The temporal sensitivity of the surveillance system (TemSSe) for Bovine Viral Diarrhea (BVD) in Danish dairy herds was evaluated. Currently, the Danish antibody blocking ELISA is used to test quarterly bulk tank milk (BTM). To optimize the surveillance system as an early warning system, we considered the possibility of using the SVANOVIR ELISA, as this test has been shown to detect BVD-positive herds earlier than the blocking ELISA in BTM tests.Information from data (2010) and outputs from two published stochastic models were fed into a stochastic scenario tree to estimate the TemSSe. For that purpose we considered: the risk of BVD introduction into the dairy population, the ELISA used and the high risk period (HRP) from BVD introduction to testing (at 90 or 365 days). The effect of introducing one persistently infected (PI) calf or one transiently infected (TI) milking cow into 1 (or 8) dairy herd(s) was investigated. Additionally we estimated the confidence in low (PLow) herd prevalence (<8/4109 infected herds) and the confidence in complete freedom (PFree) from BVD (< 1/4109).The TemSSe, the PLow, and the PFree were higher, when tests were performed 365 days after BVD introduction, than after 90 days. Estimates were usually higher for the SVANOVIR than for the blocking ELISA, and when a PI rather than a TI was introduced into the herd(s). For instance, with the current system, the median TemSSe was 64.5 %, 90 days after a PI calf was introduced into eight dairy herds. The related median PLow was 72.5 %. When a PI calf was introduced into one herd the median TemSSe was 12.1 %, while the related PFree was 51.6 %. With the SVANOVIR ELISA these estimates were 99.0 %; 98.9 %, 43.7 % and 62.4 %, respectively.The replacement of the blocking ELISA with the SVANOVIR could increase the TemSSe, the PLow and PFree remarkably. Those results could be used to optimize the Danish BVD surveillance system. Furthermore, the approach proposed in this study, for including the effect of the HRP within the scenario tree methodology, could be applied to optimize early warning surveillance systems of different animal diseases. In Denmark, Bovine Viral Diarrhea (BVD) is considered an exotic disease . An eradThe BVD virus (BVDV) can cause uterine infections in pregnant cows, abortions, stillbirths or weak calves –6. Cows Because the Danish dairy herd size has increased remarkably since the eradication program started , an evalThe time from the introduction of a pathogen into the country, until it is detected by the surveillance activities, can be defined as “high risk period” (HRP) , or “timThe probability of detecting a pathogen in a given time period has been called “temporal sensitivity” .In two previous studies we estimated the detection time for BVD in Danish dairy herds  and the Hence, the aims of the present study where (i) to evaluate the temporal sensitivity of the Danish BVD surveillance system (TemSSe) with fixed HRPs, according to different routes of BVDV introduction into Danish dairy herds , (ii) to investigate surveillance optimization, by taking into account the risk of BVDV introduction to the country, the herd size, and the antibody ELISA used on BTM; and (iii) to estimate (at country level) the confidence in low herd prevalence (PLow) and in complete freedom from BVD (PFree).To estimate the TemSSe of the Danish surveillance system, we developed a stochastic scenario tree model Section , Fig. 1 H), (II) the prevalence of infected animals within a herd , (III) the relative risk (RR) of infection in the population´s strata, and (IV) the sensitivity (Se) of the diagnostic test used [The sensitivity of national veterinary surveillance systems (SSe) can be evaluated through use of stochastic scenario tree models –15. Infoest used .H and PU, is correctly classified by the surveillance system [Thus the SSe represents the probability that a population, infected with the assumed Pe system –15. Furte system  it is usWe developed a stochastic scenario tree Fig.  in an ExH was set at 0.2 % (≈ 8 infected herds out of 4109 Danish dairy herds present in October 2010), or as 0.02 %, indicating that we aimed at detecting the first infected herd. The 0.2 % PH is similar to the limit set by the World Animal Health Organization (OIE) to substantiate officially free status for infectious bovine rhinotracheitis (IBR) [The Pis (IBR) , which cis (IBR) , 18. FurU was here defined as the threshold prevalence of antibody positive milking cows, at which the BTM was classified positive by the ELISA used . For that reason, we estimated the temporal sensitivity of the surveillance system (TemSSe) for two different ELISAs, with a HRP of 90 or 365 days. Those two time intervals lead to TemSSe testing quarterly (current system) or testing one year after BVDV introduction into the Danish dairy population (as a worst case scenario), respectively. The effect of introducing one PI calf or one TI milking cow into naïve Danish dairy herd(s), on the final TemSSe was also investigated.P animals , 19. TheMoreover, dairy herds were divided into two levels of risk: herds with import of live cattle (ImpoCattle category) or without (NoImpoCattle category) Fig. , based oWe used data from the last trimester of 2010, when approximately 4109 herds (N) delivered milk and were tested in the BTM. Hence, we considered a single surveillance component, which includes the whole population of Danish dairy herds.NoImpoCattle and PrPImpoCattle, respectively   with the adjusted relative risk (ARRj) of infection [NoImpoCattle and RRImpoCattle) were adjusted to maintain the specified relativity (weight) and yet average to one over the whole population [The probability that a herd was infected with BVD was represented by the effective probability of infection in the category (EPIle) Fig. . The EPInfection , 14. Thepulation .NoImpoCattle = 1, while for the ImpoCattle category we set RRImpoCattle = RImpoCattle/RNoImpoCattle. Where, the numerator and denominator represented the risk of BVD introduction for the ImpoCattle and NoImpocattle category, respectively  that at least one BTM positive herd was detected in the BTM surveillance system was:Furthermore, the component unit sensitivity was calculated as the weighted sum of the probabilities with a positive outcome at the end of each limb of the stochastic scenario tree Fig. . TherefoNoImpoCattle and PTRImpoCattle represented the probability that the threshold prevalence of seropositive cows (PU) was already reached within the milking group at the day of BTM testing . Data from 2010, on herd size, milk deliverance, imports/exports of live cattle, imports of semen and embryos, were obtained from the Danish Cattle Federation for all Danish dairy cattle herds.Moreover, for both herd categories, we estimated the percentage of imported goods from countries without or with endemic BVD Table . We madeImpoCattle and RNoImpoCattle) was calculated by use of a stochastic model, which was previously developed by Foddai et al. [The annual risk of BVDV introduction in each risk category , we used the same assumptions and input values described in Foddai et al. .ImpoCattle) was estimated using five published stochastic scenario trees [Hence, the risk of BVDV introduction in the ImpoCattle category  of BVDV introduction into the NoImpoCattle category was estimated using the same formula (Eq. The risk (Rmula Eq.  without U within the milking group) needed for BVD detection by BTM testing within a fixed HRP, was estimated using a stochastic simulation model; which was previously developed to simulate within-herd BVD dynamics and to estimate the detection time in Danish dairy herds, according to the ELISA used on BTM [The prevalence of antibody positive milking cows was assumed to increase over time . The prod on BTM .The PTR is affected by the route of BVDV introduction to the herd , the herd size, the threshold prevalence of the antibody ELISA used, and the time elapsed between BVDV introduction and day of testing (HRP). Thus, the PTR was calculated as the number of model iterations out of 500, where the threshold prevalence was reached, at 90 or 365 days after the introduction of one PI calf or one TI milking cow, into a naïve Danish dairy herd. A total of 500 iterations appeared sufficient to simulate the within-herd disease dynamics in the study by Foddai et al. [NoImpoCattle and PTRImpoCattle) to represent the variability, between herds of different sizes within each risk category.The estimation of PTR was carried out for the minimum, most common, and maximum herd size within each herd category. Thereafter, the PTR values were used in the stochastic scenario tree Fig. , Eq. 3 iIn the NoImpoCattle category, the herd size was minimum 1, median 123 and maximum 1185 cows . For the smallest herd we did not run the simulation model, because we considered the threshold prevalence as reached soon, after the first infectious animal was introduced into the herd. Therefore, for this herd size we used PTR 100 %. In the ImpoCattle category, the herd size was minimum 24, median 180 and maximum 1070 cows.Furthermore, in Foddai et al. , it was ®BVDV-Ab ELISA  [In a previous study , the SVA Sweden) –23 was s Sweden) , 25, whiThe threshold prevalence has been estimated at 50 % for the Danish blocking ELISA  and at 6With a cut-off blocking % (bl%) of 50, the sensitivity (Se) of the Danish blocking ELISA on BTM is 100 % , while fH and PU) represents a hypothetical level of infection in the country. If a single positive unit is found, the country would lose the “free status”.The scenario tree methodology –15 is usH. In the latter case, we did not exclude that in reality few positive herds could be present in the country.In our study, we estimated the NPV of the surveillance system to show the PFree, but we also estimated the confidence (PLow) that the prevalence of infected herds is below the PH = 0.2 %, while to estimate PFree the TemSSe was obtained using PH = 0.02 %. The NPV of the surveillance system was:Thus, to estimate PLow we used the TemSSe based on PWhere PLow (or PFree) is the confidence that the prevalence of infected herds was below the assumed design prevalence 0.2 % (or 0.02 %) at the beginning of the surveillance period, since no BVD cases have been detected with the investigated HRP.PriorPInf is the probability that the country was “infected” with the assumed design prevalence, at the beginning of the surveillance period. This input was set to 50 %, which corresponds to a conservative uninformed prior .Adj)  Eq. , by takiThe annual median PIntro for Danish dairy herds has been estimated as 10.7 %  . This PIThus, PLow and PFree were estimated for each infection scenario , HRP (90 or 365 days) and ELISA used.The importance of each input parameter was investigated by using the regression mapped values in @Risk and the amount of change in the output due to a plus 1 standard deviation for each input was estimated.H = 0.02 %), since PIs are the main source of BVDV spread between and within herds [The reference scenario was defined as: all dairy herds tested with the Danish blocking ELISA (current system) one year after introduction of a PI calf into a single dairy herd  was 4.8 % , while in the ImpoCattle category (RImpoCattle) it was 5.1 % .The median annual risk of BVDV introduction in the NoImpoCattle category (RImpoCattle) was calculated as the ratio between the two median risk estimates . Thus, the RRImpoCattle was simulated from a Pert distribution with minimum 1, mode 1.03 and maximum 1.07, to calculate the ARRNoImpoCattle  in small herds than in large herds, ii) for BVDV introductions through one PI calf than through one TI milking cow, and iii) with HRP of 365 days than with HRP of 90 days. Moreover, the SVANOVIR ELISA had higher PTR values than the Danish blocking ELISA , and the BTM was tested 90 days later with the blocking ELISA; to 97.2 % when one PI calf was introduced into the smallest herd (24 cows), and the BTM was tested one year later with the SVANOVIR Table .In the NoImpoCattle category the PTR ranged from 0 %, e.g. when a PI calf or a TI cow was introduced into the largest herd (1185 cows), and the BTM was tested 90 days later with the blocking ELISA; to 100 % when one infectious animal was introduced into a herd of a single cow Table .H = 0.2 %), with the Danish blocking ELISA (current surveillance system), the median TemSSe ranged from 64.4 % to 98.2 %. The related median PLow estimates were 72.5 % and 97.6 %, respectively  higher for the SVANOVIR ELISA  for the blocking ELISA. With a HRP of 365 days the opposite situation was observed, and the TemSSe and the PLow were remarkably higher for the SVANOVIR  than for the blocking ELISA (Table H = 0.02 %), and all Danish dairy herds were tested with the blocking ELISA, the median TemSSe ranged from 12.1 % to 39.3 %. The related median PFree estimates were 51.6 % and 55.7 %, respectively  higher for the SVANOVIR ELISA than for the blocking ELISA Table .NoImpoCattle). When this input was increased with 1 standard deviation the TemSSe increased between 0 and 11.4 %. The second input in order of importance was the PTR distribution used in the ImpoCattle category (PTRImpoCattle). In that case, the increase caused on the TemSSe ranged between 0 and 0.03 %. The other inputs caused a change lower than 0.03 %.According to the sensitivity analysis, the input with the highest impact on the estimated TemSSe was the PTR distribution used in the NoImpoCattle category  probability of BVDV introduction (PIntro) into the Danish dairy population. Increasing the PIntro of 1 standard deviation caused a decrease in the PFree between 0 and 3.1 %. All the other inputs caused a change lower than 0.006 %.For the PFree, the most important input was still the PTRTo evaluate the BVD surveillance system in Danish dairy herds, we followed the concepts from Martin et al. . AdditioThe way we adapted the scenario tree model allowed us to estimate the TemSSe. Thurmond  suggesteIn this way, we could evaluate if the surveillance system can actually function as an early-warning system, or if optimization was needed, to increase the probability of detecting infected herds (TemSSe) within the aimed time period.To our knowledge, this is the first study, where the impact of the HRP is included in the evaluation of a surveillance system using stochastic scenario trees. We believe that this approach should be considered, especially when early-warning surveillance systems are established for slowly spreading diseases, as is the case of BVD in large dairy herds after introduction of TI animals , 19.Conclusions on surveillance sensitivity and disease status at national level should be related to a specific time period, when the pathogen could have been introduced into the country. Hence, in our case, the TemSSe and PLow/PFree should be related to the period when the BVDV could have been introduced into the Danish dairy herd(s).H = 0.2 % as a reasonable cut-off level. Such design prevalence has been set up by the OIE to substantiate officially free status from IBR [Between December 2011 and December 2012, no dairy herds have been found positive in Denmark. Therefore, we can assume that very few (≤ 8) or no dairy herds were infected in the country at the beginning of 2012 and became BTM positive. Currently, there is no regulated between-herds design prevalence to substantiate BVD status at country level . Thus wefrom IBR , which hfrom IBR .If BVDV was introduced by a PI calf in at least 8 dairy herds, the median probability of detecting at least one of these herds after one year by BTM testing, would be > 95 % with both ELISAs. According to the European Food Safety Authority (EFSA) this could be considered as an acceptable level of confidence  . The conOn the other hand, if the objective of the surveillance system was to detect BVD by 90 days after introduction of a PI calf in 0.2 % dairy herds, then the SVANOVIR ELISA could be preferred, because only that test showed median TemSSe and PLow higher than 95 % Table . PI animAs we showed with the PTR values Table , outbreaH = 0.02 % (corresponding to 1/4109 infected herds), the TemSSe and the related PFree were <95 %, with both ELISAs  and a HRP of 90 days was used, the TemSSe and the related PLow/PFree were similar or slightly higher for the blocking ELISA than for the SVANOVIR Table . Under tThus, when 1) the herd size is very small (e.g. <50 cows), 2) the threshold prevalence of the test used is low, and consequently 3) the PTR is around 100 %, the TemSSe would become more dependent on the Se of the test used on BTM, than on the PTR. Then there is less need to consider the threshold prevalence of the test used and the PTR parameter . In contrast, using a higher BTM testing frequency, before the threshold prevalence is reached in the milking group, would be inefficient. In the latter case, changing the test (rather than increasing the testing frequency), could increase the TemSSe without increasing the costs of the surveillance system .In this study, we showed that the TemSSe was higher for HRP of 365 days than for HRP of 90. This was due to two main reasons: a) the longer the time an infectious animal is kept in the herd, the higher the probability that such an animal causes an outbreak with seroconversion of several milking cows, and b) within the first 90 days from BVDV introduction, no new PI calves could be born in the herd from recently infected cows (TI).The first observation is also valid for other diseases, while the second is peculiar to the epidemiology of BVD. In fact, PI calves are born from PI cows or from susceptible cows, which become infected within the first four months of pregnancy , 5. In tWhen PIs are present in a herd, the immunization of other herd mates occurs quicker than when only TI animals are present . In factThese observations are in accordance with our results. In most of the iterations where we introduced a TI and the within herd BVDV spread was simulated, the outbreak died out, before the threshold prevalence was reached. Thus, in those scenarios, the TemSSe was low Table  due to tTherefore, the approach proposed in this study, allowed us to evaluate the Danish BVD surveillance system taking into account all the main epidemiological characteristics of the disease.In the sensitivity analysis, we confirmed that the PTR is an important parameter to consider, when the temporal sensitivity of the surveillance system is estimated.Moreover, we showed how an increase in the probability of BVDV introduction into the Danish dairy population (PIntro) could cause a decrease in the PFree. Testing imported animals at the border, could reduce the PIntro and could increase the confidence in freedom (PFree), as previously argued for bovine tuberculosis .Our estimates  could be considered as conservative, since we assumed that herds became infected by introduction of one BVDV positive animal only (a PI or a TI). In reality, more infected animals could be introduced to one herd at the same time leading to higher values of PTR and TemSSe.Moreover, the PTRs were estimated for three herd sizes within each herd category. A more precise modeling for all herd sizes would have required to run the simulation at least 1185 times, to determine the PTR for each dairy herd size (from 1 to 1185 cows). Because this was not feasible, to include uncertainty, we set the PTR values as Pert distributions within each risk category , compared to the current situation, where the Danish blocking ELISA is used. Those results could be considered to optimize the BVD surveillance system in Danish dairy herds and to substantiate freedom from disease in the Danish dairy population. Moreover, in this study, we showed a novel idea on how to include the effect of the high risk period within the stochastic scenario tree methodology. By using this approach surveillance systems of different animal diseases could be evaluated and optimized as early warning systems.ImpoCattle, adjusted relative risk of infection for the ImpoCattle category; ARRj, adjusted relative risk of infection for the risk category “j”; ARRNoImpoCattle, adjusted relative risk of infection for the NoImpoCattle category; BTM, bulk tank milk; BVD, bovine viral diarrhea; BVDV, bovine viral diarrhea virus; EFSA, European food safety authority; ELISA, enzyme-linked immunosorbent assay; EPIImpoCattle, effective probability of infection for the ImpoCattle category; EPIj, effective probability of infection for the category “j”; EPINoImpoCattle, effective probability of infection for the NoImpoCattle category; HRP, high risk period; IBR, infectious bovine rhinotracheitis; ImpoCattle, dairy herds which import live cattle; NoImpoCattle, dairy herds which do not import live cattle; NPV, negative predictive value of the surveillance system; PAnim, annual risk of BVDV introduction due to import of live cattle ; PEmb, annual risk of BVDV introduction due to import of embryos (estimated for each category); PFree, confidence in complete freedom from BVD (PH < 0.02 % or <1/4109 infected herds); PH, between-herds design prevalence; PI, persistently infected cattle; PIntro, probability of BVDV introduction into the Danish dairy population; PLow, confidence in low herd prevalence (PH < 0.2 % or < 8/4109 herds); PriorPInf, prior probability that the country is infected at the assumed PH at the beginning of the surveillance period; PriorPinfAdj, prior probability that the country is infected at the assumed PH at the beginning of the surveillance period adjusted for the PIntro; PrPImpoCattle, proportion of dairy herds which import live cattle; PrPNoImpoCattle, proportion of dairy herds which do not import live cattle; PSem, annual risk of BVDV introduction due to import of semen (estimated for each category); PTrim, annual risk of BVDV introduction due to hoof trimmers practicing abroad (estimated for each category); PTRImpoCattle, probability that the threshold prevalence is reached within the ImpoCattle herds on the day of testing; PTRNoImpoCattle, probability that the threshold prevalence is reached within the NoImpoCattle herds on the day of testing; PTruck, annual risk of BVDV introduction due to trucks used abroad (estimated for each category); PU, within-herd threshold/design prevalence of antibody positive milking cows; RImpoCattle, risk of BVDV introduction for the ImpoCattle category; RNoImpoCattle, risk of BVDV introduction for the NoImpoCattle category; RRImpoCattle, risk of BVDV introduction for the ImpoCattle category relative to the RNoImpoCattle; RRNoImpoCattle, relative risk of BVDV introduction for the NoImpoCattle category ; Se, test sensitivity; SSe, surveillance system sensitivity; TemSSe, temporal surveillance system sensitivity; TI, transiently infected cattleARR


N-terminal region and are called the N-RASSF proteins. In contradistinction to them, RASSF1 to RASSF6 are referred to as the C-RASSF proteins. The C-RASSF proteins have the RA domain in the middle region and the Salvador/RASSF/Hippo domain in the C-terminal region. RASSF6 additionally harbors the PSD-95/Discs large/ZO-1 (PDZ)-binding motif. Expression of RASSF6 is epigenetically suppressed in human cancers and is generally regarded as a tumor suppressor. RASSF6 induces caspase-dependent and -independent apoptosis. RASSF6 interacts with mammalian Ste20-like kinases (homologs of Drosophila Hippo) and cross-talks with the Hippo pathway. RASSF6 binds MDM2 and regulates p53 expression. The interactions with Ras and Modulator of apoptosis 1 (MOAP1) are also suggested by heterologous protein-protein interaction experiments. RASSF6 regulates apoptosis and cell cycle through these protein-protein interactions, and is implicated in the NF-κB and JNK signaling pathways. We summarize our current knowledge about RASSF6 and discuss what common and different properties RASSF6 and the other C-RASSF proteins have.Humans have 10 genes that belong to the Ras association (RA) domain family (RASSF). Among them, RASSF7 to RASSF10 have the RA domain in the  C-terminus. This C-terminal domain is named the Salvador/RASSF/Hippo (SARAH) domain, because Salvador and Hippo also have this domain. As described later, Salvador and Hippo are the founding members of the tumor suppressor Hippo pathway [N-terminal region and lack the SARAH domain [Humans have 10 genes that are collectively called Ras association domain family (RASSF) ,3. Among pathway ,5. In coH domain . Based oH domain . Thus thC-terminus   and MDM2 (an E3 ligase) are verified to interact with RASSF6 at the endogenous level ,9 Figur. MST1/2 H domain . More inDrosophila Hippo pathway and Drosophila C-RASSF. The mosaic analysis to search for oncogenes in Drosophila revealed that the mutations of hippo, salvador, mats, and warts exhibit the similar cell overproliferation phenotype [Hippo and Warts encode protein kinases. Hippo phosphorylates and activates Warts. Salvador and Mats function as adaptors and assist in the Hippo-mediated activation of Warts. That is, these four genes are not only genetically related but their products also physically interact with each other and form the signaling pathway that controls cell proliferation and apoptosis. This pathway was named the Hippo pathway. Thereafter, Yorkie was identified as a Warts-interacting protein [Drosophila has only one C-RASSF, dRASSF [hippo mutant lacking the SARAH domain  kinases. In the in vitro kinase assay, MST kinases are co-expressed with C-RASSF proteins. The immunoprecipitated MST kinases are used on the beads or after the elution for the in vitro kinase assay. The co-expression of C-RASSF proteins tends to stabilize and increase MST kinases, which affects the results. Consequently, we detect some inconsistency among these papers.There are several reports about the effect of the mammalian C-RASSF proteins on MST kinases . The resin vitro kinase assay and in the autophosphorylation in vivo [in vitro kinase assay and stabilizes MST2 in cells [in vitro kinase assay, RASSF1A activates MST2 [N-terminal region of RASSF1A and the C-terminal region of RASSF6 activates MST2, supporting that the activation of MST2 is specific for RASSF1A and is mediated by the N-terminal region of RASSF1A. Overall RASSF1A is likely to activate MST2 in vivo and the Hippo pathway  and JNK inhibitor suppresses RASSF6-induced apoptosis [RASSF6 induces apoptosis in caspase-dependent and caspase-independent pathways . It indupoptosis . RASSF2 poptosis . Therefoin vitro. However, whether all the other C-RASSF proteins directly bind Ras proteins has been a matter of debate. Here, we focus on RASSF6. When we prepared the cell lysates with the buffer containing 3 M urea to fully solubilize RASSF6 and used the lysates after dialysis for the immunoprecipitation, we could not detect the interaction of RASSF6 with Ras proteins, while Nore1 could bind Ras proteins under the same condition [et al. demonstrated that RASSF6 binds Ki-Ras in a GTP-dependent manner and that farnesylation is required [C. elegans, RSF-1 depletion blocks the multivulvar phenotype caused by the dominant active Ras mutant [Researchers concur on the point that Nore1 binds Ras proteins . Endogenondition . Allen erequired . In the required . Accordirequired . Nore1 drequired . Togethes mutant . This obs mutant . This fiin vitro [The Nore1 deletion mutant without the RA domain and the SARAH domain can suppress the anchorage-independent cell growth of human lung cancer A549 cells, suggesting that Nore1 has a function independent of Ras and the Hippo pathway . Likewisin vitro , but it in vitro . Once RAin vivo, and that the SARAH domain of Nore1 inhibits Ras-induced apoptosis [in vitro, whereas RASSF1A activates it [N-terminal region of RASSF1A and the C-terminal region of RASSF6 activates MST2 [et al. reported that MST1 stabilizes RASSF2 and that RASSF2 activates MST1 in vivo, which is opposite to our in vitro observation [et al. showed that RASSF2 stabilizes MST2 [in vivo activation of MST1. RASSF3, like RASSF6, regulates p53 expression via MDM2. In alveolar rhabdomyosarcoma associated with Pax3-FOXO1, RASSF4 is highly expressed and leads to YAP1 activation by the suppression of the Hippo pathway [In the field of the C-RASSF protein studies, RASSF1A and Nore1 play leading roles. Other C-RASSF proteins have been studied after RASSF1A and Nore1. This is true for RASSF6, too. Many, including us, have compared RASSF1A and RASSF6 in their properties as a tumor suppressor, their ability to interact with MDM2, their involvement in TNF-α signaling and inflammation, as well as comparing them with Nore1- in the Ras-induced apoptosis. In most cases, the studies have revealed that RASSF6 behaves like RASSF1A and Nore1. However, RASSF6 is distinct from RASSF1A in the relationship with the Hippo pathway. Upon a certain cue, RASSF1A activates MST2 and triggers the Hippo pathway-dependent pro-apoptotic process (poptosis . The strpoptosis . Consequpoptosis . In our vates it . Interestes MST2 . Song etervation . Howeverzes MST2 , we spec pathway . As we hbona fide tumor suppressor, the experiments using RASSF6 knock out mice are necessary but the clinical data support the importance of RASSF6 in cancers. RASSF1A, RASSF2, RASSF4, and RASSF5 are reported to be silenced by high promoter methylation [C. elegans, RSF-1 is necessary for the multivulvar phenotype, which is observed by the hyperactivation of EGF-Ras signaling. We have to ask whether RASSF6 and other C-RASSF proteins are activated in response to the physiologically activated GTP-bound form of Ras and yield cellular consequences other than apoptosis and cell cycle arrest.To conclude that RASSF6 is a hylation ,46,47. Thylation ,48,49,50hylation . Instead


Ube3a gene. Ube3a is expressed nearly exclusively from the maternal chromosome in mature neurons. While imprinting in neurons of the brain has been well described, the imprinting and expression of Ube3a in other neural tissues remains relatively unexplored. Moreover, given the overwhelming deficits in brain function in AS patients, the possibility of disrupted Ube3a expression in the infratentorial nervous system and its consequent disability have been largely ignored. We evaluated the imprinting status of Ube3a in the spinal cord and sciatic nerve and show that it is also imprinted in these neural tissues. Furthermore, a growing body of clinical and radiological evidence has suggested that myelin dysfunction may contribute to morbidity in many neurodevelopmental syndromes. However, findings regarding Ube3a expression in non-neuronal cells of the brain have varied. Utilizing enriched primary cultures of oligodendrocytes and astrocytes, we show that Ube3a is expressed, but not imprinted in these cell types. Unlike many other neurodevelopmental disorders, AS symptoms do not become apparent until roughly 6 to 12 months of age. To determine the temporal expression pattern and silencing, we analyzed Ube3a expression in AS mice at several time points. We confirm relaxed imprinting of Ube3a in neurons of the postnatal developing cortex, but not in structures in which neurogenesis and migration are more complete. This furthers the hypothesis that the apparently normal window of development in AS patients is supported by an incompletely silenced paternal allele in developing neurons, resulting in a relative preservation of Ube3a expression during this crucial epoch of early development.Angelman Syndrome (AS) is a devastating neurodevelopmental disorder characterized by developmental delay, speech impairment, movement disorder, sleep disorders and refractory epilepsy. AS is caused by loss of the Ube3a protein encoded for by the imprinted  Ube3a gene[Ube3a is maternally imprinted in the brain, such that it is expressed nearly exclusively from the maternal chromosome while the paternal chromosome is epigenetically silenced[Ube3a imprinting is regulated by a neuron-specific anti-sense Ube3a transcript that prevents transcription of Ube3a from the paternal chromosome[Ube3a results in approximately 30% of wild type (WT) protein level is present at P5[Ube3a maternally deficient (m-/p+) mice. This suggests that there is an early period of preserved Ube3a expression in (m-/p+) mice, from an unsilenced paternal allele, might serve to protect normal neurodevelopment and cellular function.Angelman syndrome (AS) is a neurodevelopmental disease characterized by developmental delay, speech impairment, movement disorders, sleep disorders and refractory epilepsy. These sbe3a gene. Ube3a i silenced. Ube3a ihromosome,6. Unlikhromosome, Furtherent at P5 in Ube3aUbe3a, dUbe3a, has shown that loss of this protein results in slowed dendritic growth and reduced dendritic branching in peripheral nerves[Ube3a is imprinted in peripheral or spinal nerves, this would provide a reason to evaluate the consequences of loss of Ube3a in neurons outside of the brain.To date, Ube3a expression in neural tissue outside of the brain has been largely unexplored. However, work with the drosophila homolog of al nerves. Additioal nerves. This suUbe3a imprinting is relaxed in early postnatal cortex and Ube3a is not imprinted in astrocytes or oligodendrocyte[Ube3a silencing in development[Ube3a imprinting in neurons is more widespread than previously appreciated, while non-neuronal cells express Ube3a biallelically.Much of the work presented here builds upon previously published work showing that endrocyte. Howevervelopment,8,12,13.Ube3a deletion mice, derived by Jiang were used for all studies[Ube3a m+/p+) were crossed with AS females (Ube3a m−/p+) to produce WT and AS littermates. PCR analysis was performed on tail tissue (harvested prior to P10) or ear punches (harvested after P10) to determine genotypes. PCR primers used as followed: P1 (Common), 5′-CCAATGACTCATGATTGTCCTG-3′; P2 (WT reverse), 5′-TCAAACATTCCAAGTTCTCCC-3′; and P3 (mutant), 5′-TGCATCGCATTGTGTGAGTAGGTGTC-3′. PCR protocol: 94°C for 3 minutes,  x 30 cycles. We utilized a modified protocol as the previously published PCR primers have errors in the primer sequences compared to the curated sequence[l studies. Mice we sequence. The proThis study was carried out in strict accordance with recommendations in the Guide for the Care and use of Laboratory Animals of the National Institute of Health. All procedures were approved by the Vanderbilt Institutional Animal Care and Use Committee (IACUC) under protocol number M/11/059.2HPO4 (1.25 mM), MgCl2 (1.3 mM), CaCl2 (2.0 mM), glucose (10 mM), and NaHCO3 (25 mM) and dissected to isolate cortex, subcortex and cerebellum. All tissue was flash frozen in liquid nitrogen and stored at -80°C until being prepared for Western blot.Brain tissue was collected from mice aged 0 to 42 days. Mice were decapitated and the brains were removed quickly, chilled in ice cold artificial cerebrospinal fluid (aCSF) containing NaCl (125 mM), KCl (2.5 mM), NaTissue was homogenized with a sonicator (QSonica) in a modified RIPA buffer containing (50 mM Tris (pH = 7.4), 150 mM NaCl, 1% NP-40, 0.2% sodium deoxycholate, 1 mM EDTA) with protease inhibitors (Sigma). Protein concentration was determined by a Bradford assay (Bio-Rad), and samples were diluted to a final concentration of 1 to 3 μg/μL in loading buffer containing β-mercaptoethanol. Samples were denatured at 60° C for 10 minutes before being loaded onto a 10% SDS gel (Bio-Rad). Proteins were transferred in a Tris-glycine transfer buffer onto Immobilon-FL PVDF membranes (Millipore). Membranes were then blocked in 4% milk overnight at 4° C. Membranes were incubated with primary antibody in PBS-T with 0.1% Tween-20 (PBS-T) for 2 hours at room temperature. Primary antibodies used: Ube3a 1:2000 , Ube3a 1:1000 , GFAP 1:1000  and actin 1:50000 . Membranes were washed at least three times for 10 minutes in PBS-T and then incubated with fluorescent secondary antibodies at 1:10000  in PBS-T for 1 hour at room temperature. Membranes were then washed three times for at least 10 minutes with PBS-T. Imaging was performed on a LI-COR Odyssey fluorescence scanner and data was analyzed with Odyssey imaging software.Ube3a null littermates, we found that the Sigma Ube3a antibody is highly selective, recognizing no non-specific bands in the 100kD range in any tissue. In contrast, the Cell Signaling antibody recognized non-specific bands in the same molecular weight range as Ube3a in cortical, cardiac and spinal cord lysates. However, no non-specific bands were detected in the sciatic nerve or liver, kidney or lung (Ube3a m-/p+), paternal deletion (Ube3a m+/p-) and Ube3a null mice we found the expected pattern of paternal deletion being indistinguishable from WT, a small residual protein content in AS mice and tissue from Ube3a null mice having no protein detected in the 100kD range with either antibody. However, there was a non-specific band that migrates at a slightly higher molecular weight than Ube3a detected by the Cell Signaling antibody  and compared it to the best characterized antibody against Ube3a (Sigma Anti-Ube3a Clone 330). Each antibody was run against a panel of tissues including lysates from brain, spinal cord and sciatic nerve as well as several non-neurally derived tissues including liver, heart, lung and kidney. When comparing the banding pattern produced in WT lysates to  or lung . We nextantibody . Therefo2. Two-thirds media changes were done on DIV3 and DIV6, with 5 μg/mL insulin supplemented at DIV6. Following a pre-shake at 50 RPM for 45 minutes to remove microglia, on DIV9, oligodendrocyte precursors were isolated by shaking overnight at 220 RPM to make both enriched oligodendrocyte and astrocyte cultures. Following shaking, suspended oligodendrocyte precursors were plated into PLL coated 8-well chamber slides at a density of 20,000 cells/well in OL media  and cultured for 7–9 days at 37° C and 8.5% CO2. Astrocytes remaining in the T25 flask were washed with PBS and dissociated with 0.25% trypsin in Hank’s buffered saline solutions (HBSS). Dissociated cells were plated in PLL coated 24-well plates at a density of 30,000 cells per well for IF or into 6-well culture dishes for Western blot. Cells were grown for 2–3 days at 37° C in 5% CO2 before being fixed or harvested for Western blot.Primary astrocyte and oligodendrocyte cultures were derived with modification of a shaking protocol described previously. FollowiCultured astrocytes and oligodendrocytes were fixed with 4% PFA for 30 minutes and washed 2x with PBS before being blocked with 5% goat serum and 0.1% Triton X-100. Primary antibodies  were diluted in blocking buffer and samples were incubated overnight. Secondary antibodies were diluted in blocking buffer (Goat Anti-Rat Alexa-488 (Life Technologies), Goat Anti-Rabbit Alexa-488 (Life Technologies) and Goat Anti-Mouse 680LT (Li-Cor), all 1:500) and incubated for one hour at room temperature. Cells were washed twice with PBS and coverslipped with Vectashield with DAPI prior to imaging. All imaging was performed on an Evos-FL microscope.All statistical analyses were performed using GraphPad Prism  software. Unless otherwise specified, an unpaired t-test was used to compare two age-matched groups. A one-way ANOVA with Tukey’s post hoc test was used to compare the residual paternal Ube3a expression in AS mice at different ages.Ube3a throughout the neuraxis, we first confirmed the imprinting of Ube3a in P42 murine cortical lysates. We next determined that Ube3a is highly expressed in the spinal cord and sciatic nerve in WT mice and it is significantly reduced in AS mice. As expected, paternal Ube3a is expressed at very low levels, approximately 5–10% of WT in AS mice in all neural tissues assayed  tissue  tissue . These dUbe3a imprinting occurs primarily in neurons, recent evidence suggests Ube3a is not imprinted in glial cells [Ube3a is not imprinted in astrocytes.A growing body of clinical evidence has suggested that myelin dysfunction may be a contributing factor to morbidity in a variety of neurodevelopmental syndromes–18. Receal cells ,22. In aUbe3a is imprinted in oligodendrocytes, expression of Ube3a was evaluated in primary cultures of mature oligodendrocytes. Myelin basic protein (MBP)-positive mature oligodendrocytes from WT mice show robust expression of Ube3a in the nucleus as well as high levels of expression in the cytosol, in contrast to astrocytes which demonstrated a somewhat reduced immunoreactivity in the cytosol compared to the nucleus  Comparison of Ube3a antibodies from Sigma (raised in Mouse) and Cell Signaling (raised in Rabbit) in a panel of tissues known to express Ube3a. WT and KO tissue from tissue was assayed. Top panel shows specificity of Sigma Ube3a with no immunoreactivity at 100 kD in KO tissue in any tissue. Bottom panel shows lack of specificity for Cell Signaling Ube3a with nonspecific bands present at around 100 kD in lysates from cortex, spinal cord, and heart. b) Comparison of antibodies run against WT, paternal deficient Ube3a, AS and KO tissue. As expected, both antibodies have the expected result of WT and paternal deficient sample being nearly indistinguishable, significantly reduced expression in AS tissue and no expression in KO tissue. As noted, a faint non-specific band is recognized by the Cell Signaling antibody.(TIF)Click here for additional data file.


Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural information available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from  Ras of complex protein domains are GTP binding domains found in the ROCO family of proteins. Civiero et al. summarize recent advances in our understanding of the biology of these domains and efforts to target them in human disease Dictyostelium discoideum (The Ras of complex proteins (ROC) domain was first established as a distinct protein domain family following the identification of a guanosine triphosphate (GTP) binding motif in a series of large multidomain proteins in the Amoeba scoideum . Since 2D. discoideum that contains nine domains  or M. Barkeri (archaea) COR domains against myxobacteria sequences reveal no significant domain conservation. Although it is unclear whether ROC domains originated in prokaryotes or were instead horizontally transferred from eukaryotes, their presence in both archaea and bacteria suggest an ancient origin   . Comparefficking  lipid rafficking , and exofficking , supportTrichoplax adhaerens, the only species of the phylum placozoa and the most ancient metazoan known, reveals the presence of multiple ROCO genes. T. adherens is a simple, disc-shaped organism with two epithelial layers covering an inner layer of fiber cells and no apparent nerve, muscle, or sensory cells (T. adherens (D. discoideum GbpC protein in chemotaxis and the role of human ROCO proteins in processes related to phagocytosis in response to host infection (MASL1 and LRRK2), it can be speculated that slime molds and placozoa have independently acquired multiple ROCO genes in a process of convergent evolution.A bioinformatic analysis of ROC domains in ry cells . Using madherens . As showadherens . Based oEctocarpus siliculosus by using bioinformatic searches  exhibit a repetitive intro-exon structure where each LRR is encoded by a 72-nucletide/24-amino-acid-long individual exon also present in noncoding regions. This striking arrangement suggesting highly dynamic exon shuffling, together with the remarkable expansion of the Ectocarpus ROCO family, hints that Ectocarpus ROCO proteins may be involved in immune response mechanisms. A role of ROC domains in immune response mechanisms is gaining attention among human ROCO proteins; for example, MASL1 and LRRK2 have been shown to be upregulated upon pathogen infection , is involved in leaf patterning processes including leaf symmetry and venation patterning  studies of tterning . Due to The evolutionary history of ROCO genes in animals has been thoroughly reconstructed by Marin . ProtostLRRK2 gene. A study by Giesert et al. examined splicing of LRRK2 in the mouse brain, uncovering evidence of altered splicing of exon 5 and a novel exon 42 (located within the kinase domain of this protein)  . Of direprotein) . AlthougD. discoideum ROCO protein GbpC  in addition to their ROC domains. An early observation was that manipulating the GTP binding properties of LRRK1 had a major impact on the kinase activity of this protein, leading the authors to propose a model for LRRK1 function, and by implication the ROCO proteins . In thisD. discoideum ROCO protein Gbpc, which possesses a C-terminal guanine exchange factor (GEF) domain in addition to its ROC and kinase activities. Examination of Gbpc biology suggests that this GEF domain interacts with and regulates the ROC activity of this protein, adding further complexity  located in the ROC-COR interface leading to a reduction of GTPase activity (Due to their low affinity (in the range of μM) for nucleotides  and to tactivity . In addiactivity  bind theactivity .D. discoideum studies have been carried out on GbpC to address this issue, investigating the contribution of the various domains of GbpC to the chemotaxis phenotype , this protein does not possess an ROC domain (Drosophila LRRK ortholog (dLRRK2) has been the subject of detailed investigations, with knockout and targeted mutation models developed. An observation directly relevant to the biology of the ROC domain is that dLRRK KO results in a neurodegenerative phenotype , and the close association between point mutations in these genes and human cancer, substantial efforts have been made to correct their oncogenic activity (Following the identification of the Ras genes (Hactivity . As it bactivity . Recent activity , directlactivity  and disractivity . Most reactivity . While iq (Heterotrimeric G proteins are involved in the control of a range of cellular functions, and similar to Ras the pathways within which they function have been implicated in oncogenesis. Efforts to target G protein α subunits, the subunit of the heterotrimeric G protein complex responsible for the binding and hydrolysis of GTP, have resulted in the derivation of a number of compounds that modulate the release and exchange of guanosine nucleotides. Examples include suramin  and imidq . This prWhat is revealing about the experience of targeting G proteins and Ras is that advances have been achieved not by directly targeting the active site of these proteins but by acting on protein-protein interactions or regulatory mechanisms. For the ROCO proteins, this is hindered by the lack of validated interacting proteins that modulate guanosine nucleotide hydrolysis or exchange. Candidate GAPs and/or GEFs have been identified for LRRK2; however, the spatial details of these relationships remain obscure . FurtherFrom a standing start in 2002, our understanding of ROC domain structure and function has advanced at a startling rate, primarily driven by the realization of the role that ROC-domain-containing proteins play in human disease. It is likely that the coming years will provide even greater insights into the function of this domain, in particular with higher resolution structural data in the context of multidomain fragments of ROCO proteins. The urgent requirement for novel therapies to treat the disorders linked to proteins containing ROC domains highlights the need to examine the feasibility of targeting ROC biology, despite the huge challenge that this represents, and this is sure to be a major focus of research into the proteins in the future.


The dcGO is a comprehensive resource for protein domain annotations using a panel of ontologies including Gene Ontology. Although increasing in popularity, this database needs statistical and graphical support to meet its full potential. Moreover, there are no bioinformatics tools specifically designed for domain ontology analysis. As an add-on package built in the R software environment, dcGOR offers a basic infrastructure with great flexibility and functionality. It implements new data structure to represent domains, ontologies, annotations, and all analytical outputs as well. For each ontology, it provides various mining facilities, including: (i) domain-based enrichment analysis and visualisation; (ii) construction of a domain (semantic similarity) network according to ontology annotations; and (iii) significance analysis for estimating a contact  network. To reduce runtime, most analyses support high-performance parallel computing. Taking as inputs a list of protein domains of interest, the package is able to easily carry out in-depth analyses in terms of functional, phenotypic and diseased relevance, and network-level understanding. More importantly, dcGOR is designed to allow users to import and analyse their own ontologies and annotations on domains  and RNAs (from Rfam) as well. The package is freely available at CRAN for easy installation, and also at GitHub for version control. The dedicated website with reproducible demos can be found at  PLOS Computational Biology Software ArticleThis is a Proteins are of modular design, with structural units called domains The dcGOR package is designed in a general way that allows for representing and analysing three bits of information: domains, ontologies and annotations. For it to be applicable in domain-centric annotations, the backend is its built-in data that is pre-compiled from the latest version of the dcGO database The dcGOR is exclusively implemented on the R software environment. Three S4 classes are defined: ‘InfoDataFrame’ for domains, ‘Onto’ for ontologies and ‘Anno’ for annotations. The class ‘InfoDataFrame’ is used to store domain information. Since an ontology is organised as a directed acyclic graph , the class ‘Onto’ represents the ontology as a directed graph in which both adjacency matrix and node/term information are defined. For annotations, the class ‘Anno’ is defined to accommodate a sparse annotation matrix and additional metadata on domains and terms. All these classes have their class-specific S4 methods. This design of data representations greatly simplifies domain ontology analyses. dcEnrichment conducts enrichment analysis based on the hypergeometric/binomial distribution or Fisher's exact test dcEnrichment reports ontology terms that are enriched in this input domain group. To account for the ontology DAG, it also implements several algorithms that were originally applied to GO visEnrichment visualises the top significant terms in the context of the ontology DAG to aid intuitive interpretation.The function dcDAGdomainSim first calculates semantic similarity between terms, which is then used to derive similarity between domains. All popular IC-based semantic similarity measures dcDAGdomainSim has several methods to calculate similarity between pairs of domains, including 3 best-matching (BM) based methods: average, maximum, and complete. For a term in either domain, all these BM-based methods first calculate maximum similarity to any terms in the other domain. For more detail, the reader is referred to this review Semantic similarity is a type of comparison to assess the degree of relatedness between two entities (here domains) in meaning of their annotations dcDAGdomainSim), the function dcRWRpipeline performs random walk with restart (RWR) for estimating contact strength and significance between two input groups of domains (as seeds). It is based on the earlier work Given a domain network . This ensures that only the valid part of the ontology (in terms of domain annotations) is used properly. The function dcConverter is able to convert an object between newly defined graph classes and the one used in packages ‘igraph’ In addition to the analyses above, dcGOR also has several auxiliary functions for data load, annotation propagation, graph class conversion, and fast computation. The function The most common use case is to analyse a list of protein domains of interest. As a proof of principle, I use two interesting lists of domains  to demonstrate the functionalities supported in the dcGOR package, particularly enrichment analysis and network analysis. Also, I show how users can benefit from this package to analyse their own domains, ontologies and annotations. All these examples are reproducible following step-by-step demos on the package website, from which results can also be found.de novo gained during the evolution of the human lineage. To shed insight into these domains in the relevance to functions, phenotypes and diseases, I use dcEnrichment to perform enrichment analysis using all domains in Metazoa as the background. GO Biological Process (GOBP) enrichments suggest that they are of functional relevance to ‘multicellular organismal development’ and ‘toll-like receptor signalling pathway’; First, I analyse a list of SCOP domain superfamilies that have been gained by the human genome since the ancient ancestral ‘Metazoa’ . According to this report dcDAGdomainSim to construct a domain network according to domain-centric annotations by DO. This is done via calculating the semantic similarity between pairs of domains (dcRWRpipeline to estimate the contact strength and significance between sets of domains. The example domain set used here is a GO Molecular Function (GOMF) term and its annotated domains  this network has a functional preference. Taken together, this example greatly encourages domain-centric approaches to genome evolution, function and phenotype/disease.To further understand the relevance of these 58 domains to diseases, I use Next, I extend the analysis to a list of Pfam domains that tend to occur in diverse domain architectures; this tendency is called ‘promiscuous’. In this study dcBuildInfoDataFrame, dcBuildOnto and dcBuildAnno) are able to create objects newly defined in the package (http://supfam.org/dcGOR/demo-Customisation.html) provides detailed instructions on how to analyse (starting with input files) the InterPro2GO mapping Unique to this package, dcGOR supports customised analysis using data files provided by users. From input files , three functions ( package Table 1.Software S1). For ease of installation (R package dependencies), it is distributed as part of CRAN, http://cran.r-project.org/package=dcGOR. For ease of version control, it is also distributed at GitHub, https://github.com/hfang-bristol/dcGOR. The details on documentations and demos can be found at http://supfam.org/dcGOR. As missed in most R packages, online documentations and demos are user-friendly; users can see both illustrated codes and executed outputs. This will dramatically reduce the learning curve and promote the wide adoption as users can exactly reproduce what they see.As open-source software, the dcGOR package is freely available under the GPL-2 license  and Rfam RNA families as well, and to support customised analysis. For example, users can analyse domains with different definitions, such as the partner members of the InterPro consortium Software S1Package ‘dcGOR’ (version 1.0.3) including source code, documentation and data.(GZ)Click here for additional data file.


Three studies investigated whether exposure to materialistic cues would increase perceptions of personal relative deprivation and related emotional reactions. In Study 1, individuals who were surveyed in front of a luxury store reported higher levels of personal relative deprivation than those surveyed in front of an ordinary building. In Study 2, participants who viewed pictures of luxurious goods experienced greater personal relative deprivation than those viewed pictures of neutral scenes. Study 3 replicated the results from Study 2, with a larger sample size and a more refined assessment of relative deprivation. Implications of these findings for future studies on relative deprivation and materialism are discussed. We encounter materialistic cues on a daily basis. When you walk out, you see shopping malls with grand electronic billboards standing against the sky, and prestige cars roaring past the street. If you turn on the TV or surf the Internet, you see entertainment shows demonstrating upper class lifestyle, and commercials on all kinds of goods urging you to buy. It is no doubt that materialistic signs have a significant impact upon individuals’ psychology and behavior. In recent years we witness an increasing interest into identifying the psychological implications of signs of wealth and material possessions e.g., . For ins1. Rather than a consequence of absolute inadequacy of economic resources, personal relative deprivation is evoked by the perception that one is unjustly worth-off than others. Relative deprivation is an important yet somewhat understudied concept in social psychology. It has been linked to a variety of consequences, ranging from physical health and psychological well-being, to inter-group attitudes and collective action . The brief survey was conducted in downtown Nanjing, the capital city of Jiangsu Province, China.Forty-seven men and 42 women participated in this survey. Their ages ranged from 15 to 65 years , or in front of an ordinary building without any sign . Whereas those interviewed before the Prada store were on their way to the luxury stores, those interviewed after the Prada store were walking in the opposite direction, before they were stopped by the experimenters.strongly disagree) to 5 (strongly agree). We calculated a measure of relative deprivation by subtracting the answers from 6, so that higher scores indicate higher levels of relative deprivation. We then recorded participants’ age and SES. To assess SES, we asked participants to indicate their placement on a scale ranging from 1 to 10, where 1 represents the group with lowest levels of education and income in the society and 10 the highest. Participants were then given an opportunity to ask questions, and were thanked for participation.Pedestrians walking alone were stopped at the appropriate locations by two female experimenters and asked to participate in a brief survey conducted by the social psychology laboratory of Nanjing University. In the materialistic-cue condition, the experimenters positioned themselves so that participants faced one of the display windows of the Prada store during the interview. In the two control conditions, participants faced the ordinary building. They were not able to see the luxury stores or the signs during their interviews. Participants were asked the critical question “To what extent do you agree that you have got what you deserve compared to others?” Participants responded to this question on a 6-point scale ranging from 0  = 1.70, p = 0.19 (Table 1). We then conducted a one-way ANOVA to determine whether materialistic cues influenced individuals’ perceptions of relative deprivation. The main effect of the survey location was significant, F = 3.94, p = 0.023, F = 3.17, p = 0.047, p = 0.023. The comparison between those surveyed in front of the Prada store and those before it yielded a significant result, MD = 0.73, 95% CI = , p = 0.014, Cohen’s d = 0.71. However, no significant difference was found between those surveyed in front of the Prada store and those after it, p = 0.126.No significant difference in SES was found among the three groups, t = 0.09, p = 0.93. Therefore, the effect of materialistic cues on relative deprivation seemed not to vary according to participants’ SES.We further examined whether participants’ SES would moderate the effect of materialistic cues on relative deprivation. A hierarchical regression was conducted with age, gender, SES, and survey location  entered in the first block and the interaction between SES and survey location entered in the second block. The interaction term was non-significant, β = 0.01, Generally, those interviewed right in front of a luxury store reported higher levels of relative deprivation than those interviewed without any luxurious signs in their immediate environment. However, the difference in relative deprivation between those interviewed in front of the luxury store and those who had just passed it did not reach significance. It was probably due to the fact that for those who had just come across a line of luxury stores , the visual impact of the material signs might still linger in their minds.We expected that individuals with higher SES would be less influenced by materialistic cues than those with lower SES. However, this hypothesis was not supported. Material pursuits are virtually endless. For many people, no matter how wealthy they already are, they may still have financial aspirations that are not fulfilled. Hence, materialistic cues may invoke a sense of discontent regardless of individuals’ SES. It should be noted that participants’ subjective SES may have also been influenced by the situation they were interviewed. Indeed, those interviewed in front of the luxury store tended to report lower SES than those interviewed before or after the store, though the differences did not reach statistical significance. In the next study, we avoided this problem by assessing SES before the experimental manipulation. Study 1 assessed only the cognitive component of relative deprivation. However, emotional reactions such as anger and resentment are seen as an indispensable element of relative deprivation too . In StudIn this study, we sought to replicate the findings from Study 1 with a different procedure. Specifically, we exposed participants to pictures of luxurious goods in laboratory, and examined whether such materialistic cues would have similar effects as the materialistic priming employed in Study 1. Moreover, we also included another potential moderator, trait materialism, in our investigation.Trait materialism is a relatively stable individual difference variable characterized by revolving one’s life round acquiring material possessions and viewing material possessions as signs of success and happiness . Trait mSD = 1.90). Participants were recruited from a large subject pool. They received a small gift (worth around US$ 5) for their participation.Seventy-two graduate and undergraduate students  participated in this study. Their mean age was 20.93  depicted objects more luxurious or expensive than those presented to the control group , t(50) = 8.53, p < 0.001. However, the objects in the pictures were matched in attractiveness and interest, ts(50) < 1.10, ps > 0.30.In a pilot study, 52 undergraduate students were asked to evaluate the pictures used in the study. Specifically, participants indicated how luxurious or expensive, attractive, and interesting the objects depicted in the pictures were. It was confirmed that pictures presented to the materialistic-cue group  to 7 (very difficult)2. They also indicated how pleasant and depressed they felt after viewing the pictures [from 1  to 7 (very much)]. The two items (r = -0.44) were combined to form a positive affect index. Then, participants were asked to fill out a set of ostensibly unrelated questionnaires assessing their feelings at that time, which included our measure of state relative deprivation.To buttress our cover story, after picture recognition, participants rated whether the pictures were difficult to memorize on a scale from 1 . Moreover, as a measure of objective SES, participants also rated their parents’ highest levels of education completed and their annual household income. Education was coded into four categories: (i) did not finish high school, (ii) high school graduate or some college, (iii) college graduate, or (iv) postgraduate degree. Fathers’ median level of educational attainment was college graduation, and mothers’ median level of educational attainment was high school graduation or some college. Annual income was assessed using six categories: (i) below CNY 50,000, (ii) CNY 50,000-100,000, (iii) CNY 100,000-300,000, (iv) CNY 300,000-600,000, (v) CNY 600,000-1000,000, (vi) CNY 1000,000 and above. Participants had a median annual household income of between CNY 100,000 and 300,000.We administered two measures of SES. First, participants completed the MacArthur Scale of subjective SES e.g., . In thisr = 0.58, p < 0.01), and were standardized and averaged to compute an overall measure of SES.The subjective  and objective  measures of SES correlated significantly , happiness , and success . Participants responded to these items on a 7-point Likert scale, ranging from 1 (Strongly disagree) to 7 (Strongly agree). Cronbach’s α for this scale was 0.81.Trait materialism was measured with the 18-item materialism scale developed by Strongly disagree) to 7 (Strongly agree). Answers on the two items were averaged (r = 0.31).The two items to assess personal relative deprivation were “Right now, I think I have got what I deserve compared to others” (reverse scored), and “Right now, I feel resentful and angry toward the situation that many people are better-off than me.” Participants responded to the items on a 7-point Likert scale, ranging from 1  < 1.71, ps > 0.09. However, participants in the materialistic-cue group reported lower levels of positive mood, t(70) = 2.74, p = 0.008, Md = 0.70, 95% CI = , d = 0.64, and higher levels of relative deprivation, t(70) = 3.20, p = 0.002, Md = 0.69, 95% CI = , d = 0.76, than those in the control group. The difference in relative deprivation between the two groups remained significant when SES, trait materialism, or both variables were controlled for3. It also remained significant when the effect of mood was partialled out, F = 5.46, p = 0.0224.Descriptive statistics are shown in t = -0.67, p = 0.51, for the interaction between trait materialism and experimental condition, β = -0.09, t = -0.28, p = 0.79.Two hierarchical regressions were performed to examine whether SES or trait materialism moderated the effect of materialistic cues on relative deprivation. In the regressions, the first block contained the main effects, and the second block included the interaction term. Both interactions were found to be non-significant; for the interaction between SES and experimental condition, β = -0.24, Participants who had just been exposed to materialistic cues reported higher levels of personal relative deprivation than those exposed to neutral scenes. Moreover, the discrepancy cannot be fully accounted for by the general emotional state they experienced. These results corroborated our hypothesis that materialistic cues would boost personal relative deprivation. Again, the effect of materialistic cues was independent of individuals’ SES.Contrary to our prediction, we failed to find a significant moderating effect of trait materialism. In other words, materialistic cues evoked a sense of relative deprivation even among those who generally had low levels of materialistic aspirations. The visual impact of materialistic cues seemed to be powerful enough to temporarily override individuals’ chronic tendencies and to evoke inflated feelings of inadequacy and resentment in all. However, the validity of such conclusion may be questionable due to the small sample size involved. We conducted Study 3 to replicate the effect of materialistic cues on relative deprivation, and further examine the interaction between trait materialism and materialistic cues.Study 3 was a replication of Study 2, with a larger sample size and a more stringent measure of relative deprivation.SD = 1.58). Participants were recruited from a large subject pool. They were paid CNY30 (around US$ 5) for their participation.One hundred and twenty graduate and undergraduate students  participated in this study. The mean age was 20.70 (strongly disagree) to 7 (strongly agree). Cronbach’s α for this scale was 0.64.The materials, procedure, and measures involved in this study were all the same as those used in Study 2, except that relative deprivation was measured with the personal relative deprivation scale . The scaTable 3 presents the descriptive statistics. No significant difference was found in SES, trait materialism, or positive mood between the two groups, ts(118) < 1.07, ps > 0.29. However, as predicted, participants in the control group reported lower levels of relative deprivation than those in the materialistic-cue group, t(118) = 2.63, p = 0.010, Md = 0.40, 95% CI = , d = 0.48. The difference in relative deprivation between the two groups remained significant when SES, trait materialism, and mood were controlled for, F = 5.63, p = 0.019.t = -0.19, p = 0.85; for the interaction between trait materialism and experimental condition, β = -0.07, t = -0.71, p = 0.48).Results from hierarchical regressions showed that neither SES nor trait materialism had significant moderating effects on the association between materialistic cues and relative deprivation  increased individuals’ sense of relative deprivation. After viewing material signs, individuals are more likely to believe that they are unjustly worse-off than others, and feel resentful about the situation. This effect was not significantly moderated by either SES or trait materialism.Although abundant research has been accumulated on the negative association between materialism and well-being (see Previous research from our laboratory has shown that personal relative deprivation increases materialism . The curWhy, then, do materialistic cues induce a sense of relative deprivation? We believe that the key to this phenomenon lies in the interplay between humans’ innate propensity and prevailing social norms. Human beings have an inherent preference for material possessions, and a materialistic culture legitimates and encourages materialistic aspirations. In a society where people are disproportionately judged by their material possessions, material goods do not only mean life necessities, but also signal social acceptance and status . It may This analysis alludes to potential cultural differences. Relative deprivation may not be increased at the sight of materialistic cues in societies where materialism is not so enthusiastically endorsed, or where wealth is more fairly distributed. However, given the irresistible attractiveness of material possessions to the human mind, we believe that the effect of materialism on relative deprivation would also be found in many other societies.We did not find the hypothesized moderating effect of trait materialism. One explanation may be that materialistic situations are powerful enough to override this individual difference. Therefore, even individuals who generally do not focus their attention on material possessions may be unable to resist the lure of luxury goods. The effect of materialistic cues on relative deprivation was not moderated by participants’ SES either. This may be due to the fact that we used material goods that are highly desirable but beyond what most people can afford. Therefore, those with relatively high SES would still feel relative deprived when exposed to these goods. Future studies may investigate whether the effect of materialistic cues on relative deprivation would hold in more aﬄuent societies. Future studies may also examine whether individual differences in power and narcissism, variables that are tied to individuals’ sense of entitlement, would moderate the effect of materialistic cues on relative deprivation e.g., .We investigated only the instant effect of materialistic cues on personal relative deprivation. It remains open whether the effect of materialistic cues would retain if people are constantly involved in materialistic environments. We think two opposite tendencies may be equally possible. First, individuals may be habitualized to materialistic cues. The level of relative deprivation they experience may return to baseline or remain constant after reaching a certain point. As an alternative possibility, the effect of materialistic on relative deprivation may be accumulated, leading to deleterious consequences such as depression, interpersonal conflict, or acts of sabotage. Longitudinal studies conducted in natural settings may provide an answer to this question. If the latter were the case, the society should find ways to prevent such consequences, such as promoting fair distribution of social wealth, reducing the income gap, cutting down advertising expenditures, and inhibiting conspicuous consumption.There are several limitations in the current studies. First, although the measures of relative deprivation used in Studies 1 and 2 have face validity, reliability and construct validity of these items need to be established. We used an existing scale to measure relative deprivation in Study 3. However, the internal consistency was lower than optimal. Nevertheless, we believe that the consistent results across the three studies have provided substantial support for our hypothesis. Second, most of the participants in the studies were young. We don’t know whether similar effects would be obtained among older samples. Given the fluctuating importance of acquiring material possessions across the life span, we suspect that the effect of materialistic messages may show an inverted-U shaped association with age. Third, one reason why we did not find significant moderating effect of SES might be that participants involved in our studies had low-to-medium SES on average. Those who have extremely high SES may not experience relative deprivation when seeing materialistic cues. On the contrary, they may even feel satisfied and proud. Fourth, the current studies focused solely on relative deprivation, a subjective cognitive and emotional state. It is worthwhile to examine potential behavioral consequences, such as gambling tendency and compulsive buying. Future studies may investigate the adverse effects of relative deprivation as induced by materialistic cues and find ways to alleviate them.We observed in three studies that after viewing materialistic cues, participants experienced heightened levels of relative deprivation. These studies broaden our understanding of the antecedents of relative deprivation, and shed light on the mutual reinforcement of materialism and relative deprivation.This research is approved by the Institutional Review Board of Nanjing University.HZ designed the study and drafted the paper. WZ did the majority work on data collection and data analysis.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.


Preliminary model of G protein of rabies virus stable at 12 ns with molecular dynamics was obtained.The RABVG ectodomain is a homotrimer, and trimers are often called spikes. They are responsible for the attachment of the virus through the interaction with nicotinic acetylcholine receptors, neural cell adhesion molecule (NCAM), and the p75 neurotrophin receptor (p75NTR). This makes them relevant in viral pathogenesis. The antigenic structure differs significantly between the trimers and monomers. Surfaces rich in hydrophobic amino acids are important for trimer stabilization in which the C-terminal of the ectodomain plays an important role; to understand these interactions between the G proteins, a mechanistic study of their functions was performed with a molecular model of G protein in its trimeric form. This verified its 3D conformation. The molecular modeling of G protein was performed by a I-TASSER server and was evaluated via a Rachamandran plot and ERRAT program obtained 84.64% and 89.9% of the residues in the favorable regions and overall quality factor, respectively. The molecular dynamics simulations were carried out on RABVG trimer at 310 K. From these theoretical studies, we retrieved the RMSD values from C Lyssavirus, Ephemerovirus, and Vesiculovirus. The genus Lyssavirus includes rabies virus. The viral genome consists in a single and negative-stranded nonsegmented RNA, which encodes five proteins: nucleoprotein, matrix protein, phosphoprotein, glycoprotein, and the viral-dependent RNA polymerase [Rabies is a 100% fatal disease caused by the rabies virus (RABV) that affects the central nervous system [lymerase , 3.The glycoprotein (RABVG) rabies virus is comprised of four domains: signal peptide (SP), ectodomain (ED), transmembrane (TM), and a cytoplasmic domain (CD) , 5. The The RABVG in each peak is anchored in the plasma membrane and the lipid envelope by the transmembrane domain of 22 amino acids from 439 to 461 residues . The C-tOn the other hand, the RABVG ectodomain is a homotrimer that contains a transmembrane domain. Each monomer has 439 residues, and the trimers are commonly called spikes. These spikes are responsible for the attachment of the virus through the interaction with nicotinic acetylcholine receptors, neural cell adhesion molecule (NCAM), and the p75 neurotrophin receptor (p75NTR). This makes them relevant for viral pathogenesis –15. In aThe RABVG C-terminal has 44 amino acid cytoplasmic domains that interact with the matrix protein to complete the viral assembly .The RABVG protein induces an immune response due to its multiple antigenic domains. Hence, RABVG is the major contributor to RABV pathogenicity . The anthttp://www.rcsb.org). Various physical and chemical parameters of primary structure analysis were computed using the ProtParam online tool [This study was designed to predict the 3D model of RABVG protein under an iterative threading assembly refinement algorithm implemented in I-TASSER . This waine tool . The secine tool . The DiAine tool  was usedine tool , MEMSAT3ine tool .C-score). After generating the 3D model, structure and stereochemical analysis were performed using different evaluations and validation tools. The Psi/Phi Ramachandran plot was obtained using PROCHECK [Z-scores, which indicate the overall model quality and confirm that the predicted structure is within the range of scores as found in the native proteins. The ProSA web tool [Z-scores. Furthermore, the generated model was submitted in the protein model database (PMDB) (https://bioinformatics.cineca.it/PMDB/) with PMDB identifier PM0079619.Finally, the 3D model of RABVG was generated using the I-TASSER online server . This gePROCHECK . This asweb tool  was usedWith the monomer structure in hand, we attempted to make the trimer interact with protein-protein docking studies to predict the protein complex formed in a protein-protein interaction. These docking studies used the ClusPro server –31 that We used the PDBsum Generate server  to underThese MD simulations employed NAMD 2.6   by applyThis study was initiated to perform structure-based sequence analysis studies on RABVG. The protein sequence was retrieved using accession AGN94258.1 from the NCBI protein. The primary structure analysis showed that the RABVG protein had a molecular weight of 58487.3 Daltons and a theoretical isoelectric point (pI) of 7.83. The instability index (II) is computed to be 38.05. This classifies the protein as stable. The negative grand average of hydropathicity (GRAVY) shows a value of −0.173 indicating that the protein was hydrophilic according to other reports .β-sheets, 7 beta hairpins, 3 beta bulges, 21 strands, 5 α-helices, 10 helix-helix interacs, 38 beta turns, and 10 γ-turns. Secondary structural features are shown in Sequence and secondary structure analyses of RABVG revealed that it has 6 Disulfide connectivity was predicted to be within 1–8, 2–7, 3–12, 5–11, 6–10, 9–19, 13–17, 14–18, and 15-16. The TMHMM, MEMSAT3, and MEMSAT-SVM programs identify a transmembrane region between amino acids 460 and 480. The C-terminal is possibly located in the cytoplasm. The N-terminal—including 19 amino acids from the signaling peptide—is located in the extracellular region .http://zhanglab.ccmb.med.umich.edu/I-TASSER/) online server and the best predicted structure with the maximum confidence score (C-score −2.18) was selected (http://cluspro.bu.edu/)server to get a trimer complex for visualizing the protein interactions  whit 2J6J and 2CMZ crystals; for closely related protein sequences with identity higher than 40%, the alignment is almost always correct. Regions of low local sequence similarity become common when the overall sequence identity is below 40% , 39. In selected  monomer ractions .The central trimerization domain has a significant contribution to the structural stability of the G protein trimer, due to the formation of 6 alpha helices. The different conformations  of G protein are pH-dependent, several acidic residues  are brought close together in the postfusion bundle of six helices, suggesting that the acid residues play molecular switches role in their deprotonated forms, and this should destabilize the central six-helix bundle and thus allow the refolding of G back toward its prefusion conformation similar to VSV G protein [These residues ensure a correct activation of G and ensure that the stability of the 6 chain helices has to be tightly regulated since both its destabilization and overstabilization are detrimental to the virus.The glycoprotein of rhabdovirus is the target of neutralizing antibodies; its antigenicity and antigenic sites of G have been extensively studied in VSV and RABV . The antThey have described various regions of the protein G which have an important role in membrane fusion for the internalization of the virus; the region between amino acids 118 and 139 was generally considered to represent an internal fusion peptide for VSV G. However, other studies demonstrated that amino acids 395–418 have a significant influence on fusion, and additional studies identified region 145–164, termed p2-like peptide, as being a pivotal domain in facilitating glycoprotein G-mediated membrane fusion . The staThe structural organization of G is very similar to that of VSV G. This similarity extends from the N-terminal part to at least the end of helix G of domain II. It includes both the PH domain and the fusion domain (109 residues of the fusion domain), as well as the trimerization domain, and reveals a clear structural homology between the two proteins.β sheet at the top of the molecule (domain I), a central, mostly α-helical domain that is involved in the trimerization of the top of the molecule (domain II), a neck domain that has the characteristic fold of pleckstrin homology (PH) domains (domain III), and the elongated fusion domain that makes the trimeric stem of the molecule (domain IV). The C-terminal part of G corresponds to AA 411 to 422 in VSV and 410 to 455 in RAVBG [G has an altogether different structural organization from those of both class I and class II viral fusion proteins described so far. The polypeptide chain of G folds into four distinct domains : a laterin RAVBG .The top lateral domain I contains about 90 residues in two segments (1 to 17 and 310 to 383 for VSV and 1 to 17 and 311 to 383 for RABV). Domain II is made of three segments . Domain III is inserted within domain II. It is made of two segments (36 to 50 and 181 to 258 for VSV and RABV) and has the fold of a PH domain. Domain IV (51 to 180 for VSV and RABV) is inserted in a loop of the PH domain .These residues ensure a correct activation of G and show that the stability of the 6 chain helices is tightly regulated because both its destabilization and overstabilization are detrimental to the virus.The number of residues involved in residue-residue interactions and a model of the interactions between each monomer denominated A-B, B-C, and C-A are shown in This trimer model shows the number of interactions across two interfaces as well as details of the individual residue-residue interactions across these interfaces .The interactions analysis in the 3D structure was obtained by a PDBsum server; for these hydrogen bonds and other nonbonded, disulfide bridges of CYS between two side chains or the formation of an amide bond (–CO–NH–) between side chains of Lys and a dicarboxylic aminoacid (Glu or Asp) were considered. The 3D structure presents electrostatic interactions between Glu 293 in chain A with Lys 297 in chain B, Glu 300 of chain A with Lys 313 of chain C, Lys 297 of chain A with Glu 300 of chain C, Lys 148 on chain B with Glu129 on chain C, and Lys298 on 25 chain C with Glu 286 and 293 on chain B. These stabilize the structure .Z-score and Ramachandran plot. The stereochemical quality of the RABVG 3D structure was checked with a Ramachandran plot by analyzing the backbone dihedral angles residue by residue. The result showed that 89.9% of the residues were in the favorable region  indicates that the RABVG trimer is stabilized at 12 ns and remains constant until the end of the simulation (Molecular dynamics (MD) simulations were carried on RABVG trimer at 310 K. From these theoretical studies we retrieved the RMSD values from Cmulation .α-helices. During the MD simulation, no structural uncoiling was observed. At 310 K, the RMSF values go from 3 to 15 Å obtained from 12000 to 7500 frames; RMSF are similar for all chains except Ala87 to Thr100, Pro136 to Thr147, Phe173 to Asn201, and Ser422 to Val435 residues of chain C  identify the regions responsible for the fluctuations during the MD simulations. The areas with higher fluctuations correspond to beta-loop-beta chains residues and turn regions. Those with lower fluctuations are regions of  chain C .The regions with the most fluctuations have not been described as important areas to maintain the structural stability of the G protein trimer. This does not generate significant changes within the main trimer binding regions.In the residues 125 to 131 conformational change of chain C after 10 ns was detected and is stable from 40 ns to 80 ns when molecular dynamic end .During molecular dynamic simulations, the structure of the G protein remained stable after 12 ns. It maintained the trimer bound and conserved key interactions to maintain the stability of the structure.The molecular modeling of G protein was performed by a I-TASSER server and was evaluated via a Rachamandran plot and ERRAT program obtained 84.64% and 89.9% of the residues in the favorable regions and overall quality factor, respectively.The interactions between residues, 274 to 293, are directly linked to the structure of the prefusion and postfusion of Glycoprotein. These interactions are important to maintain these structures.This is important for structural stability of the G protein trimer. It might be a good target for antiviral compounds because such modifications would change the helical conformation and be detrimental to the virus.The fluctuations that occurred during the molecular dynamics do not affect the stability of the structure of G protein trimer. Protein G structural stability was obtained by molecular dynamics analysis at 12 ns.


We previously reported that arterial infarction of vital organs after lobectomy might occur only after left upper lobectomy and be caused by thrombosis in the left superior pulmonary vein stump. We hypothesized that changes in blood flow, such as blood stasis and disturbed stagnant flow, in the left superior pulmonary vein stump cause thrombosis, and this was evaluated by intraoperative ultrasonography.From July 2013 to April 2014, 24 patients underwent lobectomy in the Steel Memorial Muroran Hospital. During the procedure, an ultrasound probe was placed at the pulmonary vein stump and the velocity in the stump was recorded with pulse Doppler mode. The peak velocity and the presence of spontaneous echo contrast in the stump were evaluated. After the operation, the patients underwent contrast-enhanced CT within 3 months.The operative procedures were seven left upper lobectomies, four left lower lobectomies, seven right upper lobectomies, and six right lower lobectomies. Blood flow was significantly slower in the left superior pulmonary vein stump than in the right pulmonary vein stumps. However, that was not significantly slower than that in the left inferior pulmonary vein stump. Spontaneous echo contrast in the pulmonary vein stump was seen in three patients who underwent left upper lobectomy. Of the three patients with spontaneous echo contrast, two patients developed thrombosis in the left superior vein stump within 3 months after the operation. There was no patient who developed arterial infarction.In patients who underwent left upper lobectomy, intraoperative ultrasonography to evaluate blood flow and the presence of spontaneous echo contrast in the left superior pulmonary vein stump may be useful to predict thrombosis that may cause arterial infarction.The online version of this article (doi:10.1186/s13019-014-0159-8) contains supplementary material, which is available to authorized users. Arterial infarction of vital organs, such as the brain, kidneys, and intestines, is a very important and serious complication after pulmonary lobectomy. There have been 12 reported cases of arterial infarction of vital organs after lobectomy . All 12 What is the cause of arterial infarction after LUL? Of the previously reported 12 cases, six cases showed a thrombus in the left superior pulmonary vein (LSPV) stump . TherefoWhat is the cause of thrombosis in the LSPV stump? As for thrombosis in the pulmonary artery (PA) stump, there have been some reports that thrombosis developed more frequently after right pneumonectomy than after left pneumonectomy because the right PA stump was anatomically longer than the left PA stump -16]. We. We16]. This study was approved by the ethics committee of the Steel Memorial Muroran Hospital. Informed consent was obtained from all patients and documented in a writing form before the surgery.From July 2013 to April 2014, 36 patients underwent lobectomy in the Steel Memorial Muroran Hospital. The following patients were excluded: four patients who underwent right middle lobectomy (RML); two patients whose respiratory status exacerbated on one-lung ventilation; two patients who underwent long surgery for more than 6 hours; two patients with allergy to contrast dye; one patient with interstitial pneumonia; and one patient whose PV was divided in the pericardium. Finally, 24 patients were the subjects of this study. In these patients, blood flow in the PV stump during the operation was evaluated by the method described below, and they underwent contrast-enhanced chest computed tomography (CT) within 3 months after surgery.For all patients, except those with large tumor size, lymph node metastases, chest wall invasion, bronchial invasion, or pulmonary vessel invasion on preoperative images, video-assisted thoracoscopic surgery (VATS) with a 7-cm, small thoracotomy, 3-cm window, and two ports was performed. For the other patients, open chest surgery with a 15 to 20-cm thoracotomy was performed.Under one-lung ventilation in the lateral position, the operation was performed as follows. First, the PV was divided using a linear surgical stapler. Second, a few branches of the PA were divided by ligation or linear staplers. Third, the bronchus was divided using a linear stapler. Fourth, dissection of mediastinal lymph nodes was performed. Then, blood flow in the PV stump was evaluated by ultrasonography using the method described below. Finally, a chest drainage tube was inserted, and the operation was completed after closure of all wounds.In patients receiving warfarin, the warfarin was discontinued from 7 days before surgery, and then they were started on intravenous heparin for up to 6 hours before surgery. On postoperative day (POD) 1, heparin was re-started if there was no sign of bleeding. On POD 7, oral warfarin was re-started after removal of the epidural catheter. If the prothrombin time entered the therapeutic range, heparin was stopped.In the patients receiving antiplatelet drugs, cardiovascular or neurosurgical specialists were consulted to determine whether that drug could be discontinued. If antiplatelet drugs could be discontinued, they were discontinued from about 7 days before surgery. In high-risk patients, heparin was administered preoperatively based on the advice of a specialist. Perioperative management of heparin was as previously described. Oral antiplatelet drugs were re-started after removal of the epidural catheter, and then heparin was discontinued.Blood flow in the PV stump was evaluated using an ultrasound console  with a 33-mm, Large Animal Flexible Transducer . A small amount of saline was infused into the thoracic cavity. An ultrasound probe was placed at the PV stump under thoracoscopic guidance Figure . The proFirst, the velocity in the PV stump was recorded with pulse Doppler mode Figure . The samAnalysis of the blood flow was performed using the Mann Whitney U test. All statistical analyses were performed using Stat View 5.0 software . A p value of less than 0.05 was regarded as significant.The characteristics of the 24 patients are shown in Table The blood flow was significantly slower in the LSPV stump  than in the right superior PV stump  and the right inferior PV stump (median, 20.9 cm/s; range, 16.1 to 22.7 cm/s; p = 0.015; Figure SEC was seen in the PV stump in three patients who underwent LUL Figure . In the Of the three patients with SEC, two patients developed thrombosis in the LSPV stump within 3 months after the operation Figures  and 5. TWe hypothesized that changes in blood flow, such as blood stasis and disturbed stagnant flow, in the long stump caused thrombosis in the LSPV stump after LUL . That isOf the 12 cases that had been reported with infarction of vital organs after lobectomy, one patient was found to have SEC in the LSPV stump on echocardiography . PatientWhen does thrombosis in the LSPV stump develop after LUL? In paroxysmal Af, if it continues over 48 hours after onset, there is a need for anticoagulant therapy for defibrillation . This meHow is thrombosis in the LSPV stump diagnosed? Generally, the D-dimer test is considered to be useful for diagnosing a thrombus. However, in our previous report, we demonstrated that the D-dimer level was normal in patients with thrombosis in the LSPV stump . Thus, bWhat is the treatment for thrombosis in the LSPV stump after LUL? Because it may easily enter the systemic circulation and cause arterial embolism, we recommend that anticoagulant therapy be started immediately. Additionally, based on the results of the present study, we also recommend anticoagulant therapy if intraoperative ultrasonography reveals SEC in the LSPV stump. If arterial embolism unfortunately develops, surgical removal of the thrombus should be considered. Ohira et al. reported that a case with thrombus in the LSPV stump developed cerebral infarction and underwent surgical removal of a thrombus .Asai et al. reported that a case developed syncope after segmentectomy of the left upper division and showed a thrombus in the LSPV . In thisThe limitation of this study was that intraoperative ultrasonography was performed in the lateral decubitus position. To more closely approximate daily conditions, blood flow in the PV stump was evaluated after inflation of the affected lung. However, blood flow in the PV stump in the lateral decubitus position might not reflect that in the usual position for ultrasonography.By evaluating blood flow in the PV stumps using intraoperative ultrasonography, blood flow was found to be significantly slower in the LSPV stump than in the other three PVs. The patient who showed SEC in the LSPV stump intraoperatively developed thrombosis in the LSPV stump after LUL. In patients who undergo LUL, intraoperative ultrasonography to evaluate blood flow and the presence of SEC in the LSPV stump may be useful to predict thrombosis.


Streptococcus pneumoniae Neuraminidase A (NanA) is a multi-domain protein anchored to the bacterial surface. Upstream of the catalytic domain of NanA is a domain that conforms to the sialic acid-recognising CBM40 family of the CAZY (carbohydrate-active enzymes) database. This domain has been identified to play a critical role in allowing the bacterium to promote adhesion and invasion of human brain microvascular endothelial cells, and hence may play a key role in promoting bacterial meningitis. In addition, the CBM40 domain has also been reported to activate host chemokines and neutrophil recruitment during infection.Crystal structures of both apo- and holo- forms of the NanA CBM40 domain (residues 121 to 305), have been determined to 1.8 Å resolution. The domain shares the fold of other CBM40 domains that are associated with sialidases. When in complex with α2,3- or α2,6-sialyllactose, the domain is shown to interact only with the terminal sialic acid. Significantly, a deep acidic pocket adjacent to the sialic acid-binding site is identified, which is occupied by a lysine from a symmetry-related molecule in the crystal. This pocket is adjacent to a region that is predicted to be involved in protein-protein interactions.S. pneumoniae.The structural data provide the details of linkage-independent sialyllactose binding by NanA CBM40 and reveal striking surface features that may hold the key to recognition of binding partners on the host cell surface. The structure also suggests that small molecules or sialic acid analogues could be developed to fill the acidic pocket and hence provide a new therapeutic avenue against meningitis caused by  Streptococcus pneumoniae is a human pathogen responsible for respiratory tract infections, septicaemia and meningitis. Several virulence factors contribute to colonization and early infection processes ; RMSD 1.7 Å for 169 Cα’s [S. pneumoniae NanB [PDB ID: 2VW0]; RMSD 2.0 Å for 170 Cα’s [M. decora NanL [PDB ID: 2SLI]; RMSD 2.0 Å for 176 Cα’s [V. cholerae NanH [PDB ID: 1W0P]; RMSD 3.1 Å for 134 Cα’s  was used to solve the SpCBM structure by molecular replacement with the PHASER program from the CCP4 suite [The leech P4 suite , 26. RefP4 suite  from theP4 suite . After fP4 suite . RefinemSpCBM [The meta-PPISP server was used to predict potential protein-protein interaction sites on SpCBM . The serSpCBM –33. For Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 4ZXK, 4C1W and 4CIX for the apo structure, the 3’SL complex and the 6’SL complex, respectively.