Cells were then cultured in the medium supplemented with indicated doses of different mnps for 24 h. the final concentrations of mnps in the culture medium were fixed at 3.125, 6.25, 12.5, and 25 m in the experiment . Addition of 10 l of mtt (0.5 mg / ml) solution to each well and incubation for 3 h at 37 c was followed to produce formazan crystals . Then, the supernatant was removed and the products were lysed with 200 l of dmso . The absorbance value was recorded at 590 nm using a microplate reader . The absorbance of the untreated cells was used as a control and its absorbance was as the reference value for calculating 100% cellular viability . U87 mg cells (1 10 per well) were seeded in 12-well tissue culture plates and allowed to attach overnight . The cells were washed twice with serum - free dmem and incubated with the cu - labeled mnps (2 ci per well, final concentration approximately 6 nm) in 400 l of serum - free dmem at 37 c . The specific binding of the probes with u87 mg cells was determined by coincubation with rgd (30 g per well). After 1, 2, and 4 h, the cells were washed three times with cold pbs and lysed with the addition of 200 l of 0.2 m naoh . The radioactivity of all fractions was counted using a perkinelmer 1470 automatic gamma - counter . The uptake (counts per minute) was expressed as the percentage of added radioactivity . For studying the pai properties of mnps, different concentrations of mnps aqueous solutions ranging from 0.625 to 20 m were filled into polyethylene capillaries and then the capillaries were laid on the surface of solidified agarose gel . The capillaries were further covered with thin 1% agarose gel to make the surface smooth . For the particle s sensitivity in living body, mnps aqueous solutions with different concentrations from 5 m to 80 m were mixed with matrigel at 0 c and then subcutaneously injected on the lower back of mice . Toronto, canada) with a laser at excitation wavelength of 680 nm and a focal depth of 10 mm was used to acquire photoacoustic and ultrasound images . Mri experiments were performed at 25 c in a magnetic resonance (mr) scanner (siemens 1.0 t). To simulate the biological environment, agarose gel, prepared in 300 l of the pcr tube using secondary distilled water as the solvent for dissolving the agarose, was used to demonstrate the magnetic signal . The bottom of the tube was first covered with a layer of 1% agarose gel . When agarose gel was cooled, the mixtures of mnps and aqueous solution of agarose (ratio 1:1 by volume) with iron concentrations at 62.5, 125, 250, 500, and 1000 m fe (amount to 1.25, 2.5, 5, 10, 20 m mnp), were then filled into the intermediate portion of the pcr tube respectively while the sample was hot . After cooling, another 1% agarose gel was covered on the top layer of the cube . The tubes were placed into the mr scanner and a number of mr sequences were run, spin echo for r1 determination (tr: 503000 ms; te: 5.5 ms, flip angle 30; fov: 3.5 cm, matrix: 256 256; slice thickness: 1 mm). The luminance values of the resulting image were obtained through the imagej software processing, thereby calculating the r1 value . For measurement the mnps detection sensitivity in living subject, fe - chelated mnps aqueous solution with different concentrations from 1.25 m to 20 m were mixed with matrigel at 0 c and then subcutaneous injected on the lower back of mice . Tr: 700 ms, te: 5.2 ms; fov: 3.5 cm, matrix: 256 256; slice thickness: 1 mm . All animal experiments were performed in compliance with the guidelines for the care and use of research animals established by the stanford university animal studies committee . Female athymic nude mice (nu / nu) in 46 weeks old were obtained from the charles river laboratories (boston, ma, usa) and kept under sterile conditions . U87 mg cells suspended in 100 l of pbs were inoculated subcutaneously in the shoulder of nude mice . When the tumors reached 0.50.8 cm in diameter, the tumor bearing mice were subjected to in vivo multimodality imaging (pai, mri and pet) and biodistribution studies . Mice bearing tumor (u87 mg) were anesthetized with 2% isoflurane in oxygen and placed with lateral position . Mri was performed using the same instrument, protocols and conditions as in the phantom mri study . Pai was carried out using the same vevo lazr pai system as the in vitro study . Quantification analysis of pa and mr signals was performed on the pai and mri images . Because the enhanced signals of mri and pai in tumors were dispersed heterogeneously, the enhanced signal regions in tumors on the pai and mri images were used as rois to analysis the signal change with injection time (comparing the signal intensity at 4 h injection with 0 h injection). Small animal pet imaging of tumor - bearing mice was performed on a siemens inveon micropet - ct . Mice bearing u87-mg tumors were injected with cu - labeled mnps (110.0 5.0 ci) via the tail vein . At different times after injection (2, 4, and 24 h), the mice were anesthetized with 2% isoflurane and placed prone near the center of the fov of the scanner . All the small animal pet images were reconstructed using irw4.0 software by two - dimensional ordered - subsets expectation maximization (osem) algorithm . The radioactivity uptake in the tumor and normal tissues was calculated using a region of interest (roi) drawn over the whole organ region and expressed as a percentage of the injected radioactive dose per gram of tissue (% id / g).
Numerous reports have documented opposing effects of the gs - coupled d1r and gi / o - coupled d2r on biological processes such as adenylyl cyclase activity and camp formation (hyttel, 1978; onali et al ., 1985) and however, it has also been demonstrated that the concomitant activation of both d1r and d2r is essential to achieve certain other behavioral, biochemical, and electrophysiological effects . For instance, past studies have shown coactivation of the d1r and d2r as being necessary for maximal stereotyped and locomotor behavior (walters et al ., 1987; white et al ., 1988; deboer and abercrombie, 1996), and more recently it was reported that to evoke neural and behavioral phenotypes of cocaine sensitization, both d1r and d2r stimulation is required (capper - loup et al ., 2002). Similarly, at a cellular level, combined administration of specific d1r and d2r agonists was shown to potentiate immediate early gene expression (lahoste et al ., 1993; keefe and gerfen, 1995). For example, while the systemic administration of a dopamine d1r agonist induced the expression of c - fos and zif268 in the dopamine - depleted striatum, and a d2r agonist decreased zif268 expression, both agonists together significantly enhanced gene expression compared to the d1r agonist alone (keefe and gerfen, 1995). Electrophysiological evidence has also demonstrated that simultaneous administration of d1r and d2r agonists could synergistically inhibit the neuronal activity of nac neurons (white and wang, 1986; hu et al ., 1990), and restoration of long - term depression of synaptic transmission following dopamine depletion occurred by dopamine administration or coadministration of specific d1r and d2r agonists but not by either selective agonist alone (calabresi et al ., 1992). Although the mechanisms for the d1r and d2r interaction have been suggested to occur by receptor activation on discrete neurons (gerfen et al ., 1995; lahoste et al ., 2000), or changes in d2r - mediated cholinergic interneuron activity with subsequent impact on d1r - expressing striatonigral neuronal transmission (di chiara et al ., however, an interaction between the receptors occurring within the same cell may have been involved in some instances . Msn connections in neonatal striatal cultures have been reported to exhibit modulation by both d1r and d2r agonists (geldwert et al ., 2006) and the selective activation of d1r or d2r in single striatal neurons differentially modulated tetrodotoxin - sensitive sodium channels (aizman et al ., 2000 the synergistic potentiation of the arachidonic acid release by coadministration of d1r - selective and d2r - selective agonists has also been observed in cells expressing both receptors (piomelli et al ., 1991). Given the functional linkage between d1r and d2r, neuroanatomical studies began to address the question of dopamine receptor segregation and many of these studies have now shown, both at the mrna level and the protein level, that d1r and d2r coexist in a fraction of striatal msns . In situ hybridization studies examining d1r and d2r mrna in serial rat brain striatal sections have shown overlap of d1r and d2r transcript expression in the same neurons (meador - woodruff et al ., 1991; lester et al ., 1993) and double in situ hybridization has been used to report colocalization of d1r and d2r mrna in some striatal neurons in primate (aubert et al ., 2000). D1r / d2r mrna coexistence was also reported using single cell reverse transcription pcr in neonatal neurons (surmeier et al ., 1992, 1996) and immunolabeling has been a widely used tool for visualizing striatal d1r / d2r coexpression at the protein level in both neonatal striatal cultures (shetreat et al ., 1996;; deng et al ., 2006; hasbi et al ., 2009; perreault et al ., it had been noted that while neurons expressing solely mrna for the d1r or d2r were abundant in sp or enk mrna respectively, neurons containing mrna for both d1r and d2r expressed both sp and enk mrnas (surmeier et al ., 1996). (2010), using highly validated antibodies, it was shown that the d1r and d2r were expressed exclusively in neurons containing both dyn and enk . Although a small minority of dyn / enk neurons did not express d1r or d2r, these findings indicated that sp / dyn enk coexpression could be a useful marker for identifying neurons coexpressing d1r and d2r . Interestingly, unlike the controversy surrounding the localization of d1r and d2r, sp / dyn enk coexpression in striatal neurons has been widely accepted, and overlap of sp / dyn and enk mrna or protein in a proportion of striatal projection neurons has been reported for a number of species (penny et al ., 1986; gerfen and young, 1988; anderson and reiner, 1990; besson et al ., 1990; enhanced green fluorescent protein (egfp)-tagged promoter elements of d1r and d2r in bacterial artificial chromosome (bac) transgenic mice is one such method, and has been used to quantify the proportion of striatal neurons expressing the receptors within the striatonigral and striatopallidal pathways (valjent et al ., 2009). (2008) it was reported that while the majority of d1r and d2r were segregated to these discrete pathways, an estimated 17% of nac shell msns exhibited receptor colocalization with ~6% d1r / d2r coexpression in cp . Although there has been doubt as to the reliability of bac transgenic mice in providing an accurate representation of dopamine receptor expression due to unlabeled neurons (shuen et al ., 2008), another group was able to show that 100% of msns in these mice expressed d1r, d2r or both (matamales et al ., 2009) and that the proportion of neurons showing coexpression were similar to that observed in the bertran - gonzalez study . Fluorescence activated cell sorting (facs), another relatively novel method, is used for sorting a heterogeneous mixture of biological cells and is based upon the fluorescent characteristics of each cell . Essentially, this method employs the fluorescent properties of the neurons in the bac transgenic drd1a - egfp or drd2-egfp mice to purify d1r - expressing and d2r - expressing msns, and is often used to assess specific characteristics of these two neuronal subtypes . The quality of the purification process can then be verified by assessing mrna content of the each sample . Using this process, it was recently shown that while purified d1r - positive neurons from whole striatum displayed enrichment of d1r mrna, d2r mrna was also evident, albeit in small amounts (lobo et al ., 2010). Similarly, d2r - positive neurons also displayed evidence of minimal d1r mrna being present . In addition, using bac transgenic mice with cre recombinase being driven by the d1r or d2r promoters, and using double immunofluoresence to stain for cre and enk, this group also showed that a fraction of striatal d1r - expressing neurons were also positive for enk . In recent years, technological advances have provided novel approaches for assessing dopamine receptor colocalization . Enhanced green fluorescent protein (egfp)-tagged promoter elements of d1r and d2r in bacterial artificial chromosome (bac) transgenic mice is one such method, and has been used to quantify the proportion of striatal neurons expressing the receptors within the striatonigral and striatopallidal pathways (valjent et al ., 2009). (2008) it was reported that while the majority of d1r and d2r were segregated to these discrete pathways, an estimated 17% of nac shell msns exhibited receptor colocalization with ~6% d1r / d2r coexpression in cp . Although there has been doubt as to the reliability of bac transgenic mice in providing an accurate representation of dopamine receptor expression due to unlabeled neurons (shuen et al ., 2008), another group was able to show that 100% of msns in these mice expressed d1r, d2r or both (matamales et al ., 2009) and that the proportion of neurons showing coexpression were similar to that observed in the bertran - gonzalez study . Fluorescence activated cell sorting (facs), another relatively novel method, is used for sorting a heterogeneous mixture of biological cells and is based upon the fluorescent characteristics of each cell . Essentially, this method employs the fluorescent properties of the neurons in the bac transgenic drd1a - egfp or drd2-egfp mice to purify d1r - expressing and d2r - expressing msns, and is often used to assess specific characteristics of these two neuronal subtypes . The quality of the purification process can then be verified by assessing mrna content of the each sample . Using this process, it was recently shown that while purified d1r - positive neurons from whole striatum displayed enrichment of d1r mrna, d2r mrna was also evident, albeit in small amounts (lobo et al ., 2010). Similarly, d2r - positive neurons also displayed evidence of minimal d1r mrna being present . In addition, using bac transgenic mice with cre recombinase being driven by the d1r or d2r promoters, and using double immunofluoresence to stain for cre and enk, this group also showed that a fraction of striatal d1r - expressing neurons were also positive for enk . Although the existence of d1r- and d2r - coexpressing msns is now generally accepted, little is yet known about the functional relevance of these neurons, most likely as a result of the methodological difficulties attempting to isolate them . However, it has been shown that while all msns express mrna for the glua1 and glua2 subunits of the ampa receptor (chen et al ., 1998; stefani et al ., 1998; vorobjev et al ., 2000), only sp / enk neurons preferentially express mrna for glua3 (stefani et al ., 1998) a finding which the authors posited as being suggestive of distinctive postsynaptic glutamatergic mechanisms in the neurons . A role in neurotransmission is supported by evidence documenting that striatal projection neurons coexpressing d1r and d2r or sp and enk terminate in a number of regions including gp and epn (gpi), as well as substantia nigra and ventral tegmental area (deng et al ., 2006; wang et al ., 2006, 2007), two regions rich in dopamine cell bodies . In addition, a region - specific distribution of d1r / d2r - dyn / enk neurons throughout the rat basal ganglia and mesolimbic pathway has been reported, being present in nac (figure 2) and cp, as well as gp and ventral pallidum and epn (perreault et al ., 2010). Specifically, it was estimated that the fraction of d1r - expressing neurons also expressing the d2r in nac core and shell was ~25% and ~35% respectively, whereas in cp the percentage was much lower being only ~7% . In gp and vp, while the total number of d1r neurons was quite low compared to other regions, a high percentage of these neurons (~60% and ~30%) also contained d2r . Similarly, in epn the fraction of d1r containing neurons also expressing the d2r was relatively high (~50%). The dopamine d1 and d2 receptor are colocalized with dynorphin and enkephalin in rat nucleus accumbens shell . (a, b) confocal images showing d1r and d2r colocalization with dynorphin (dyn) or enkephalin (enk; white arrows). (c, d) neurons coexpressing dyn and enk also expressed the d1r or the d2r (white arrows). In basal ganglia, the coexpression of d1r and d2r has been shown to occur both within neuronal cell bodies and selectively at presynaptic, but not postsynaptic terminals, as evidenced by d1r and d2r coexpression with the presynaptic marker synaptophysin, but not the postsynaptic marker psd-95 (perreault et al ., 2010). Presynaptic colocalization of d1r and d2r on a fraction of varicosities has also been reported in neonatal striatal cultures (wong et al ., 1999; geldwert et al ., 2006; mizuno et al ., 2007) and electrophysiology studies have demonstrated that presynaptic dopamine receptors on msn terminals could modulate gabaergic inhibitory postsynaptic currents (ipscs; delgado et al ., 2000; interestingly, in one of these studies it was shown that while the d2r agonist quinpirole predominantly mediated inhibition of autaptic connections in msns, the d1r agonist skf 38393 mediated either inhibition or facilitation (geldwert et al ., 2006). Although the authors did not directly address the seemingly discrepant effects of the d1r agonist on gaba transmission, it has been shown that skf 38393 activates two biologically different signaling pathways, the camp pathway and phospholipase c (plc)-phosphoinositide (pi) pathway (undie and friedman, 1990; undie et al ., 1994). Although it has been suggested that these two pathways are linked directly to d1r (undie et al ., 1994), it is now known that in msns that coexpress d1r and d2r, the receptors can form a heteromeric complex, the dopamine d1d2 receptor heteromer, and it is this complex that directly activates the plc - pi pathway (lee et al ., 2004; rashid et al ., 2007). Dopamine receptors exist as receptor homomers and can additionally form heteromeric receptor complexes that often exhibit pharmacological and cell signaling properties distinct from their constituent receptors (lee et al ., 2004; rashid et al ., 2007; a physical interaction between the d1r and d2r was first shown by coimmunoprecipitation from rat and human striatum (lee et al ., 2004), and was the first evidence of a dopamine heteromeric receptor complex in brain . These findings were later confirmed by fluorescence resonance energy transfer (fret) a tool commonly used for the identification of receptor oligomers . Although first performed in cells (so et al ., 2005; dziedzicka - wasylewska et al ., 2006), quantitative fret in situ has now been utilized to verify the presence of dopamine d1d2 receptor heteromers in neonatal cultured rat striatal neurons (hasbi et al ., 2009). Similarly, this method has now been employed to show, in vivo, that in adult rat nac, cp (hasbi et al ., 2009; 2010) and gp (figure 3) the d1r and d2r existed in close <50100 proximity indicative of d1d2 receptor heteromerization . However, the propensity for striatal neurons to exhibit d1d2 heteromers was region - dependent, with the majority (> 90%) of d1r / d2r - coexpressing neuronal cell bodies in nac core and shell showing robust d1d2 heteromer formation, but only ~25% of d1r / d2r neurons expressing the d1d2 heteromer in cp (perreault et al ., 2010) this suggests that d1r and d2r can coexist as homomers in the same cell without forming heteromers . It was also shown in the neuropil of both nac and cp the presence of d1d2 heteromers at presynaptic terminals, a finding that suggests a possible involvement of synaptic d1d2 receptor heteromer in gaba release in both regions . Fluorescence resonance energy transfer (fret), using alexa 488 and alexa 350 was used to identify interactions between endogenous d1r and d2r in neurons of rat globus pallidus . An interaction between the d1r and d2r was evident, with a relatively high mean fret efficiency (efficiency of energy transfer between the donor and acceptor fluorophores) of ~22% . The receptor antibody - linked fluorophores were calculated to be in close proximity with a relative distance of 57 nm (5070) indicative of d1d2 receptor heteromer formation . The role of the d1d2 heteromer in vivo is just beginning to be elucidated, however several lines of evidence have emerged implicating the complex as a potential therapeutic target in disorders involving elevated dopamine transmission, such as schizophrenia and drug addiction . Specifically, it has been postulated that abnormal regulation of calcium signaling may constitute the central dysfunction that is responsible for generating the psychopathology of schizophrenia (lidow, 2003). Although the d1r and d2r have not been shown individually to be directly coupled to calcium signaling, coactivation of both receptors within the dopamine d1d2 receptor heteromer by concurrent administration of d1r and d2r agonists, or the selective d1d2 heteromer agonist skf 83959, resulted in a novel gq - plc - linked increase in intracellular calcium release (lee et al . In addition, dopamine d1d2 heteromer activation by skf 83959 also induced striatal calcium calmodulin kinase ii (camkii) phosphorylation (rashid et al ., 2007; ng et al ., 2010) and brain - derived neurotrophic factor (bdnf) expression (hasbi et al ., 2009) two proteins that have been linked to schizophrenia (weickert et al ., 2003; issa et al ., 2010; jindal et al ., 2010; novak and seeman, 2010; wong et al ., 2010; carlino et al .,, an upregulation of striatal d1d2 heteromeric activity was seen following repeated amphetamine treatment in rats and in human gp of patients with schizophrenia (perreault et al ., 2010). Given that amphetamine sensitization is often employed as an animal model for schizophrenia (featherstone et al ., 2007), but no common biochemical marker linking the two has previously been documented, it was inferred that the sensitized state of the d1d2 heteromer may provide the first neuropharmacological correlate between increased dopamine neurotransmission and its functional consequence . As antipsychotics invariably also target the d1d2 heteromer, these findings strongly suggest that further research examining the d1d2 receptor heteromer as a potential drug target in schizophrenia is warranted . In addition to an involvement in schizophrenia, camkii and bdnf have also been shown to play a major role in cocaine addiction (anderson et al ., 2008;, 2010; wang et al ., 2010). In one particularly compelling study, it was shown that cocaine reinstatement in rats depended upon an interaction between nac dopamine and glutamate systems that was mediated by camkii (anderson et al ., 2008). More specifically, cocaine reinstatement increased activation of nac shell camkii, which subsequently led to the phosphorylation of the glua1 subunit of the ampa receptor at ser, and increased cell - surface expression of glua1-containing ampa receptors . Interestingly, in contrast to the effects of acute selective activation of the d1d2 heteromer in nac, more prolonged selective activation has been reported to reduce total camkii levels in this region and additionally reduce phosphorylation of glua1 at ser (perreault et al ., 2010), a finding effectively linking the d1d2 heteromer to the reward pathways of the brain . Dopamine receptors exist as receptor homomers and can additionally form heteromeric receptor complexes that often exhibit pharmacological and cell signaling properties distinct from their constituent receptors (lee et al ., 2004; rashid et al ., 2007; a physical interaction between the d1r and d2r was first shown by coimmunoprecipitation from rat and human striatum (lee et al ., 2004), and was the first evidence of a dopamine heteromeric receptor complex in brain . These findings were later confirmed by fluorescence resonance energy transfer (fret) a tool commonly used for the identification of receptor oligomers . Dziedzicka - wasylewska et al ., 2006), quantitative fret in situ has now been utilized to verify the presence of dopamine d1d2 receptor heteromers in neonatal cultured rat striatal neurons (hasbi et al ., 2009). Similarly, this method has now been employed to show, in vivo, that in adult rat nac, cp (hasbi et al ., 2009; perreault et al ., 2010) and gp (figure 3) the d1r and d2r existed in close <50100 proximity indicative of d1d2 receptor heteromerization . However, the propensity for striatal neurons to exhibit d1d2 heteromers was region - dependent, with the majority (> 90%) of d1r / d2r - coexpressing neuronal cell bodies in nac core and shell showing robust d1d2 heteromer formation, but only ~25% of d1r / d2r neurons expressing the d1d2 heteromer in cp (perreault et al . This suggests that d1r and d2r can coexist as homomers in the same cell without forming heteromers . It was also shown in the neuropil of both nac and cp the presence of d1d2 heteromers at presynaptic terminals, a finding that suggests a possible involvement of synaptic d1d2 receptor heteromer in gaba release in both regions . Fluorescence resonance energy transfer (fret), using alexa 488 and alexa 350 was used to identify interactions between endogenous d1r and d2r in neurons of rat globus pallidus . An interaction between the d1r and d2r was evident, with a relatively high mean fret efficiency (efficiency of energy transfer between the donor and acceptor fluorophores) of ~22% . The receptor antibody - linked fluorophores were calculated to be in close proximity with a relative distance of 57 nm (5070) indicative of d1d2 receptor heteromer formation . The role of the d1d2 heteromer in vivo is just beginning to be elucidated, however several lines of evidence have emerged implicating the complex as a potential therapeutic target in disorders involving elevated dopamine transmission, such as schizophrenia and drug addiction . Specifically, it has been postulated that abnormal regulation of calcium signaling may constitute the central dysfunction that is responsible for generating the psychopathology of schizophrenia (lidow, 2003). Although the d1r and d2r have not been shown individually to be directly coupled to calcium signaling, coactivation of both receptors within the dopamine d1d2 receptor heteromer by concurrent administration of d1r and d2r agonists, or the selective d1d2 heteromer agonist skf 83959, resulted in a novel gq - plc - linked increase in intracellular calcium release (lee et al ., 2004; in addition, dopamine d1d2 heteromer activation by skf 83959 also induced striatal calcium calmodulin kinase ii (camkii) phosphorylation (rashid et al ., 2007; ng et al ., 2010) and brain - derived neurotrophic factor (bdnf) expression (hasbi et al ., 2009) two proteins that have been linked to schizophrenia (weickert et al ., 2003; issa et al ., 2010; jindal et al ., 2010; novak and seeman, 2010; wong et al ., 2010; carlino et al ., 2011). Finally, of particular relevance, an upregulation of striatal d1d2 heteromeric activity was seen following repeated amphetamine treatment in rats and in human gp of patients with schizophrenia (perreault et al ., 2010). Given that amphetamine sensitization is often employed as an animal model for schizophrenia (featherstone et al ., 2007), but no common biochemical marker linking the two has previously been documented, it was inferred that the sensitized state of the d1d2 heteromer may provide the first neuropharmacological correlate between increased dopamine neurotransmission and its functional consequence . As antipsychotics invariably also target the d1d2 heteromer, these findings strongly suggest that further research examining the d1d2 receptor heteromer as a potential drug target in schizophrenia is warranted . In addition to an involvement in schizophrenia, camkii and bdnf have also been shown to play a major role in cocaine addiction (anderson et al ., 2008; lobo et al ., 2010; mcginty et al ., 2010; wang et al ., 2010). In one particularly compelling study, it was shown that cocaine reinstatement in rats depended upon an interaction between nac dopamine and glutamate systems that was mediated by camkii (anderson et al ., 2008). More specifically, cocaine reinstatement increased activation of nac shell camkii, which subsequently led to the phosphorylation of the glua1 subunit of the ampa receptor at ser, and increased cell - surface expression of glua1-containing ampa receptors . Interestingly, in contrast to the effects of acute selective activation of the d1d2 heteromer in nac, more prolonged selective activation has been reported to reduce total camkii levels in this region and additionally reduce phosphorylation of glua1 at ser (perreault et al ., 2010), a finding effectively linking the d1d2 heteromer to the reward pathways of the brain . It is now known that the agonist skf 83959 directly activates calcium signaling via the dopamine d1d2 receptor heteromer, but does not activate the d1r camp pathway or the gi - coupled d2r (rashid et al ., 2007). Therefore findings from studies assessing the effects of skf 83959 would not likely have been derived from functional interactions between d1r or d2r homomers within the same neuron, or between the d1r - expressing striatonigral and d2r - expressing striatopallidal populations of msns . However, for many years it was believed that the effects of the agonist skf 83959 were mediated through its actions solely at the d1r . Reports had demonstrated that skf 83959 had a substantial affinity for the d1r (andringa et al ., 1999; neumeyer et al ., 2003), however the physiological actions of skf 83959 at the d1r were the subject of much debate as the drug exhibited antagonistic properties on adenylyl cyclase activity (arnt et al . 2003) yet mimicked certain behavioral characteristics of d1r agonism such as grooming or the induction of circling behavior (deveney and waddington, 1995; gnanalingham et al ., 1995; waddington et al ., 1995; zhen et al ., 2005; perreault et al ., however, skf 83959 was also linked to plc activation and pi hydrolysis in brain (panchalingam and undie, 2001; jin et al ., 2003; zhen et al ., 2005; rashid et al ., 2007), a pathway that had also been observed to be induced with a number of other d1r agonists in brain tissue (undie et al ., 1994; desai et al ., 2005) but, interestingly, not in cells expressing only the d1r (lin et al ., 1995). Given the ability of d1r agonists to directly induce pi hydrolysis in brain, but not in cells expressing solely the d1r, this suggests that d1r agonists that stimulate this pathway do so through agonist activity at the d1d2 receptor heteromer . For example, the d1r agonists skf 81297 and skf 38393 are still often employed as selective d1r agonists, despite evidence demonstrating their ability to induce behavioral and neurochemical effects characteristic of dopamine d1d2 heteromer activation, such as grooming and activation of plc leading to intracellular calcium release (molloy and waddington, 1987; undie and friedman, 1990; undie et al ., 1994; furthermore, the d5 receptor has also been shown to induce a calcium signal through plc activation or extracellular calcium influx (so et al ., 2009), further confounding the pharmacological profile of these agonists . As such, the interpretation of both past and future results should take into account that activation of individual dopamine receptors by these compounds most likely occurs in tandem with d1d2 receptor heteromer activation on the physiologically relevant subset neurons coexpressing both the d1r and d2r . The appropriate selection of dopaminergic drugs is therefore essential to effectively isolate the specific complex, or signaling pathway, under investigation . For the most part, studies agree that the d1r and d2r are predominantly segregated to discrete populations of msns in striatum and, additionally, that functional cross - talk between the d1r and d2r is critical in mediating some of the physiological effects of dopamine, such as the synergistic effects of d1r / d2r activation on immediate early gene expression . However, there is also an abundance of evidence showing that a proportion of msns in striatum coexpresses the d1r and d2r, and the physiological importance of these neurons is now beginning to become apparent . Specifically, unlike d1r - only and d2r - only striatal msns, d1r / d2r - coexpressing striatal projection neurons have been reported to terminate in regions within both the striatonigral and striatopallidal pathways, as well as regions containing dopamine neuronal cell bodies . These findings, as well as the demonstration of d1r / d2r coexpression at presynaptic terminals, indicate that receptor coexpression may have a unique physiological function at a local level, via msn msn synaptic connections, as well as distal effects through their efferent projections (figure 4). This would likely contribute to the regulation of thalamic neurotransmission, perhaps with the purpose of maintaining homeostatic balance between the direct and indirect pathways . It is possible that this may occur, at least in part, through the regulation of postsynaptic glutamate transmission in the basal ganglia (stefani et al ., 1998), as these neurons exhibit a unique expression phenotype of the ampa glua subunits . In addition, these d1r / d2r msns also express the dopamine d1d2 receptor heteromer, a novel receptor complex that links dopamine receptor activation directly to calcium signaling and bdnf production in vivo . Together, the evidence indicates that msns coexpressing the d1r and d2r in the basal ganglia embody a physiologically relevant, and functionally active, subset of neurons . We propose that d1r / d2r - coexpressing msns represent a third major dopamine receptor neuronal pathway, in addition to the striatonigral and striatopallidal pathways, with the potential to provide a novel approach to drug discovery in basal ganglia disorders . Model of d1 and d2 receptor - coexpressing projections in the basal ganglia circuitry . Previously reported (solid lines; deng et al ., 2006; wang et al ., 2006, 2007) projection sites of d1r / d2r or sp / enk - coexpressing neurons and putative projections of d1r / d2r - dyn / enk neurons (dashed lines), based on the reported regional distribution of neuronal cell bodies and presynaptic d1r and d2r colocalization (perreault et al ., 2010). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
With an increased use of magnetic resonance imaging (mri) for characterization of abnormal tissue, especially with regard to breast lesions, there is a concomitant demand for tissue markers that are visible and relatively harmless in this setting . Tissue markers are commonly used in association with biopsy procedures to mark the location in soft tissue for future surgical procedures . For example, markers can be used to localize a tumor and allow for the monitoring of growth or recurrence of a lesion [13]. Specifically, breast biopsy tissue markers are generally used for tumor localization for subsequent tumor resection [2, 3]. For tissue markers made from metal, there are possible issues related to performing mri procedures in patients with these implants, particularly with the use of high - field - strength (i.e., 3 tesla) mr systems [4, 5]. Therefore, the purpose of this investigation was to assess a newly developed, metallic tissue marker using standardized testing techniques to characterize magnetic field interactions, mri - related heating, and artifacts at 3 tesla . These testing procedures are vital to ensure patient safety in the mri setting when a metallic implant is present [4, 5]. A newly developed, metallic tissue marker (achieve marker, 3-mm length; 12-gauge, 99.9% gold material, carefusion, vernon hills, il) (figure 1) was selected for assessment of mri issues at 3 tesla . The features of this marker include that it is the same size as the biopsy that is obtained and has a configuration (i.e., an irregular surface allowing for growth of tissue around it) that is specially designed to prevent migration once it is deployed in tissue . The marker is supplied in a delivery device that approximates the same size of the biopsy device (achieve biopsy device, carefusion, vernon hills, il), thus, permitting precise placement . Translational attraction was determined at 3 tesla for the tissue marker using the deflection angle method, as previously described [613]. The marker was suspended from a 20-cm length of light - weight string (less than 10% of the weight of the marker) that was attached at the 0-degree position on a protractor that was fixed on a test apparatus [613]. The apparatus was positioned in a 3 tesla mr system (excite, hdx, software 14x.m5, general electric healthcare, milwaukee, wi) at the point of the highest patient accessible spatial gradient magnetic field for the 3-t mr system [613]. The value of the spatial gradient magnetic field at this point was determined using a gaussmeter (extech 480823 electromagnetic field and extremely low frequency meter; extech, nashua, nh) and was found to be 720 gauss / cm, occurring at an off - axis position, 74-cm from isocenter of the mr system [713]. The deflection angle from the vertical position to the nearest 1 degree was measured three times, and the mean value was calculated [613]. Magnetic field - induced torque was determined qualitatively for the tissue marker using a standardized test, as previously described [710, 12]. This test consisted of placing the marker on a flat plastic grid with millimeter markings at a 45-degree orientation relative to the direction of the 3 tesla static magnetic field . The apparatus was then placed at the center of the mr system, where the effect of torque on metallic objects is known to be the greatest [710, 12]. Movement of the marker with respect to rotation relative to the static magnetic field was carefully observed and this procedure was repeated three times to encompass full 360 degrees of rotation for the implant . A mean value of torque was calculated for the marker with it being oriented along its short and long axes, according to the following scale: 0: no torque; + 1: mild or low torque, the implant moved slightly but did not align with the magnetic field; + 2: moderate torque, the marker eventually aligned with the magnetic field; + 3: strong torque, the marker aligned rapidly with the magnetic field; + 4: very strong torque, the implant shows very rapid and forceful alignment with the magnetic field [4, 1115]. The tissue marker was assessed for mri - related heating using a 3 tesla/128-mhz mr system (excite, hdx, software 14x.m5, general electric healthcare, milwaukee, wi). Involved the use of a plastic american society for testing and material (astm) international phantom that was filled 10-cm with gelled - saline (i.e., 1.32-g / l nacl plus 10 g / l polyacrylic acid in distilled water) to simulate human tissue [710, 14]. Using this formulation, the room temperature (22c) electrical conductivity of the gelled - saline the tissue marker was placed in the phantom at a position known to have the highest uniform electric field tangential to the implant to ensure extreme heating conditions based on an analysis of the astm international phantom and the mri conditions used for this evaluation [710, 14]. Because of the lack of perfusion, this testing methodology is an example of extreme mri - related heating for a metallic implant [710, 14]. Temperatures were recorded on the tissue marker using a fluoroptic thermometry system (model 3100, lumasense technologies, santa clara, ca) [710]. Fluoroptic thermometry probes (model sff-2, lumasense technologies, santa clara, ca) were placed on the ends of the marker to obtain representative temperature recordings insofar as the ends of an implant are known to heat the most during mri - related heating [5, 710]. Mri was conducted at 3 tesla using transmit / receive radiofrequency (rf) body coil . The mri parameters were selected to generate an mr system reported, whole body averaged specific absorption rate (sar) of 2.9-w / kg . The land marking position (i.e., the center position or anatomic region for the mri) and section locations were selected to encompass the entire tissue marker . The phantom with the tissue marker was placed in the 3 tesla mr system and given time to equilibrate for more than 24 hours . The room temperature of the mr system was measured before and after the experiment to confirm that no change greater than 0.2c occurred during this time . Proper fluoroptic thermometry probe positioning relative to the marker was confirmed immediately before and after mri . The highest temperature changes recorded for the temperature probes attached to the tissue marker are reported, herein . Background temperatures were also recorded in the gelled - saline - filled astm international phantom by measuring temperatures in the same fluoroptic thermometry probe positions and at the same time intervals as those used when measuring the temperatures for the tissue marker as part of the mri - related heating evaluation [710, 14]. The highest temperature change obtained from this evaluation is also reported . The marker was attached to a plastic frame and placed into a plastic phantom filled with gadolinium - doped, saline, which provided a high signal background [610]. Mri was conducted using transmit / receive rf head coil (i.e., for increased signal - to - noise) and the following pulse sequences: t1-weighted, spin echo, repetition time, 500-msec; echo time, 20-msec; matrix size, 256 256; section thickness, 10-mm; field of view, 24-cm; number of excitations, 2; bandwidth, 16 khz, and gradient echo pulse sequence (gre), repetition time, 100-msec; echo time, 15-msec; flip angle 30; matrix size, 256 256; section thickness, 10-mm; field of view, 24-cm; number of excitations, 2; bandwidth, 16 khz [610]. The imaging planes were positioned to encompass the long axis and short axis of the tissue marker . The image display parameters (i.e., window and level settings, magnification, etc .) Were selected and applied in a consistent manner to obtain accurate measurements of the sizes of the artifacts . Planimetry software provided with the mr system was used to measure (accuracy and resolution 10%) the cross - sectional areas of the largest artifact size for each pulse sequence and imaging plane associated with the tissue marker [610]. Notably, this standardized technique of evaluating artifacts has been used in many prior studies that characterized artifacts for metallic implants and, therefore, provides an acceptable means of comparison [510]. Evaluation of magnetic field interactions for the tissue marker yielded a mean deflection angle of 2 0 degrees . There was no torque (i.e., rotational alignment) exhibited by the marker during the qualitative evaluation (i.e., mean torque value, 0, no torque). The mri - related heating evaluation revealed that the highest change in temperature rise was 1.7c with the tissue marker present in the phantom . By comparison, therefore, the temperature rise associated with the presence of the metallic tissue marker in the phantom was 0.1c . Artifacts appeared as low signal intensity signal voids that were relatively small in relation to the size and shape of the marker, with no apparent distortion of the mr images . The gre pulse sequence (figure 2(a)) produced larger artifacts than the t1-weighted, spin echo pulse sequence (figure 2(b)) for the metallic tissue marker . A tissue marker may be placed in the patient in association with the removal of a tissue sample and acts as a landmark to represent the location of tissue removal for future surgical reference [2, 3, 16]. A biopsy frequently removes the entire lesion; therefore, it has become commonplace to insert a marker so that the site can be easily detected at a later time [2, 3, 1517]. With regard to markers used for breast tissue, in 2013, it was expected that 232,340 new cases of female invasive breast cancer would be diagnosed in the united states . With the prevalence of stereotactic mri and ultrasound - guided biopsies accompanying these diagnoses, there is a need for a tissue marker that is permanently visible when using these modalities, that will not migrate, and does not present a risk to patients . Recently, a new tissue marker was developed with these important features and this implant underwent evaluation in the present investigation . With any metallic implant, concerns arise related to the safety aspects and other potential problems in association with the use of mri [4, 5]. The standard of care when screening patients prior to performing mri is to determine if the individual has a metallic implant and, if that is the case, the mri - specific labeling information must be reviewed (i.e., typically found in the instructions for use for the product) and carefully followed to ensure patient safety . The labeling information presents the particular parameters that are deemed acceptable for a given implant, according to its mr conditional information (i.e., an item that has been demonstrated to pose no known hazards in a specified mr environment with specific conditions of use indicated). Importantly, the conditions stated in the labeling are derived from in vitro mri tests that are conducted to assess the mri issues for the implant [4, 5, 19]. In the present study, a newly developed, metallic tissue marker underwent mri testing using standardized test procedures in consideration of current clinical mr systems, insofar as 3 tesla was utilized for the assessment of magnetic field interactions and a relatively high, whole body averaged sar level was applied during the heating evaluation . While other tissue markers have undergone mri testing [6, 8, 2022], to our knowledge, the evaluation of several of these markers did not follow the accepted standardized tests as recommended by the united states food and drug administration (i.e., following the documents from the astm international). Therefore, the relative risks of using mri in patients with those particular tissue markers are currently unknown . The metallic tissue marker that underwent evaluation exhibited a 2-degree deflection angle and no torque at 3 tesla . Thus, there are no risks associated with movement or displacement of this marker in a 3 tesla or less mri environment . These results are not surprising because the material (gold) used to make this implant has a low magnetic susceptibility value and, therefore, the associated magnetic field interactions will be minor [5, 24]. The exposure of a metallic object to mri has the potential to cause heating under certain conditions [4, 5, 15]. Therefore, as part of proper mri testing that ensures patient safety, a standardized in vitro evaluation is performed to characterize the temperature rises for a metallic implant [4, 5, 15]. The recorded temperature changes for the tissue marker indicated that the highest temperature change was only 0.1c above the highest background temperature when mri was conducted at a relatively high whole body averaged sar level (2.9-w / kg). Obviously, this minimal temperature rise will not pose a risk to the patient with this implant . Although a higher whole body averaged sar level could have been used (i.e., the current upper limit is an sar, 4-w / kg), this was not believed to be necessary because the experimental setup involved a nonperfused gelled - saline medium and, thus, created an extreme case for implant heating . The presence of a metallic implant in a patient undergoing mri will typically cause low intensity signal loss and, in severe cases, distortion of the image [4, 5, 710, 20, 21]. A primary factor that impacts the size of a metal - related artifact is the magnetic susceptibility of the material . The artifacts observed for the tissue marker made from gold (99.99%) appeared as localized signal loss that were relatively small in size in relation to its size and shape (please refer to figures 2(a) and 2(b)). The gradient echo pulse sequence (i.e., representing an extreme condition for mri - related artifacts), which is often used for evaluation of the breast or other tissues, produced larger artifacts than the t1-weighted, spin echo pulse sequence . Notably, an investigation by genson et al . Assessed a range of tissue markers and found that these markers created signal voids that were two to six times the diameter of the marker, which is potentially problematic because subsequent lesion detection may be compromised if the extent of the artifact is too extensive . Because of the low magnetic susceptibility value for gold, which is lower than the other materials that have been previously used for metallic markers, this new marker appears to be ideally suited for mri applications where the extent of the artifact is an important consideration [6, 2022]. Findings from mri tests conducted on a metallic tissue marker indicated that this implant is acceptable or (i.e., using current mri labeling terminology) [19, 24] for a patient undergoing an mri examination at 3 tesla or less . Furthermore, the mri test results for this tissue marker can be applied to other gold markers made from other wire gauges (range 12 to 18 gauges) that have the same or smaller dimensions because the findings will obviously be less than those found for the marker that was tested . This guideline has been applied in prior mri evaluations of other implants including aneurysm clips, hemostatic clips, and otologic implants that have undergone mri testing [7, 12, 25].
Traditionally, post bronchodilator forced expiratory volume in 1 second (pb fev1) is used to assess the severity of chronic obstructive pulmonary disease (copd). However, it has now been shown that functional exercise capacity is a strong predictor of survival following pulmonary rehabilitation and so assessment of functional status is very important for proper prescription of medical therapy and rehabilitation programs . Van stel et al . Suggested that 6 min walk test (6mwt) was an easy to perform procedure and was able to predict functional status in all patients with chronic respiratory disease; however, pinto - plata et al . Argued that walking by itself might not reflect the true functional status in these patients as a person has to do several other activities in addition to walking . A sit - to - stand test (stst) was proposed as a better alternative to 6mwt, but it lacks universal application, more specifically in countries like india where people resort to squat (sit on the floor) rather than sitting on chair . A more practical approach for them will be to assess the functional ability to stand from the squatting position, i.e., squat - to - stand test (sqtst). Therefore, a study was undertaken to compare sqtst with pb fev1, 6mwt, and stst and to assess whether it is a feasible and effective tool in evaluating the functional status of the copd patients . All patients attending the outpatient department of respiratory medicine, national institute of medical sciences, jaipur, with clinical history, consistent with copd, were recruited . Twenty age- and sex - matched, healthy and nonsmoking attendants / hospital staff were also recruited to serve as controls . Was also taken from all the patients and controls after duly explaining the study protocol . All the recruited patients were evaluated in detail, including present and past clinical history, physical examination, complete blood counts, random blood sugar, renal and liver function tests, two sputum smears for acid - fast bacillus using ziehl neelsen staining, skiagram chest posteroanterior view, and a standard electrocardiogram . Patients showing obvious pulmonary or other system abnormalities such as active pulmonary tuberculosis, malignancy, diabetes mellitus, coronary artery disease, stroke, and renal or hepatic disease were excluded . The remaining patients were subjected to spirometry including the reversibility test as per the american thoracic society guidelines using an rms helios spirometer . Three attempts were made, and the best was selected and recorded to obtain the cases of forced vital capacity (fvc), fev1, and fev1/fvc ratio . A repeat spirometry was performed 20 min after inhaling 200 g of salbutamol to obtain pb fvc, fev1, and fev1/fvc ratio . All patients with fev1/fvc ratio <70% and fixed airway obstruction on spirometry (pb improvement in fev1 of <200 ml or in fev1/fvc of <12%) were included in the study, but patients with history of wheeze, chest tightness, eye allergy, nasal allergy, or skin allergy, suggesting bronchial asthma, those suffering from osteoarthritis and oxygen saturation <90%, were also excluded from the study . The body mass index (bmi) of the study patients was calculated as body weight in kg / height in meters square and was classified as follows: underweight <18.5 kg / m, normal 18.525.0 kg / m or overweight> 25 kg / m as per the world health organization criteria . Pack years of smoking was calculated as: number of bidi packs smoked / day number of years smoked (where a bidi pack was calculated as number of bidies/20). The study patients were further subjected to combined risk assessment, which included the modified british medical research council questionnaire, history of exacerbations in the past 2 years, and history of exacerbations needing hospitalization in the past 2 years and categorized as risk category a, b, c, or d as per gold guidelines . All the patients and controls were then subjected to 6mwt, stst, and sqtst (a modified form of stst, used for the first time for rural patients) as follows: the participants were instructed by the command start for them to stand from their squatting position and they then asked to go to their squatting position without any delay, repeating these steps as many times as possible in 1 min at a self - selected speed which was felt safe and comfortable by them or until asked to stop . In case a patient was unable to stand unaided, support of a wide block of one feet height was allowed . The patient's functional status was recorded as (a) unable to stand even with support, (b) able to stand with support only, and (c) number of times he was able to stand in 1 min without support . Since normal ranges of 6mwt in meters (m), stst, and sqtst are not available for universal use, the mean of the respective parameters minus twice the standard deviation in normal controls was used as cutoff for normal values for these parameters . Data so obtained were tabulated and assessed for statistical significance using student's t - test, anova test / test, and fisher's exact test, as and when applicable . Ninety stable copd patients and twenty controls could be studied, between july 2014 and january 2016 . The basic parameters of the study patients and the controls are shown in table 1 . The mean age of the patients in the four categories was similar to that of the controls (p = 0.0059). The mean age was higher in category b patients, but the differences from other categories were statistically insignificant (p = 0.058). Males outnumbered the females in all the risk categories but the sex - wise distribution was also fair (p = 1.000). The duration of illness was higher in category a patients as compared to the rest, but the differences from other categories were statistically insignificant (p = 0.823). Sixty - eight patients were current smokers and the rest 22 were reformed or ex - smokers . There were no differences in the copd categories with regard to the smoking status (p = 0.913), but the mean pack years of smoking was lower in category a patients as compared to the rest (p = 0.914). The mean bmi and pb fev1% were significantly lower in category d patients as compared to the rest (p = 0.000). Basic parameters of the patients and controls the mean of 6mwt, stst, and sqtst in controls were 244 + 30.15 m, 27.05 + 7.89, and 17.8 + 5.38, respectively, so the respective cutoff values were derived as 184 m, 11, and 07 . The mean distance walked by category d patients was lower as compared to the rest (f = 1.47, p = 0.109), but the difference was statistically insignificant . On the contrary, the stst and sqtst values were significantly lower in category d patients as compared to the rest (p = 0.000). Eight patients desaturated (sao2 <90%) at the end of sqtst in group d as compared to 3 and 2 in group c and b, respectively, but all recovered of their own in due course . None of the patients in group a or the controls desaturated . The study of functional status is very vital in assessment of severity of copd as it has been shown to be a strong predictor of survival following pulmonary rehabilitation . 6mwt and more recently stst have been used in functional assessment of copd, but for rural population, squatting is also an important routine activity . This study was, therefore, undertaken to assess the feasibility and efficacy of sqtst in evaluation of the functional status of the copd patients residing in rural areas . Data of this study were found to be valid for statistical comparisons as the controls in the study were age and sex matched, and the distribution of the patients in various risk categories of copd was fair . The mean age of the patients, sex, duration of illness, smoking status, and pack years of smoking did not correlate to severity of copd in this study (p> 0.5). Have reported more dyspnea, lower exercise tolerance, and higher incidence of severe exacerbations in the elderly (p <0.05). Movahed and milne could observe a direct correlation between amount of expectoration / cough and the duration or amount of smoking . Some of these observations, made at variance from the current study, can be explained on the basis of differences in the disease progression in copd in individual patients of different studies . It has been observed that smoking patterns of the cases, such as depth of smoking and the holding time, may play a more crucial role in pathogenesis of copd than the duration or pack years of smoking . Further, it was significantly lower in category d patients as compared to the rest of the risk categories in this study (p = 0.000). Harik - khan et al . Reasoned that the nutritional abnormality and weight loss in copd are caused due to decreased caloric intake and increased basal metabolic rate . Stated that copd produced malnutrition due to loss of fat as well as fat - free components . The mean distance covered in 6 min (6mwt) was significantly lower in copd patients as compared to controls (p = 0.003). Further, it progressively declined with progression of the severity of the disease, but at the cutoff distance of 184 meters, there was a significant overlap in different categories and the correlation was rather poor (p = 0.109). Suggested stst as a superior alternative to 6mwt, the former being less stressful, easier to apply, and more sensitive for the patient's clinical status, compared to the latter . In the present study, further, at a cutoff of 11, all patients in category a were classed as normal and all patients in category d as abnormal . Thus, stst was found to be superior to 6mwt in the present study also . In this study, cutoff, none of the category a patients had abnormal sqtst and none in category d was classed as normal . Further, the mean sqtst value was highest in category a and lowest in category d as compared to rest (p 0.000). Thus, a strong correlation was observed between sqtst and disease severity (p = 0.000). Further, a strong correlation was also observed between sqtst and stst (p = 0.000), but its correlation with 6mwt was moderate (p = 0.004) and to that with fev1%, was poor (p = 0.091). The major limitations of the study are the small number of patients and exclusion of patients with evident hypoxia due to fear of further drop in oxygen saturation . In spite of these limitations, sqtst was found to be an easy, feasible, and effective tool to study the functional status in stable, rural copd patients, as the test is very close to their routine daily activities . Whether the findings of this study are applicable to other population groups who routinely resort to squat or not needs to be studied further . Sqtst is an easy, feasible, and effective tool to study the functional status in stable copd patients of rural india as it is physiologically more close to their routine daily activities . Further, bmi of the patients was observed to be an important surrogate marker of the functional status in these patients.
The use of oral medications offers many advantages, because these medications act directly on the airways and require administration of lower doses, with no gastric changes . 2 using the proper inhaler technique ensures sufficient deposition of drug particles in the distal airways, optimizing drug effectiveness and reducing possible side effects . One of the determinants of the effectiveness of inhaled medications is the ability of the patient to adhere to good inhaler techniques . 3 for some patients, that can be difficult, and the prescription of medication should therefore always be accompanied by appropriate inhaler technique training delivered by a health professional . Thus, it is possible to reduce the number of inhaler technique errors and minimize the clinical consequences of poor drug delivery . The first therapeutic aerosol devices were developed in the 1950s 4 and consisted of nebulizers and atomizers containing anticholinergics for treatment of asthma . 5 despite the long time since development and the wide use of these devices, inhaler technique errors continue to be common among respiratory patients, 6 reducing the benefits of inhaled medications . In chile, 7 observed that only 12.5% of the mothers of hospitalized infants have a correct inhaler technique . However, it is unknown whether this trend is reflected in adult patients, given that the elderly are more likely to make inhaler technique errors . 8 this promotes the assessment of inhaler technique by age group, because tailoring education to the needs of each patient could significantly improve disease management . The purposes of the present study were to assess inhaler technique in pediatric and adult patients with asthma; to determine the most common errors in each group of patients; and to compare the results between the two groups . This was a descriptive cross - sectional study conducted in the region of valparaso, chile, between march and may of 2014 . The sample consisted of male and female patients with a diagnosis of asthma based on spirometry, in accordance with the global initiative for asthma criteria . 9 the ages of the participants ranged from 5 to 90 years, and the sampling was non - probabilistic (purposive). Patients had to meet the following inclusion criteria: being enrolled in and attending follow - up visits as part of an asthma program in clinics in the region of valparaso, regardless of smoking status; having received a prescription for a bronchodilator and having been instructed on the proper use of their inhaler (practical demonstration by a nurse, physician, or kinesiologist at each follow - up appointment); and being able to self - administer inhaled medication . We excluded patients who had a respiratory comorbidity or any concomitant condition that could directly affect inhaler technique (prostration, oxygen dependence, or altered cognitive status). All participants gave written informed consent, and the study was approved by the research ethics committee of the university of santo toms at via del mar school of kinesiology . For comparative purposes, the patients were divided into two groups: pediatric patients (5 - 18 years); and adult patients (19 - 90 years). The volunteers were recruited during their follow - up appointments at the health facilities . On that occasion, they were scheduled to undergo assessment one week later . On the assessment day, they were asked to use their inhaler as usual . The medication administered was albuterol (100 g; fesema(r); laboratorio etex, santiago, chile), used as rescue medication and delivered with an mdi . Inhaler technique was assessed using a protocol described by melani, 10 as shown in table 1 . This protocol documents the performance of ten essential inhaler technique steps by means of closed dichotomous response options (well performed / poorly performed). All assessments were made by two investigators with ten years of experience in the follow - up of asthma patients . After assessment, all patients were given supplemental instruction on inhaler technique by a health professional, in the form of a demonstration . Insert the inhaler into the spacer5 . Hold the inhaler upright with the mouthpiece at the bottom during use6 . Hold breath for 10 seconds on the basis of a study of inhaler technique in pediatric patients, 11 which reported an 89.1% completion rate for " hold breath for 10 seconds ", we calculated that, in order to achieve an alpha of 5%, a statistical power of 80%, and an estimation error of 6%, a sample size of at least 104 patients was required . Allowing for a loss of 10%, we determined that the minimum sample size needed was 115 patients . For data analysis, we used descriptive statistics, calculating the number of errors per patient and the percentage of completion for each step of the protocol . Differences between the percentages of errors made by each group were determined by the equivalence test for two proportions . We excluded seven patients, for the following reasons: prostration (n = 2); oxygen - dependence (n = 2); alzheimer's disease (n = 2); and sequelae of pulmonary tuberculosis (n = 1). The final sample therefore consisted of 263 patients: 135 pediatric patients and 128 adult patients . The general characteristics of the participants are shown in table 2 . In the pediatric group, the most well - represented age group was the 13- to 18-year group, with 63 patients, whereas the 61- to 75-year group was predominant, with 51 patients, in the adult group . Characteristic age, years n male gender,% mean tobacco consumption,% age, years fev1 fvc fev1/fvc pediatric patients 5 - 6 887.56.0 0.582 20100 972 90.07 - 8 1376.97.0 0.581 1598 870 80.09 - 10 2147.69.0 0.581 799 770 90.011 - 12 3050.012.0 0.579 1499 1071 50.013 - 18 6347.614.0 0.579 19101 872 730.0adult patients 19 - 30 1258.323 0.778 10101 1871 825.031 - 45 650.034.0 0.682 9102 1569 1133.346 - 60 2548.051.0 0.680 1099 1970 728.061 - 75 5149.067.0 0.781 1298 1871 929.476 - 90 3452.979.0 0.579 1595 1569 914.7total263100.0 anumber of patients in each age group . The most common errors in the pediatric group were failing to execute a 10-s breath - hold after inhalation (in 8.1%) and failing to continue to inhale after actuation (in 6.1%). In the adult group, 53.1% failed to exhale before using the inhaler, whereas 46% failed to execute a 10-s breath - hold after inhalation . Table 3.frequency and percentages of inhaler technique errors observed in the pediatric and adult groups . Type of error pediatric group adult group n% n% failing to exhale before using the inhaler53.76853.1failing to hold breath for 10 s118.1 * 5946.0failing to take only 1 puff at a time43.03728.0failing to continue to inhale after actuation of the inhaler86.13526.5failing to actuate the inhaler in the first half of inhalation43.03022.7failing to shake the inhaler before use00.02518.9failing to inhale gently and deeply while actuating the inhaler43.01410.6failing to insert the inhaler into the spacer10.7118.6failing to hold the inhaler upright with the mouthpiece at the bottom during use00.021.5anumber of patients who made the error . P <0.001 (equivalence test for two proportions). Number of patients who made the error . 0.001 (equivalence test for two proportions). Table 4 shows the frequency of correct and incorrect inhaler technique, by patient age group . In the 61- to 75- and 76- to 90-year age groups, the frequency of incorrect technique was greatest (48 and 35 patients, respectively). Significant differences were found in the frequency of incorrect technique between the pediatric and adult groups . Age (years) correct technique incorrect technique pediatric patientsnn5 - 6 537 - 8 1219 - 10 14711 - 12 191113 - 18 4914%73.426.6 adult patients 19 - 30 9331 - 45 0646 - 60 12461 - 75 34876 - 90 135%9.490.6 p <0.05 (equivalence test for two proportions). The results of the present study show that most of the pediatric patients used correct inhaler techniques . The most common errors were failing to execute a 10-s breath - hold after inhalation (in 8.1%) and failing to continue inhaling after actuation of the device (in 6.1%). Among the adult patients, the most common errors were failing to exhale before using the inhaler (in 53.1%) and failing to execute a 10-s breath - hold after inhalation (in 46%). 11 stated that poor inhaler technique in older patients might be due to cognitive impairment and their inability to retain the instructions received from the medical team . It is important to point out that, although the protocol used in our study is a guide for correct inhaler technique in adult patients, we found that pediatric patients appear to have better inhaler technique . 12 studies of inhaler technique have established that the most common errors are, in order of incidence, as follows: poor coordination between actuation and inhalation; an insufficient breath - hold after inhalation; excessive inhalation flow; failing to shake the canister vigorously before use; failing to continue to inhale after actuation; pressing the canister down several times during the course of a single inhalation; exhaling during actuation; and failing to hold the inhaler upright . 13 other authors have observed that the rate of inhaler technique errors decreases when devices other than mdis are used . 14 however, drug delivery effectiveness is similar when inhaler technique is correct, 15 regardless of the device used . 6 10 under this criterion, the results of our study allow us to state that the most frequent error made by the pediatric patients (made by 13 subjects) would moderately affect drug deposition in the lung, whereas the most frequent error made by the adult patients (made by 90 subjects) would slightly affect this deposition . As for specific consequences, the poor inhaler technique observed in the two groups of patients can affect drug delivery to the distal airways and prevent drug deposition on the respiratory epithelium . The clinical implication of these results is that, in patients with poor inhaler technique, there is a waste of inhaled medication . Consequently, there would be an increase in the economic costs associated with the disease, an increase in the risk of side effects, and a reduction in treatment effectiveness . 18 all participants in our investigation regularly attend their follow - up appointments, and, at each such opportunity, they are instructed in the proper use of their medications . These mistakes are considered either unintentional (patients not noticing that their inhaler technique is poor) or intentional (patients knowingly using the incorrect inhaler technique). 18 in addition, there is clear evidence that inhalers are underused among asthma patients . 19 in this aspect, one of the limitations of our study was that we did not delve into the causes of the observed errors, which would have made it possible to provide each patient with specific supplemental instruction on the proper administration of the medication . In our sample, we found that approximately 30% of the adult patients were smokers . Although this proportion is lower than observed among adults in chile (40.6%), 20 it is significant, given that smoking affects asthma control . 21 therefore, tobacco consumption in these patients would further increase the difficulty in controlling the disease . Recent studies suggest that better results would be obtained by tailoring inhaler prescription to suit the characteristics and functional capabilities of each patient . 22 it has been observed that even individuals with correct inhaler technique can make errors if they are reassessed over time, 23 which makes it mandatory to provide patients with ongoing education in the administration of inhaled medications . Many times there are factors that hinder this learning process, such as limited duration of appointments, a lack of knowledge on the part of health care personnel about the correct steps of the inhaler technique, and the technical language used in teaching the technique . 16 therefore, it is necessary to use new methods to provide patients with supplemental instruction on correct inhaler use, such as videos or illustrative leaflets that can promote the retention of information by patients . 24 in addition, it is necessary that supplemental instruction on inhaler use protocols be properly provided to health personnel and included in asthma clinical guidelines, which rarely address the administration of inhaled medications . 25 in conclusion, we found that most pediatric asthma patients appear to have correct inhaler technique . However, the same does not seem to be true for approximately 90% of adult patients, among whom the most common error was failing to exhale before using the inhaler . We suggest that asthma patients, especially those who are older, should be given supplemental instruction on inhaler technique through the use of new methods, so that they can administer their medications properly . La tcnica inhalatoria es un conjunto de procedimientos mediante el cual se administra un frmaco al sistema respiratorio . Se caracteriza por ser utilizada como primera lnea para tratar las enfermedades pulmonares . Su correcta ejecucin garantiza un mayor depsito del frmaco en la va area distal, optimizando sus efectos teraputicos y disminuyendo los efectos secundarios . Los objetivos de este estudio son describir la ejecucin de la tcnica inhalatoria en un grupo de pacientes asmticos peditricos versus un grupo de pacientes asmticos adultos, definir los errores ms comunes en cada grupo de pacientes y comparar los resultados entre ambos grupos . Se evalu la tcnica inhalatoria segn un protocolo de diez pasos en 135 pacientes asmticos peditricos y 128 pacientes asmticos adultos . Se encontr que el error ms comn en los pacientes peditricos fue no realizar una apnea de 10 s despus de la inhalacin, mientras que en los pacientes adultos el principal error fue no exhalar completamente antes de aplicar el inhalador . Se determin que los pacientes asmticos peditricos cumplen con la mayora de los pasos para una correcta tcnica inhalatoria, lo que no se observa en los pacientes adultos . La tcnica inhalatoria (ti) es un conjunto de procedimientos mediante el cual se administra un frmaco al sistema respiratorio . Se caracteriza por ser utilizada como primera lnea para tratar las enfermedades respiratorias, siendo el inhalador de dosis medida (idm) uno de los dispositivos comnmente utilizados por los pacientes . 1 el uso de medicamentos inhalados ofrece muchas ventajas, ya que ellos actan directamente en la va area y requieren menor dosis en su administracin, con ausencia de alteraciones a nivel gstrico . 2 la correcta ejecucin de la ti permite un mayor depsito de partculas en la va area distal, mejorando la eficiencia del frmaco y disminuyendo los posibles efectos adversos . Uno de los factores determinantes de la eficiencia del medicamento inhalado es la manera en la que el paciente realiza la ti . 3 para algunos pacientes puede resultar difcil la ejecucin de sta, por lo que al momento de prescribir el medicamento debe acompaarse siempre de un adecuado entrenamiento en la ti por parte de un profesional . De esta manera se logra reducir el nmero de errores cometidos durante la ejecucin de la ti y aminorar las consecuencias clnicas de una mala administracin . Los primeros aerosoles teraputicos fueron creados durante la dcada de 1950, 4 que consistieron en nebulizadores y atomizadores con frmacos anticolinrgicos para tratar el asma . 5 a pesar del tiempo transcurrido desde su creacin y su amplia utilizacin, los errores en la ti siguen siendo comunes entre los pacientes respiratorios, 6 menoscabando los beneficios del medicamento inhalado . En chile, sols et al . 7 observaron que slo un 12,5% de las madres de lactantes hospitalizados ejecuta correctamente la ti . Sin embargo, se desconoce si esta tendencia se mantiene en los pacientes adultos, considerando que en la tercera edad existe un riesgo ms alto de cometer errores en la ti . 8 este hecho motiva a investigar de qu manera realizan la ti los pacientes segn grupo etario, considerando que al adaptar la educacin a las necesidades de cada paciente podra mejorar considerablemente el manejo de la enfermedad . Los objetivos del presente estudio son describir la ejecucin de la ti en un grupo de pacientes asmticos peditricos versus un grupo de pacientes asmticos adultos, definir los errores ms comunes en cada grupo de pacientes y comparar los resultados entre ambos grupos . Estudio de tipo descriptivo transversal, realizado en la regin de valparaso, chile, entre los meses de marzo y mayo del ao 2014 . La muestra estuvo constituida por pacientes asmticos de ambos sexos diagnosticados mediante espirometra segn los criterios establecidos por la global initiative for asthma (gina). 9 las edades de los participantes estuvieron comprendidas entre 5 y 90 aos, quienes fueron seleccionados de forma no probabilstica (intencional). Los pacientes debieron cumplir los siguientes criterios de inclusin: pacientes asmticos inscritos y controlados en consultorios pertenecientes a la regin de valparaso, fumadores o no fumadores, con indicacin mdica de broncodilatador e instruidos previamente en el uso correcto de su inhalador (demostracin prctica en cada control por parte de la enfermera, mdico o kinesilogo), capaces de auto realizarse la ti . Se excluyeron pacientes que tuviesen comorbilidad respiratoria o alguna condicin asociada que interfiera directamente con la ejecucin de la ti (postracin, dependencia de oxgeno, estado cognitivo alterado). Todos los participantes firmaron un consentimiento informado aprobado por el comit de tica perteneciente a la escuela de kinesiologa de la universidad santo toms, sede via del mar . Para efectos comparativos se dividieron en dos grupos: pacientes peditricos (5 - 18 aos) y pacientes adultos (19 - 90 aos). Los voluntarios fueron reclutados cuando asistieron a sus controles en sus respectivos centros de salud . En esa oportunidad se les cit a evaluacin una semana despus . El da de evaluacin se les solicit que ejecutaran la ti de la manera habitual . Todos los voluntarios utilizaron su espaciador personal con vlvulas (adecuado segn la edad del paciente). El medicamento administrado correspondi al idm que usaban de rescate (salbutamol, 100 g; fesema; laboratorio etex, santiago, chile). Durante la evaluacin, se observ la ejecucin de la ti segn el protocolo descrito por melani, como se muestra en la tabla 1 . Este protocolo registra la realizacin de los diez pasos fundamentales de la ti mediante preguntas cerradas con respuesta dicotmica (bien ejecutado / mal ejecutado). Todas las observaciones fueron realizadas por dos evaluadores, con diez aos de experiencia en control de pacientes asmticos . Realizar una apnea de 10 segundos posterior a la evaluacin, todos los pacientes fueron reforzados en la correcta ejecucin de la ti, mediante una demostracin por parte del profesional de salud . Basados en un estudio de ti en pacientes peditricos 11 en el que se reporta un 89,1% de cumplimiento para el tem " realizar apnea de 10 segundos ", con un alfa del 5%, una potencia estadstica del 80% y un error de estimacin del 6%, se calcul un tamao muestral necesario para el presente estudio de al menos 104 pacientes . Considerando un 10% de prdida, se estim un tamao mnimo muestral de 115 pacientes . Para el anlisis de los datos se utiliz estadstica descriptiva, calculando el nmero de errores por cada paciente y el porcentaje de cumplimiento de cada paso del protocolo . Las diferencias entre los porcentajes de errores cometidos por cada grupo se obtuvieron mediante el test de comparacin de dos proporciones . El nmero total de pacientes seleccionados fue de 270 . Se excluyeron dos pacientes postrados, dos pacientes oxgeno dependientes, dos pacientes con diagnstico de alzheimer y un paciente con secuelas de tuberculosis pulmonar . Las caractersticas generales de los participantes se observan en la tabla 2 . En el grupo peditrico el mayor nmero de sujetos (n = 63) corresponde a pacientes con edades entre 13 y 18 aos . En el grupo de pacientes adultos el mayor nmero de sujetos (n = 51) se encuentra entre los 61 y 75 aos . Caractersticas edad, aos n sexo masculino,% promedios consumo de tabaco,% edad, aos vef1 cvf vef1/cvf pacientes peditricos 5 - 6 887,56,0 0,582 20100 972 90,07 - 8 1376,97,0 0,581 1598 870 80,09 - 10 2147,69,0 0,581 799 770 90,011 - 12 3050,012,0 0,579 1499 1071 50,013 - 18 6347,614,0 0,579 19101 872 730,0pacientes adultos 19 - 30 1258,323 0,778 10101 1871 825,031 - 45 650,034,0 0,682 9102 1569 1133,346 - 60 2548,051,0 0,680 1099 1970 728,061 - 75 5149,067,0 0,781 1298 1871 929,476 - 90 3452,979,0 0,579 1595 1569 914,7total263100,0 anmero de pacientes segn rango de edad . Valores expresados en media de . Porcentaje del valor predicho . En la tabla 3, se observan los tipos de errores cometidos por el grupo de pacientes peditricos y el grupo de pacientes adultos . Los errores ms comunes en el grupo de pacientes peditricos fueron no realizar la apnea de 10 segundos despus de inhalar (en 8,1%) y no continuar inhalando despus de pulsar el idm (en 6,1%). En el caso de los pacientes adultos se observa que un 53,1% no exhala antes de aplicar su inhalador, mientras que un 46% no realiza una apnea de 10 segundos posterior a la inhalacin . Tabla 3.frecuencia y porcentajes de los errores en la tcnica inhalatoria observados en el grupo de pacientes peditricos y en el grupo de pacientes adultos . Tipo de error grupo peditrico grupo adulto n% n% no exhalar antes de aplicar el inhalador53,76853,1no realizar apnea de 10 segundos118,1 * 5946,0no administrar slo 1 puff a la vez43,03728,0no continuar inhalando despus de activar el inhalador86,13526,5no activar el inhalador en la primera mitad de la inhalacin43,03022,7no agitar el inhalador antes de usarlo00,02518,9no inhalar suave y profundamente mientras activa el inhalador43,01410,6no posicionar correctamente la aerocmara10,7118,6no sostener el inhalador vertical, con la boquilla hacia abajo durante el uso00,021,5anmero de pacientes que cometieron el error sealado . p <0,001 (test de comparacin de dos proporciones). En la tabla 4 se observa la frecuencia de entre los rangos de 61 a 75 y de 76 a 90 aos de edad se observan la mayor cantidad de maniobras incorrectas de inhalacin (48 y 35 maniobras, respectivamente). Se verificaron diferencias significativas entre el porcentaje de ejecuciones incorrectas de los grupos peditrico y adulto . Edad (aos) ejecucin correcta ejecucin incorrecta pacientes peditricosnn5 - 6 537 - 8 1219 - 10 14711 - 12 191113 - 18 4914%73,426,6 * pacientes adultos 19 - 30 9331 - 45 0646 - 60 12461 - 75 34876 - 90 135%9,490,6 * * p <0,05 (test de comparacin de dos proporciones). Los resultados obtenidos del estudio muestran que la mayora de los pacientes peditricos ejecutan la ti de manera correcta . Los errores ms comunes fueron no realizar la apnea de 10 segundos (en 8,1%) y no continuar inhalando despus de activar el dispositivo (en 6,1%). Entre los pacientes adultos, los errores ms comunes fueron no exhalar antes de activar el inhalador (en 53,1%) y no realizar una apnea de 10 segundos despus de la inhalacin (en 46%). Crompton et al.relatan que la deficiente calidad de la ti observada en los pacientes mayores puede estar determinada por un deterioro cognitivo y su incapacidad para retener las instrucciones recibidas desde el equipo mdico . Es importante indicar que el protocolo utilizado en nuestro estudio est orientando a la correcta inhalacin del paciente adulto, y a pesar de ello se observa que los pacientes peditricos son los que mejor ejecutan la ti . 12 estudios sobre la ti establecen que los errores ms comunes por orden de incidencia son: mala coordinacin entre pulsacin del dispositivo e inspiracin; periodos de apnea tras la maniobra demasiados cortos; flujo inspiratorio excesivo; no agitar bien el cartucho antes de usarlo; interrumpir la inhalacin despus de la activacin; presionar el cartucho varias veces durante una nica maniobra respiratoria; exhalar durante el disparo y no colocar el inhalador en posicin vertical . 13 otros estudios han observado que la tasa de errores en la ti disminuye al utilizar un dispositivo distinto al idm . 14 sin embargo, la eficacia en la entrega del medicamento es similar cuando la ti se ejecuta correctamente, 15 independiente del dispositivo utilizado . Una mala ejecucin de la ti trae consigo consecuencias clnicas que van desde pequeas hasta crticas . 6 10 bajo este criterio, los resultados observados en nuestro estudio permiten afirmar que la mayor frecuencia (13 sujetos) de los errores cometidos por el grupo de pacientes peditricos incidira moderadamente en el depsito del medicamento en el pulmn, mientras que en los pacientes adultos la mayor frecuencia (90 sujetos) de los errores cometidos incidira levemente en este depsito . Respecto a las consecuencias especficas, la mala ti observada en ambos grupos de pacientes puede afectar el ingreso del medicamento a la va area distal e impedir su adherencia al epitelio respiratorio . 16 las implicancias clnicas de estos resultados indican que en los pacientes con mala ti se produce un desperdicio del medicamento inhalado . En consecuencia aumentara el costo econmico asociado a la enfermedad, incrementara el riesgo de sufrir efectos secundarios y disminuira la efectividad del tratamiento . La educacin de los pacientes respiratorios es un factor crtico en el correcto uso de sus frmacos . 17 los programas de educacin al paciente asmtico mejoran sustancialmente la adherencia y la ti . 18 todos los participantes de nuestra investigacin asisten regularmente a sus controles de salud y en cada oportunidad se les enfatiza el uso correcto de sus medicamentos . A pesar de ello, los resultados muestran que persisten errores en la utilizacin del inhalador . Estas equivocaciones se consideran involuntarias, cuando el paciente no advierte que la ti est mal ejecutada, o intencionales, cuando el paciente conoce la ti correcta pero no la ejecuta por omisin . 18 adems, existe evidencia de una clara subutilizacin de estos dispositivos entre los pacientes asmticos . 19 en ese aspecto, una de las limitaciones de nuestro estudio fue no profundizar en las causas de los errores observados, lo que hubiese permitido reforzar de manera ms especfica en cada paciente la correcta administracin del medicamento . A pesar que esta cifra es inferior al porcentaje de poblacin adulta fumadora en chile (40,6%), 20 es un porcentaje importante de pacientes considerando que el hbito tabquico interfiere en el control del asma . 21 por lo anterior, el consumo de tabaco en estos pacientes agregara una mayor dificultad al control de la enfermedad . Estudios recientes sugieren que se obtendran mejores resultados si la prescripcin de inhaladores se ajustara a cada paciente segn sus caractersticas y capacidades funcionales . 22 se ha observado que incluso personas que realizan correctamente la ti pueden cometer errores nuevamente al ser reevaluadas en una segunda instancia, 23 lo que obliga a educar constantemente a nuestros pacientes en la administracin de sus medicamentos inhalados . Existen factores que muchas veces dificultan este proceso de aprendizaje, entre ellos el escaso tiempo que existe para atender a cada paciente, el desconocimiento de los pasos correctos de la ti por parte del personal de salud y el lenguaje tcnico que se utiliza para ensear . 16 por lo anterior, es necesario utilizar nuevas metodologas para reforzar el correcto uso de los inhaladores, por ejemplo videos o folletos ilustrativos que promuevan la retencin de informacin en el paciente, 24 adems de reforzar el protocolo correctamente en el personal de salud y en las guas clnicas de asma que escasamente hacen referencia a la administracin de medicamentos inhalados . 25 como conclusin se determin que la mayora de los pacientes asmticos peditricos realiza la ti de manera correcta, mientras que aproximadamente el 90% de los pacientes adultos la ejecuta de manera incorrecta, siendo el error ms comn no exhalar antes de aplicar el inhalador . Se sugiere reforzar en los pacientes asmticos, especialmente en los de mayor edad, la ti a travs de nuevos mtodos para lograr una correcta administracin de sus medicamentos.
The success of radiotherapy in eradicating a tumor depends mainly on the total radiation dose given, but the tolerance of the normal tissues surrounding the tumor limits this dose . There is a significant variation between patients regarding the severity of toxicity following a given dose of radiotherapy . As a consequence, chronic side effects from radiotherapy are often irreversible and can decrease health - related quality of life as well as limit treatment intensity in radical radiotherapy regimens . Quantification of acute and chronic toxicity is therefore crucial in the assessment of the therapeutic benefit of radiotherapy . Various studies worldwide have attempted to identify common genetic variations associated with the development of radiation toxicity as a step in the process of identifying such a subset of toxicity prone patients [2, 3]. Studies among prostate cancer patients have identified single nucleotide polymorphisms (snp) in candidate genes, mostly involved in the dna damage response, including ataxia - telangiectasia mutated (atm), transforming growth factor beta 1 gene (tgfb1), x - ray repair cross - complementing protein 1 gene (xrcc1), x - ray repair cross - complementing protein 3 gene (xrcc3), and superoxide dismutase 2 gene (sod2) among others that are associated with the development of toxicity in response to radiation therapy [47]. One study using a genome - wide approach has also identified variants associated with the development of long term side effects of radiation therapy . Polymorphisms in genes responsible for dna damage signaling and repair might modulate dna repair capacity and, therefore, affect cell and tissue response to radiation and influence individual radiosensitivity [9, 10]. The tumor suppressor protein p53 encoded by the tumor protein 53 gene (tp53), a key regulator of cellular responses to genotoxic stress, is stabilized and activated after dna damage . The rapid activation of p53 by ionizing radiation is largely dependent on the atm kinase . The protein p53 is phosphorylated by atm shortly after dna damage, resulting in enhanced stability and activity of p53 [12, 13]. The murine double minute 2 (mdm2) oncoprotein is a pivotal negative regulator of p53 . In response to ionizing radiation the increase in mdm2 basal levels induces degradation and blockade of transcriptional activity of p53 protein [14, 15]. The functional p53 polymorphism, characterized by c> g change at the second position of codon 72 (rs1042522), results in arg> pro amino acid substitution . It has been shown that 72arg allele may induce apoptosis with faster kinetics than pro72 allele . On the other hand, the pro72 variant seems to be more competent in inducing cell cycle arrest and dna repair . Therefore, pro72 could be associated with a lower incidence of side effects to radiotherapy by being more efficient in cell cycle arrest allowing repair of radiation - induced damage . The p53 polymorphism at codon 47 (rs1800371) is also functionally significant, since it has been demonstrated that the ser47 variant has a decreased ability to induce apoptosis compared with the pro47 p53 allele . The tp53 pin3 insertion (rs17878362) was chosen based on a study by tan et al . 2006, which reported no association between this insertion and the development of radiation toxicity; however, we found that it was important to confirm the lack of association in our study population . The remaining intronic polymorphisms (rs35117667 and rs17883323) were selected because they are located in nearby regions to the studied polymorphisms and also because they have not been evaluated by any study so far . The mdm2 single - nucleotide polymorphism t309 g (rs2279744) is located in the promoter region of mdm2 . As demonstrated in a different study, it increases the promoter affinity for the stimulatory protein (sp) 1, intensifying mdm2 expression, and subsequent attenuation of the p53 pathway . We hypothesized that the increased degradation of p53 by mdm2 could lead to adverse effects of radiation therapy . Mutations and polymorphisms of the atm gene have been investigated as possible causes of normal tissue radiation sensitivity in breast cancer patients . Breast cancer patients undergoing radiotherapy presented with a higher incidence of adverse chronic effects (fibrosis) when the exon 39 polymorphism d1853n (rs1801516) were present . Atm protein presents kinase activity induced by ionizing radiation, and it is involved in dna damage detection and in the control of cell cycle progression . The purpose of this study was to examine the hypothesis that the presence of atm codon asp1853asn (5557g> a) polymorphism (rs1801516), tp53 pro72arg (rs1042522), tp53 pro47ser (rs1800371), 3 intronic tp53 polymorphisms (rs17878362, rs17883323, and rs35117667), or mdm2 309t> g polymorphism (rs2279744) (table 1) is predictive for the development of adverse radiotherapy responses among prostate cancer patients . It was hypothesized that polymorphisms in these genes would influence dna repair capacity, modulating responses to radiation and the risk of acute or chronic radiation - induced side effects . We explored any possible association of acute and chronic skin, urinary, and rectal functional outcomes with atm and tp53 polymorphisms using the radiation therapy oncology group (rtog) morbidity criteria . The present study was performed with patients from the associao de combate ao cncer em gois (accg) who were recruited between january 2009 and december 2010 . The 48 eligible patients had histologically confirmed prostate cancer and underwent conventional external beam radiation therapy with curative intent at the department of radiotherapy of arajo jorge hospital in goinia, gois, brazil . The treatment was performed using a daily dose of two grays and high - energy photons (15 mv) to obtain deeper penetration and better dose uniformity . Patients whose followup was less than 24 months were not included on chronic toxicity evaluations . The study was approved by the internal ethical committee, and all patients agreed to participate in the study by signing an informed consent before the biological sample was collected . The clinical information of the patients, such as treatment type, radiation dose, pretreatment symptoms, age and comorbidities, such as diabetes and vascular disease and radiation - induced injury, were obtained retrospectively from the medical record of each patient . Acute and chronic radiation - related toxicities were graded using the european organization for research and treatment of cancer / radiation therapy oncology group (eortc / rtog) morbidity criteria . Patients who developed rtog grading level 2 or more were classified as having a high grade (hg) adverse response, and those with rtog grading level 1 and level 0 responses were classified as low grade (lg) responses . This study used a candidate gene approach to search for associations between genetic variants such as single nucleotide polymorphisms and radiation treatment toxicity . For genetic analysis, a consent form was presented to the patient prior to the collection of their clinical data and blood sample, and all patients included agreed to sign the consent form for this research study . Dna was isolated from peripheral blood through the iprep purification system developed by invitrogen corporation, ca, according to the manufacturer's protocol . Polymerase chain reaction (pcr) was used to amplify each fragment using specific primers (table 2) designed from the genomic sequence obtained from the national center for biotechnology information (http://www.ncbi.nlm.nih.gov/). The optimal annealing temperature for each primer, pcr was performed at a 50 l reaction mixture containing 100 ng of dna, 10 m of each primer, 0.2 l of accuprime taq high fidelity, and a reaction buffer containing 600 mm tris - so4, 180 mm (nh4)2so4, 20 mm mgso4, and 2 mm of each deoxynucleoside triphosphate (invitrogen, ca). The pcr profile consisted of an initial melting step of 10 minutes at 95c, followed by 40 cycles of 30 seconds at 95c, 30 seconds at 54.3c for atm, at 61.3c for tp53 or at 60.3c for mdm2 amplification, 1 minute at 72c, and a final elongation step of 7 minutes at 72c . The pcr reactions were first subjected to purification using the purelink quick gel extraction and pcr purification combo kit (invitrogen, ca), treated with exo sap - it (usb corporation, cleveland, ohio, usa) to remove dntps and primers and then underwent nucleotide primer extension reaction . The sequencing reactions (bigdye terminator v. 3.1 ready reaction cycle sequencing kit; applied biosystems, foster city, ca) were subjected to purification using bigdye xterminator (applied biosystems, foster city, ca) for a complete removal of ddntps and underwent capillary electrophoresis on an applied biosystems 3130xl dna analyzer . The data were analysed by the seqscape software v2.6 (applied biosystems, foster city, ca). Each polymorphism was tested for deviation from hardy - weinberg equilibrium by comparing the observed and expected genotype frequencies using the chi - square test with one degree of freedom . Analyses were performed using the sigmastat version 15.0 statistical software package (spss, inc ., the association between atm tp53 and mdm2 polymorphisms and adverse response to radiotherapy was estimated by chi - square test or fisher's exact test with one degree of freedom . Of 48 patients, 29.2% had family history of cancer, 47.9% had hypertension, 12.5% were diabetic, and 54.2% had a history of smoking during some time in their lives . A total of 45 patients (93.7%) had tumor gleason score higher than five . 25 patients (52.1%) had mean prostate specific antigen (psa) higher than 10 ng / ml before treatment and in 77.1% of the patients, psa decreased after radiotherapy . Twelve patients (25%) underwent concomitant hormonal therapy, and seven (14.6%) had undergone prior prostatectomy . The average radiation dose in the 50 patients was 70.1 grays (gy), and the treatment lasted for an average of 61.5 days . The patients were classified as having an adverse response, rtog high grade, or not having an adverse response (rtog low grade). Tables 4 and 5 show the number of patients having acute or chronic adverse effects of radiotherapy, respectively . There were no significant differences between patients who developed severe acute or chronic radiation toxicities and those who did not in relation to age, smoking status, gleason score, family history of cancer, hypertension or diabetes, and radiotherapy dose and patients who underwent concomitant hormonal therapy or had prior prostatectomy (p> 0.05). From the initial group of 48 patients eligible for the present study, we analysed polymorphisms in the genes atm, tp53, and mdm2 . For the atm and mdm2 snps, all patients were successfully genotyped . For tp53 gene, we analysed four polymorphisms in the pcr amplified fragment . It was possible to verify the presence or absence of this polymorphism in all 48 patients, while data regarding tp53 polymorphisms, at codon 47 and intronic regions 16250 and 16225, were analysed in 47 patients, and region 16273 of intron three was analysed in 46 patients . For chronic analysis, three patients were excluded because their followup was less than 24 months . The frequencies of polymorphisms for chronic associations are shown in table 7 . In univariate cross - table analysis, the atm asp1853asn polymorphism was not associated with occurrence of either acute or chronic toxicity after radiotherapy (p> 0.05). The intronic polymorphism located on region 16273 with a homozygous change of c> t was significantly associated to the development of acute skin toxicity rtog grade two or higher (or = 0.0072, 95% ci 0.00020.227, p = 0.003) as shown in table 6 . The heterozygous change of a> c on region 16250 was strongly associated to the developing of chronic urinary toxicity rtog grade two or higher (or = 0.071, 95% ci 0.0060.784, p = 0.032). None of the remaining tp53 polymorphisms, including the one located on codon 72, were significantly associated with the presence of acute or chronic skin, gastrointestinal, and urinary toxicities (tables 6 and 7). Our analysis showed, as expected, that clinical features were not determinants of acute or chronic toxicities of the prostate adjacent tissues when submitted to radiotherapy (p> 0.05). These results confirm the importance of investigating the genetic profile of patients undergoing radiotherapy in order to find if genetic differences are involved or not involved in the development of radiation toxicities during and after treatment . There is a belief that the genomic revolution triggered by full sequencing of the human genome and technological development of large - scale methodologies announces the future of personalized medicine . In oncology, this progress should increase the possibility to predict individual patient responses to radiotherapy and chemotherapy . (2002) hypothesized that the radiation sensitivity of normal tissues should be associated with a so - called trait - dependent complex which adds the effect of many genetic determinants and single nucleotide polymorphisms that could correspond in part to the frequency and severity of these components . Some suggestive evidence was obtained from the correlation between the atm codon asp1853asn (5557g> a) snp and the high risk of the development of radiotherapy - induced acute skin complications in breast cancer patients [21, 25, 26]. For prostate cancer, atm sequence alterations including codon 1853 polymorphism were associated with the development of radiation - induced proctitis, although no relation was shown between this marker and bladder or rectal toxicities arising from the radiation therapy in canadian prostate cancer patients . Our findings showed that asp1853asn polymorphism was not associated with the development of any acute or chronic adverse effects in patients with prostate cancer treated with radiotherapy (p> 0.05). Further investigations will be necessary to verify the association of other atm snps in more representative sample groups in order to establish whether single nucleotide polymorphisms or gene haplotypes may actually contribute to the toxicity of normal tissue . The single base polymorphism in the promoter of mdm2 gene (snp309; rs2279744) increases gene expression, and it is shown to associate with accelerated tumor formation in both hereditary and sporadic cancers . The increase of mdm2 basal levels will also increase the degradation of p53 tumor suppressor protein and may stop its transcriptional activity leading to increased apoptosis [14, 15, 28, 29]. The hypothesis that increased apoptosis could cause necrosis of normal tissue leading to radiotherapy side effects was not confirmed by our results . Among the prostate cancer patients of this study, no association of the investigated mdm2 polymorphism (rs2279744) and the incidence of radiotherapy - induced acute and chronic effects were found, thus indicating no major contribution in this cohort of prostate cancer . Another study also showed that the mdm2 snp309 polymorphism was not found to be involved with clinical pathologic variables, recurrence risk, and overall survival outcome in prostate cancer . Many studies have showed the association between the tp53 pro72arg polymorphism and cancer susceptibility [3234]. An association between the risk of acute skin toxicity and tp53 pro72 carriers in those with the cdkn1a 31ser genotype in a subset of normal weight patients treated with radiotherapy for breast cancer has been shown . Another study found evidence that genetic polymorphisms in the atm - p53 pathway can influence susceptibility to developing radiation - induced pneumonitis in lung cancer patients after radiotherapy . Despite the strong hypothesis for involvement of this gene, the association between the pro72arg polymorphism and the development of normal tissue reaction to radiotherapy in prostate cancer patients has not been fully investigated previously . This was the first study to evaluate the acute and chronic radiation toxicities in patients with prostate cancer and tp53 pro72arg polymorphism . No association was found in the studied group, even though 27 (54%) patients presented the combination arg / arg, which has been described as a more predisposed to apoptosis genotype [16, 36]. Another polymorphism in tp53 gene, at codon 47 (rs1800371), is also functionally significant . Codon 47 encodes proline in wild type p53, but in a small subset of individuals it can encode a serine . It was showed that the serine 47 variant has up to 5-fold decreased ability to induce apoptosis compared with wild type p53 . However, on our study, the percentage of patients with this polymorphism was not statistically sufficient to find a relationship with the adverse effects of radiotherapy, even though it is a variant that induces apoptosis . There is no other literature data of association between this polymorphism and toxicity induced by radiation therapy . Our findings showed that a polymorphism located in the region 16273 of intron 3 of tp53 (rs35117667) was associated with high risk of developing acute skin adverse effects (or: 0.0072, 95% ci 0.00020.227, p = 0.003). The present study is the first to observe the frequencies of this polymorphism in a specific population and also the first to associate this polymorphism with radiotherapy . There is also evidence of association between the intronic polymorphism at position 16250 (rs17883323) and chronic high grade toxicity of urinary tract (or: 0.071, 95% ci 0.0060.784, p = 0.032). This polymorphism has not been previously studied in a clinical population, according to searches on scientific websites . Both intronic polymorphisms (rs35117667 and rs17883323) it is now necessary to evaluate the molecular basis of the association between these intronic polymorphisms and the risk for radiotherapy side effects . An intronic polymorphism is not implicated directly in an altered protein; however, it may lead to alternative splicing or it can be related to micrornas [3840]. A protein modified by these processes may be associated, for example, an increase of apoptosis . Gemignani and colleagues in 2004 reported that an insertion / duplication of 16 base pairs in intron 3 of tp53 gene, the so called tp53 pin3 (rs17878362) described by lazar et al . (1993), is associated with an increased risk of developing colorectal cancer (or 1.55, 95% ci 1: 10 to 2: 18, p = 0.012). Tan et al . (2006) reported no association between the tp53 pin3 (rs17878362) insertion and the development of adverse effects of radiotherapy (p> 0.05). (2006), which also found no association between this polymorphism and the risk of toxicity after radiotherapy . The frequency of the snp suggest the importance of a case - control study to assess the association between this polymorphism and the development of prostate cancer, and thus at this stage the functional significance of tp53 pin3 polymorphism remains poorly explored . Until now, most of studies in this field have been carried out using candidate genes, as was done in the present study . These studies can provide valuable information; however, they are limited and have not yet succeeded in providing a predictive test to identify radiosensitivity in patients . In response to lack of success in candidate gene studies and the subsequent recognition that this approach is too limited in scope, radiogenomic investigators have now begun a much broader search to identify genes and snps associated with radiation response . Several large - scale genome wide association (gwa) studies have been initiated in which radiotherapy patients are being genotyped for large numbers of common snps [8, 43]. The results of this study hint to a few candidate genes; however, our study was limited by the small sample size and therefore low statistical power to detect associations . This study shows the importance of genetic investigations as a useful tool for individualization of radiotherapy strategies . It is anticipated that over the next few years, snps correlated with susceptibility for the development of adverse effects resulting from radiotherapy will be identified from gwa studies . The data provided by studies in radiogenomics will open new perspectives for interpretation of the results of candidate gene studies such as ours and can be the basis for the development of a predictive assay that may be useful to personalize and optimize cancer treatment.
Environment - friendly biocatalysts are now rapidly substituting the conventional harsh chemical methods for the synthesis of important fatty acid esters used in many chemicals, medicines, cosmetics, and foods [15]. The attention towards tremendous use of microbial lipase (triacylglycerol acylhydrolase, ec 3.1.1.3) was exploited in the past decade leading to the easy hydrolysis / synthesis of esters at ambient condition with an advantage of precise selectivity . Such reactions mediated by biocatalysts have advantages like mild reaction conditions, one step synthesis without protection and deprotection steps, and easy application to food processing [68]. A lipase catalyzes a reversible reaction and the direction and equilibrium of the reaction is determined by the activities of the substrates, products, temperature, and pressure . Enzymes immobilization is the inherent advantage to isolate the biocatalyst from the reaction product and reuse it in order to increase the process productivity [1012]. Immobilization by adsorption has been most widely used for immobilization of various enzymes [13, 14]. Highly porous inorganic matrices such as silica aerogels with differing balances of hydrophobic and hydrophilic functionalities have been successfully used for the immobilization of enzymes . Silica aerogels can be considered as solid solvent for the enzymes that are able to provide hydrophobic / hydrophilic characteristics differing from those prevailing in the liquid surrounding the aerogels . The present study is focused on the effect of various parameters on silica - immobilized lipase - catalyzed synthesis of isopropyl acetate in n - heptane (scheme 1) as a model system for the study of esterification . Nano3, k2hpo4, kcl, mgso47h2o, feso47h2o, and (nh4)2so4 (s.d . Fine - chem, mumbai); yeast extract and gum acacia (himedia laboratory, mumbai); p - nitrophenyl palmitate (p - npp) alkanes (n - pentane, n - hexane, n - heptane, n - nonane, n - hexadecane and n - heptadecane), and silica gel 60 (0.0400.063 mm, 230400 mesh) were from lancaster synthesis, england; glacial acetic acid; triton x-100 (qualigens fine chemicals, india); isopropyl acetate was obtained from acros organics (new jersey, usa); licl, ki, kno3, sucrose, isopropanol, and molecular sieve (3 1.5 mm) were procured from e. merck pvt . Ltd ., bacillus cereus mtcc 8372 was originally isolated from a soil sample by selective enrichment technique at 55c . The culture was maintained by repeated subculturing on a mineral - based (mb) medium supplemented with 1% (v / v) cotton seed oil (as a sole c - source). / l); nano3 3; k2hpo4 0.1; mgso47h2o 0.5; kcl 0.5, feso47h2o 0.01, and yeast extract 4.0, ph 7.5 0.1 . Seed culture of b. cereus was prepared by inoculating 50 ml of broth with a loop full of culture . The culture was allowed to grow for 36 h at 55c under continuous shaking at 160 rpm . Thereafter, 10% (v / v) of 36 h old seed culture was used to inoculate 1000 ml of the production medium (50 ml each in 250 ml capacity erlenmeyer flasks). The seeded production medium was incubated at 55c and 160 rpm for 48 h (orbitek shaking incubator, aid electronics, chennai, india). The culture broth was centrifuged after 48 h post inoculation at 10,000 g for 10 min at 4c (sigma 3k30, germany). 1 . The protein content was measured . Henceforth, this filtrate broth was referre to as crude lipase . The required amount of ammonium sulfate was added to the crude lipase to achieve 80% (w / v) saturation . The precipitates sedimented by centrifugation at 12,000 g at 4c for 30 min were reconstituted in minimum amount of tris buffer (0.05 m, ph 8.5) and were extensively dialyzed against the same buffer . The purification of the dialyzed lipase enzyme was performed on an octyl - sepharose column (amersham pharmacia, sweden). The specific activity of the purified enzyme was compared with the crude enzyme and fold purification was calculated . The reaction mixture contained 80 l of p - npp stock solution (20 mm p - npp prepared in isopropanol), 20 l of test sample (lipase) and tris buffer (0.05 m, ph 8.5) to make final volume to 3.0 ml . The reaction mixture was incubated at 55c for 10 min in a water bath, and lipase activity was assayed at 410 nm . One unit (iu) of lipase activity was defined as micromole(s) of p - nitrophenol released per minute by hydrolysis of p - npp by one ml of soluble enzyme or 1 g of matrix bound enzyme at 55c (weight of matrix included) under assay conditions . The matrix was first washed with distilled water (thrice) followed by tris buffer (0.05 m, ph 8.5) before immobilization . Approximately, 4 ml (~0.82 iu) of the purified enzyme was then added to silica (1 g), and the suspension was incubated for 1 h at 37c in a glass vial . The unbound lipase was removed by five washings with tris buffer (0.05 m, ph 8.5). Finally, immobilized matrices were kept suspended in tris buffer at 4c till further use . In each of the assays, 20 mg immobilized enzyme preparation (matrix) was used . The volume of the supernatant, unbound protein and lipase activity were estimated using standard methods . The bound protein in matrix was determined by subtracting unbound protein in the supernatant from the total protein used for immobilization . A reference profile was prepared using varying concentrations of isopropyl acetate (20100 mmol / l) in n - heptane (retention time 0.91 min). The reference curve was plotted between the molar concentration (mmol / l) of isopropyl acetate and the corresponding area under the peak . The retention time for isopropanol and acetic acid is 0.79 min and 1.03 min, respectively . The sample was analyzed with glc using a packed column (10% se-30 chrom whp, 2-meter length, mesh size 80100, internal diameter 1/8 inches, netel chromatograph, thane, india). Glc was programmed for oven temperature 75125c, ramp rate 25c min, injector temperature 135c, fid temperature 145c, and holding time 2 min at 125c . The synthesis of isopropyl acetate was studied by taking different amount of immobilized lipase (25125 mg / reaction volume) in 2.0 ml of reaction mixture containing 100 mm and 75 mm each of isopropanol and acetic acid in n - heptane at 55c in 9 h under shaking (160 rpm). It was studied by keeping the concentration of one of the reactants (isopropanol or acetic acid) at 100 mm and varying the concentration of another reactant (25100 mm) in the reaction mixture in n - heptane . The esterification was carried out for 9 h at 55c under continuous shaking using silica - bound lipase, and the amount of ester formed was determined by glc . The reaction mixture comprised silica - immobilized lipase, isopropanol (100 mm), and acetic acid (75 mm) in n - heptane . The glass vials were incubated at 55c in a water bath incubator shaker for 15 h. at 3-hour intervals, the solvent phase (2 l) was sampled and analyzed by glc for the presence of isopropyl acetate . The effect of temperature (45, 55, 65, and 75c) on the synthesis of isopropyl acetate was studied . The reaction mixture containing isopropanol (100 mm) and acetic acid (75 mm) in n - heptane was catalyzed by silica - immobilized lipase at each of the selected temperatures for 9 h. the amount of ester synthesized was determined by glc . The molecular sieves (3 1.5 mm) were added at the concentration from 25 to 150 mg per reaction volume as mentioned above and synthesis of isopropyl acetate in the reaction mixture was determined . In the reaction mixture, (2.0 ml) n - heptane that was initially employed as a solvent phase was replaced with n - alkanes of varying c - chain length, that is, n - pentane, n - hexane, n - heptane, n - octane, n - nonane, n - hexadecane, and n - heptadecane . The silica - bound lipase was added to the reaction mixture, and the reaction was performed for 9 h at 55c . The immobilized lipase with molecular sieves was used for the synthesis of isopropyl acetate in n - nonane in a batch reaction up to 8 cycles of 9 h each at 55c . After first use, the biocatalyst was recovered by decanting the reaction mixture and was washed thrice in excess of n - nonane . The immobilized lipase and the substrates were pre - equilibrated separately in containers containing different saturated salt solutions; licl, ki, kcl, and kno3 with aws of 0.12, 0.689, 0.869, and 0.960, respectively . The silica - bound lipase was added to the reaction mixture and reaction was performed for 9 h at 55c without addition of molecular sieves . Standard deviation (sd) and standard error of means (sme) were calculated from data obtained for three replicates for each of the parameters studied . The purification of microbial lipases using hydrophobic interaction chromatography has been recently well established [21, 22]. The purified lipase by octyl - sepharose column was used for the enzyme immobilization . The bound lipase exhibited an activity 2.1 iu / g matrix (weight of matrix included). Different amounts of bound lipase were used and the progress of the ester synthesis was monitored by glc . Maximum esterification (67.0 0.1 mm) was achieved when 25 mg of silica - bound lipase was used (table 1). However, further increase in the amount of bound lipase failed to further enhance the rate of esterification . An increase in the quantity of biocatalyst concentration resulted in a decrease in the apparent enzyme activity in the production of ethyl acetate to an increase in diffusion limitation, a problem that may be minimized in large - scale experiments by optimal biocatalyst and bioreactor design . At a fixed concentration of acetic acid (100 mm), an increase in the concentration of isopropanol (25100 mm) at 55c promoted the synthesis of ester (21.0 0.2 to 52.0 0.2 mm). However, when the concentration of isopropanol was kept fixed (100 mm), there was a marked increase in ester synthesis (27.0 0.1 to 66.0 0.1 mm). It appeared that the ester was optimally synthesized when alcohol and acid were used at 100 mm and 75 mm, respectively, in n - heptane under continuous shaking for 9 h at 55c (figure 1). Thus, in the subsequent reactions, isopropanol and acetic acid were used at 100 mm and 75 mm concentration, respectively . In a previous study, high concentration of acetic acid (0.4 to 0.7 m) inhibited sp435 lipase activity resulting in low conversion yields for acetate esters . The presence of fatty acid (acetic) at higher concentration could damage the hydrolytic layer of the enzyme structure causing lipase deactivation during esterification process . Thus, b. cereus mtcc 8372 lipase appeared to be vulnerable to high concentration of acetic acid in reaction medium just like other lipases . Due to toxicity of (acetic) acid in higher concentration on lipase activity in enzymatic acetylation, the use of acids as an acyl donor in transesterification and direct esterification reactions was previously attempted with little or no success [16, 17]. It was studied at a temperature of 55c in n - heptane under continuous shaking (figure 2) up to 15 h. the synthesis of ester was time dependant, and maximum amount of isopropyl acetate (66.0 0.1 mm) was produced after 9 h of reaction when isopropanol and acetic acid were used at 100 mm and 75 mm, respectively, in n - heptane . An increase in temperature of reaction mixture might interfere with the porosity, hydrophobic character and diffusion of the reactants and/or products at the catalytic site of enzyme or hydrogel . The reaction temperature above or below 55c decreased the ester production (figure 3). This might be on account of denaturation of the lipase as well as alteration in the 3d structure of lipase . Thus, 66.0 0.2 mm of isopropyl acetate synthesis was achieved at 55c in 9 h in a batch reaction . It appeared that temperature has an important effect on the physical state of substrate dispersion in an organic solvent . Higher temperature and liquefaction can be assumed to make the substrate more acceptable for the enzyme . Moreover, immobilization facilitated dispersal of enzyme on a solid surface to provide far greater interfacial area and accessibility of substrate relative to the use of enzyme powders in low water reaction media . The choice of an appropriate solvent system that keeps the reactants dissolved and did not interact with the enzyme, matrix / support, or any of the reactants is very important in achieving efficient esterification . Uses of n - octane or n - nonane have nearly similar effect on the amount of ester formed under similar conditions . The maximum conversion of reactants into ester was recorded in n - nonane (67.0 0.2 mm) at 55c . There is an increase in ester synthesis from n - pentane (39.0 0.2 mm) to n - nonane (67.0 0.2 mm) followed decrease in ester synthesis from n - hexadecane (53.0 0.3 mm) to n - heptadecane (57.0 0.1 mm). Among various n - alkanes, n - nonane was considered best for ester synthesis in a water - free system (figure 4). As an n - alkane with a lower or a higher c - chain than n - nonane was used as a reaction medium, a gradual decrease in rate of isopropyl acetate synthesis was noticed . As the log p value of an n - alkane decreased corresponding to decrease in the c - chain length of the alkanes, the hydrophobicity of the alkanes, that is, alkanes also decreased in that order . The esterification reaction results in formation of water as a by - product of the reaction, and its removal using molecular sieves might enhance the synthesis of ester by pushing the reaction equilibrium in the forward direction [26, 27]. However, when the effect of the presence of a molecular sieve was studied by adding molecular sieve at concentrations of 25 to 150 mg per reaction volume, a gradual increase in the synthesis of isopropyl acetate was noticed up to 100 mg (73.0 0.2 mm), and any further increase in the amount of molecular sieves resulted in a decrease (figure 5). The silica - bound lipase when repetitively used to perform esterification under optimized conditions (25 mg immobilized lipase, 55c reaction temperature, 9 h reaction time, 100 mg molecular sieves) in n - nonane resulted in 30 0.2 mm isopropyl acetate after 8th cycle of esterification (figure 6). The decrease in the yield of the synthesis of ester may be due to a leakage of enzyme to the reaction medium . Generally, all lipases showed an increase in ester synthesis as aw increased until an optimum aw, whereby thereafter the ester synthesis decreased as the aw further increased (table 2). At saturated salt solutions of licl, there was maximum ester synthesis (72.0 0.1 mm) that was however lower that the isopropyl acetate was produced without the use of any of the salts for choosing the aw . Effect of each of the chosen water activities except licl was inhibitory in the isopropyl acetate synthesis . These results suggest that, at these water activity values, enzymes are fully hydrated (too much water), and thus the enzyme is rendered to have less rigidity and therefore conversion rate diminishes . In the present study, we have shown that a systematic approach could be devised on the basis of optimization of reaction parameters and solvent engineering to obtain optimal synthesis of an ester of interest . Isopropyl acetate was successfully synthesized by lipase - catalyzed esterification in a short time of 9 h at 55c in n - heptane under continuous shaking (160 rpm) using bound lipase (25 mg). The immobilized enzyme could be repetitively used for 6 cycles with more than 50% yield of ester.
More leisure time for hobbies, hiking, and trekking has led to more mushroom exposures . Increased demands for uncultivated food or gastronomic delights have contributed to more mushroom poisoning . Although severe mushroom poisonings and fatalities remain infrequent, the proportions of fatal mushroom poisonings among confirmed mushroom ingestions seems to be increasing (1). In addition to increasing poisoning of known toxic mushroom, new mushroom syndromes have been described (23). The severe patient with r. subnigricans mushroom poisoning was reported to present with rhabdomyolysis, severe electrolyte disturbance (hyperkalemia, hypocalcemia), acute renal failure, respiratory failure, pulmonary edema, ventricular tachycardia, and circulatory shock (45). In this case report, we present a patient poisoned by r. subnigricans mushroom complicated with rhabdomyolysis, acute kidney injury (aki), severe hypocalcemia, respiratory failure, ventricular tachycardia, and cardiogenic shock, leading to death . A 51-year old man had a breakfast cooked with wild mushrooms that he had gathered one day earlier during summer vacation in august, 2010 at the jujak mountain located on the province of jeollanam - do, the southern area of korea . He had no particular past medical or social history other than the occasional ingestion of wild mushrooms during recreational hiking . He did not have a history of trauma, infection, other known underlying cause, or medication use that could explain the occurrence of rhabdomyolysis . He shared the meal with his wife and son who did not eat any more mushrooms due to nausea after ingesting a single piece of mushroom . Six hours later, he began to complain of vomiting, diarrhea, and myalgia . He visited a local hospital where his general condition worsened and systolic bp fell to 60 mmhg requiring dopamine treatment and endotracheal intubation . Twenty - eight hours after ingestion of the mushrooms, he was transferred to the intensive care unit of our hospital . At the time of admission, he was alert, but acute ill - looking . Blood pressure was 80/56 mmhg, heart rate 121/min, respiratory rate 25/min, and body temperature 36.3c . Laboratory tests showed the following: hemoglobin 15.9 g / dl, hematocrit 45.9%, leukocyte count 20,740/l with neutrophil 89.6%, platelet 253,000/l, c - reactive protein (crp) 8.36 mg / dl, blood urea nitrogen (bun) 38.5 mg / dl, serum creatinine 2.84 mg / dl, serum osmolarity 314 mosm / kg, sodium 140 meq / l, potassium 4.7 meq / l, chloride 100 meq / l, ionized calcium 0.75 mmol / l, glucose 187 mg / dl, cholesterol 221 mg / dl, albumin 4.0 g / dl, aspartate aminotransferase (ast) 1,214 u / l, alanine aminotransferase (alt) 343 u / l, total bilirubin 0.51 mg / dl, alkaline phosphatase 92 iu / l, creatine kinase (ck) 69,121 u / l, ck - mb> 500 ng / ml, lactate dehydrogenase (ldh) 2,196 u / l, troponin i 1.86 ng / ml, n - terminal pro brain natriuretic peptide (nt - pro bnp) 7,414 pg / ml, amylase 1,930 u / l, lipase 18 u / l, prothrombin time (pt) 10.2 seconds . (inr 0.86), activated partial thromboplastin time (aptt) 28.2 seconds, urine osmolarity 341 mosm / kg, sodium 22 meq / l, potassium 59.2 meq / l, chloride 14 meq / l, creatinine 123.8 mg / dl, myoglobin 51.7 ng / ml, and fractional excretion of sodium 0.36% . Urine analysis showed ph 5.0, specific gravity 1.020, protein 2 +, ketone 2 +, blood 4 +, rbc 5 - 9/high - power field, and wbc 0 - 1/high - power field . Arterial blood gas analysis was ph 7.04, paco2 69 mmhg, pao2 172 mmhg, and hco3 18.7 mmol / l . The enlarged left ventricle (lv), severe lv systolic dysfunction (ejection fraction 30%), and akinesia of lv mid to apex wall suggesting stress induced cardiomyopathy were noted on echocardiography . He received ventilator support, hemodynamic monitoring, continuous venovenous hemodiafiltration, and conservative care including intravenous fluid and electrolyte repletion . Seventy hours after ingestion of the mushrooms, bun 41.9 mg / dl, serum creatinine 3.46 mg / dl, ast 3,271 u / l, alt 1,254 u / l, total bilirubin 1.55 mg / dl, ck 121,397 u / l, ck - mb> 500 ng / ml, ldh 6,102 u / l, troponin i 11.77 ng / ml, amylase 2,779 u / l, lipase 601 u / l, pt 42.1 seconds . Ventricular tachycardia developed, and he died due to cardiogenic shock 72 hours after ingestion of the mushrooms . The leftover samples of the mushrooms that he had ingested were sent to agricultural microbiology division, national academy of agricultural science, rural development administration, suwon, korea . Those were identified as r. subnigricans by their gross morphology (fig . The worldwide incidence of mushroom poisoning appears to be increasing due to increased human demands for uncultivated food or hallucinogenic mushrooms . The proportion of severe and fatal mushroom poisonings among confirmed mushroom ingestions has demonstrated statistically significant increases . Deaths, although rare, occurred most commonly from the ingestion of amanita species, primarily amanita phalloides, in adult amateur mushroom hunters (16). In addition to increasing poisons following ingestions of known species, formerly edible or new mushroom species are causing newly recognized syndromes from mushroom poisonings . Several new syndromes in mushroom poisoning have been described since the early 1990s (123): accelerated nephrotoxicity (non - orellanine - induced renal failure), erythromelalgia, rhabdomyolysis, and delayed neurotoxicity (convulsive or non - convulsive encephalopathy). Rhabdomyolysis is a new type of mushroom poisoning caused by ingestion of tricholoma equestre (1347). Some of these cases with rhabdomyolysis are associated not only with renal dysfunction and electrolyte disturbance (hyperkalemia, hypocalcemia), but also with respiratory and cardiac complications (arrhythmia, cardiovascular collapse) leading to death (4). An outbreak of russula subnigricans poisoning with rhabdomyolysis occurred in taiwan (5). The most severely ill patient presented with rhabdomyolysis, severe electrolyte disturbance (hyperkalemia, hypocalcemia), respiratory failure, acute renal failure, pulmonary edema, ventricular tachycardia, and circulatory shock . Japan has also experienced several cases of fatal poisoning with rhabdomyolysis caused by ingestion of this mushroom (311). R. eccentrica, r. nigricans and r. subnigricans have been recorded in korea (12). Poisonous r. subnigricans can be mistaken with non - poisonous, edible r. eccentrica and r. nigricans . R. subnigricans can be found associated with broad - leaved evergreen tree habitat which is present and restricted to the southern area of korean peninsula . This report of our case represents the first record of r. subnigricans poisoning with rhabdomyolysis in korea . Rhabdomyolysis, the dissolution of skeletal muscle, is a rare but potentially fatal condition . The common causes of rhabdomyolysis are trauma, exertion, muscle hypoxia, infections, body temperature changes, metabolic and electrolyte disorders, drug and toxins, and genetic defects . The incidence of rhabdomyolysis in acute poisonings was the highest in poisonings with opiates, pesticides, neuroleptics, anticonvulsants, and ethyl alcohol (1314). In our case, severe rhabdomyolysis with aki, severe hypocalcemia, respiratory failure, ventricular tachycardia, and cardiogenic shock were noted throughout the clinical course . There was no history of trauma, strenuous exercise, seizures, and loss of consciousness . The possible cause of rhabdomyolysis after ingestion of the mushroom may be the mushroom toxin, such as cycloprop-2-ene carboxylic acid, isolated from r. subnigricans . This compound may be responsible for fatal rhabdomyolysis as a toxic trigger for some biochemical reactions (1011). The mechanism involved in the pathogenesis of rhabdomyolysis is depletion of atp within the myocyte, leading to an unregulated increase in intracellular calcium, resulting in disintegration of the myocyte . It usually results from calcium entering the damaged myocyte and from the precipitation of calcium phosphate in necrotic muscle . Intrarenal vasoconstriction, direct and ischemic tubular injury, and tubular obstruction all may play a role in myoglobin - induced aki (13). Cardiogenic shock due to severe lv systolic dysfunction might be caused by the mushroom toxin (451516). General management of mushroom poisoning is fluid resuscitation, early gastric decontamination by both gastric lavage and multiple dose activated charcoal, and baseline laboratory assessment . Since mushroom - poisoned patients often experience vomiting and diarrhea, emetics and cathartics are rarely indicated . Treatment should be directed by the patient's clinical manifestation (117). The mushroom poisoning with rhabdomyolysis is usually associated with volume depletion that is due to the sequestration of water in damaged muscles . Therefore, the early, aggressive repletion of fluids is the main step of management ., early hypocalcemia should not be treated unless it is symptomatic or unless severe hyperkalemia is present, since the calcium load could increase the precipitation of calcium phosphate in necrotic muscle (13). Continuous venovenous hemofiltration or hemodiafiltration has shown some efficacy in removing myoglobin (18). However, the evidence is from case reports, and the effect on outcomes is unknown . If renal or hepatic function is worsening, patients should be transferred to the hospital equipped for hemodialysis and kidney or liver transplantation (1). Intra - aortic balloon counterpulsation can be considered as a final treatment option in the mushroom poisoning with cardiogenic shock (15). Correct categorization and better understanding are essential for the safe and healthy consumption of mushrooms (19). Since mushroom toxicity may be dose - related, overconsuming mushrooms should be avoided (1). Furthermore, r. subnigricans should be considered in the mushroom poisoning with rhabdomyolysis . To confirm the exact diagnosis, leftover raw or cooked mushrooms should be collected and sent to a mycologist for gross and spore identification.
It can involve any site of gastrointestinal tract, but the most common site is the mesentery of the small bowel . Its biological behavior is intermediate between benign fibrous tissue proliferation and fibrosarcoma . Sometimes, it is often confused with submucosal tumor or malignant neoplasm of gastrointestinal tract, especially gastrointestinal stromal tumors (gists). We described a case of mf which induced obstruction of colon and ureter simultaneously and resulted in a positive positron emission tomography (pet) scan . A 49-year - old woman was admitted for constipation and left lower quadrant abdominal pain for seven days . Physical examination revealed heperactive bowel sound and palpable mass at left lower quadrant of abdomen . Initial complete blood cell counts and blood chemistry results were as follows: white blood cell count, 8.210/l (normal, 4.510/l-10.510/l); hemoglobin, 12.7 g / dl; platelet count, 26410/l; blood urea nitrogen, 24.4 mg / dl; creatinine, 1.1 mg / dl . 2) showed near - complete obstructive mass at sigmoid colon covered with normal mucosa . Abdominal ct scan disclosed not only an about 5.65.2 cm sized well circumscribed, locally infiltrative soft tissue mass in left pelvic cavity, but also marked left hydroureteronephrosis and atrophic change of left kidney (fig . 3), suggestive of longstanding ureteral obstruction . Pet scan revealed the corresponding pelvic mass with focal increased activity with a maximum standardized uptake value (suvmax) of 4.1 (fig . Light microscopy shows ordely arrangement of uniform fibroblasts associated with moderate amouts of collagen (fig . 5). Immunohistochemistry disclosed that the lesion were negative for cd34 and c - kit, whereas were positive for actin, vimentin . Mf is a rare, benign fibrous lesion found in the bowel mesentery or the retroperitoneum . The mesentery of the small bowel is the most common site.1 mf tend to be locally invasive and to recur locally, but do not metastasize.1,2 it can occur spontaneously or after surgical trauma and also is associated with hormonal therapy, familiar polyposis, or gardner's syndrome.1,3 the diagnosis of mf is based on clinical suspicion, which depends on the location or local effect of tumor . The role of imaging is to define the degree of extension to local structure and tumor relationship to neurovascular structure.4,5 pathologic confirmation was made by microscopic examination and immunohistochemistry . However, gists are potentially misdiagnosed as mf, because of their features such as large size, infiltration of adjacent structures and mitotic activity.6 their diagnostic discrimination is essential because of their very different biological behaviors and their therapeutic strategies.7 the absence of cd34 and s100 expression supports the fibromatous nature of the lesion and may be helpful in discriminating mf from gist.8 in addition, recently a report showed that -catenin separates mf from gist and sclerosing mesenteritis.9 our case showed that the lesion were negative for cd34, whereas positive for -catenin, which were compatible with fibromatosis . The preferred treatment of mf is wide local surgical excision with a margin of uninvolved tissue.1,3 in the current case, unusually, the mass involved sigmoid colon and ureter at once and result in more aggressive features such as colonic obstruction with local invasion and hydroureteronephrosis . Pet scan showed hot uptake in corresponding mass, considered as submucosal tumor of malignant neoplasm such as gist . First, however rare, mf is considered as one of the causes of stenosis of the colon in patients with a history of pelvic surgery . Second, pet scan is not helpful for differential diagnosis of other malignant neoplasm from this disease, which is correspond with recent report that mf has a positive interpretation of fluorodeoxyglucose pet scan.10 third, -catenin is useful to differentiate between mf and gist.
Acute coronary syndrome is among the most common diseases that prevail in various societies nowadays . So, based on statistics, in the us, about 1.5 million people get affected by myocardial infarction (mi) annually, of whom a high percentage is hospitalized in health centers . It imposes huge economic burden to these societies due to partial disability of these patients . In iran, previous research showed the mortality rate of the patients due to cardiovascular disease was 2545% in eastern mediterranean and middle east countries including iran . Cardiovascular diseases in iran account for 45% of mortality, 26% of life years wasted, and 10.4% of disease burden . Among the risk factors for cardiovascular diseases, hyperlipidemia, hypertension, and hyperglycemia are common . Hyperglycemia due to diabetes is one of the major factors for increase in mortality and morbidity caused due to cardiovascular diseases . Prevalence of diabetes among the hospitalized patients with mi is 1020% of which 30% accounts for undiagnosed diabetes and 35% for the individuals with glucose tolerance disorder . In fact, 85% of the patients with acute coronary syndrome experience a degree of blood glucose tolerance disorder . Therefore, diabetes mellitus is known to be the main cause for mortality due to high risk of atherosclerosis, and thus, the risk of mortality in diabetic patients is twofold more than in non - diabetic individuals . Acute mi, accompanied with hyperglycemia, leads to increase of necrotic area and prevalence of heart failure and mortality . One of the factors that reduces the development and prevalence of microvascular complications including sclerosis is appropriate control of blood sugar . Nowadays, the interventions to improve the prognosis in mi patients are conducted through two methods of metabolic modulation and metabolic control . Previous studies conducted on metabolic modulation focused on the potentially useful effects of insulin and potassium during acute stress, regardless of the level of blood sugar . This strategy is based on infusion of a steady dosage of glucose - insulin potassium (gik). On the other hand, metabolic control lies on the usage of insulin to reduce blood sugar level to an already determined level in order to decrease the negative effects of hyperglycemia and to make the best of the useful effects of insulin which may be injected by intravenous or subcutaneous methods . Insulin prescription method should be performed with the lowest risk of hypoglycemia as it may lead to cardiac injury and dangerous arrhythmia . The published guidelines of european society of cardiology (esc) and european association for the study of diabetes (easd) suggest insulin infusion to control blood sugar in all patients with acute coronary artery syndrome with history of diabetes . Hypoglycemia is a major complication of insulin infusion compared to subcutaneous insulin injection, but previous research showed that iatrogenic hypoglycemia is not accompanied with high risk of mortality in patients after insulin therapy . Checking the blood sugar by the nurses and conducting continuous insulin infusion protocol concurrently result in ideal control of blood sugar among critical patients in cardiac care unit (ccu), but subcutaneous insulin injection needs a longer time . In the patients with insulin infusion, it is possible to return the patients to their former diet therapy (through modification of lifestyle, diet, and/or oral insulin agents) after acute period of the disease . Meanwhile, among the patients without appropriate control of blood sugar, subcutaneous insulin injection is needed not only during hospitalization but also after discharge . Therefore, the nurses in ccus are responsible for preservation of patients blood sugar level based on insulin protocol which starts with a physician order . In this protocol, the nurses are permitted to control patients blood sugar with the lowest need of physician order . The research has also shown that it is possible to achieve ideal blood sugar level without incidence of hypoglycemia through comprehensive nursing care as well as appropriate nutrition . The american association of diabetes has emphasized on the importance of blood sugar control in diabetic patients and considers nurses function essential in successful administration of protocols, taking medical orders, precise monitoring, and educational programs of blood sugar control . Outcomes of hyperglycemia control through insulin infusion compared to conventional methods have been considered in various studies . Among these outcomes, mean blood sugar level at the time of beginning insulin infusion and the level of hypoglycemiacan be mentioned . As the conventional method in ccus in iran is usage of a subcutaneous insulin injection chart as well as insulin infusion in acute period of the disease to achieve the aforementioned outcomes better and faster, the present study compared the effects of insulin infusion method to those of conventional method (subcutaneous insulin injection) on blood sugar control and the outcomes such as hypoglycemia, and on achieving the target blood sugar in diabetic patients with acute coronary syndrome in ccus . This is a clinical trial conducted on patients with acute coronary syndrome with history of diabetes mellitus and hospitalized in ccu of saee hospital in khomeinishahr, iran . The patients were explained about the research, its goals, and conditions . Among the selected patients, those who were willing to attend entered the study after filling a written consent form . Inclusion criteria were age over 18 years and diagnosis of acute coronary syndrome at the time of admission to ccu with approval of a specialist . Not inclusion criteria were high - risk patients and unusual hypoglycemia (like known insulin secretion tumors or history of frequent and idiopathic hypoglycemia), pregnancy, renal and hepatic failure, or liver transplantation . Exclusion criteria were imminent death (expected heart arrest within <24 h), patients decision on withdrawal from intervention during the study, and not achieving the target blood sugar level 24 h after beginning insulin infusion . In the present study, the . The number of the subjects obtained was 32 in each group by confidence interval of 95%, test power of 80%, and d = 0.7 (after consultation with a statistician). Subject drop was considered with regard to the exclusion criteria, and each dropped out subject was replaced by another new subject . Five subjects were left out of the study due to expedition to a more equipped center, one patient due to his withdrawal of taking part in the study, and two subjects as a result of not giving consent . The glucometer and the infusion pump were calibrated before sampling began . Just after patients admission to ccu, their blood sugar was checked by an accucheck glucometer device, and concurrently, a blood sample was sent to laboratory for random blood sugar check . The patients with blood sugar> 180 mg / dl were randomly assigned to the study group (insulin infusion) and control group (subcutaneous insulin injection). The earlier blood sugar control medications (metformin, pioglitazone etc .) Of all patients were stopped before the study began . Insulin infusion was prescribed for the subjects based on east jefferson protocol in the study, and subcutaneous insulin injection in the control group was administered by a cardiologist . For patients in the study group, a venous infusion solution made from 100 units human regular insulin and 100 ml normal saline (0.9%) at a ratio of 1:1 was administered . Patients blood sugar was checked each hour using the glucometer, and the protocol of insulin infusion level (units per hour) was changed based on the last measurement value of blood sugar based on the measured blood sugar level . In case of no reduction observed in the blood sugar level, compared to the latest measurement, the column was changed by one to right (e.g. From column 5 to column 6), and if blood sugar was less than 140 mg / dl, the column was changed by one to left (e.g. From column 5 to column 4), and infusion level was regulated based on the new column . When patients blood sugar level was in the range of target value (140180 mg / dl) and/or the blood sugar titer was less than the former one, insulin infusion was continued based on the same column . When patients blood sugar was not in the target level (140180 mg / dl) for at least 4 h, insulin infusion was changed to subcutaneous insulin injection two times a day or other blood sugar control methods . If the blood sugar was not in the target level for 24 h, insulin infusion was stopped and the patient was excluded from the study . Blood sugar was checked four times a day in this group (three times before meals and one time before sleep). Based on patients nutrition timetable in the hospital, patients blood sugar was checked at 06:00 h, 12:00 h, 18:00 h, and 24:00 h for 48 h, and human regular insulin was injected to patients in this group through subcutaneous method based on their blood sugar level . After 48 h, infusion was stopped based on the chart and changed to subcutaneous insulin injection twice a day or to other conventional methods of blood sugar control (blood sugar control with diet therapy or blood sugar lowering pills). In both groups, if blood sugar was less than 8 mg / dl, blood sugar (daily fasting blood suger (fbs), bs) was checked every 12 h in all patients until discharge . All the obtained data (all the measured blood sugar levels for each patient and the nursing interventions conducted such as stopping infusion, interventions for hypoglycemia, etc .) Were recorded for each patient separately by the researcher or the project cooperator in the data collection form . Each data collection from yielded the time interval to reach target sugar level and the onset number of patients hypoglycemia . Data were analyzed by descriptive and analytical statistical tests such as central indexes, dispersion index, frequency distribution, independent t - test, paired t - test, and chi - square through spss . The ethical and scientific contents of this study have been approved by isfahan university of medical sciences . The ethical and scientific contents of this study have been approved by isfahan university of medical sciences . Both groups were identical concerning demographic characteristics based on chi - square test and independent t - test [table 1]. Mean patients blood sugar was 289.6 (108.9) mg / dl in the insulin infusion group before intervention and 275.03 (77.5) mg / dl in the subcutaneous insulin injection group, which showed no significant difference (p = 0.54). Identical baseline characteristics of the patients in study and control groups mean patients blood sugar was 153.6 (44.5) mg / dl in the study group and 180.7 (76.3) mg / dl in the control group after intervention . It was significantly less in the study group compared to the control group (p = 0.04). Mean blood sugar levels at the end of intervention compared to before beginning the intervention were significantly reduced in study (p <0.001) and control (p <0.001) groups, of which the reduction was significantly lower in the study group compared to control (p = 0.04). Mean patients blood sugar during hospitalization period was 189.2 (25.5) mg / dl in the study group and 217.9 (65.25) mg / dl in the control group, which showed a significant difference (p = 0.02). Mean time interval to reach target blood sugar level was 4.75 h in the study group and 36.94 h in the control group, for which independent t - test showed a significant difference (p <0.001) [table 2]. Incidence of hypoglycemia was 32.2% in the study group and 25% in the control group, for which chi - square test showed no significant difference (p = 0.57). Investigation showed that the mean blood sugar level during the entire hospitalization period (all blood tests except those of admission and discharge) was 189.2 (25.5) mg / dl in the insulin infusion group and 217.9 (65.1) mg / dl in the subcutaneous insulin injection group . Zimmerman (2004) showed that the mean blood sugar level in the insulin infusion group during hospitalization was 114 (66) mg / dl and it was 183 (39) mg / dl in the subcutaneous insulin injection group . The subjects in zimmerman's study had lower level of blood sugar during hospitalization compared to the present study, which can be a result of lower target level of blood sugar in zimmerman's study (80150 mg / dl), participation of the patients both with and without diabetes mellitus in his study, and usage of a different protocol . On the other hand, blood sugar level showed a significant difference during hospitalization after intervention in both groups in the above study, which is consistent with the present study . Patients mean blood sugar level in the study group was 253 (95.6) mg / dl before intervention and 133.5 (43.9) mg / dl after intervention, which shows a significant effect of insulin infusion with protocol on the blood sugar level (p <0.001). This finding is in line with that of the present study . In the present study, 31.2% of the patients in the study group and 25% of the patients in the control group developed hypoglycemia . In dilkhush's study (2005) on 30 patients hospitalized in the icu, frequency distribution of hypoglycemia was reported to be 0.4% in the study group . But in osborne's study conducted on the effect of evaluation of nurses role on administration of insulin infusion column protocol among the patients hospitalized in the icu, frequency distribution of hypoglycemia was reported as 0.9% . In nice - sugar study (2009) on two different target blood sugar levels with insulin infusion and its outcomes in ccu patients, the frequency distribution of hypoglycemia among the patients in the insulin infusion group with target blood sugar level of 140180 mg / dl was reported as 0.5% and in the other group with a target level of 81108 mg / dl, it was reported to be 6.8% . As observed, the frequency distributions of hypoglycemia obtained by studies conducted on these patients are different from those of insulin infusion group in the present study, which could have resulted from the difference in subjects number, type of the used protocol, target blood sugar, environment, and the type of the disease between those studies and the present study . But there was no significant difference in the incidence of hypoglycemia in both groups in the present study . In zimmerman's study, the incidence of hypoglycemia was reported to be 16.1% in the insulin infusion group and 98% in the subcutaneous insulin injection group . In the above study, there was no significant difference between two groups (p = 0.098), which is consistent with the present study . In zimmerman's study, there was also a significant difference between the two groups concerning the time interval to achieve target blood sugar (p <0.001), which was 2.1 h in the study group and 9.4 h in the control group, while they were 4.75 h versus 36.93 h in the present study . The difference can be due to patients lower mean blood sugar in zimmerman's study and the various protocols used in these studies . (2004) reported the mean time interval to achieve target blood sugar as 10.1 (4.6) h in insulin infusion, which could have resulted from lower target blood level in their study and the difference in the ward where the patients were hospitalized, as well as the protocols used in these two studies . Meanwhile, there was a significant difference in the time interval to achieve target blood sugar in these studies just like that observed in the present study . With regard to the existing studies, it can be concluded that blood sugar control with use of insulin infusion protocol in acute coronary syndrome patients with history of diabetes mellitus is more efficient and effective . As appropriate control of blood sugar among ccu patients affects the length of their hospitalization, treatment costs, and the disease outcomes, it is suggested to facilitate use of insulin infusion protocol in ccus through education of nurses as the administrators of the protocol . One of the limitations of the present study was the short length of the study and conducting the study by just the researcher and her colleague . It is suggested that nurses in charge of the patients should conduct further studies after receiving education to investigate the effect of blood sugar control on the delayed outcomes.
The patient was a 23-year - old woman, a known case of arnold - chiari malformation with peripheral neuropathy and muscular atrophy, who presented with headache, drowsiness, decreased vision, and severe gait dysfunction lasting for several years . Brain magnetic resonance imaging (mri) confirmed a hypointense - signal mass in the left hemisphere of the cerebellum causing mass effects on the fourth ventricle, which shifted it, accompanied with dilation of third and lateral ventricles . Hypertensive cerebrospinal fluid (csf) form of hydrocephaly was seen in the supratentorial region, but the fourth ventricle was normal . The possibility of aqueduct stenosis or obstruction was mentioned (fig . 1). Cervical spine mri revealed a normal spinal column with herniation of the cerebellar tonsil (fig . 2). Brain magnetic resonance imaging . A hypointense signal mass in the left hemisphere of the cerebellum causing mass effects on the fourth ventricle, which shifted it, accompanied with dilation of third and lateral ventricles . Tonsillar herniation to the cervical spine is seen . With the impression of tonsillar herniation and cerebellar tumor in the sense of arnold - chiari syndrome, external ventricular drainage with external reservoir a cream - white tight mass was seen, which could not be extracted by suction and thus was totally resected . Simultaneously, regarding the arnold - chiari malformation and tonsillar herniation, the patient underwent suboccipital decompression and microscopic laminectomy of c2 vertebrae with excision of foramen magnum and duraplasty . Gross examination revealed multiple pieces of irregular cream tissue totally measuring 2.5 2.0 0.7 cm . Microscopic assessment confirmed a granulomatous inflammation composed of aggregation of epithelioid histiocytes associated with giant cells and lymphocyte cuffing foci of caseating necrosis compatible with tuberculoma . Postoperative spiral chest computed tomography (ct) scan showed a normal pulmonary parenchyma without evidence for pulmonary tuberculosis, thus, the patient had a primary extrapulmonary cerebellar tuberculoma (fig . The patient has been followed to now; the neurological symptoms were alleviated 6 months subsequent to the surgery . Chiari malformations are rare congenital anomalies with an estimated prevalence of 0.1 to 0.5%5; however, the true frequency is unknown . In most cases, due to the small posterior fossa the main pathogenesis of this malformation remains the subject of debate and involves patients presenting with a wide spectrum of clinical symptoms.1 arnold - chiari syndrome is usually detected prenatally or at birth, as it is nearly always associated with lumbosacral or thoracic myelomeningocele.1 weakness, stridor, apneic spells, aspiration, and dysphagia are the common manifestations in infancy,6 followed by progressive hydrocephalus in childhood.1 it may be associated with other syndromes like syringomyelia and scoliosis.7 8 our case presented with headache, drowsiness, decreased vision, and severe gait dysfunction lasting for several years . Arnold - chiari syndrome should be considered in any fetus or newborn with clinical evidence of a spinal myelomeningocele . Neuroimaging plays the main role in confirming the diagnosis, and mri is the best imaging modality for evaluation.9 mri of the brain along with the entire spinal cord (cervical) is appropriate to demonstrate downward displacement of the inferior cerebellar vermis and medulla through the foramen magnum into the upper cervical canal . Despite these typical findings, brain mri in our case showed an unusual mass in the left cerebellar hemisphere causing mass effect . The patient underwent the most common procedure for arnold - chiari malformations, which is posterior decompression via suboccipital craniotomy with duraplasty.10 11 simultaneously, regarding the cerebellar mass with the primary impression of cerebellar tumor, posterior fossa craniotomy and resection of the mass were performed . Postoperative histopathologic evaluation showed that the mass was in fact a granulomatous tuberculoma . Because no pulmonary involvement was found in either clinical manifestation or pulmonary ct scan, it was diagnosed as a primary extrapulmonary cerebellar tuberculoma mimicking a cerebellar tumor . Although tuberculosis is considered primarily a pulmonary disease, it can affect any organ system . However, central nerves system (cns) involvement is so rare, it is associated with potentially devastating complications; it affects both immunocompetent and immunologically incompetent populations ., only a few numbers of cases with cerebellar tuberculoma have been reported, and most of them are secondary to the pulmonary involvement12 and occurred in immunocompromised patients.13 14 primary cerebellar tuberculoma is rare in an immunologically incompetent patient like our case . To our knowledge, this is the first reported case of primary cerebellar tuberculoma mimicking a posterior fossa tumor in a patient with arnold - chiari malformation . Depending upon the involved part of the cns, various spectrums of clinical manifestations intracranial tuberculoma may present as either a solitary or multiple lesions in the brain parenchyma . A ring - enhanced area on ct scan or mri is a characteristic appearance, but when there are no accompanied clinical manifestation or laboratory findings for tuberculosis, it would be so difficult to differentiate it from other cns tumors . Despite being rare, cns involvement of tuberculosis always should be kept in mind in any patient with neurological complaints from regions with a high endemic rate of tuberculosis, either in those being immunocompetent or immunologically incompetent.
A 55-year - old man presented with difficulties in hand coordination and dressing himself that had first appeared in early 2006 . It took him a long time to put on his clothes because he had difficulty distinguishing between the front and back of clothing, and needed assistance with buttoning and zipping up his clothing . A severe memory disturbance that had also developed a neurological examination showed typical cortical signs including severe apraxia, cortical sensory loss, myoclonus, and alien - limb phenomenon that predominantly affected the right arm . There were no prominent visuospatial problems, including simultanagnosia, visual inattention, oculomotor apraxia, or optic ataxia . A detailed neuropsychological assessment revealed prominent verbal and visual memory deficits with marked frontal executive dysfunctions (table). The patient scored 17/30 on the korena version of mini - mental state examination, with the subscore for time orientation being 3/5 and a delayed three - word recall of 0/3 . He exhibited an abnormal digit span on attention tests and showed severe ideomotor and ideational apraxia on several praxis tasks . His performance on copying in the rey - osterrieth complex figure test was impaired . On the seoul verbal learning test, he was able to recall two items (<percentile 1) with a 20-minute delayed recall . He also scored poorly in delayed recall in the rey - osterrieth complex figure test (0/36, percentile 2). His performances on the tasks of controlled oral - verbal fluency and stroop test were also severely impaired . Brain mri performed 18 months after the onset of the symptoms revealed significant cortical atrophy in both parietal areas that were more prominent on the left side with diffuse cortical atrophic changes in t1-weighted images (fig . Brain pet showed prominent asymmetric (left - dominant) hypometabolism in both parietal areas, with significant metabolic deficits in the left temporal lobes (fig . Cognitive impairments such as severe amnesia and visuospatial abnormalities were initially thought to be a rare or late presenting trait in cbd, with cognitive functions being relatively spared until the late stages of cbd and higher mental function being relatively preserved in cbd patients.1,7 clinical descriptions of cbd (mostly from movement disorder clinics) have emphasized motor manifestations such as parkinsonian features, apraxia, myoclonus, gaze palsies, and alien - limb phenomenon . Research focused on the motor symptoms may have led to the notion that cognitive impairment or dementia occurs only in a few patients with cbd.6,7 postmortem pathological studies of cbd show neuronal loss, swollen achromatic neurons, and diffusely stained tau - positive astrocytic plaques . These changes typically involve the cortical and subcortical areas.2,3 asymmetric cortical atrophy involves mainly the superior parietal and frontal lobes, with smaller effects in the temporal and occipital lobes.4 several recent studies have documented that cognitive dysfunctions and language disturbances in the early stage of the disease course are not rare manifestations in cbd patients.8,9 however, the current findings related to episodic memory functioning in cbd are not described well by comprehensive cognitive assessments . Our patient showed prominent memory impairment in several cognitive domains upon a detailed neuropsychological evaluation and history taking by his caregiver . The results of the word - list learning test as a verbal memory task indicated severe impairment of encoding, resembling the learning process frequently seen in patients with alzheimer's disease (ad). Very few case studies have found abnormalities with respect to episodic memory test using the story recall test in patients with cbd.10 - 12 in general, cbd patients perform better on story recall and word list tasks than matched ad patients.11,13 the impairment of episodic memory appears to be less severe in cbd patients than in ad patients . In ad, poor strategic processes in frontal lobe dysfunctions or disruption of frontal - subcortical circuits leads to episodic memory impairment . However, the pattern of memory deficits in our patient differed from that typical of ad.12 the prominent memory deficits in our case can be explained by additional cortical hypometabolism in the left temporal area . However, there were no prominent visual complaints with typical presentations of balint's syndrome in our case . Although significant visuospatial and constructive dysfunctions were observed when our patient was asked to draw interlocking pentagons and rey - osterrieth figures, those deficits were augmented by severe hand apraxia . Our patient also showed severe frontal subcortical circuit deficits when asked to perform several tasks of executive functioning . The frontal lobe dysfunctions could be explained by the significant hypometabolism in both frontal areas . In summary, our patient presented with severe episodic memory impairment and frontal executive dysfunctions at an early stage of cbd . However, other neurodegenerative diseases such as ad or other focal dementia syndromes associated with parkinsonism cannot be completely ruled out without a postmortem pathologic diagnosis.

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