Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress. The family of Herpesviruses is divided into three subfamilies alpha-, beta-, and gamma-herpesviruses varying in cell tropism, pathogenicity, and the site of latency . NumerouHerpes virions carry a linear double stranded DNA genome, packaged in an icosahedral capsid, which is surrounded by a tegument protein layer, and a host-derived membrane equipped with numerous viral glycoproteins. The herpesviral replication cycle starts upon docking of virions to the host cell followed by entry due to fusion of the virion membrane with the host membrane  membrane protein . Importantly, the nucleocapsid periphery was intensely decorated suggesting an efficient association of pUL53 with the nucleocapsid surface in preparation for transport of nucleocapsids to the nuclear periphery and subsequent NEC formation. Proteins that may functionally and/or physically link pUL31 with capsids ,14 incluThe question arises about the potential role of pUL31 in replication compartments and in association with nucleocapsids. In the nucleoplasm of infected cells, several intermediates of capsid assembly are distinguished, A capsids that lack a viral genome and scaffold, B capsids that are nonproductive intermediates, and C capsids that represent mature capsids with a packaged genome . PreferrLittle is known how capsids are released from replication compartments ,85. InteCapsids assembled and matured in the nuclear interior need to reach the nuclear periphery for primary envelopment and egress to the cytoplasm where they receive their final envelope. In particular during early phases of infection, the densely packed chromatin represents a major barrier. As viral infection progresses, the nuclear architecture is dramatically changed ,86. ViraInitial reports suggested that capsids use the nuclear acto-myosin system for directed movement from replication compartments to the nuclear periphery ,11,87. THaving traversed the chromatin, the nuclear lamina that lines the INM likely represents another obstacle for egressing capsids. The nuclear lamina forms a dense meshwork composed of various filamentous lamins and of integral membrane lamina-associated proteins (LAPs) ,91, a stRecruitment of various cellular kinases to the lamina and the NEC has been reported during infection suggesting that cellular kinases play a complex and potentially redundant role during nuclear egress ,95. ImpoThe NEC represents the crucial hub at the INM to execute primary envelopment of nucleocapsids. Several lines of evidence support its multiple roles in binding of nucleocapsids, membrane vesiculation and rearrangement of the nuclear envelope ,94. ProtPreviously, mutational analysis revealed details on the interaction and function of the NEC partners of several herpesviruses. A library of charged cluster mutations identified residues in pUL34 involved in recruiting pUL31 and in NEC function . SimilarIn 2015, five crystal structures of three different herpesviral NECs, of Herpes simplex virus 1  part of the NEC while pUL31 provides the membrane-distal (MD) part ,45,68. AThe MD part of the NEC composed of the C-terminal domain of pUL31 is oriented towards the lumen of the coated vesicle suggesting that this part of pUL31 contacts the capsid surface ,39,45,68Nucleocapsids with primary envelopes, or intermediates thereof, are rarely observed in the perinuclear space and do not accumulate during wildtype infection. This suggests that both primary envelopment and de-envelopment are rapid processes. HSV1 and PrV mutant strains with defects in the pUS3 kinase accumulate primary enveloped capsids in the perinuclear space of the infected cell ,121,122 Nuclear egress is a process critically required to release nucleocapsids to the cytoplasm for a productive viral replication. For efficient nuclear egress, both NEC proteins have to reach the nucleus to get into contact with the mature nucleocapsid. To this end, the NEC needs to be primarily active at the INM and upon availability of the nucleocapsid. Altogether, this strongly points to a tight regulation of nuclear egress and in particular the NEC activity. Based on the combined data of many labs, I would like to present the capsid-escort-model that describes an orchestrated sequence of events for a highly regulated and efficient nuclear egress of herpesviral nucleocapsids: following their cytoplasmic synthesis, pUL31 and pUL34 orthologs take separate and distinct transport routes to the nucleus A,B. PremA first level of regulation of the NEC is important in the cytoplasm where naturally both proteins co-exist. Evidence is increasing that both pUL34 and pUL31 are regulated to prevent premature interaction. In all NEC structures, pUL31 is characterized by a hook-like extension that embraces pUL34 forming extended interfaces (In the infected nucleus, two possibilities obviously exist how capsids and NECs could come into contact. The membrane-anchored pUL34 may readily recruit the imported pUL31 to form the NEC and NEC seeds at the INM. Docking of capsids at such NECs pre-existing at the INM is expected to invoke a signal transferred to the MP part resulting in conformational changes, NEC oligomerization and ultimately budding (Alternatively, as shown by Funk et al., 2015  and receThe underlying molecular processes of nuclear egress that most likely involve conformational alterations of the NEC partners are expected to be complex and likely regulated by cellular and viral kinases ( and refe


The persistence of atmospheric circulation anomalies over East Asia shows a winter to winter recurrence (WTWR) phenomenon. Seasonal variations in sea level pressure anomalies and surface wind anomalies display significantly different characteristics between WTWR and non-WTWR years. The WTWR years are characterized by the recurrence of both a strong (weak) anomalous Siberian High and an East Asian winter monsoon over two successive winters without persistence through the intervening summer. However, anomalies during the non-WTWR years have the opposite sign between the current and ensuing winters. The WTWR of circulation anomalies contributes to that of surface air temperature anomalies (SATAs), which is useful information for improving seasonal and interannual climate predictions over East Asia and China. In the positive (negative) WTWR years, SATAs are cooler (warmer) over East Asia in two successive winters, but the signs of the SATAs are opposite in the preceding and subsequent winters during the non-WTWR years. A considerable amount of climatology research focuses on winter temperatures. Winter is the season most obviously affected by global warming; therefore, research on temperature change during the winter helps scientists better understand climate warming. Additionally, disastrous winter climates are closely related to temperature changes that influence people’s lives. Local large-scale atmospheric circulation anomalies are an important factor affecting the winter weather and climate over East Asia. They are closely related to the intensity of the East Asian winter monsoon, which is closely linked with the Siberian High –5. ThereThe persistence of atmospheric circulation anomalies has strong seasonal dependence over East Asia, which is called winter to winter recurrence (WTWR). WTWR was first discovered in mid-latitudinal sea surface temperature anomalies (SSTAs), which recur from one winter to the next with no persistence during the intervening summer –12. BecaHowever, the interannual variability of the atmospheric WTWR over East Asia and its impact on winter temperatures remains unclear. Zhao and Li  analyzedThe remainder of this manuscript is organized as follows. The datasets used are described in Section 2, and the WTWR of the atmospheric circulation anomalies over East Asia and the interannual variability of WTWR are examined in Section 3. Atmospheric circulation anomalies during WTWR and non-WTWR years are presented in Section 4. The impact of the WTWR on winter temperature anomalies is analyzed in Section 5. A summary and discussion are provided in Section 6.The primary datasets used in the present paper are from the National Center for Environmental Prediction–National Centers for Atmospheric Research (NCEP–NCAR) reanalysis data . ObserveLet us first examine the geographical distribution of the WTWR of anomalous atmospheric circulation over East Asia. Lag correlation analysis is performed at each grid point of the sea level pressure anomalies (SLPAs), which obtains more accurate recurrence areas compared to the selection of regions or specific spatial patterns. At one grid point, there is a WTWR year if the lag correlation coefficient for the starting month of the winter season (February) drops to an insignificant level during summer and recurs during the following winter (January to March). This method was explained in more detail by Zhao and Li . Fig 1 sThe above lag correlation was used in previous studies to define the SSTAs WTWR . Thus, fThe characteristic patterns of atmospheric circulation anomalies in WTWR and non-WTWR years are examined through composite analysis. Therefore, the seasonal variations in atmospheric circulation anomalies over Eurasia have significant differences between WTWR and non-WTWR events. Generally, a WTWR event is characterized by the recurrence of the anomalous Siberian High and East Asian winter monsoon (EAWM). The Siberian High strengthens (weakens) in the first winter of the positive (negative) cases, which is accompanied by a strengthened (weakened) winter monsoon in East Asia. The state is the same in the second winter. For the non-WTWR events, the Siberian High also strengthens (weakens) in the first winter of the positive (negative) cases. In the second winter, however, the Siberian High weakens (strengthens) and is accompanied by a weakened (strengthened) winter monsoon in East Asia. During winter, atmospheric circulation anomalies are controlled by the EAWM. Obviously, WTWR of anomalous atmospheric circulation is important to the winter climate in East Asia.Meridional wind is a crucial aspect of the EAWM –21 becauFor WTWR and non-WTWR events, SATAs also show characteristic differences over China . During (t+1)th year  over the period of 1952–2015, and SLPAs(t) are the SLPAs in the winter of the tth year. Because the value of the F-test is 11.1, exceeding the significant value of 7.1 at the 0.01 level, the equation is statistically significant. Therefore, SLPAs(t) can be considered a better predictor of SATAs(t+1).The WTWR of circulation anomalies is associated with that of SATAs over East Asia, implying that the former is possibly an important hint for seasonal and interannual predictions of the latter. Zhao and Li  discusseFurthermore, the WTWR of the atmospheric circulation anomalies will inevitably impact the air temperatures over East Asia and China from one winter to the next. In the positive (negative) WTWR cases, the SATAs are cooler (warmer) over East Asia in two successive winters, which are relatively weak during the intervening summer. However, for the non-WTWR events, the signs of the SATAs are opposite in the preceding and following winters. Thus, the WTWR of anomalous atmospheric circulation contributes to the recurrence of winter air temperature anomalies over East Asia, which is useful information for improving seasonal and interannual climate prediction in East Asia and China.Due to the large meridional extent of the EAWM, the climate anomalies associated with the EAWM are not consistent between the mid-high latitudes and the low latitudes of East Asia. Several studies have revealed the presence of two distinct modes associated with the EAWM variability. Kang et al. –25 firstThere are studies indicating the discrepancy between the different reanalysis datasets –29. TherPrevious studies have shown that the EAWM experienced a decadal change around the mid-1980s , 30 and Zhao and Li  have inv


Even though the caesarean section is an essential component of comprehensive obstetric and newborn care for reducing maternal and neonatal mortality, there is a lack of data regarding caesarean section rates, its determinants and health outcomes among tribal communities in India.The aim of this study is to estimate and compare rates, determinants, indications and outcomes of caesarean section. The article provides an assessment on how the inequitable utilization can be addressed in a community-based hospital in tribal areas of Gujarat, India.Prospectively collected data of deliveries (N = 19923) from April 2010 to March 2016 in Kasturba Maternity Hospital was used. The odds ratio of caesarean section was estimated for tribal and non-tribal women. Decomposition analysis was done to decompose the differences in the caesarean section rates between tribal and non-tribal women.The caesarean section rate was significantly lower among tribal compared to the non-tribal women  respectively. The 60% of the differences in the rates of caesarean section between tribal and non-tribal women were unexplained. Within the explained variation, the previous caesarean accounted for 96%  of the variation. Age of the mother, parity, previous caesarean and distance from the hospital were some of the important determinants of caesarean section rates. The most common indications of caesarean section were foetal distress (31.2%), previous caesarean section (23.9%), breech (16%) and prolonged labour (11.2%). There was no difference in case fatality rate  and incidence of birth asphyxia  comparing the tribal and non-tribal women.Similar to the prior evidences, we found higher caesarean rates among non-tribal compare to tribal women. However, the adverse outcomes were similar between tribal and non-tribal women for caesarean section deliveries. The maternal and neonatal health outcomes among tribal and indigenous communities are worse compared to non-tribal populations in low-middle income countries –2. The aThe clinical indications of caesarean section among rural tribal women have not been studied in India. In general, there is a lack of data on indications and outcomes of caesarean section among tribal communities –7. As thWe used the data of all women who delivered at the Kasturba Maternity Hospital (KMH), a hospital managed by SEWA Rural, a voluntary, not-for-profit organization. KMH has been providing maternal and neonatal health services in this area since 1980 along with community-based services in surrounding areas . The hosThe study is based on secondary analyses of data which was primarily collected for delivery and monitoring of services at the hospital. The data is part of the hospital program to provide quality health services to the remote and tribal areas of Gujarat. The ethical approval for the use of this data has been obtained from SEWA Rural Institutional Ethics Committee (IEC). The SEWA Rural IEC reviewed this data and allowed its use for analyses and publication. The IEC have also waived the need for the informed consent of the patient, given that the anonymity of the patient will be maintained. Any personal information of the patient was removed from the datasets before analysis.We used the data of all women who delivered at the Kasturba Maternity Hospital (KMH), a hospital managed by SEWA Rural, a voluntary, not-for-profit organization. KMH has been providing maternal and neonatal health services in this area since 1980 along with community-based services in surrounding areas . The hosThe tribal population native to the study area is known as Vasava, and belongs to the Bhil tribe. As with other tribal communities, Vasava tribe has its own language and customs which are different from the mainstream culture; though this is changing as they are increasingly connected with the mainstream culture. Agriculture is the primary occupation with a large percentage of the population working as landless labourers .KMH is a 200-bed tribal-friendly first referral unit providing CEmONC care to surrounding villages. The CEmONC services at the hospital include services from full-time clinicians including obstetricians, anaesthetists and internists. Along with the clinician, the hospital has a functional operation theatre, blood storage centre, ultrasound, and inpatient facility . The misThe hospital maintains a prospective registry for all admissions that is maintained by care providers and subsequently digitized by trained staff. A team of care providers including gynaecologists ensured the accuracy of the entire dataset, including that of indications and outcomes of all deliveries. Women who delivered from April 2010 to March 2016 were included in the study. The definition of tribal is as per the specifications from the government of India . The desThe deliveries were categorized as caesarean section and vaginal deliveries, and are the primary dependent variables. The potential determinants of caesarean section were maternal age, parity, maternal education, gestational week, haemoglobin status, government scheme, distance from the health facility and child’s gender. The adverse outcomes of deliveries examined for this analyses were still birth, low birth weight (less than 5.5 lbs/2500 grams), survival status of the neonate at the time of discharge from the hospital  and birth asphyxia (no cry after delivery).Rate, indication and outcomes of caesarean section were represented by percentages and counts comparing tribal and non-tribal women. Still birth rate and neonatal death rate are expressed per 1000 total (live + still) deliveries and per 1000 live births respectively. Logistic regression was used to estimate the odds ratio caesarean section by background characteristics as potential determinants. The odds ratio was adjusted for Mother’s age, Mother’s education, Parity, Mother’s haemoglobin, Gestational week, Child’s gender, Number of ANC visits by mother and Distance from the health facility. The odds ratio of caesarean section was estimated for tribal and non-tribal women. Decomposition analysis was done to decompose the differences in the rates of caesarean section between tribal and non-tribal women. Fairline modification of Blinder-Oaxaca decomposition for the logit models was used . VariousThe total number of deliveries were 19923 . The number of vaginal deliveries and those via caesarean section by ethnicity are given in The caesarean section rates for tribal and non-tribal women were 9.4% (n = 1300) and 15.6% (n = 950) respectively . The gapIncreasing age of the mother was related to increasing caesarean section rates in tribal women . EducatiDecomposition analysis decomposed differences found between tribal and non-tribal women to other selected variables . OverallThe most common indications for caesarean section were foetal distress followed by previous caesarean section and breech presentation . Among nThere was no difference in the case fatality rate among neonates comparing the tribal and non-tribal in caesarean section deliveries . PercentThis is one of the few studies to compare the rates, indications and outcomes of caesarean section between tribal and non-tribal women in India. Studies in the past have shown lower caesarean section rate in India compare to other Asian countries –31. StudPrevious caesarean section, foetal distress and mal-presentation of the foetus (Breech and Transverse Lie) were the most common clinical indication for caesarean section in our study. Our results are relatively consistent with other studies in India and other countries ,37. SimiThere are a few lessons learned regarding how to develop tribal friendly health care facility for reducing the inequity of the health services in the tribal area. Some of the government sponsored policies that enabled the KMH to develop tribal friendly hospital, such as the grant-in-aid scheme and Chiranjivi Scheme could be explored for replicating in other tribal predominant states in India –41. At tThere are some limitations of this study. This study was done in the context of a tribal friendly hospital. The findings may not be generalized to other areas of India. Most of the women (90%) who had still birth were admitted with no foetal heart rate, which may cause higher still birth rate in vaginal delivery. It would have been useful to compare other outcomes of the caesarean section such as APGAR  score along with the intra-operative and post-operative complications. Other mother related complication and delays in receiving services were also not available. The information was not available for the analysis. The study is based on maternal admissions; some women may have more than one delivery during the period of study.It is one of the few studies which have compared indications and outcomes of caesarean section among tribal and non-tribal women in India. The caesarean rates were higher among non-tribal compared to tribal women. However, the adverse outcomes are similar among the tribal women compared to the non-tribal women who underwent the caesarean section. The previous caesarean section, maternal age, maternal education, parity and number of ANC visits are some of the important determinants of caesarean rates. The previous caesarean section among non-tribal and foetal distress in tribal was the most common indication of caesarean. Further research investigating and addressing the factors identified in this study are recommended.S1 File(XLSX)Click here for additional data file.


A major issue of machinery fault diagnosis using vibration signals is that it is over-reliant on personnel knowledge and experience in interpreting the signal. Thus, machine learning has been adapted for machinery fault diagnosis. The quantity and quality of the input features, however, influence the fault classification performance. Feature selection plays a vital role in selecting the most representative feature subset for the machine learning algorithm. In contrast, the trade-off relationship between capability when selecting the best feature subset and computational effort is inevitable in the wrapper-based feature selection (WFS) method. This paper proposes an improved WFS technique before integration with a support vector machine (SVM) model classifier as a complete fault diagnosis system for a rolling element bearing case study. The bearing vibration dataset made available by the Case Western Reserve University Bearing Data Centre was executed using the proposed WFS and its performance has been analysed and discussed. The results reveal that the proposed WFS secures the best feature subset with a lower computational effort by eliminating the redundancy of re-evaluation. The proposed WFS has therefore been found to be capable and efficient to carry out feature selection tasks. Condition monitoring and fault diagnosis is essential for a wide range of mechanical components to ensure optimal performance. A bearing is a common mechanical component that has an appreciable impact on machine integrity. Vibration signal analysis has been proven to be the most effective method for rotating machinery fault diagnosis. Its effectiveness, however, is highly dependent on the knowledge and experience of the operator . There hThe feature selection approach can generally be classified into three categories: the filter, wrapper, and embedded methods. Wrapper feature selection alternatives are usually combined with machine learning classifiers to develop a heuristic mechanism that aims to provide an optimal input for targeting optimization functions by considering the options available within a search space boundary. This is performed by the renowned genetic algorithm (GA) ,9, partia priori knowledge [Various feature selection crossover combinations such as the hybrid filter-wrapper method have been implemented, with a twofold aim: To refine the feature selection performance and reduce the disadvantages introduced by individual techniques –27. Nonenowledge . In addinowledge  likely jThis paper proposes an improved WFS method that aims to select the fittest feature subset with minimum computational resources via selecting potential candidates only through unique feature combinations. This provides the advantage of avoiding the unnecessary consideration of repetitive feature combinations and previously eliminated candidates. In this section, the necessities of the feature selection in automated machinery fault diagnosis and the limitations and drawbacks of the WFS method have been discussed in detail. The methodology for the bearing data collection, from the feature extraction to the proposed selection strategy, is described in the following section. The performance of the proposed WFS method is discussed based on the k-fold cross-validated classifier performance and compared to the recently published Max-Relevance-Max-Distance (MRMD) technique.The following part of this paper describes the methodology of the bearing data collection, the feature extraction and the proposed WFS strategy in greater detail.The bearing conditions dataset used in this study was downloaded from the Case Western Reserve University Bearing Data Centre website with the intention of specifically representing ball bearings in healthy and faulty conditions . The test rig consisted of a 2-horse power (HP) motor, a torque transducer and a dynamometer. The arrangement of the test rig was used to simulate different bearing conditions . The motA total of 400 sets of time series vibrations were extracted from the raw continuous vibration signal collected from a 7-mil fault diameter with a 1-HP load. Then, the 400 sets of vibration data were divided into two sets of data, one of which was used to establish the relationship between the input and output of the machine learning model (training phase), while the other set was used to validate the trained machine learning model (testing phase). The distribution of the vibration dataset employed in this study is tabulated in In this section, the time series vibration data from Section 3 is subjected to statistical analyses. The features obtained, namely, the skewness factor, kurtosis factor, crest factor, shape factor, impulse factor and margin factor, were converted from the corresponding equations in Since there was a total of 100 samples for each bearing condition, 50% of the samples were randomly selected as training data to synthesize the machine learning model, while the remaining 50% of the samples were used to validate the trained machine learning model.In this study, an improved WFS method was proposed for performing the feature selection task. The proposed WFS method employed the SVM as a classifier in feature selection. The performance of each feature was based on SVM classifier training accuracy after multi-fold cross-validation appraisal  in pursuFurther investigation has been conducted using a recently published feature selection technique in order to validate the proposed WFS method. The MRMD technique was selected after it demonstrated a good balance between classifier accuracy and stability when subjected to an image processing dataset ,32. Its The aim of this study was to improve the capability of the WFS method for selecting the best feature subset with a reduced computational effort. The analysis of the results revealed that the proposed WFS is capable of selecting the most representative feature subset for the bearing dataset. In addition, this study also confirmed that entering all the extracted features into the machine learning algorithm does not guarantee the best classification performance. Thus, feature selection plays a vital role in ensuring the optimum performance of a classifier. The proposed WFS method also reduces the number of feature combinations needing to be evaluated by avoiding the re-evaluation of identical feature combinations. This reduced the computational effort required by two thirds. In sum, the main advantage of the novel, state-of-the-art WFS method introduced here is its ability to select the best feature subset using less computational effort. This is essential when analysing a large number of inputs. This proposed WFS method should be embedded into machine learning algorithms in order to improve their performance. A further improvement of the proposed WFS method can focus on the selection of image related visual features.S1 Data File(MAT)Click here for additional data file.


Papio anubis) at Gashaka Gumti National Park, Nigeria. Two social networks including all independently moving individuals  were created based on aggressive and grooming behaviour. We used knockout simulations based on the random removal of individuals from the network in order to investigate to what extent the exclusion of juveniles affects the resulting network structure and our interpretation of age-sex specific social roles. We found that juvenile social patterns differed from those of adults and that the exclusion of juveniles from the network significantly altered the resulting overall network structure. Moreover, the removal of juveniles from the network affected individuals in specific age-sex classes differently: for example, including juveniles in the grooming network increased network centrality of adult females while decreasing centrality of adult males. These results suggest that excluding juveniles from the analysis may not only result in a distorted picture of the overall social structure but also may mask some of the social roles of individuals belonging to different age-sex classes.In recent years there has been much interest in investigating the social structure of group living animals using social network analysis. Many studies so far have focused on the social networks of adults, often excluding younger, immature group members. This potentially may lead to a biased view of group social structure as multiple recent studies have shown that younger group members can significantly contribute to group structure. As proof of the concept, we address this issue by investigating social network structure with and without juveniles in wild olive baboons ( Marmota flaviventris) increase the cohesion of the affiliative network )uricatta), horses s ferus) , ring-taa nasua) ). Our rea nasua)  and as sa nasua) –43,87 ana nasua) . Howeverops sp.) .Thus, although the exact nature of the effects of the inclusion/exclusion of juveniles will depend on the species and the group sex ratio as well as the behaviour investigated, our results suggest that the exclusion of an entire age-class can have important implications for a variety of social network applications. For example, many studies on the patterns of disease spread in social networks do not include juveniles  [Ateles geoffroyi) [belied marmots [Age and sex-specific social roles have been described for a large variety of species ,44,58. Hus patas ). Moreovus patas , our stu vulpes) , spider offroyi)  and yell marmots . ExcludiAs predicted, the social network position of males was not strongly affected by the removal of juveniles in the grooming network, due to the fact that adult males rarely groomed juveniles, a finding that corresponds to other studies on primates –100. Howper se are limited. Instead, the aim of our study was to demonstrate that even though incomplete networks can still provide robust results [It is important to emphasise that the simulated knockouts we conducted here are of course not equivalent to the physical removal of individuals from the group ,8,21. Th results  excludinIn conclusion, using olive baboons as an example, we have provided proof of the concept that the inclusion of juveniles can change the resulting structure of the social networks compared to adult-only networks. This influence is present (but differs in direction and extent) across social behaviours and a range of network metrics. The results of our study thus suggest that a bias in incomplete sampling of social groups, such as omitting juveniles. can lead to an incomplete or distorted representation of age and sex specific social roles in animals.S1 Table(DOCX)Click here for additional data file.S2 TableSignificant results are in bold.(DOCX)Click here for additional data file.S3 TableNumbers in the first column represent the number of removed individuals.(DOCX)Click here for additional data file.S4 TableNumbers in the first column represent the number of removed individuals.(DOCX)Click here for additional data file.S5 TableNumbers in the first column represent the number of removed individuals.(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 TextFig A. Density plot of simulated distributions of in degree network metrics of juveniles and adults in the grooming network.Fig B. Density plot of simulated distributions of out degree network of juveniles and adults in the grooming network.Fig C. Density plot of simulated distributions of in strength network of juveniles and adults in the grooming network.Fig D. Density plot of simulated distributions of out strength network of juveniles and adults in the grooming network.Fig E. Density plot of simulated distributions of betweenness centrality of juveniles and adults in the grooming network.Fig F. Density plot of simulated distributions of clustering coefficient of juveniles and adults in the grooming network.Fig G. Density plot of simulated distributions of in degree of juveniles and adults in the aggression network.Fig H. Density plot of simulated distributions of out degree of juveniles and adults in the aggression network.Fig I. Density plot of simulated distributions of in strength of juveniles and adults in the aggression network.Fig J. Density plot of simulated distributions of out strength of juveniles and adults in the aggression network.Fig K. Density plot of simulated distributions of betweenness centrality of juveniles and adults in the aggression network.Fig L. Density plot of simulated distributions of clustering coefficient of juveniles and adults in the aggression network.(DOCX)Click here for additional data file.


Danio rerio) species has recently been recognized as an innovative experimental system. In fact, the transparent body and incomplete immune system of zebrafish embryos are especially useful for evaluating patient-derived tumor tissue interactions in healthy hosts. In conclusion, ex vivo systems represent an important tool for cancer research, but samples require correct manipulation to maximize their translational value.The use of patient-derived primary cell cultures in cancer preclinical assays has increased in recent years. The management of resected tumor tissue remains complex and a number of parameters must be respected to obtain complete sample digestion and optimal vitality yield. We provide an overview of the benefits of correct primary cell culture management using different preclinical methodologies, and describe the pros and cons of this model with respect to other kinds of samples. One important advantage is that the heterogeneity of the cell populations composing a primary culture partially reproduces the tumor microenvironment and crosstalk between malignant and healthy cells, neither of which is possible with cell lines. Moreover, the use of patient-derived specimens in innovative preclinical technologies, such as 3D systems or bioreactors, represents an important opportunity to improve the translational value of the results obtained. In vivo models could further our understanding of the crosstalk between tumor and other tissues as they enable us to observe the systemic and biological interactions of a complete organism. Although engineered mice are the most common model used in this setting, the zebrafish ( Primary cancer cell cultures are ex vivo cell populations recovered directly from fresh surgically resected tissue samples . These sCancer is a dynamic disease and represents the second cause of mortality in humans, mainly due to the presence of metastatic disease at diagnosis and the failure of clinical treatments . Tumors The physical properties and functions of the ECM serve as an anchor point for the cells and as direct modulator of cellular behavior. This cell–matrix crosstalk regulates pathways involved in cell proliferation, differentiation and migration, creating a favorable microenvironment for cancer progression and metastatic propagation , 11. TheAmong stroma components, fibroblasts play a central role in tumor growth, motility, angiogenesis, and metastatic dissemination , 19–21. Developed over a decade ago, the cancer stem cell (CSC) theory posits that the tumor has its own stem cells with self-renewal ability and ascribes the origin of these malignant cells to alterations of regulation pathways through genetic mutations of the healthy stem cell population . CSCs reThe cancer crosstalk with healthy components includes many other cell populations, e.g. MSCs, adipocytes, endothelial progenitor cells (EPCs), macrophages and myeloid-derived suppressor cells (MDSCs), all of which are involved in various stages of the disease , 46–49. Up to now, in vitro and in vivo cancer research has largely depended on the use of immortalized cell lines, which are usually cultured on monolayer supports for in vitro assays or xenografted onto immunocompromised animals for in vivo evaluations –53. CellHowever, cancer cell lines also have some limitations. The homogeneity of cell line populations and the high number of passages in monolayer systems make these models a far cry from the actual disease. Cell line cultures do not faithfully reproduce the molecular crosstalk, cell–cell interactions and tissue morphology that characterize the tumor microenvironment , 55, resIncreasing interest has recently been shown in the ex vivo culture of patient-derived tumor samples , 56, 57.Moreover, the availability of fresh samples enables single tumors to be studied and facilitates comparisons between lesions in the same or different parts of the body . For exaThe use of primary cancer cultures in preclinical studies, which requires the approval of the local Ethics Committee, is dependent on the availability of surgical material . A key fThe first step after surgical dissection consists in tissue dissociation to obtain a single cell suspension . The thrThe number of cells obtained depends on the amount and condition of surgical tissue available. Faili et al. compared different digestion approaches to analyze the viability rates of 20 tissue samples of resected colorectal cancer . They reThe composition of primary cultures varies, mainly on the basis of the tissue of origin. Different cell populations do not have the same ability to grow for long periods on in vitro devices or are not capable of attaching onto synthetic surfaces. Hematopoietic and stromal cells are often present after sample digestion . For exaAnother critical area for the correct management of primary cultures is the preservation of the tumor microenvironment following the use of early passages for downstream analyses. A small number of passages does not generally cause phenotype alteration in cell lines but have a drastic impact on the nature of the primary culture. In fact, early cell passaging leads to changes in gene expression, proliferation rate and drug response that enrich some subclones but not others, altering the high translational value of this model. Thus, the first steps of primary culture manipulation are of prime importance if useful translational results are to be achieved .As described above, the molecular crosstalk with non malignant cells and the presence of stem-like subclones are important aspects that reflect the nature of the tumor. Although the heterogeneity of cell populations contributes to mimicking the tumor microenvironment, elements such as nutrient perfusion or 3D structures cannot be reproduced by ex vivo samples. Some of these disadvantages can be overcome by using innovative in vitro and in vivo systems, described below. Indeed, new approaches are constantly being developed to improve the potentialities of primary cultures in different areas of preclinical research. However, the choice of the model must be correlated with the aim of the study to maximize the advantages of using cells directly recovered from patients.We have already seen the advantages and disadvantages of working with cell lines. Whilst they are undoubtedly an important tool for cancer research, their limitations must be borne in mind when analyzing results. Furthermore, commercial cell lines are not available for all stages of disease or for all tumor types , making The establishment of an immortalized cancer cell line includes three key events: (1) selection of cancer cells from stromal cells; (2) cell immortalization; and (3) morphological and biomolecular characterization of the cell line. The tumor fraction must also be separated from the various components of the original tissue. The rapid proliferation of fibroblasts favored by cancer cell-secreted growth factors is one of the main problems of this process , 82. GenImmunomagnetic separation is another method used to isolate cancer cells from fresh tissue, peripheral blood or bone marrow . SaalbacDifferent approaches have been validated to develop immortalized phenotypes . InfectiThe immmortalization of primary cells can be also performed using in vivo models, usually of mice. The microenvironment of live tissue promotes cancer cell growth mainly because of the high number of epithelial components and the consecutive crosstalk with the tumor . Human cThe last step in this process is the morphological characterization of the new line which is needed to validate the cancer phenotype and to exclude cross-contamination by other cell components . This isTumor lesions are dynamic masses of malignant and healthy cell populations in continuous co-evolution and supported by an extracellular 3D matrix that is constantly being remodeled. These alterations in the tissue structure are driven by the nature of the disease which modifies the matrix scaffold . Thus, tSynthetic matrices have been extensively studied as tools for the reproduction of cancer architecture, and polymer hydrogel systems are one of the resulting technologies. These engineered gels possess some of the properties of the tumor niche, e.g. stiffness. One option for synthesis is the use of end-functionalized multiarm polyethylene glycol (PEG) macromers to generate an inert and hydrophilic platform through the chemical crosslinking of functionalized polymers . Jiglared,l-lactide) (PDLLA), poly(l-lactic-co-glycolic acid) PLGA, polystyrene (PS), poly(methyl-methacrylate) (PMMA), poly(caprolactone) (PCL) and polyurethane (PU), non-polymeric materials , or substances derived from biological samples [Biomimetic porous scaffolds belong to the technologies used in tissue engineering applications. The nature of the matrix composition can be selected from a pattern of synthetic polymers, i.e. poly, we use the term ‘spheroids’ to refer to all spherical cancer models. These 3D cultures originate from cell–cell interactions that drive the aggregation processes of clusters or single cells in spherical structures. The spherical nature of these systems leads to the development of inner and superficial zones with different phenotypic and biological features [Spherical cancer models made their appearance four decades ago and are still used today . As therfeatures . For exafeatures . Primaryfeatures . Their rfeatures . Qureshyfeatures .Different protocols have been validated for the generation of spheroids on the basis of the tumor site . For priUnlike cell lines, ex vivo material cannot be totally dissociated into single cells. The process of finely cutting and crushing the culture in flasks containing medium supplemented with FBS is sufficient to guarantee the compaction and remodeling of cells into spheres . A strai3 in size which are then seeded in culture flasks coated with 0.75% agar plunged in medium supplemented with an excess of nonessential amino acids [Another method used to generate spheroids is the direct culture of tissue fragments in which the tumor tissue is cut into pieces of roughly 0.3–0.8 mmno acids . Dependino acids . Fragmenno acids .Finally, the main contribution of all of these systems is that they permit the interaction between cancer cells and 3D structures. Some techniques enable all the cell populations of patient tissue to be maintained, e.g. spheroids originating from directly by undigested fragments. Other systems show a preferential selection of malignant components, such as collagen-based scaffolds. Overall, 3D cultures enable us to study the stem-like properties of cancer cells. Sphere-forming assays and CSC enrichment offer a suitable platform to study the impact of cancer stem-like components on resistance to clinical treatments, one of the most important aims of preclinical research.The recapitulation of tumor-stroma crosstalk using primary tumor cell populations and a 3D tissue structure lacks a series of biochemical and biophysical stimulations that influence biological processes. Some of these missing parameters can be added and monitored using bioreactor systems, which are devices designed to influence the physiological properties of a cell culture through controlled fluid perfusion . This moAs previously mentioned, cell-based assays generally require a large number of cells that are difficult to obtain from limited patient-derived samples. . Two staIn conclusion, bioreactors reproduce tumor microenvironment features that are impossible to recreate using common in vitro methods. Microfluid systems haven’t still highlighted the contribution of non-malignant components of primary samples. On the other hand, the possibility to prolong the time on culture and the dynamic perfusion permit to overcome some of the limitation of ex vivo static methods.The use of animal models has long been considered a fundamental tool to better understand the many aspects of human diseases, including cancer. In vivo models provide more realistic data than those obtained from in vitro experiments in that, as complete organisms they reproduce many of the tissues and molecular interactions with malignant cells, resulting in a more faithful reproduction of the human scenario . In thisA major contribution to experimental cancer research has been made by genetically engineered mouse models (GEMMs). The anatomy, genetics and physiology of the mouse and human species are well conserved because of the common mammalian origin . The use®, or subcutaneous implantations of 17β-estradiol (E2) pellets in experiments with ER-positive cell populations, support cell growth and increase the engraftment rate [Surgical resection tissue and biopsies are the most widely used materials for PDXs but the injection of cells obtained from drainage fluid is also an option. Mice must be immunocompromised before being injected with human cells. There are several strains of immunodeficient mice such as NUDE (nu), SCID (scid), NOD-SCID and NOD/SCID/IL2λ-receptor null (NSG) which differ on the basis of the lack of one or multiple functional immune components, e.g. T cells, B cells or NK cells , 138. Thent rate –151.The PDX approach is particularly useful in tumors for which there are no tests available for the detection of markers of sensitivity or resistance to chemotherapy. In a similar scenario, treatment efficacy in mice injected with PDX cells provides important clinical information that facilitates the choice of therapy. Dong et al. performed renal capsule transplantations of non-small cell lung cancers (NSCLC) from untreated patients . The rapSuch results can also be used to optimize the design of clinical trials. For example, PDXs in GEMM hosts help to identify the subsets of patients who are more sensitive to new drugs and combinations. Furthermore, biologic and genetic analyses of responsive and resistant mice subpopulations facilitate the discovery of new biomarkers associated with therapeutic outcome. Berlotti et al. assessed the response to cetuximab, an anti-EGFR antibody, in 85 metastatic colorectal cancer PDXs and also evaluated genotypic differences . HER2 amMurine models also have limitations related mainly to the nature of the implant and to the characteristics of the host chosen as PDX recipient. As previously mentioned, some tumors have a low engraftment rate, i.e. hormone receptor-positive tumors, which puts them at a distinct disadvantage because of the small quantity of material often obtained from primary culture. Moreover, murine stroma in the implant site progressively substitute the human components, altering the tumor phenotype. Some of these changes lead to modifications in paracrine regulation and in the natural crosstalk between the disease and the niche microenvironment, impoverishing the translational value of the model. Finally, the length of time required for the development of tumor masses and the high cost of supporting animal facilities makes this model unfeasible for the majority of preclinical studies.Danio rerio) from Bangladesh and North East India [In the 1960s, Georges Streisenger was the first to describe the potential of a small tropical fish , makes the embryo stage highly suitable for detecting neoangiogenic events stimulated by cancer crosstalk processes , 170. A The fluorescent circulatory system of these transgenic lines and the transparency of the embryo body are also useful for assessing the metastatic potential through the detection of extravasated cells. The validation of the zebrafish system as a tool for measuring tumor invasiveness has been performed with both cell lines and primary cultures , 176. Thy442) [nacre mutant with arrested transcription of mifta gene involved in melanocyte development, and roy orbison (roy) characterized by a complete lack of iridophores and sparse melanocytes [−/−; nacre−/−) shows a completely transparent phenotype of the adult body. Although casper zebrafish are still not used for primary culture transplantation, they remain a potentially useful option for the future.Recently, adult zebrafish have begun to be used for PDXs . The posy442) , 182. Thanocytes , 183. ThDespite the aforementioned advantages of the zebrafish model, its use as a xenograft platform is not without problems. The relatively recent introduction of this animal system has left the market unprepared, and there are still few commercially available zebrafish antibodies or specific molecular kits. The incomplete immune system of embryos prevents the rejection of PDXs but also reduces the translational power of preclinical evaluations given that immune cells are involved in several cancer processes. For example, the efficacy of immunotherapeutic drugs, which interact with lymphoid cells, cannot be assessed. Furthermore, the zebrafish genome does not include all the human genes involved in tumor pathways, e.g. BRCA1, p16, IL6, LIF etc. . Thus, fTranslational preclinical research is acknowledged as a valuable investigational tool through which to improve our understanding of the cancer process. For decades, the gold standard for this kind of research was cell line experimentation. However, the length of time that cells are maintained in culture on monolayer supports and the processes used to obtain an immortalized phenotype both contribute to altering the original nature of the cell population. Closer collaboration between clinicians and researchers, together with improved laboratory methodological approaches, have led to primary cultures becoming a promising new option in the area of cancer research. This system has the advantage of maintaining the original phenotype of the lesion and of preserving the original tumor features, both essential for the reproduction of the tumor microenvironment. The complexity of the manipulation methods and the generally small quantity of biological material available make the management of primary cultures more complicated than that of cell lines. New approaches have been developed to overcome some of these disadvantages. 3D cultures and bioreactors represent innovative models for the implementation of microenvironment elements that cannot be reproduced by ex vivo samples. Each system offers potentialities in different aspects of tumor biology. Thus, the choice of model takes on an essential importance to guarantee the greatest advantages from primary cultures.In conclusion, although primary cultures represent an excellent preclinical tool for the reproduction of cancer in in vitro systems, correct sample manipulation based on the processing method selected is essential to maintain the acknowledged advantages of the model.


P/2, ascribed to the anisotropic shrinkage of the chiral nematic structure.Cellulosic liquid crystalline solutions and suspensions form chiral nematic phases that show a rich variety of optical textures in the liquid crystalline state. These ordered structures may be preserved in solid films prepared by evaporation of solvent or suspending medium. Film formation from aqueous suspensions of cellulose nanocrystals (CNC) was investigated by polarized light microscopy, optical profilometry and atomic force microscopy (AFM). An attempt is made to interpret qualitatively the observed textures in terms of the orientation of the cellulose nanocrystals in the suspensions and films, and the changes in orientation caused by the evaporative process. Mass transfer within the evaporating droplet resulted in the formation of raised rings whose magnitude depended on the degree of pinning of the receding contact line. AFM of dry films at short length scales showed a radial orientation of the CNC at the free surface of the film, along with a radial height variation with a period of approximately  Cellulosic liquid crystalline materials, both cellulose derivatives ,2 and cePolarized light microscopy, supplemented by scanning probe microscopy and electron microscopy provides an accessible tool to observe the structure of these ordered suspensions and films. Here, we present some observations on the chiral nematic self-organization of cellulose-based systems, with emphasis on the aqueous suspensions of cellulose nanocrystals (CNC) and of films formed from CNC by evaporation. The factors that govern phase separation, chiral nematic pitch, mechanical properties and applications of CNC and other cellulosic nanocrystals have been reviewed elsewhere, ,6,7 and Above some critical concentration, suspensions of cellulose nanocrystals (CNC) form chiral nematic phases . The liqP/2, where P is the pitch of the chiral nematic structure. The orientation of the CNC rods is shown in the idealized sketch in A variety of disclinations  where fiAs the concentration of CNC is increased, the chiral nematic phase is first observed in a biphasic region, where the isotropic and chiral nematic phases co-exist . On stanAs the concentration of CNC in the suspensions increases, the chiral nematic pitch should in principle decrease to values of the order of visible light, where iridescent colours should be visible. However, before such concentrations are reached, freezing-in of the pitch due to gelation and glass formation may occur .While CNC suspensions don’t usually achieve pitch values small enough to reflect visible light, molecularly dispersed solutions of cellulose derivatives often show iridescent colours due to reflection of circularly polarized light of visible wavelengths, although the colours take a long time to form, presumably because the polymer chains take a long time to disentangle and adopt chiral nematic order. Thus, for the (hydroxypropyl)cellulose (HPC)-water system , the effAn example of the reflected light from a blue HPC sample illuminated by white light against a black background is shown in et al. [Cellulose and cellulose derivatives have long been exploited for their film-forming properties. Examples include cellophane, the first transparent food wrap, and cellulose acetate, whose use as base for photographic films has been replaced by its use as protective and passive optical elements in liquid crystal flat-screen displays. If these film-forming properties could be combined with their tendency to form chiral nematic solutions and suspensions, the unique optical properties of chiral nematic liquid crystals would become available in a cheap easily handled solid film, based on an essentially renewable resource. To date, the cellulose derivatives have proved difficult, in general forming viscous solutions and solids that are slow to develop chiral nematic organization. CNC suspensions in water are less viscous, and chiral nematic suspensions and films are more readily prepared. However, control of the orientation and the CNC in dry films remains challenging. In this work, we focus on CNC films produced by simple evaporation of water from a sessile droplet of CNC suspension on a flat surface. Hoeger et al.  showed bFor samples sandwiched between microscope slide and cover glass, evaporation can only occur from the edge of the sample, resulting in a concentration gradient decreasing from the edge to the centre of the sample. This gradient will influence the orientation of the nanocrystals at the point where they are frozen into the solid. Other factors will also affect the orientation and texture of the film produced on drying . When plComplex textures are observed for dry films of 8% CNC suspensions allowed to dry between microscope slide and cover glass . For thiThe edge-wise evaporation of water from a sample of CNC suspension between glass slide and cover glass is slow and impractical on a larger scale. What happens if the droplet is left uncovered? Aqueous droplets containing low concentrations of rod-like CNC have been reported to display a nematic-like arrangement at the edge of the ring . The oriEvaporation of suspensions above the critical concentration for liquid crystal formation showed a more dramatic effect. Preliminary observations of the evaporation of droplets starting from CNC concentrations high enough to form chiral nematic suspensions showed the formation of films with a remarkable 3-Dimensional iridescent pattern . The confluid CNC suspensions, but iridescence is often observed for films made from such suspensions [The drying process initially causes a reduction in chiral nematic pitch, if for no other reason that the concentration of chiral centres increases with increasing concentration. Thus, there are no reports of the reflection of circularly polarized light from pensions ,19,20,21An example of the texture of an iridescent film is shown in et al. [et al. [The freely dried droplets show another difference from edge evaporation, in that the progression of colours from edge to centre is reversed. For covered droplets, evaporation from the edge leads to a higher concentration and hence a shorter pitch at the edge, as shown for example in et al.  and Duma [et al. . However [et al. ; evidentP/2) are much reduced on drying  images of the surface of the dried droplets were obtained. As a first observation by means of optical profilometry, there appear to be concentric rings of material, with raised edges. However, the rings appeared to be discontinuous, and the sample signal was very noisy. It seemed that the oriented and birefringent nature of the CNC film was interfering with the optical interferometric measurements used by the instrument. To avoid this problem, samples were coated with gold to provide an optically homogeneous surface, and the samples were scanned with the optical profilometer . In thisThe heights of steps and cracks are shown in the X and Y profiles across the film  are of tet al., nevertheless addition of low volatility low surface tension components such as PG to CNC suspensions would be expected to alter contact line pinning and hence the orientational order of the resulting film.The contrast between the relatively high ring of CNC deposited at the edge of the evaporating droplet for a pinned contact line  and the The profilometric measurements reported above demonstrate the large scale deviations from planarity generated by colloidal forces during evaporation from anisotropic suspensions of CNC. At much higher magnifications, the orientations of individual nanocrystals in solid films may be observed. For films cast from dilute CNC suspensions, below the critical concentration for liquid crystal formation, the orientation of the nanocrystals is essential random , despiteThe surface orientation of CNC in films cast from suspensions above the critical concentration for chiral nematic phase formation is quite different. Atomic force microscope (AFM) images of a typical area of a film cast from an 8 wt % CNC suspension on a mica substrate are shown in P/2 in height along the helicoidal axis, where P is the pitch in the final dry film. This type of anisotropic swelling and shrinkage is well-known on a larger length scale; the orientation of the cellulose microfibrils along the secondary wall of wood pulp fibres causes them to shrink on drying much more laterally than longitudinally, with important consequences for the bonding and properties of paper sheet [A possible explanation is sketched in er sheet .This model is clearly oversimplified; the drying process will generate in-plane as well as out-of-plane shrinkage and the chiral nematic axis may well be at an angle to the evaporating surface. However, where clear fingerprint patterns are detected optically near a free surface, a corresponding surface topology may be expected. The effect resembles the theoretical predictions of nanoscale undulations and disclination lines for appropriately anchored chiral nematic liquid crystals, driven by director surface gradients and elastic constants . Howeverw/w, 700 mL) at 45 °C for 45 min, after which the reaction was quenched by 5 L of deionized water  and left to settle for 2 h. The suspension was repeatedly concentrated and washed by centrifugation  until the supernatant appeared turbid, the suspension was then extensively dialysized against deionized water until the effluent remained at neutral pH . Following the removal of excess acid, the suspensions were dispersed by sonication using a Vibracel sonicator . An ice bath was used to prevent overheating.All the CNC suspensions were prepared as described previously . BrieflyTwo batches of CNC, labelled SA and SB were prepared. The main difference was in the ion exchange treatment used to remove excess ion content from the suspensions. Batch SA was stirred over Dowex Marathon MR-3  for 24 h. Batch SB was also stirred over Dowex Marathon MR-3 for 24 h, but then the mixed bed resin was filtered off and replaced by Dowex Marathon C, a strong acid cation exchange resin that had previously been rinsed repeated with ethanol to remove colour. The second resin treatment was to ensure that the resin was in acid form. The suspensions were next filtered through glass microfibre filters (0.45 μm pore size) to remove any particulate impurities introduced by the sonicater tip, and concentrated by means of a Labconco RapidVap vacuum evaporation system. The concentration of the suspensions was determined gravimetrically by drying small volumes in an oven at 105 °C. Sample SA was used as a 5.2 wt % suspension, and sample SB was concentrated to give 6 wt % (SB-6%) and 8 wt % (SB-8%) suspensions.The CNC samples were stored at −4 °C for at least 2 weeks until the suspensions had separated into isotropic and anisotropic phases. Small amounts of each sample were sealed in glass micro-slides  and kept standing vertically for 2–3 days before imaging by polarized light microscopy.Photographs of the liquid crystalline suspensions and the dried films prepared from CNC were taken with a Nikon Eclipse LV100POL polarized light microscope and DS-Fi1 camera . A 530 nm red wave plate was inserted between crossed polars to indicate CNC orientation . Some imCNC suspensions were drop cast onto glass , black acrylic (for reflected light imaging) or mica surfaces (for atomic force microscopy). The suspensions were allowed to evaporate undisturbed at ambient conditions  for 12 h to give disc-shaped solid films on the glass, acrylic or mica surfaces.The topography of mm-scale areas of the dry CNC films was investigated by optical profilometry. CNC films deposited on glass substrates were scanned with an optical profilometer . The initial results were noisy, but the quality of the topographic images was much improved by coating the surface of the CNC film with a 20 nm thick layer of gold by means of a NexDep Ebeam Evaporator .L-lysine (0.01 wt % in water) was placed onto a freshly cleaved mica surface , rinsed with water, followed by a drop of dilute CNC suspension (~0.005 wt %). The samples were left to dry in Petri dishes under ambient conditions. For images of CNC orientation in films cast from chiral nematic suspensions, a droplet of 8 wt % suspension was allowed to evaporate on the mica surface. A Multi-Mode-3 AFM  was used in tapping mode to image the samples, with probes obtained from NanoScience .The topography of the CNC film surfaces at much smaller length scales was investigated by AFM. To prepare very dilute CNC samples on mica for AFM imaging of individual nanocrystals, a drop of poly-Starting with a dilute suspension of CNC, well below the critical concentration for liquid crystal formation, the CNC tend to align radially at the edge of the droplet.For more concentrated suspensions that are initially above the critical concentration, some radial initial orientation of CNCs at the edge of the film is observed. This is in line with recent observations of the orientation of CNCs in shear flow, where the acid-form CNCs self-oriented across the flow direction .Drying droplets of concentrated CNC display a topography governed by evaporation-driven mass transfer (the “coffee-stain effect”).The magnitude of the coffee-stain effect is greatest for strongly pinned droplets, where the height near the edge of the dry film is several times the height in the middle of the film .When pinning of the contact line is weaker, a “stick-slip” situation is observed, with layers of increasing thickness are laid down by the receding contact line. The layers give a complex optical texture; radial cracking of the drying film was sometimes observed.P/2, ascribed to the anisotropic shrinkage of the chiral nematic structure.The surface topography of dry films at short length scales showed a radial orientation of the CNC at the free surface of the film, along with a radial height variation with a period of approximately The rich liquid crystalline textures displayed by the evaporation of water from CNC suspensions depend on the interplay of concentration gradients that influence chiral nematic pitch, director orientation and gelation with the effects of surface orientation and the elastic constants normally associated with liquid crystalline phases. The qualitative results here are very preliminary, but hopefully indicate some of the variables involved in the formation of chiral nematic solids from cellulose nanocrystal suspensions.Simple evaporation of a droplet of aqueous CNC suspension leads to a surprising range of self-organizing phenomena:


The capacity of the liver to convert the metabolic input received from the incoming portal and arterial blood into the metabolic output of the outgoing venous blood has three major determinants: The intra-hepatic blood flow, the transport of metabolites between blood vessels (sinusoids) and hepatocytes and the metabolic capacity of hepatocytes. These determinants are not constant across the organ: Even in the normal organ, but much more pronounced in the fibrotic and cirrhotic liver, regional variability of the capillary blood pressure, tissue architecture and the expression level of metabolic enzymes (zonation) have been reported. Understanding how this variability may affect the regional metabolic capacity of the liver is important for the interpretation of functional liver tests and planning of pharmacological and surgical interventions. Here we present a mathematical model of the sinusoidal tissue unit (STU) that is composed of a single sinusoid surrounded by the space of Disse and a monolayer of hepatocytes. The total metabolic output of the liver (arterio-venous glucose difference) is obtained by integration across the metabolic output of a representative number of STUs. Application of the model to the hepatic glucose metabolism provided the following insights: (i) At portal glucose concentrations between 6–8 mM, an intra-sinusoidal glucose cycle may occur which is constituted by glucose producing periportal hepatocytes and glucose consuming pericentral hepatocytes, (ii) Regional variability of hepatic blood flow is higher than the corresponding regional variability of the metabolic output, (iii) a spatially resolved metabolic functiogram of the liver is constructed. Variations of tissue parameters are equally important as variations of enzyme activities for the control of the arterio-venous glucose difference. Glucose homeostasis is one of the central liver functions. The liver extracts glucose from the blood when plasma glucose levels are high and produces glucose when plasma glucose levels are low. To fulfill this function the liver is organized in smallest functional units, the sinusoidal tissue units (STUs). These STUs consist of a single sinusoid surrounded by linear arranged hepatocytes. Liver zonation describes the spatial separation of metabolic pathways along the STUs. As blood flows through the sinusoid the plasma nutrient and hormone composition changes and in conjunction with the heterogeneous endowment of metabolic enzymes this leads to big differences in the metabolic performance of hepatocytes depending on their position within the sinusoid. This makes liver zonation and blood flow two central determinants for the functional output of the liver. In this work we present a tissue model of hepatic carbohydrate metabolism that combines liver zonation and microperfusion within the STU. We show that structural properties, enzymatic properties and regional bloodflow are equally important for the understanding of liver functionality. With our work we provide a true multi-scale model bridging the scale from the cellular to the tissue level. In mammals, the liver is the central organ for the control of plasma glucose. To restrict variations of plasma glucose to the range between 3 mM (after exercise or moderate fast ) and 10 The majority of metabolic functions of the liver are confined to hepatocytes. These cells form tightly connected cell layers that are separated from the liver capillaries (sinusoids) by the space of Disse . The traThe importance to include blood perfusion and the tissue architecture into a quantitative estimate of liver metabolism has been stressed in an earlier modeling work of Chalhoub und Belovich . HoweverTo better understand the role of blood perfusion and tissue structure for the metabolic performance of the liver, we have developed a multi-scale tissue model of the so-called sinusoidal tissue unit (STU). We define a single STU by an ensemble of a single sinusoid, the accompanying space of Disse and the adjacent layers of hepatocytes. The sinusoids form the microvascular bed linking the hepatic portal vein and artery with the hepatic central vein. The exchange of substances between the blood, the space of Disse and the hepatocytes leads to a progressive alteration of plasma composition along the STU, continuously changing from the portal region  to the central region connected to the liver vein.Importantly, hepatocytes at different spatial positions of the STU display a differential endowment with metabolic enzymes. Higher amounts of the glycolytic enzymes glucokinase (GK), PFK2/FBP2, phosphofructokinase 1 (PFK1), pyruvate kinase (PK) are found in the pericentral region, while higher enzyme levels of the gluconeogenetic enzymes glucose-6-phosphate phosphatase (G6PP), fructose1,6bisphosphatase (FBP1), mitochondrial pyruvate carboxylase (PCmito) and PEPCK are found in the periportal region . ConsistIn a previous work , we consThe reaction scheme for the glucose metabolism of a single hepatocyte is depicted in The kinetic parameters of metabolic enzymes were taken from the literature. For each parameter, the experimental source is cited in supplements 1 and 2. If for an enzyme-kinetic parameter more than a single measurement was available, we used one representative value that fits with the majority of the reported values and that—whenever possible—was obtained in an enzyme assay that reported consistent values for other kinetic parameters. Mathematical terms in the rate law related to allosteric enzyme effectors which are not included in the model were neglected. Their average contribution is thus indirectly contained in the fitted Vmax values. The only free adjustable parameters of the model are the maximal enzyme activities (Vmax) which depend on the expression level of the enzyme protein and numerous other factors . Vmax values were determined by adjusting simulated steady-state and time-dependent metabolite profiles and flux distributions to experimental data obtained with isolated hepatocytes in culture or the perfused liver (details are given in ).The tissue transport of water, nutrients and blood is described by a compartment model. within the sinusoid by a compartment model. The STU is dissected into N discrete adjacent domains where N is the number of hepatocytes. Each domain subdivides into three compartments representing a single hepatocyte and the spatially associated part of the sinusoid and the space of Disse. Within the 3N compartments the concentrations of metabolites and hormones are constant. The exchange of metabolites and hormones between adjacent compartments are given by the following elementary processes: (i) Pressure-dependent directional convective flow of blood within the sinusoid from the periportal to pericentral pole, (ii) diffusion within the sinusoid, (iii) pressure-dependent directional convective flow from the sinusoid lumen to the space of Disse, (iv) diffusional exchange between the sinusoid lumen and space of Disse, (v) pressure-dependent directional convective transport of fluid in the space of Disse, (vi) diffusion within the space of Disse, (vii) active transport of metabolites between the space of Disse and the cytosol of hepatocytes.Lateral blood flow in the vessel compartment is described by the Hagen-Poiseuille law for fluid flow through a cylinder. Water flow in the space of Disse is described by the Hagen-Poiseuille law for a hollow cylinder. Exchange of water between the vessel and the space of Disse is driven by hydrostatic and oncotic pressure difference between the blood vessel and the space of disse. The blood-born substances considered in our model are the metabolites glucose and lactate, the hormones insulin and glucagon and the indicator substances erythrocytes, albumin and water. The mathematical description of the tissue model and a complete list of all tissue parameters is given in Hormonal regulation of metabolic fluxes is on the short term brought about by changes in the ratio of the phosphorylated and de-phosphorylated form of interconvertible metabolic enzymes. The phosphorylation states of the key regulatory enzymes of hepatic carbohydrate metabolism are controlled by the concentrations of the hormones insulin and glucagon. Insulin and glucagon concentrations in the plasma are determined by the glucose-dependent secretion rates of these hormones from the pancreas  and their plasma clearance. We used the same empirical functions developed in  to relatPlasma concentrations of insulin and glucagon are usually determined in peripheral blood samples. However, pancreatic hormones released in the portal vein are cleared during their passage through the liver and the body which results in a gap between reported plasma concentrations and the true intra-hepatic hormone concentrations Comparison of measured hormone concentrations in the portal vein, central vein and systemic circulation revealed that the extra-hepatic body clearance accounts for about 50% of total hormone clearance . We tookHepatic hormone clearance occurs preferentially through binding of the hormones to their respective receptors and subsequent internalization. We thus linearly scaled the rate of hormone clearance to the signaling strength of the hormone. A detailed description of the functions and their construction is given in The main gluconeogenetic precursors lactate, external and internal amino acids and glycerol are lumped together into one generic precursor "lactate"ATP producing and utilizing processes are not included, the adenine nucleotides ATP, ADP and AMP, the uridine nucleotides UTP and UDP and inorganic phosphate Pi are kept constant at their normal values. The same holds for the pyridine nucleotides NAD and NADH.The contribution of monosaccharides other than glucose  to the hepatic carbohydrate metabolism is not considered.Coupling of oxaloacetate (OA) with reactions of the citric acid cycle  is not includedBranching of the glycolytic triose phosphates (DHAP) to the lipid precursor glycerol phosphate is neglectedThe representation of the sinusoid as a straight tube aligned by a single layer of linearly arranged cells is certainly a gross simplification of the true microanatomy of the liver.With 20 hepatocytes per STU, the model is given by a system of 889 first-order differential equations which was numerically integrated by using the ODE15S solver of the MatLab package, Release R2011b, The MathWorks, Inc., Natick, Massachusetts, United States. The numerical simulation yielded stable solutions at variable number of cells (15–25) and variable parameter values.For the interested reader, an SBML file of the cellular metabolic model is available from the authors.The perfusion CT study was performed using a multi-slice scanner .The CT protocol consisted of a non-enhanced abdominal low-dose CT , which was obtained to localize the liver porta. Subsequently, a scan range of 6.9 cm z-axis coverage was planned over the involved liver, followed by a VPCT using an adaptive spiral scanning technique in shuttle mode. Perfusion parameters were: 80 kV; 100/ 120 mAs ; collimation, 64 0.6 mm with z-flying focal spot; and 26 CT-whole coverages of the liver volume within a total scan time of 40 s. The mean radiation exposure for liver perfusion measurements was 7.5 mSv. Contrast medium was administered by using a dual-head pump injector . For this purpose, 50 mL Ultravist 370  were injected in all patients irrespective of patients’ weight at a flow rate of 5 mL/s in an antecubital vein followed by a saline flush of 50 mL NaCl at 5 mL/s. All images were transferred to an external workstation  for analysis. An abdominal scan in portal vein phase was also obtained.All data sets were transferred to a dedicated workstation  and quantitative data evaluation was performed with a commercial software .CT perfusion analysis software is based on the maximum slope model using time density curves (TDC) to determine perfusion. Calculation of arterial liver perfusion (ALP) and portal venous perfusion (PVP) according to the dual blood supply of the liver by hepatic artery and portal vein is done by using the time of peak splenic enhancement as a separation point of arterial and portal-venous phase by drawing region of interest (ROI) in portal vein and spleen respectively. Arterial TDC for ALP is calculated dividing maximum arterial slope by maximum aortic enhancement. Portal-venous TDC for PVP is calculated dividing maximum portal-venous slope by maximum portal-vein enhancement. Hepatic Perfusion index (HPI) in % represents ALP divided by the sum of ALP and PVP.Automated motion correction and noise reduction of the datasets were applied by using an integrated motion correction algorithm with non-rigid deformable registration for anatomic alignment. Regions of interest (ROI) were placed in the abdominal aorta, portal vein, and spleen. For the HCC lesions quantitative perfusion parameters BF, BV, ALP, PVP and hepatic perfusion index (HPI) were obtained. Additionally, arterial liver perfusion (ALP), portal venous perfusion (PVP) and hepatic perfusion index (HPI) were registered also in the non-involved liver parenchyma at more sites including the right and left liver lobes. This was done drawing a Volume of interest (VOI); an automatic pixel based recognition algorithm allowed for encompassing whole tumor volume along the z-axis. Subsequently, a separate VOI was drawn encompassing the maximum perfused site within the tumor which was visually determined using the ALP-color coded map. BF, ALP and PVP is indicated as ml per 100ml of tissue per minute, BV is indicated as ml per 100ml of tissue and HPI is indicated in percent.All data generated or analyzed during this study are included in this published article.max values) of the MH to the arithmetic mean of the Vmax values of the hepatocytes that are spatially closest to the portal and central pole . The ratios of the Vmax values of the PPH and PCH were put to the measured ratios of protein abundances. All Vmax-values are given in The various hepatocytes lining the sinusoids display a heterogeneous endowment with metabolic enzymes. PP and EPC are the enzyme abundances of the enzyme in the first and the last cell and PP+ EPC)/2. Hence, the Vmax values of the enzymes in this cell, Vmax (N/2), were chosen such that that the best concordance between simulated metabolite concentrations and fluxes and measured values in the whole organ was achieved. Once a numerical value for Vmax (N/2) is known, the Vmax values of the enzyme in the other cells can be calculated from the relationFor the enzyme endowment of the individual hepatocytes lying between the PPH and PCH we assumed that the protein abundances of the key regulatory enzymes see  change lFirst we simulated the glucose exchange flux for the PPH, PCH and MH at plasma glucose concentrations in the physiological range between 3 mM and 12 mM  and compNext we simulated the dynamic changes of intrahepatic glycogen during a starvation-refeeding cycle . ExperimFor the calibration of the blood flow model we compared calculated time courses of various indicators with the experimental data obtained by the indicator dilution technique in an in situ perfused phenobarbital-treated rat liver  Fig 3A–. A solubFrom the average length of sinusoids (≈ 300 μm) and the mean blood flow velocity one can estimate that the transition time of red blood cells trough a liver sinusoid is about 1 second. However, the experimentally determined dilution curve for red blood cells has a width of about 40 seconds. This delay is due to the time required by the red cells to be transported from the injection site to the sinusoids and from the sinusoids to the collecting veins. To take this into account, we approximated the temporal plasma profile of all indicators by the mean dilution curve of the red cells.To take into account random variations in tissue structure and blood pressure, we simulated indicator dilution curves for a multitude of liver sinusoids while randomly sampling the trans-sinusoidal pressure difference Δp and the structural parameters vessel diameter and thickness of the space of Disse from the respective observed distributions see . The larDuring the passage of blood through the liver, the plasma concentrations of glucose and hormones are continuously changing. We simulated the intra-sinusoidal alterations in plasma glucose and hormone concentrations at different fixed portal glucose concentrations in the physiological range of 3–15 mM. The corresponding portal hormone concentrations were calculated by means of the glucose-hormone transfer (GHT) function see . The conThe simulations were repeated 100 times with randomly and independently sampled structural, morphological and metabolic parameters. Structural and morphological parameters were drawn from their measured distributions , while pA convenient measure to assess the contribution of the liver to the homeostasis of the plasma glucose is the portovenous glucose concentration difference. The mean and variance of the computed and the clearance of the hormones insulin and glucagon are shown in Interestingly, the set-point for the STU lies at approximately 7.5 mM and is therefore even higher than the set-points of the isolated periportal and pericentral hepatocyte see . While tThe grey-shaded areas in We further analyzed the distribution of glucose within the various compartments of the STU and the contribution of individual hepatocytes to the net glucose balance. Depending on the portal glucose concentration, the glucose concentration along the sinusoid decreases or increases in a nonlinear manner . IntriguAs shown in Next, we simulated the impact of a starvation-refeeding cycle on the dynamic changes of the glycogen content in the individual hepatocytes of the STU . As for The amplitude of variations of the cellular glycogen content decrease from PPH to the PCH. In the PPH, the synthesis and degradation of glycogen PPH occurs with a significantly higher rate compared to the PCH. This implicates for hepatocytes of the periportal zone a higher glycogen content under euglycemic conditions but lower glycogen content in hypoglycaemia.PV elicited by a small (= 5%) change of the model parameter p , i.e.ε = 0.05.To check the relative influence of metabolic parameters and tissue parameters on the performance of the STU, we carried out a sensitivity analysis of the model . The senPV. Intriguingly, the sensitivity analysis shows that variations of the tissue architecture have an equally important influence of the metabolic performance of the liver than variations of protein abundances.The relative impact of individual metabolic enzymes is dependent on the metabolic state , similar as found in  for the The blood flow within different regions of the liver may vary up to a factor of three . We9  and glucose uptake rates  add up to a whole liver uptake rate of—131.8 μmol/g/h and production rate of 75.8 μmol/g/h, respectively. Interestingly, the variance of the glucose exchange fluxes is smaller than the variance of the blood flow values  and systemic parameters . To assess the functional variability introduced by variances in these parameters, we conducted a multitude of simulations by randomly and independently varying structural, morphological and metabolic parameters. The simulations in Interestingly the set-point for the STU lies at approximately 7.5 mM and is therefore higher than the set-point for the periportal and pericentral hepatocyte alone. While the set-points for the individual hepatocytes were calculated with the GHTF given in appendix 3, the relation between plasma glucose and plasma hormones changes along the sinusoid as glucose is exchanged (either taken up or produced) and hormones are degraded. Therefore the actual relation between glucose and hormones (and thereby the position of the set-point) for a cell within the STU (and the STU itself) does depend on the metabolic activity of the preceding cells resulting in the observed shift.PV with respect to variations of each model parameter. This analysis revealed two important peculiarities in the control of the hepatic glucose metabolism. First, the impact of variances in the expression of metabolic enzymes on ΔGluPV is highly dependent on the metabolic state of the liver. For example, the enzyme pyruvate carboxylase exerts considerable control on ΔGluPV at low plasma glucose concentrations, but is unimportant at high glucose concentrations. This is in agreement with findings in  glucose provided no evidence for zone-dependent differences in the glycogen repletion of 24h fasted conscious rats [Relating our model simulations of the glycogen metabolism to experimental data it has to be noted that strikingly different glycogen distributions in the liver have been reported in the literature depending on the species , assay , method of glycogen quantification  and day-time of sampling. For example, Bartels et al.  found gous rats . This maous rats  accordinous rats . Moreoveous rats . We likeous rats ) reporteous rats ), studyiOur simulations shed some light on a novel mechanism which could be involved in the rapid adjustment of hepatic glucose metabolism to sudden changes of plasma glucose. As can be seen in in silico approach will provide a 3D metabolic functiogram of the liver of individual patients that provides valuable information on the available metabolic reserve capacity in distinct liver regions.Finally, as first step towards a model-based construction of metabolic liver 'functiogram', we have used the model to convert the regional blood flow of the liver into regional glucose production- and utilization rates in hypo- and hyperglycemia, respectively. Interestingly, the metabolic heterogeneity appears to be smaller than the perfusion heterogeneity although there is still 25% - 42% variability in regional glucose exchange rates. The next step will be to apply our approach to unravel the regional metabolic capacity of steatotic, inflammatory, fibrotic and cirrhotic livers. This will require the re-parametrization of the tissue model with histological parameters of the diseased tissue and proteomics-based protein levels of metabolic enzymes. In combination with transient elastography and perfusion CT, such an In summary, we presented a model of sinusoidal glucose metabolism by extending a previously established model of hepatic glucose metabolism to include liver zonation. We developed a realistic sinusoidal blood flow model taking morphological and systemic parameters into account thereby bridging the scale from the cellular to the tissue level. Taking into account variations in the tissue parameters as well as variations in the enzyme abundance we assessed the variability of sinusoidal glucose input-output relationship occurring among different livers sinusoids thereby assessing inter-organ variability. Swelling, cirrhosis and restriction of blood flow contribute to progression of liver disease thus allowing for interesting future uses of the model as a valuable tool to analyze liver functionality especially under disease conditions.S1 Supplement(DOCX)Click here for additional data file.S2 Supplement(DOCX)Click here for additional data file.S3 Supplement(DOCX)Click here for additional data file.