Background: Nonoperative management (NOM) of liver trauma is currently rather the rule than the exception. However, the current evidence presents subgroups of patients at higher risk for NOM failure. These patients must be treated more cautiously regarding the NOM approach. Method: A case report of 3 polytrauma patients (Injury Severity Score>17) with high–degree liver trauma managed nonoperatively. Results: The first case presented is the one of a polytrauma patient with degree Ⅳ liver injury and impaired mental status. It was a high risk for NOM failure because there was an angiographically hemostasis. The second case is one of a polytrauma patient who became hemodynamically stable after the administration of 2000 ml of fluid intravenously. There was a nonoperative approach with angiography and embolization of degree Ⅳ liver injury. Despite the success of the nonoperative treatment, there was an important hepatic necrosis following embolization. The third case is one of a polytrauma patient with a degree Ⅳ hepatic injury. Success was accomplished in NOM without an angiography. Conclusions: Nonoperative management of liver injuries can be applied safely even in high degree hepatic trauma. In hemodynamically metastable patients or impaired mental status patients, the nonoperative approach can be applied successfully, but the trauma surgeon must be very cautious. Introduction: The prevalence of the blunt liver trauma has increased over the past 3 decades . It is nThe liver, the most voluminous solid organ of the abdomen is frequently injured by both, blunt and penetrating abdominal trauma. Although in the urban setting the penetrating abdominal wounds may be more common, about 85% of liver lesions are produced by blunt trauma . The reqOver the past 20 years, there have been fundamental changes in the liver trauma approach. There was a major role in the recognition that most liver injuries stop bleeding spontaneously –5. MoreoCase 1: A 14–year–old male patient was admitted for a traffic related accident. The patient was mechanically ventilated through an orotracheal tube at the accident site.  In the emergency room  the blood pressure (BP) was of 90/65 mm Hg, the heart rate (HR)=120 bpm, and the oxygen saturation (SpO2) 97%. On clinical examination the patient had two traumatic marks at the head , as well as thoracic and abdominal trauma. The hemodynamic stability was maintained by intravenous crystalloids administration. FAST ultrasound  made by the imagist physician showed perihepatic fluid. Thoracic Computed Tomography (CT) showed right hemothorax  and bilateral lung basal contusions  ( AIS= 3) .Abdominal CT showed degree Ⅳ (OIS) liver laceration, AIS= 4, at the level of the Ⅶ and the Ⅷ Couinaud segments, with minimum hemoperitoneum . Although the patient was tracheally intubated and mechanically ventilated, being intravenously sedated, the nonoperative management of the liver injury was decided due to the patient's hemodynamic stability.Due to the high degree of the liver injury, it was decided that an angiography should be carried out. The active bleeding detected in the right hemiliver was embolized .The post–embolization clinical course was favorable, the patient being discharged 20 days after admission. Case 2: A 28–year–old male patient was admitted after a workplace accidental explosion. During the primary survey, the patient was fully conscious, with Glasgow Coma Scale (GCS) = 15, BP = 80/55 mm Hg, HR = 115 bpm and respiratory rate (RR) = 27/min, SpO2 = 96%.On the clinical examination the patient experienced a minor craniocerebral trauma, a facial trauma with open wounds at this level (AIS = 2), thoracic trauma with penetrating wounds at the level of the right thorax, produced by metallic fragments, multiple wounds in the left upper limb caused by metallic fragments (AIS = 3).In the emergency room, the patient received resuscitating crystalloids intravenous fluids. During clinical and imagistic examination, the patient received 2000 ml of crystalloids solutions having a secondary blood pressure rate of 110/60 mm Hg.The FAST ultrasound made by the imagist physician did not show free intraperitoneal fluid. The Chest X–Ray showed right pneumothorax with metallic fragments in the right thorax and in the right superior abdominal quadrant  Iiver laceration, AIS = 4, at the level of the right hemiliver, Ⅴ, Ⅶ, Ⅷ Couinaud segments , it was decided that a nonoperative management of the liver injury with early angiographic embolization of arterio–venous fistula  should bIn the post–embolization period, the patient experienced an important hepatic necrosis: AST = 1720 UI/L, ALT = 1927 UI/L, Total Bilirubin = 6.20 mg/d . On clinThe 10 days post embolizaton follow-up abdominal CT showed a favorable development of the liver laceration area . Due to The follow–up CT scan performed 6 weeks later showed a favorable development of the liver lesion . Case 3: A 38–year–old patient was admitted after an accidental fall. In the emergency room, the patient experienced right thoracic and right upper quadrant abdominal pain. His BP was of 110/70 mm Hg, HR = 95 bpm, RR = 32/min, SpO2 = 95 %, GCS = 15.FAST ultrasound made by the imagist physician showed an average amount of free intraperitoneal fluid  in the right pleural cavity. Chest X–Ray showed right hemothorax with pneumothorax, multiple rib fractures and right clavicle fracture.Thoracic CT scan: right pneumothorax with mass effect on mediastinal structures, right hemothorax , upper lobe laceration of the right lung, bilateral basal pulmonary contusions , C Ⅱ – C Ⅶ unilateral flail chest, C Ⅱ – C X rib fractures  (AIS = 4) .Abdominal CT: Contusion of the Ⅵ, Ⅶ and Ⅷ liver segments, with a deep liver laceration adjacent to the hilum area  hemodynamic stability or patients who become stable after a moderate amount of fluid replacement, (2) accurate CT characterization of the liver lesions and a small amount of free intraperioneal fluid, (3) the absence of other abdominal lesions requiring laparotomy, (4) < 4 units of transfused blood . One of the major fears of trauma surgeons dealing with nonoperative management is the possibility to initially miss an injury. Miller et al. showed that the frequency of associated abdominal injuries is of 5% in liver trauma and of 1,7% in spleen trauma . The assInitially, it was thought that the patients with an impaired mental status are not suitable candidates for nonoperative management, but today, it has been proven that the failure rate of nonoperative approach is not higher in this subgroup of patients ;16. As the trauma surgeons became familiar with the nonoperative therapy, the hepatic injuries of increased gravity were approached nonoperatively. High degree liver lesions, with a large amount of hemoperitoneum can be managed nonoperatively if the patient is hemodynamically stable . HoweverAlthough traditionally the patients with blunt solid abdominal organs trauma were restricted to bed rest and light physical activity, there is no data to support this recommendation. The timing of mobilization of patients with blunt solid organ injuries does not seem to contribute to delayed hemorrhage requiring laparotomy . The current medical literature shows that degree Ⅲ or lower liver injuries in hemodinamically stable patients should not repeat computed tomographic scan before discharge, assuming the low rate of failure in these patients ;20. In cThe complications of liver lesions nonoperatively treated are hemobilia 0.2 – 0.3%), delayed hemorrhage (<3%), liver abscess and biloma (< 0.5%) and extrahepatic bile duct injury  – 0.3%, ;22.The process of hepatic repair after blunt trauma follows a predictable pattern: hemoperitoneum usually resolves within 1 week, subcapsular hematomas in 6 – 8 weeks and liver laceration in 3 weeks . HepaticBy increasing the frequency of selective nonoperative management use in abdominal trauma, another important issue emerges: the education of the residents in abdominal emergency surgery. The use of modern imagistic tools decreased the nontherapeutic laparotomy rate as a result of positive diagnostic peritoneal lavages from 35% to 14%. However, as the nonoperative experience grows, the resident's opportunity for operative experience decreases . Lucas and Ledgerwood highlight the inherent challenges of achieving psychomotor skills for the hemostasis of solid organ injury in an era of nonoperative therapy . This stConclusions: Selective nonoperative management of blunt hepatic lesions is the treatment modality of choice in hemodinamically stable patients, irrespective of the degree of injury. It is associated with a low overall morbidity and mortality and the result does not increase the length of stay, need for blood transfusions and bleeding complications compared with operative management. In polytrauma setting, the nonoperative management of liver injuries can be challenging. However, it can be quite satisfying to be able to successfully manage patients with severe and multiple traumatic injuries in nonoperative fashion. Modern imagistic tools can accurately depict various patterns of hepatic parenchymal injuries as well as associated bowel or pancreatic injuries that require an emergency laparotomy. Reduced incidence and decreased therapeutic laparotomies for liver injury have created a training vacuum for future trauma surgeons.


With regard to the analysis of spontaneous movements, a differential profile emerged early in development among the three genotypes. While only slight coordination difficulties are exhibited by heterozygous pups, all indices of motor development appear delayed in reeler mice. Overall, our results evidence a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reelin mutant pups.Reelin is a large secreted extracellular matrix glycoprotein playing an important role in early neurodevelopment. Several genetic studies found an association between RELN gene and increased risk of autism suggesting that reelin deficiency may be a vulnerability factor in its etiology. Moreover, a reduced reelin expression has been observed in several brain regions of subjects with Autism Spectrum Disorders. Since a number of reports have documented presence of vocal and neuromotor abnormalities in patients with autism and suggested that these dysfunctions predate the onset of the syndrome, we performed a fine-grain characterization of the neonatal vocal and motor repertoire in reelin mutant mice to explore the developmental precursors of the disorder. Our findings evidence a general delay in motor and vocal development in heterozygous (50% reduced reelin) and reeler (lacking reelin gene) mutant mice. As a whole, an increased number of calls characterized heterozygous pup's emission. Furthermore, the typical ontogenetic peak in the number of calls characterizing wild-type pups on postnatal day 4 appeared slightly delayed in heterozygous pups (to day 6) and was quite absent in reeler littermates, which exhibited a flat profile during development. We also detected a preferential use of a specific call category ( A number of studies implicate the glycoprotein reelin in the etiology of several neurodevelopmental disorders such as schizophrenia Spontaneous reeler mutant mouse  presents lamination defects in cerebral cortex, hippocampus and cerebellum. The cerebellum shows a decreased number of Purkinje cells Unlike Rl mice, levels of reelin in heterozygous mice (Het) are reduced by 50% compared to wildtype (Wt) and the lamination defects in the CNS are absent. This molecular depletion produces a range of subtle neurobehavioral consequences including increased impulsivity and disinhibited behavior Reduced reelin expression has been observed in several brain regions of subjects with autism Moreover, several genetic studies found an association between RELN gene and increased risk of autism To address the hypothesis that Het and Rl mice display motor and vocal alterations Our results evidence a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reelin mutant pups.B6C3Fe heterozygous female and male mice, originally purchased from Jackson Laboratories (USA) were bred in our laboratory. Two females were housed with one male in (33 cm×13 cm×14 cm) Plexiglas boxes, with sawdust bedding and a metal top. After two weeks of mating, male mice were removed, dams were housed individually in Plexiglass cages (33×13×14 cm), and daily checked for delivery. Mice were maintained on a reversed 12∶12 h light∶ dark cycle (lights on at 18∶30 h). The temperature was maintained at 21±1°C, relative humidity (60±10%). Mice genotype was determined at weaning (pnd 25) by RT-PCR on tail samples. Mice are weaned into cages of same sex pairs. After weaning mice were housed in pairs within Plexiglass cages (27×21×14 cm), with sawdust bedding. Animals were provided drinking water and a complete pellet diet  ad libitum. Pups were tattooed on the paw with animal tattoo ink  by loading the ink into a 30G hypodermic needle and inserting the ink subcutaneously through the needle tip into the center of the paw. The procedure was performed at two days of age, immediately after behavioral testing. The procedure causes only minor brief pain and distress and does not require the use of anesthesia. All procedures were in accordance with the European Communities Council Directive (86/609/EEC) and formally approved by Italian Ministry of Health.Litters chosen for testing contained more than six pups. Body weights and body temperatures of pups were measured after the ultrasonic vocalization test on pnd 2, 4, 6, 8 and 12. On each day of testing, each pup was placed into an empty glass container , located inside a sound-attenuating styrofoam box, and assessed for ultrasonic vocalizations during a three minute test. At the end of the three minute recording session, each pup was weighed and its axillary temperature measured by gentle insertion of the thermal probe in the skin pocket between upper foreleg and chest of the animal for about 30 seconds . No differences in patterns of calling were detected in a comparison of male and female pups, therefore data were collapsed across sex.An Ultrasound Microphone  sensitive to frequencies of 10–180 kHz, recorded the pup vocalizations in the sound-attenuating chamber. The microphone was placed through a hole in the middle of the cover of the styrofoam sound-attenuating box, about 20 cm above the pup in its glasses container. The temperature of the room was maintained at 22°C. Vocalizations were recorded using Avisoft Recorder software (Version 3.2). Settings included sampling rate at 250 kHz; format 16 bit. For acoustical analysis, recordings were transferred to Avisoft SASLab Pro (Version 4.40) and a fast Fourier transformation (FFT) was conducted. Spectrograms were generated with an FFT-length of 1024 points and a time window overlap of 75% . The spectrogram was produced at a frequency resolution of 488 Hz and a time resolution of 1 ms. A lower cut-off frequency of 15 kHz was used to reduce background noise outside the relevant frequency band to 0 dB. Call detection was provided by an automatic threshold-based algorithm and a hold-time mechanism (hold time: 0.01 s). An experienced user checked the accuracy of call detection, and obtained a 100% concordance between automated and observational detection. Parameters analyzed for each test day included number of calls, duration of calls, total calling time, qualitative and quantitative analyses of sound frequencies measured in terms of frequency and amplitude at the maximum of the spectrum.Complex calls displayed one component containing two or more directional changes in pitch, each ≥6.25 kHz; 2) Two-component calls consisted of two components: a main call (flat or downward) with an additional punctuated component towards the end; 3) Upward-modulated calls exhibited a continuous increase in pitch that was ≥12.5 kHz, with a terminal dominant frequency at least 6.25 kHz more than the pitch at the beginning of the vocalization; 4) Downward-modulated calls exhibited a continuous decrease in pitch that was ≥12.5 kHz, with a terminal dominant frequency at least 6.25 kHz less than the pitch at the beginning of the vocalization; 5) Chevron calls resembled an ‘inverted-U’, which was identified by a continuous increase in pitch ≥12.5 kHz followed by a decrease that was ≥6.25 kHz; 6) Short calls were punctuated and shorter than 5 ms; 7) Composite calls were formed by two harmonically independent components, emitted simultaneously; 8) Frequency steps were instantaneous frequency changes appearing as a vertically discontinuous “step” on a spectrogram, but with no interruption in time; 9) Flat calls displayed a constant beginning and the ending of the pitch frequency remained constant (≤3 kHz of each other).We classified every USVs emitted at pnd 2, 4, 6, 8, 12, in nine distinct categories, based on internal pitch changes, lengths and shapes, using our previously published categorization Call category data were subjected to two different analyses: a) genotype dependent effects on the frequency of vocalizations emitted by each subject at pnd 2, 4, 6, 8, 12; b) genotype-dependent effects on the probability of producing calls from each of the nine categories of USV, as described below under Statistical analysis.After each USV recording session, each pup was placed on its back over a flat surface, and the time needed to return to the natural position  was measured. This reflex requires complex coordination between head, trunk, and paws. The reflex was tested once with a cut-off latency of 60 s. Latencies were measured in seconds, using a stopwatch for righting reflex.Concomitant with the vocalizations recording on pnd 2, 4, 6, 8 and 12, mouse pups were also videorecorded for analysis of spontaneous movements. Frequency and duration of behavioral items were analyzed by an observer blind to mouse genotype. We used the NOLDUS OBSERVER software V 10XT  to score the videotapes. In accordance with previous studies focused on neonatal rodent behavior A mixed-model Analysis of Variance (ANOVA) with Repeated Measures was performed to analyze genotype-dependent effects on neonatal USVs and spontaneous movement responses, with the genotype (Wt vs Het vs Rl) as factor and pnd or call categories as the repeated measures.Probability of vocalizations within strain was calculated as number of calls in each category for each subject/total number of calls analyzed in each subject and standardized by angular transformation. Since no sex differences were detected, data were collapsed across sex. Post-hoc comparisons were performed using Tukey HSD Test only when a significant F-value was determined. For all comparisons, significance was set at P = 0.05.Body weight did not differ between genotypes  and, as expected, progressively increased with age  (data not shown).Changes of ultrasonic vocalizations (USVs) over time were characterized by an inverted U-shape across the first 12 postnatal days of age, as confirmed by the main effect of day  . WildtypNo differences were detected in body temperatures of Rl, Het and Wt as measured after each separation test , (data not shown).two-component calls than Wt pups . As illustrated in two-component calls than the other genotypes . No differences among genotypes were evidenced on other subtypes of calls later on in development , already on pnd 2, exhibited a differential vocal repertoire in comparison to Wt controls. Indeed, mice of both mutant genotypes emitted a high proportion of two-component calls and a low proportion of flat calls, whereas other types of vocalizations remain unchanged. This profile was still there on pnd 4. The data on pnd 6 indicate that the vocal repertoire of Het and Rl pups was represented by 50% of two-component calls. This profile was still present on pnd 8, then reducing later on. In fact, on pnd 12, Het pups still persisted in emitting primarily the complex and two-component calls, while Wt emission was characterized by a prevalence of two-component and short calls. At this age, Rl pups showed a vocal repertoire characterized by the two-component, short and complex calls, indicative of a phenotype halfway between Wt and Het pups  = 3.692, p<0.05; short, pnd 6: F = 3.328, p<0,05; flat, pnd 6: F = 3.573, p<0,05; upward, pnd 12: F = 3.367, p<0.05; complex, pnd 12: F = 7.845, p<0.001]. Specifically, on pnd 6, Wt pups emitted less two-component calls than Rl pups, more short calls than Het and Rl pups, and more flat calls than Rl pups . At pnd 12, Het pups emitted more complex and upward calls than Wt subjects .When analyzing each USV category separately on different postnatal days  = 24.290, p<0.0001], with all genotypes reaching the full development of the reflex on pnd 12. Post hoc comparisons performed on the two way interaction genotype x day  indicated that Het spent more time to turn their body than Wt and Rl pups on pnd 2 (p<0.01)  see .Curling behavior is measured by time spent from pups rolling on the back. This behavior is associated to the development of the righting reflex. Curling duration rapidly decreased across the five days of observation and disappeared on pnd 12 . A main effect of genotype was found . Post hoc comparisons performed on the interaction genotype x days of testing  showed that curling durations in Het and Rl mice were longer than Wt on pnd 2 (p<0.05), and persisted longer in Rl mice on pnd 4 (p<0.05), thus suggesting a motor coordination deficit in Het and Rl pups in getting an upright position  = 4.726; p<0.05; duration: F = 5.822; p<0.005], with Rl pups being significantly much less involved than Wt and Het subjects,  = 2,899; p<0.05; F = 2,692; p<0.05, respectively],  = 102.346; p<0.0001]. Post hoc comparisons performed on the interaction genotype x time intervals  indicated that Rl mice were more active than Wt and Het mice on pnd 12 (P<0.05), (see Time spent in 05), see .circling behavior yielded a significant main effect of genotype , with Rl mice exhibiting a lower number of circling episodes than the other genotypes at all days of testing  = 4.017; p<0.05], with Rl mice performing less nose probing than Wt and Het pups see . In genehead shaking . Post hoc comparisons performed on the genotype x time intervals interaction  revealed that Rl pups spent significantly less time in shaking their heads than Wt on pnd 2 (p<0.05) and less time than Wt and Het littermates on pnd 4 (p<0.05), (see A main effect of age was found for 05), see .head rising, side, pivoting, and immobility behaviors (data not shown).No differences between genotypes were observed for Reelin is a glycoprotein playing a role in regulating neuronal migration and brain lamination during development. The reduced or complete lack of reelin signaling impairs neuronal connectivity and synaptic plasticity leading ultimately to the cognitive deficits present in autism and schizophrenia. So far, limited studies study have investigated the contribution of reelin deficiency to the establishment of the early motor and social/communicative deficits present in these neurodevelopmental disorders + tf/J, Shank2 −/− and Mecp2 null mice, Tsc1 +/− and Tsc1−/− conditional knockouts and mice with a chromosome 15q11–13 maternal deletion or a paternal duplication Heterozygous reeler pups separated from their mothers and siblings at pnd 2, 4, 6, 8 and 12 emitted significantly more calls and for longer time than their wildtype and homozygous littermates. Moreover, a different profile of emission was detected in Het and Rl mice in comparison to wildtype pups. In fact, while wildtype pups showed a peak of emission at pnd 4, Het's peak of emission was slightly postponed to pnd 6, and profile of emission in Rl mice appeared quite flat, without a peak, indicative of a potential delay in the emotional/communicative development in this mutant line. No differences were detected in body temperature upon USVs recording excluding the possibility of unusual thermoregulation in reeler mice. An increased number of USVs has been detected also in other animal models of autism such as BTBR TOur USV data are partially in contrast with previous data collected in this model complex, composite, downward, flat, frequency steps, short, two-components, upward and chevron. To our knowledge, this is the first report about a detailed analysis of pup vocal repertoire throughout the first two postnatal weeks of age. Aim of this deeper investigation was to detect subtle alterations in normative developmental aspects that could be potentially transferred to human studies for early detection of ASD chevron, complex, downward, flat and two-components) while at pnd 12 their vocal repertoire appeared more limited (64%) and narrowed to two categories of vocalizations, such as two-component and short calls, and to a low number (29%) of chevron, complex and downward calls. This analysis allowed us to evaluate the development of the vocal profile of wildtype mice from a neonatal to a more adult-like repertoire. In fact, the call distributions showed by wildtype pups at pnd 12 are similar to what B6 males and females emitted in adult social contexts two-component (about 50%), chevron (about 20%) and complex calls (13%). At pnd 12, Het pups still persisted in emitting primarily the complex and two-component calls, while Rl pups showed a vocal repertoire characterized by the two-component, short and complex calls, indicative of a phenotype halfway between Wt and Het pups. As a whole, from a detailed analysis of vocalizations in Het and Rl pups, it was possible to detect a delay in reaching the peak-day of emission (6th vs 4th day), associated to a delay in reaching a more adult-like vocal profile. Altogether, these findings confirm and extend the presence in reelin mutant mice of quantitative and qualitative alterations in USVs already in a neonatal phase We further investigated the specific types of calls emitted by the three genotypes at all days of testing. Specifically, according to our previous paper on pup vocalizations curling and longer latency to right themselves when placed on their backs than Wt littermates. These two responses are strongly associated with each other, curling is a vigorous side to side rolling movement while on the back and it is aimed at righting. Moreover, Rl pups showed a deficit in face washing and wall climbing at pnd 12 as compared to their Wt and Het littermates. Both these behaviors require coordination and equilibrium since pup is standing up on its hindlimb and washing its face or climbing the walls of the container with forelimbs. Altogether these data suggested the presence of a motor coordination deficit in mutant mice supported by the fact that, in the first weeks of life, mutant mice show progressive loss of Purkinje cells of the cerebellum circling, nose probing and head shacking behaviors and by the hyperactivity profile displayed by mutant pups on pnd 12. As a whole, persistence of immature motor behavioral responses characterizes the Rl pups. Such motor profile seems in line with the transient delays in sensory–motor development shown by other animal models of ASD such as Mecp2-null, Foxp2, and BTBR mice body righting or head tilting) recently described in infants later diagnosed for autism A number of deviations from normative motor development has been highlighted in Het and Rl mice. When tested during the first 12 days of life, mutant mice exhibited longer From our results, it appears that Het and Rl mice showed delay in the vocal and motor development in line with the alterations in these two systems seen in children with ASD and considered early warning signs of ASD. It is worth to notice that in terms of gene-dosing the deficiency of reelin in Het pups led to an alteration mainly in the vocal emission while the total lack of reelin in Rl pups primarily induce alterations at motor coordination level. Motor deficits present in Rl pups at the end of the second postnatal week could be the first signs of the cerebellar alteration generally leading to death shortly after weaning.Altogether, our results emphasize the importance of conducting behavioral phenotyping during the early developmental period in order to document the precise onset of symptoms, to identify transient signs and provide a basis for the timing of early intervention. In conclusion, our data confirm that reelin mutant mice represent an interesting animal model for behavioral abnormalities seen in neurodevelopmental disorders like autism and schizophrenia Figure S1Results about pattern of sonographic structure among genotypes and sexPie graphs show the percentages of the different call categories for the three genotypes,  in male pups during five days of testing .(TIF)Click here for additional data file.Figure S2Pie graphs show the percentages of the different call categories for the three genotypes,  in female pups during five days of testing .(TIF)Click here for additional data file.Figure S3Production of ultrasonic vocalizations by call category. A) Frequency of ultrasonic vocalizations at pnd 4, B) pnd 8 and C) pnd 12.(TIF)Click here for additional data file.Materials S1(DOCX)Click here for additional data file.


Quercus variabilis) is one of the most widely distributed species in eastern Asia. In this study, leaf material of 528 Q. variabilis trees from 50 populations across the whole distribution  was collected, and three cpDNA intergenic spacer fragments were sequenced using universal primers. A total of 26 haplotypes were detected, and it showed a weak phylogeographical structure in eastern Asia populations at species level, however, in the central-eastern region of Mainland China, the populations had more haplotypes than those in other regions, with a significant phylogeographical structure . Q. variabilis displayed high interpopulation and low intrapopulation genetic diversity across the distribution range. Both unimodal mismatch distribution and significant negative Fu’s SF indicated a demographic expansion of Q. variabilis populations in East Asia. A fossil calibrated phylogenetic tree showed a rapid speciation during Pleistocene, with a population augment occurred in Middle Pleistocene. Both diversity patterns and ecological niche modelling indicated there could be multiple glacial refugia and possible bottleneck or founder effects occurred in the southern Japan. We dated major spatial expansion of Q. variabilis population in eastern Asia to the last glacial cycle(s), a period with sea-level fluctuations and land bridges in East China Sea as possible dispersal corridors. This study showed that geographical heterogeneity combined with climate and sea-level changes have shaped the genetic structure of this wide-ranging tree species in East Asia.The biogeographical relationships between far-separated populations, in particular, those in the mainland and islands, remain unclear for widespread species in eastern Asia where the current distribution of plants was greatly influenced by the Quaternary climate. Deciduous Oriental oak ( Quercus spp.) retreated to several refugia in the Iberian Peninsula, Italy and the Balkans during the Last Glacial Maximum  period. During the inter-glacial periods, they recolonized the northern areas from these refugia The climatic changes of the Quaternary  have exerted a profound influence on the patterns of modern plant distribution and evolution at the global scale Similarly, East Asia experienced strong climate oscillations during the glacial and interglacial periods although no massive glaciers occurred since the Quaternary Relative to European and North American continents, eastern Asia showed a higher diversity of plant species due to unique topography and climate of the eastern Asia region since Quaternary Ostryopsis davidiana Decne in north China Ginkgo biloba in south China Juniperus przewalskii in northeast Qinghai-Tibetan Plateau Cyclobalanopsis glauca in Taiwan Island Aucuba japonicaAristolochia kaempferiKirengeshoma species distributing in eastern China, southern Korea and Japan. However, the distribution ranges of these species do not cover the large part of eastern Asia, and it would be an advantage to examine how Quaternary climate impacted the retreat, migration, and re-colonization of a plant species distributed across the eastern Asia.With molecular data coupled with coalescent theory, distributional and demographic change through evolutionary time and the identification of glacial refugia have been conducted for Quercus variabilis) is one of the most widely distributed deciduous species in eastern Asia (latitude from 24° to 42° N and longitude from 96° to 140°E), and it covers Mainland China, Zhoushan Archipelago, Taiwan Island, Japanese Archipelago, and Korean Penninsula  and is an ideal species to study possible climate impact on the species distribution. There have been some small-scale investigations of population diversity by using nuclear gene or plasma gene marker for the species. For example, population genetic diversity of Q. variabilis has been investigated within Shaanxi and Henan provinces using isozyme Q. variabilis distribution Oriental oak  how the identified haplotypes are distributed within and among populations of Q. variabilis; 2) how genetic variation is distributed between and within populations; 3) is there a phylogeographic relationship among the populations, in particular, between the populations growing in the Mainland China and the peripheral populations in the Archipelagoes and Island populations in Zhoushan, Japan and Tawain, and 4) what is possible paleodistribution and population demographic history of Q. variabilis in pan-eastern Asia. With information above, we could deduce the possible glacial refugia status in the eastern Asia during LGM period and explore the possible roles of land bridges in influencing geographic distribution of the species during the glacial period.In 2008, we collected the samples of 50 Q. variabilis populations were selected across the entire natural distribution area of Q. variabilis in East Asia in 2008. Thirty-four populations were from Mainland China, three from Zhoushan Archipelago which located in East China Seas, four from Taiwan Island, three from Korea Peninsula and the remained six from Japanese Archipelago  primes  were selected according to sequencing performance and variation results , 50 mM KCl, 0.2 µM of each primer, 200 µM of each dNTP, 1.25 U of Taq polymerase (TaKaRa) in a total volume of 50 µl. PCR was conducted on a Mastercycler pro Thermal Cycler , and performed with initial denaturing of 5 min at 94°C followed by 35 cycles of 30 s at 94°C, 30s of annealing at 55°C, 90 s of elongation at 72°C, ending with a 10-min extension at 72°C. The products were checked on a 1% agarose gel, and then the products with a single band were directly sequenced in both directions by standard methods using a Taq dye deoxy terminator cycle sequencing kit on an Applied Biosystems Model 3730xl automated sequencer .Approximately 50 mg silica gel-dried leaves were grounded using Precellys 24 tissue homogenizer . Genomic DNA was extracted from leaf powder using modified hexadecetyltrimethyl ammonium bromide (CTAB) procedure based on Doyle’s  results . PolymerHd) were calculated using DnaSP v5 Multiple alignments of the sequences were obtained using the program CLUSTALW Q. variabilis populations was divided into seven regions (marked from 1 to 7 in HS), total gene diversity (HT) and two parameters of population differentiation, GST  NST (coefficient of genetic variation influenced by both haplotype frequencies and genetic distances between haplotypes), were estimated by the program PERMUT (available at www.pierroton.inra.fr/genetics/labo/Software/) NST than GST. A higher NST than GST usually indicates the presence of phylogeographic structure with similar (closely related) haplotypes, being found more often in the same area than less similar haplotypes FST between mainland and islands populations and among islands populations.In order to estimate genetic differentiation among the populations from different geographic regions, the distribution range of FST/(1−FST) was calculated in ARLEQUIN and geographical distances were estimated with the program FOSSIL, and implemented in R language.Isolation by distance (IBD) Quercus7 generations, with sampling every 2000th generation, following a burn-in of the initial 10% cycles. The phylogenetic evolutionary tree was edited in FigTree v1.3.1 software (http://tree.bio.ed.ac.uk/software/figtree).Phylogenetic evolutionary tree of haplotypes was created by BEAST 1.7.3 which is a Bayesian statistical framework DSFD and F can be expected in cases of population expansion. Based on phylogeographical structure results, the demographic history of both populations in Central-Eastern China and East Asia was investigated using mismatch distribution analysis (MDA) in ARLEQUIN, to assess whether intraspecific lineages experienced past population expansions. The sum of square deviations between the observed and expected distributions and the raggedness index of the observed distribution were used as test statistics to validate fit of the models Tajima’s Q. variabilis population demographic expansion. First, the expansion time was estimated directly from the mismatch distribution with the statistic τ (tau) and translated into absolute time in years (t), using the equation T = τ/2u u is the neutral mutation rate for the entire sequence per generation and is calculated as u = µkg, where µ is the substitution rate in substitutions per site yr−1 (s/s/y), k is the average sequence length of the cpDNA region under study , and g is the generation time in yr . The mean substitution rate of the three combined cpDNA-IGS regions was obtained from the corresponding clock-calibrated BEAST tree of Q. variabilis. Second, we employed coalescence-based skyline-plot method to date population size changes over time (Bayesian skyline plot: BSP) using the BEAST program and Tracer v1.5 software (http://tree.bio.ed.ac.uk/software/tracer/).In addition, we used two methods to estimate the time of Q. variabilis via MAXENT (version 3.3.3 k) Q. variabilis was based on 325 presence records in the Chinese mainland sourced from Chinese Virtual Herbarium (http://www.cvh.org.cn/cms/) and 150 occurrence records in Taiwan, Japan, and Korea obtained from Global Biodiversity Information Facility (http://data.gbif.org/welcome.htm), after the removal of duplicate and inexistent records according to field surveys within each pixel . Bioclimatic variables were downloaded from the WorldClim database Maximum entropy modelling technique was employed to construct pontential palaeodistribution of Q. variabilis were aligned along a total length of 1670 bp with 23 variable nucleotide sites observed  of haplotypes  while population XY located in Dabie Mountains, Henan province and HZ in Zhejiang province had four haplotypes  . The popH6 existed only in Liaotung Peninsular and Korea Peninsular whereas H10 was only found in Zhejiang province, and H1 occurred only in populations from Anhui province . The Bayesian phylogenetic tree was shown in mv) in the network frame can be recognized in the network that mv1 connects H2, H16, H19 and H21, mv2 connects H9, H19 and H21, and mv3 connects H3, H10 and H17. These median vectors might be an indication that the three genotypes were missing in the sample collection during the sampling process, or were ancestor genotypes which had been extinct A phylogenetic network was established among 26 haplotypes . The netQ. variabilis populations with more haplotypes and higher nucleotide diversity  and nucleotide diversity  or haplotype diversity . Population PG, from Beijing in Northern China  and YB, from Yunnan province in Southwestern China  had the highest haplotype diversity and nucleotide diversity, population TN  and DM  had the second highest diversities, while all other populations  had low or zero diversity  was high  was essentially zero in populations from Southeastern China and 0.294±0.09 for populations from Central-Eastern China. By contrast, inter-population differentiation was the lowest in populations from Central-Eastern China (GST = 0.690±0.095) relative to populations from other regions .The total genetic diversity for all populations (P = 0.012) between GST (0.690) and NST (0.751) in Central-Eastern. This implied that there would be a significant phylogeographical structure in the Central-Eastern China region.There was a significant difference in genetic diversity (P<0.001)  indicated that 79.5% variation was partitioned among populations and only 17.51% of the variation within populations  . HoweverFCT = 0.817–0.832). In contrast, genetic differentiation among populations within Zhoushan and Japanese Archipelagoes and Taiwan Islands was low . This may indicate that isolation by distance effect was minimal in Q. variabilis populations in eastern Asia.Results from divergence test are presented in trnL-trnF, atpB-rbcL, trnH-psbA) chronogram of Q. variabilis (Q. variabilis (PP = 1) as 4.88 Ma , and obtained a point estimate for the coalescent time  of ‘Island’ clade and ‘Inland’ clade, suggesting a Quaternary split in haplotypes between Islands and Mainland in eastern Asia. H11, widespread in Taiwan and Japan Islands, was given a divergence time after 0.42 Ma .The BEAST-derived cpDNA  and the slowest reported cpDNA substitution rates for angiosperms (1.1×10−9 s/s/y), but consistent with the notion that woody taxa and/or phylogenetic relicts should have slower rates of molecular evolution Based on the cpDNA-IGS chronogram of HRag values. For Central-Eastern China populations, demographic expansions supported by significant SSD, spatial expansions supported by nonsignificant SSD and HRag values  . East Asia populations show spatial and demographic expansions supported by uniformly significant SSD and g values . Tajima’The Bayesian skyline plot (BSP) simulated the fluctuation of populations over time. Recent population size increases were observed in Middle Pleistocene (0.78 Ma - 0.13 Ma BP) from 0.5 Ma before present . The resThe test area under curve (AUC) for the ecological niche modelling (ENM), averaged across all 10 runs, was moderately high . The predicted distribution based on the model was similar to the species actual distribution  a, with Pinus tabulaeformisOstryopsis davidianaKirengeshomaDysosma versipellisTaxus wallichianaQ. variabilis had 26 haplotypes detected, with possible 114 missing ones and three un-sampled or extinct ancestral ones, with a complicated phylogenetic network and a lacking of distinct geographic structure . One important distribution characteristic is that many chloroplast haplotypes are discontinuous, such as H4 occurred in two locations which are more than 2,655 km apart, H5 is wide spread in eastern Asia but missing in most of populations in Central-Eastern China, H11 was found in coastal areas and islands but not in the inland regions except for Louguan Tai in Northwestern China. Another two cases of disjunct occurrence were found in H2 and H8. These disjunctions may be due to long-term fragmentation of Q. variabilis forests in the past. There was massive deforestation in East Asia, particularly in Central-Eastern and Northern China started around 1360s (start of the Ming Dynasty). There was also large scale clearing of forest areas for large agricultural expansion due to population expansion. This deforestation event resulted in large fragmentation of native forests and produced many isolated small plant populations Q. variabilis, as similar distribution characteristic also could be found in other species in the Chinese mainland Haplotype variation has been studied for several plant species in East Asia and China including tructure , 2. PopuDifferent haplotypes were detected in the adjacent populations from the same region. For example, H1 and H8 occurred in the North and South of Yangtze River respectively, where Yangtze River acted as a geographic barrier. In Laotung Peninsula, H6 and H8 were found in two close populations which might be derived from separate refugia.HT = 0.888) across the 50 populations of Q. variabilis in East Asia. The long evolutionary history of the species may have accumulated such genetic diversity. The wide geographical range of this species across a geologically dynamic region may help to provide ample opportunity for isolation, drift and conservation of the mutated alleles Q. variabilis was higher among populations within regions (HT = 0.667–1.00) and across the entire distribution, but lower within population (0–0.249) in these sampled regions  indicated large population differences. There was higher genetic differentiation between the populations in Mainland China and populations in Zhoushan and Japanese Archipelagoes, Taiwan Islands, and Korea Peninsula. This observed large differentiation could be readily attributed to isolation by seas. The abundance diversity among populations could also due to increased genetic drift in smaller populations, broad ecological niches, besides isolation by distance or barrier by local mountains. As an example, Oriental oak produce big-sized oak acorns which are dispersed mainly by rodents such as Apodemus peninsulae, Niviventor confucianus and A. agrarius over limited distances Q. variabilis is also a monoecious, autophilous plants, pollinated by wind and insects. Previous molecular studies have suggested that no species-specific nucleotide differences in several chloroplast DNA intergenic spacers in Japanese oak species owing to hybridization and (or) shared ancient polymorphism Q. variabilis Japanese populations. One possible explanation is that these populations might have gone through a bottleneck effect or founder effect when the population from mainland migrated to the Japan Archipelago. Similar phenomenon could also occur in Taiwan Island and Korea Peninsula. The similarity among Japan Archipelago, Taiwan Island and Korea Peninsula populations could be supported by the affinitive and shared haplotypes between Islands, simulated palaeodistribution and population expansion analysis.We detected a high level of total genetic diversity  could represent a remnant of an ancient centre of diversity for the species in North China region is an interesting observation. Previous investigations on conifer species preferred refugia in northern China during Pleistocene glaciations Q. variabilis with the range extended above 30°N and below 40°N, a finding that is somewhat inconsistent to the inference by Harrison et al., but consistent with Qian and Ricklefs’ hypothesis Q. variabilis.Whether population PG which had the second highest nucleotide diversity  to less than 35°N (Anhui Province) comparing population distribution at present and during LGM, inferred from our ENM construction. With pollen record in the sediment profiles collected in Taihu Lake , Xu et al. showed that in Taihu Lake, before 11000–9000 BP, deciduous broadleaf species  predominated while conifer species  co-existed SF indicated a demographic expansion of Oriental oak populations in East Asia. The results were consistent with Bayesian skyline plot that the ascent curve indicated an increased population size in Middle Pleistocene, a period that saw the advance and retreat of glaciers numerous times. Based on our fossil-calibrated cpDNA phylogenies, a rapid speciation event occurred at late Quaternary and we traced the spatial expansion of Q. variabilis populations in East Asia to the last glacial cycle(s).Typical responses of plants to climate changes were adaptive evolution through migration, resulting in the alteration of geographical distribution range Q. variabilis may have distributed across the temperate area in East Asia, with the northern range to Liaoning province at 40°N at least and the south range to the northern subtropical area at about 30°N. With the climate cooling at the beginning of the Quaternary, the north range of Oriental oak distribution retracted southwards (reaching to about 30°N) while some populations  might remained in certain special geographical locations between 30 and 40°N as possible refugia. The populations in the southern tip of the distribution colonized southwards (reaching about 20°N), forming a deciduous broadleaf forests in the areas where current evergreen broadleaf forests grew. As the starting point of diversification is given at the Pleistocene with 1.45 Ma, this distribution changing was very likely to cause the divergence between ‘Island’ clades and ‘Inland’ clades.Before the LGM, Q. variabilisQ. variabilis as a common tree species at the time. Across the East China Sea, the continental shelf areas as the land-bridge connected the Mainland China and Taiwan Islands, Oriental oak could distributed across the continental shelf areas and Islands.At the peak of the LGM, the MAT was about 15°C in the south range of the distribution, and about 7°C in the central part (southern Henan and northern Hubei) of the current distribution area of Although there have been a lot of literature showing a close relationship in flora distribution between Taiwan and Mainland China Q. variabilis populations migrated northwards from Central-Eastern China, re-colonized these lost distribution range, and finally, reached the Liaoning area as the north edge of the current distribution. There is possibility that some populations in Liaotung Peninsular could be the descendant from southern Korea Peninsula refugia. While the sea level rose, the land-bridge disappeared and the sea water separated populations remaining in Japanese and Zhoushan Archipelagoes and Taiwan Island from Mainland China. The lower level of population differentiation could be explained by a strong bottleneck or founder effects during the separation of Mainland and Islands. Meanwhile, in southern part of subtropical areas, other evergreen trees became dominant species in the new forests and Oriental oak retreated and only become a companion tree in the current evergreen forests.With increasing temperature after the LGM, Q. variabilis had a different evolutionary history relative to Q. robur in Europe that Q. robur only survived in several refugia in Mediterranean areas at the LGM Q. variabilis would more likely have been preserved in more places in East Asia at the LGM. We conclude that Oriental oak might have colonized current distribution by northward recolonization and possible expansion of many refugia after the LGM. Deciduous Oriental oak, one of the most widely distributed plant in Asia, providing an ideal species for investigating the biogeography of plants across the Mainland China, Korea Peninsular, Zhoushan and Japanese Archipelagoes and the Taiwan Islands in relation to Quaternary climate using phylogeographical approaches. Oriental oak haplotypes constituted one of the most complicated phylogenetic network among the plants studied so far in the eastern Asia, with possible two distinct clades. The first clade of ‘Island’ is mainly consisted by populations in isolated Taiwan Islands, Zhoushan and Japanese Archipelagoes, and Central-Eastern coastal region of Mainland China. Populations in the second clade were mainly from Central and Western Mainland China. Our results showed that the Central-Eastern China had populations with more haplotypes, the highest nucleotide diversity and significant phylogeographical structure and was the main glacial refugia at last glacier ages. Q. variabilis was distributed as dominant tree in the areas of current evergreen forests in the Mainland China, and the continental shelf areas as the land-bridge between the Mainland and Islands of East China Sea at the LGM while its offshore refugia probably existed in southernmost Korea. Restricted seed dispersal mechanisms, geographical obstacles and human-induced fragmentation resulted in higher interpopulation differentiation across the current distribution range, and in particular, between the populations in the Mainland China and Archipelagoes and Taiwan Islands. Our findings supported the hypothesis that Q. variabilis had survived in scattered multiple glacial refugia in East Asia and experienced a range expansion and rapid speciation in response to climatic fluctuations during Quaternary.This might indicate that Information S13tables.(DOCX)Click here for additional data file.


GSTP1 is A→G at nucleotide 313, which results in an amino acid substitution  at the substrate binding site and reduced catalytic activity. We evaluated the interaction between GSTP1 Val allele and Helicobacter pylori infection, smoking and alcohol consumption, increasing the risk of gastric cancer among the Chinese population. Information on potential gastric cancer risk factors and blood specimens were collected from 618 incident gastric cancer cases and 1,830 non-cancer controls between March 2002 and December 2011 in Liaoning Province, China. GSTP1 Ile105Val was genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and polymerase chain reaction-restriction fragment length polymorphism. Serum levels of anti-H. pylori IgG were measured by ELISA. Odds ratio (OR) and 95% confidence interval (CI) were calculated using multivariate logistic regression, adjusted by sex and age. The risk of gastric cancer was significantly elevated in patients with the GSTP1 Val/Val genotype . An elevated risk of gastric cancer was observed in patients with H. pylori infection, smoking, or alcohol consumption, and together with the GSTP1 Ile/Val +Val/Val genotype  (p<0.05). The GSTP1 Val allele shows an interaction with smoking, alcohol consumption, and especially H. pylori infection for increasing the risk of gastric cancer. These findings could demonstrate new pathophysiological pathways for the development of gastric cancer.Glutathione S-transferase P1 (GSTP1) is a critical enzyme in the phase II detoxification pathway. One of the common functional polymorphisms of  Gastric cancer is one of the most frequently occurring cancers globally. A total of 989,600 new cases and 738,000 deaths are estimated to have occurred in 2008, accounting for 8% of the total number of cancer cases and 10% of cancer-related deaths RAS gene can be used to assess the risk of oncogenesis S) consist of a superfamily of dimeric phase II metabolic enzymes GSTM1 and GSTT1 genes are polymorphically deleted GSTP1, c.313A>G determines the amino acid substitution Ile(105)Val, which results in reduced activity of the Val-containing enzyme GSTM1 and GSTT1 have been investigated in cancer for a long time; however, only recently have some studies investigated the association of various cancers with GSTP1 variants. Some studies have observed a different relation between the GSTP1 polymorphism and an increased risk for oral GSTP1 variants Since the first description by Krontiris in 1985 that the polymorphisms of the It has been increasingly accepted that the etiology of most common tumors involves not only genetic and environmental causes, but also interactions between the two. The finding that the strength of association between lifestyle and cancer occurrence is influenced by genotypes (gene–environment interaction) has opened the door to genotype applications for tumor prevention.GSTP1 polymorphism influenced susceptibility to gastric cancer, as well as the interaction between the polymorphism and environmental factors  involved in gastric carcinogenesis.In this case–control molecular epidemiological study in a Chinese population we determined whether this A total of 2,448 patients with different gastric diseases were enrolled in this study, including gastric cancer (n = 618), atrophic gastritis (n = 683), and superficial gastritis as a control . All patients were consecutively recruited from March 2002 to December 2011 in Liaoning Province, Northeast China, and were surveyed about their history of any illness. Before commencement of the study, approval was obtained from the Human Ethics Review Committee of China Medical University. Written informed consents were obtained from participants in accordance with the Declaration of Helsinki and its later revision.H. pylori IgG. The blood samples were allowed to clot for 30–40 min at room temperature, and then centrifuged at 3,000 rpm for 10 min. The clots and sera were stored immediately at –20°C, and then moved into a freezer at –70°C within 3 days of collection.Cases and controls were interviewed face-to-face by trained interviewers using the same questionnaire. The diagnosis of gastric disease was established by upper gastrointestinal endoscopic examination and confirmed by histopathology. Histopathological findings were assessed according to the visual analog scale of the updated Sydney System GSTP1 Ile105Val single nucleotide polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism according to Watson et al GSTP1 was genotyped using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry platform , according to the manufacturer’s instructions. Duplicate samples  were included for the evaluation of genotyping quality and the concordance rate was 100%. Genotyping was successfully performed among 2,448 cases. No deviation from the Hardy–Weinbery equilibrium was observed . Finally, there were 550 gastric cancer cases and 550 superficial gastritis controls, and 683 atrophic gastritis cases and 683 superficial gastritis controls included for analysis; controls were frequency-matched with cases by sex and age±5 years.All genomic DNA was extracted using the standard phenol–chloroform extraction procedure. In 915 cases, H. pylori IgG antibody concentrations were each measured with ELISA . An individual was considered positive if the IgG titer was above 34 EIU.Serum t-test and Pearson’s χ2 test were used to test for differences between groups. The association between the GSTP1 polymorphism and risk of gastric cancer and atrophic gastritis was estimated by odds ratios (ORs) and 95% confidence intervals (CIs) adjusted by sex and age, together with the test for an interaction between the GSTP1 polymorphism and H. pylori infection. P≤0.05 was considered statistically significant.All statistical analysis was performed using the SPSS 17.0 statistical software . Student’s H. pylori-positive rates of the superficial gastritis control and atrophic gastritis groups, the superficial gastritis control and gastric cancer groups were 25.8% (176 of 683) vs 60.8% (415 of 683), and 24.4% (134 of 550) vs 49.3% (271 of 550), respectively, and a significant difference was observed in the atrophic gastritis group compared to the superficial gastritis controls, and the gastric cancer group compared to the superficial gastritis controls. The smoking and alcohol consumption rates were of significantly different between the superficial gastritis controls and gastric cancer cases.The frequency distributions of demographic and other selected characteristics of the participants are shown in GSTP1 genotype, we analyzed the association between GSTP1 genotype and the risk of atrophic gastritis and gastric cancer. Compared with the patients with Ile/Ile genotype, those with Ile/Val or Val/Val genotype did not harbor a significantly higher risk of atrophic gastritis. However, in the gastric cancer group, using Ile/Ile genotype as a reference (OR = 1.00), the OR for Val/Val genotype was 3.324 (95% CI = 1.790–6.172), which showed a statistically significant increase in gastric cancer risk associated with Val/Val genotype  that may cause some individuals’ susceptibility to gastric cancer and its precancerous conditions GSTP1 genotype and H. pylori IgG, smoking, or alcohol consumption in gastric cancer and its precancerous conditions. Using Ile/Ile genotype and H. pylori IgG(–) as a reference, the OR for  genotype and H. pylori IgG(+) was 4.308 (95% CI = 3.062–6.061) in atrophic gastritis subgroups, and the OR for  genotype and H. pylori IgG(+) was 3.696 (95% CI = 2.475–5.521) in gastric cancer subgroups. Using Ile/Ile genotype and non-smoking as a reference, the OR for  genotype and smoking was 0.782 (95% CI = 0.538–1.136) in atrophic gastritis subgroups, and the OR for  genotype and smoking was 1.638 (95% CI = 1.044–2.571) in gastric cancer subgroups. Using Ile/Ile genotype and non-alcohol consumption as a reference, the OR for  genotype and alcohol consumption was 0.862 (95% CI = 0.565–1.313) in atrophic gastritis subgroups, and the OR for  genotype and alcohol consumption was 1.641 (95% CI = 0.983–2.739) in gastric cancer subgroups. Association of the GSTP1 Val/Val genotype with H. pylori IgG(+), smoking, or alcohol consumption could significantly increase atrophic gastritis and gastric cancer risk  with a wide variety of electrophilic compounds, including many mutagens, carcinogens, anticancer agents, and their metabolites. Polymorphisms of GSTP1 have been reported, isoleucine (Ile) 105 valine  in exon 5 and alanine (Ala) 114 valine  in exon 6 GSTP1 Ile 105, GSTP1 Val 105 has a higher catalytic efficiency with regard to the metabolism of carcinogenic aromatic epoxides Over the past 20 years, there has been marked progress in our understanding of the role of genetic and environmental factors in the etiology of gastric cancer. GSTs are multifunctional and multigene products. They are versatile enzymes and participate in the nucleophilic attack of the sulfur atom of glutathione on the electrophilic centers of various endogenous and xenobiotic compounds. Out of the major classes of GSTs, GSTP1 has significance in the diagnosis of cancer because it is expressed abundantly in tumor cells GSTP1 was significantly associated with an increased risk of gastric cancer in the Chinese population. At present, there are few reports about the association between the polymorphisms of GSTP1 and the risk of gastric cancer. Researchers in the USA GSTP1 genotype seemed not to be associated with the risk of gastric cancer and chronic gastritis in a high-risk Chinese population. The results detected by Katoh et al GSTP1 allele Val is increasing in gastric cancer in the Japanese population, but this has not yet obtained statistical significance. We found that there was a significant difference in the GSTP1 polymorphic types between the gastric cancer cases and superficial gastritis controls. The frequency of GSTP1 Val/Val genotypes was significantly higher in the gastric cancer group, compared with Ile/Ile or Ile/Val genotypes. The analysis showed a statistically significant 3.324-fold increase in gastric cancer risk associated with the GSTP1 allele Val. This suggests that individuals from Northern China with GSTP1 allele Val have an increased risk of gastric cancer, but not atrophic gastritis (one of the precancerous conditions). However, it’s worth mentioning that in subgroups aged >60 years, an increased atrophic gastritis risk associated with Ile/Val genotypes was more evident. These findings revealed that the GSTP1 Ile/Val polymorphism could affect the stage of gastric carcinogenesis, the extent of atrophic gastritis as a precancerous lesion.In the present case–control study, we reported that the polymorphism of GSTP1 (21.1%) were significantly different from those in western people, such as European–Americans (33%) and African–Americans (42%) GSTP1-genotype-associated ethnic differences. The fact that, in our study, genotype frequencies among the population fitted the Hardy–Weinberg law further supports this view. The mechanism of the association between GSTP1 gene polymorphism and gastric cancer was not clear in our study. However, it can be supposed that GSTP1 is a major GST isoform expressed in human gastrointestinal epithelium, which can eliminate DNA oxidative products of thymidine or uracil propenal. After induction by cytochrome P450, some tumor-related carcinogens, such as benzo[a]pyrene diol epoxide and acrolein, can also be eliminated by GSTP1. The Ile→Val substitution may be associated with a higher level of DNA adducts, thus increasing the susceptibility to gastric cancer induction.Compared with other studies, we found that the allele Val frequencies of H. pylori infection, smoking, or alcohol consumption, and the GSTP1 Val/Val genotype.Furthermore, stratified analyses revealed that subgroups of smoking or alcohol consumption were more likely to have been diagnosed with gastric cancer. Our analysis supports there being an elevated risk of gastric cancer among individuals with H. pylori has been assigned as a classs I carcinogen by the World Health Organization, and acts as the initiating agent H. pylori IgG testing was a useful noninvasive strategy for testing for H. pylori. It was particularly useful in areas where the prevalence of H. pylori was high. We investigated the interaction between GSTP1 genotype and H. pylori infection in gastric cancer and its precancerous conditions. Association of the GSTP1 Val/Val genotype with H. pylori infection significantly increased gastric cancer and atrophic gastritis risk. This important finding suggests that GSTP1 genotyping and H. pylori IgG seropositivity could be used to identify individuals with a high risk of gastric cancer and its precancerous conditions. The mechanism of action might be that the H. pylori infection resulting in gastric cancer and its precancerous conditions depends on genetic polymorphisms influencing the virulence of the organism. GSTP1 Ile/Ile has a higher catalytic efficiency than GSTP1 Ile/Val or Val/Val for most environmental carcinogens, including cytokines produced by H. pylori infection. These cytokines that could not be detoxified by GSTP1 could directly induce gastric mucosal damage and eventually lead to development of atrophic gastritis and even gastric cancer. The exact molecular biology mechanisms need further exploration.GSTP1 Val/Val genotype with smoking or alcohol consumption could significantly increase atrophic gastritis and gastric cancer risk. This phenomenon might be caused by alterations in catalytic efficiency between tobacco and alcohol constituents and the polymorphic GSTP1 gene. These findings provide a possible molecular explanation for the synergistic effect of smoking and alcohol consumption on gastric cancer development. However, details of the mechanism must be verified by other well-designed experiments.Tobacco smoking and alcohol consumption are the main known etiological factors of some cancers. In this study, we observed that higher ratios of people in the gastric cancer group had consumed tobacco and alcohol , compared with the controls  . This fiGSTP1 may contribute to gastric cancer susceptibility in the Chinese population. Moreover, the combined effect of GSTP1 Val allele with environmental carcinogens  significantly increases the risk of gastric cancer development.In conclusion, our results suggest that polymorphism of


Bursaphelenchus xylophilus. Interestingly, Nematostella vectensis DicerB grouped into Dicer2 subfamily harbored a 5′ pocket, which is commonly present in Dicer1. Similarly, the 3′ pocket was also found to be conserved in all Dicer proteins with the exceptions of Schmidtea mediterranea Dicer2 and Trichoplax adherens Dicer A. The loss of catalytic residues in the RNase III domain was noted in platyhelminths and cnidarians, and the ‘ball’ and ‘socket’ junction between two RNase III domains in platyhelminth Dicers was different from the canonical junction, suggesting the possibility of different conformations. The present data suggest that Dicers might have duplicated and diversified independently, and have evolved for various functions in invertebrates.Dicers are proteins of the ribonuclease III family with the ability to process dsRNA, involved in regulation of gene expression at the post-transcriptional level. Dicers are conserved from basal metazoans to higher metazoans and contain a number of functional domains that interact with dsRNA. The completed genome sequences of over 34 invertebrate species allowed us to systematically investigate Dicer genes over a diverse range of phyla. The majority of invertebrate Dicers clearly fell into the Dicer1 or Dicer2 subfamilies. Most nematodes possessed only one Dicer gene, a member of the Dicer1 subfamily, whereas two Dicer genes (Dicer1 and Dicer2) were present in all platyhelminths surveyed. Analysis of the key domains showed that a 5′ pocket was conserved across members of the Dicer1 subfamily, with the exception of the nematode  Small regulatory RNA pathways are highly conserved mechanisms present in most eukaryotic organisms and play an important role in post-transcriptional gene regulation. The gene-regulatory function of microRNAs (miRNAs) and short interfering RNAs (siRNAs) is mainly through translational repression or degradation of cytoplasmic mRNAs by an RNA-induced silencing complex (RISC). miRNA and siRNA pathways share a common RNase III processing enzyme, Dicer, and together with other proteins it constitutes RISC for gene transcriptional repression Giardia intestinalis revealed that the PAZ domain was responsible for binding of the 3′ terminus of dsRNA Dicer proteins are present in many eukaryotic organisms including plants, fungi, and metazoans Previous studies have focused on Dicers of plants and model organisms, little is known about Dicers of invertebrates. The recent availability of genome sequences of over 34 invertebrate species from 10 phyla, including 1 choanoflagellate, 2 cnidarians, 1 placozoan, 2 annelids, 1 mollusc, 7 platyhelminths, 7 nematodes, 10 arthropods, 1 echinoderm and 3 chordates, have allowed us to perform an extensive phylogenetic analysis of Dicers.Drosophila melanogaster Dicers (NP_524453 and NP_523778), Caenorhabditis elegans Dicer (NP_498761) or Schistosoma mansoni Dicers (Smp_169750.1 and Smp_033600) as query sequences. An E-value of 1×e-10 was used as a cutoff in BLAST searches and the hits were filtered to keep only those with at least 25% identity to the query sequence. Protein functional domains were identified using Pfam database and SMART database For some well-annotated genomes, Dicer sequences were directly retrieved from the databases. In addition, BLASTP and TBLASTN were performed to search against their databases using The data sets contained a total of 58 sequences from 34 species , Pediculus humanus corporis (one gene) and Echinococcus multilocularis (three genes).The final data sets contained 58 Dicer gene sequences from two cnidarians, one placozoa, two annelids, one mollusc, seven platyhelminths, seven nematodes, eleven arthropods, one echinoderm and three chordates . No DiceTrichoplax adhaerens had the most copies of Dicer genes in our investigated species; however, all of the five Dicer genes were classed into the Dicer2 subfamily. The two cnidarians N. vectensis and Hydra magnipapillata each had only one Dicer2 gene, but possessed other Dicer genes that fell outside the two subfamilies.As shown in the Maximum likelihood tree , Dicers Taenia solium Dicer2 processed only one RNase III domain. We also observed the loss of the DEAD domain, which contains two RecA-like domains as a catalytic core and can regulate various processes involving RNA We identified the functional domains using the Pfam database and confirmed each inferred domain using the SMART database. As shown in Schmidtea mediterranea, Hymenolepis microstoma, T. solium, Echinococcus granulosus, E. multilocularis, the placozoan T. adhaerens and the nematode T. spiralis. There are two possibilities: the sequences are too divergent to be clearly recognized or they may have lost the PAZ domain during evolution. We therefore aligned the key amino acid residues in the PAZ domain of Dicers in the above species. As shown in S. mediterranea and T. adhaerens, indicating the absence of the PAZ domain in those two species. Recently studies have revealed that human Dicer anchors not only the 3′ end but also the 5′ end, and the 5′ end recognition by Dicer is important for the precise and effective biogenesis of miRNAs N. vectensis DicerB, which was classed into the Dicer2 family (A PAZ domain is an RNA-binding module found in PPD proteins (PAZ and Piwi domain proteins) and Dicer orthologs, and anchors the 2-nucleotide 3′ overhang of dsRNA with its highly conserved binding pocket 2 family .Schistosoma mansoni Dicer1, S. mediterranea Dicer1, T. solium Dicer2 and E. multilocularis Dicer RNC3.1 showed variations in this key region  were found between the two molecules, whereas only 20 hydrogen bonds/salt bridges existed at the dimer interface. In the dimer, two identical “ball-and-socket” junctions were formed at each end of the interface. The ‘ball’ was the hydrophobic side chain of Phe41 and the ‘socket’ was a cavity formed by side chains of Val52, Val56, Leu67, Ser68, and Lys71 After dsRNA binding to the 5′ and 3′ pockets, two RNase III domains of Dicer cleave targeted molecules. Based on the alignment of Dicer RNase III domain, we found that the catalytic core in most invertebrates was highly conserved . Howevery region . Comparea DicerC , indicatT. adhaerens. Dicers of invertebrates were clearly classed into two subfamilies, Dicer1 and Dicer2, except for several Dicers from cnidarians. Our results support the model of Dicer evolution in which a eukaryote Dicer may have duplicated independently. Interestingly, Dicer2 of E. multilocularis may have undergone duplication after species formation. Mature miRNAs have been identified in all the invertebrates investigated in our research with the exceptions of the choanoflagellate M. brevicollis and the placozoan T. adhaerensM. brevicollis, a close known relative of metazoans. However, T. adhaerens, a simple known metazoan, possessed five Dicer proteins and all of them belonged to Dicer2 subfamily. These Dicers may constitute an immune defense mechanism against viral infection as placozoans are exposed to a high viral load In our study, the number of the Dicer genomic loci was variable, from one in several invertebrates to five in N. vectensis DicerB and H. magnipapillata DicerB were classed into Dicer2 group, while the others fell outside the two recognized subfamilies. Recent analysis has revealed that cnidarians express species-specific miRNAs and share few miRNA families with bilateria Both cnidarians possessed multiple Dicers, and only S. mediterranea Dicer2 and T. adhaerens DicerA could lead to loss of the binding ability. But these two Dicer2 genes may function with the help of other RNA binding protein, such as Drosha N. vectensis DicerB that belonged to Dicer2 lineage. It suggests that N. vectensis DicerB may retain the bioactivities of Dicer1 as well.The recognition and cleavage of dsRNA by Dicer is a core step in miRNA and siRNA pathways. The 3′ pocket of Dicer is involved in 3′ end binding of dsRNA. The absence of key sites in the pocket in E. multilocularis Dicer RNC3.1 could reduce its catalytic activity. However, Dicer RNC3.2, a paralogue of Dicer RNC3.1, possessed all the key residues, and therefore it may compensate for the reduced activity of Dicer RNC3.1. The dimerization of RNase III domains creates a catalytic valley which can accommodate a dsRNA substrate. The two “ball and socket” junctions may be responsible in part for the accurate positioning of the catalytic residues in the valley. A Previous study suggested that the ‘ball’ consisted of the hydrophobic side chain of amino acids, and the interaction between ‘ball’ and ‘socket’ was precluded by charged side chain or the absence of any side chain in the position of the ‘ball’ After dsRNA recognition by the conserved pockets, the cleavage of dsRNA is conducted by two RNase III domains Text S1Amino acid sequences of Dicers.(TXT)Click here for additional data file.


Vaccination likely prevented 700,000–1,500,000 clinical cases, 4,000–10,000 hospitalizations, and 200–500 deaths. In April 2009, the United States began a response to the emergence of a pandemic influenza virus strain: A(H1N1)pdm09. Vaccination began in October 2009. By using US surveillance data  and vaccine coverage estimates , we estimated that the A(H1N1)pdm09 virus vaccination program prevented 700,000–1,500,000 clinical cases, 4,000–10,000 hospitalizations, and 200–500 deaths. We found that the national health effects were greatly influenced by the timing of vaccine administration and the effectiveness of the vaccine. We estimated that recommendations for priority vaccination of targeted priority groups were not inferior to other vaccination prioritization strategies. These results emphasize the need for relevant surveillance data to facilitate a rapid evaluation of vaccine recommendations and effects. On April 26, 2009, the United States declared a public health emergency in response to the 2009 pandemic influenza AH1N1)pdm09 virus pdm09 virus in mid-September 2009 ; a natioccinated ,9,10. Ficcinated .We estimated the number of clinical cases, hospitalizations, and deaths prevented in the United States that were directly attributable to the 2009–2010 A(H1N1)pdm09 virus vaccination program. These results can be used by public health officials, policy makers, and the public to evaluate this program and plan for the management of future pandemics.>65 years.Using Excel , we developed a tool to estimate the effects of the vaccination program . The estFirst, we calculated the weekly number of vaccine doses administered within each population subgroup. We then estimated, using the existing epidemic curve, the probability that a person who was vaccinated had not previously been infected with A(H1N1)pdm09 virus and had a clinical or subclinical case and the probability that a person would be infected during the remaining portion of the season. We adjusted our estimates for a 2-week delay in protection against the virus after vaccine administration . In thisDuring phase 2 of the calculation, we adjusted the probabilities of infection over time to include the number of averted events by including the event prevented  in the epidemic curve . Our original epidemic curve included the effects of the actual vaccination program; therefore, it was necessary to repeat the second phase  until the number of events in the final epidemic curve , minus the number of events prevented , exactly matched the epidemic curve that existed during the pandemic, week by week, for each population subgroup. This enabled us to estimate the direct effects of the vaccination program.The population in each ACIP-defined prioritized target group was estimated by using the National 2009 H1N1 Flu Survey (NHFS) –16 and CThree influenza surveillance systems in the United States were used to estimate the incidence and outcomes of A(H1N1)pdm09; the detailed methods are published in Shrestha et al. . The ranOur estimates of vaccination coverage were based on combined monthly data from the NHFS and the Behavioral Risk Factor Surveillance System survey ,22. Chil>65, for whom we assumed the vaccine to be 43% effective pdm09 virus are based on data for April 12, 2009–April 10, 2010, and an assumed proportion of subclinical cases; we did not include protection from previous years. For our base estimate, we assumed that 30% of all cases were subclinical; this assumption was held constant throughout the pandemic among all subgroups. Data from numerous countries and influenza challenge studies indicate that 24%–36% of the A(H1N1)pdm09 virus cases were subclinical –36. We tWe used the following equations to calculate clinical cases prevented. The equations for prevention of hospitalizations and deaths are identical, except that prior clinical or subclinical infections were not included.Interim estimated clinical cases prevented by a vaccination program  = Doses administered (using estimates from the 2 weeks prior to a specific date) × probability of not having had a prior clinical or subclinical infection  × probability of having a future clinical infection  × vaccine effectiveness Interim epidemic curve = Original epidemic curve + Estimated cases prevented (Equation 1a)Estimated cases prevented by a vaccination program  = Doses administered (using the estimates from the 2 weeks prior to a specific date) × probability of not having had a prior clinical or subclinical infection  × probability of having a future clinical infection  × vaccine effectiveness Epidemic curve without a vaccination program = Interim epidemic curve (Equation 1b) + Estimated cases prevented by a vaccination program (Equation 2a)Final check: Estimated number of clinical cases prevented by a vaccination program  = Final Epidemic Curve  – Original epidemic curve (with a vaccination program) Number needed to treat = number of doses administered/number of medical events  avertedWe conducted sensitivity analyses for 8 scenarios (see below); for each scenario the epidemiologic curve used was identical to that for our base case estimates, assuming that a vaccination program did not exist . For allTo assess the effects of accelerated vaccine uptake among specific groups, we calculated the proportion of total doses administered among each population subgroup over the course of the pandemic. We multiplied the result by the number of doses administered each week; e.g., if a subgroup received a total of 20% of the doses, we assumed that they received 20% each week.We assumed that each population subgroup had a proportionately equal demand for the vaccine. For each subgroup, we set the proportion of vaccine equal to the population proportion .We used the proportion of doses administered among each subgroup during the 2008 seasonal vaccination campaign. That is, if a population subgroup received 15% of the doses in 2008, we assumed that they used 15% of the doses each week during the 2009 pandemic).To assess the effects of providing the vaccine only to the aforementioned 2009 ACIP priority subgroups, we used the total percentage of doses administered to each group, based on the total 2009 vaccine uptake estimates, but adjusted the denominator of total doses by excluding the non-ACIP priority subgroups. We applied that percentage to the total number of doses administered each week.We estimated the effects of moving the start date of the vaccination program to begin 8 weeks to 1 week earlier. We did this by moving the date forward in increments of 1 week.>65 years of age, for which we increased the effectiveness to 55% for all outcomes. Last, we assumed vaccine effectiveness at 40% for all health outcomes in all population subgroups, except those including persons >65 years of age, for which we assumed 20% effectiveness for all outcomes.We examined the outcomes of assuming different vaccine effectiveness. We initially increased vaccine effectiveness to 85% for all health outcomes in population subgroups, except those including persons We examined the effects of assuming that, among vaccinated children 6 months–9 years of age, the first dose of vaccine was 20%–40% effective 2 weeks after administration and that vaccine effectiveness reached the levels listed in We varied the range of subclinical cases from 0% to 50%. The base estimate was 30%.We estimate that during October 3, 2009–April 18, 2010, the A(H1N1)pdm09 virus vaccination program directly prevented 712,908–1,458,930 clinical cases of A(H1N1)pdm09 infection, 3,923–10,393 hospitalizations, and 201–520 deaths ,4,5. Bas<9 years, we estimate that during the 2009 pandemic ≈81,518  A(H1N1)pdm09 infections were prevented among this population subgroup. However, by entering the same number of doses and same effectiveness, but adjusting the timing of administration by group (Scenario 1), we calculated that the number of cases prevented in this population subgroup would increase to ≈131,000 . In Scenario 2, in which we assumed children 6 months–9 years of age received 9% of all vaccines administered , cases prevented decreased to ≈58,000 . If no changes had been made to the ACIP recommendations and the rate of vaccine uptake among the different population subgroups had been similar to uptake of the 2008 seasonal influenza vaccine . This would have been ≈80% of what was estimated during the A(H1N1)pdm09 pandemic. This projected decrease in cases averted indicates that this population subgroup would not have benefitted from such a change in policy. Last, if the A(H1N1)pdm09 virus vaccine had been administered exclusively to those in the ACIP priority groups, we estimate that the number of cases that would have been prevented among children aged <9 years would be ≈186,000 ; under this assumption, 43% of this ACIP target group would be fully vaccinated, compared with an estimated 27% that actually were vaccinated.The estimated numbers of clinical cases prevented under different (assumed) prioritization strategies are shown in  vaccine ,38 , 983,671–2,004,053 clinical cases would have been prevented (≈38% more than in the base estimate). If the vaccine had been less effective , 442,971– 907,688 clinical cases would have been prevented (≈38% fewer than in the base estimate) (The vaccine administered during the 2009–2010 A(H1N1)pdm09 vaccine program was 62%% effective, and was calculated to have prevented ≈1,000,000  clinical cases. If the vaccine had been more effective  .<9 years, and we estimated that vaccination prevented 52,081–100,349 clinical cases among persons in this age group pdm09 virus vaccine. Approximately 60% of clinical cases prevented were among persons 6 months–24 years of age and among those 25–64 years, including pregnant women, who were considered at high risk for influenza-related complications. We found that the effects of the vaccination program were greatly influenced by the timing of vaccine administration and by vaccine effectiveness.Vaccine prioritization recommendations were made in July 2009 based on limited epidemiologic data, previous experience with immunologic responses to novel vaccine antigens, projections about when and how much vaccine would be initially available, and previous public engagement and expert opinion summaries about public values and preservation of societal functions ,6. Thesewww.cdc.gov/h1n1flu/updates/us/051410.htm [cited 2013 Jan 11]). We did not directly account for any vaccine-induced herd immunity. Estimates of A(H1N1)pdm09 virus vaccination coverage were based on survey data and subject to bias from low sample sizes from specific population subgroups and misclassification of vaccination status. Weekly vaccination estimates were interpolated. One of our sensitivity analyses illustrated the importance of the assumed level of vaccine effectiveness ; we assumed these to remain constant, with or without a vaccination program. We did not estimate the curve beyond April 10, 2010, which may have resulted in a slight underestimation of the effects. However, influenza-like illness data for the United States indicated that it was unlikely that many cases occurred after April 2010  vaccination programs. However, current influenza vaccine production technology is limited in how quickly large-scale vaccine production can be achieved, and the public health community cannot accurately predict the arrival of a pandemic. This study also demonstrates that the 2009 prioritization of specific subgroups in vaccine administration was not inferior to other vaccination strategies. In addition, this study highlights the need for better data on the effectiveness of influenza vaccine. Influenza vaccine effectiveness estimates vary considerably according to season, yet clearly they can greatly affect the overall results and conclusions of programs for policy makers.Future influenza pandemics are likely to differ in several ways, including in severity (patients’ signs and symptoms were mild during the 2009 pandemic), basic reproductive rate of the virus, virus subtype, subgroups affected, public acceptance of vaccination, vaccine safety profile, and vaccine effectiveness. The major factor influencing the effects of the 2009 subtype H1N1 vaccination program was that the amount of vaccine available early in the epidemic (when the effects of vaccination would be greatest) was limited. Thus, a major priority is to invest in research that can reduce production time pdm09 virus in the United States. Model allows the user to adjust many of the input variables.