Many countries are facing the burden of accelerated population aging and a lack of institutional support to meet the needs of older individuals. In developing countries, adult children are primarily responsible for the care of their elderly parents. However, out-migration of adult children is common in these countries. This study aims to explore the impact of migration on the health of the elderly left behind and their health care-seeking behavior.This paper uses data from a national survey of older persons in Thailand conducted in 2007. The analysis is confined to those who were aged 60 years or above and who had at least one child  . Logistic regression was used to assess the net effect of migration of adult children on the health of the elderly left behind and their health care-seeking behavior, after controlling for other socio-demographic and economic variables.More than two-thirds of the elderly (67%) had at least one migrant child. About three-fifths (58%) reported that they had at least one symptom of poor mental health. Almost three in five elderly (56%) rated their health as poor, and 44% had experienced at least one chronic disease. About two-thirds of the elderly (65%) got sick during the 5 years preceding the survey. An overwhelming majority of elderly (88%) who got sick during the five years preceding the survey had sought treatment for their last illness.After controlling for socio-demographic and economic variables, our study found that those elderly who had a migrant child were more likely  to have symptoms of poor mental health than those whose children had not migrated. However, no significant association was observed among physical health, such as experience of chronic disease, perceived poor health, and illness of the elderly left behind. Interestingly, however, out-migration of adult children was independently associated with higher utilization of health services. The elderly who had migrant children were more likely  than those whose children had not migrated to seek treatment for their most recent illness, after controlling for socio-demographic and economic variables.Our study provides novel evidence on an issue of special importance to countries affected by heavy out-migration of adult children, an issue that has received little attention. Out-migration of adult children was highly associated with poor mental health but it was not associated with the physical health of the elderly left behind. Out-migration of children was also highly associated with higher utilization of health facilities by the elderly. Thus, in order to decrease morbidity among the elderly as well as to maintain and enhance the well-being of families, programs should focus on alleviating the symptoms of poor mental health among the elderly left behind and aim to reduce the differences in utilization of health care-seeking behavior among elderly with children present in the community and elderly left behind. In modern times, many factors have intensified migration. Improvement in transportation infrastructure, advances in communications technology (which makes keeping in contact with left-behind over much easier and cheaper), increased trade between countries , political instability, poverty, and unemployment in economically disadvantaged areas/countries are some of the important factors that contribute to this phenomenon .The interaction between health and migration is complex and dynamic. Migration can have an impact on physical, mental, and emotional health and well-being of migrants themselves, on those left behind in the place of origin, and on those at the destination . The conIn many places, and especially in most developing countries, the growing proportion of aging individuals challenges public and social institutions responsible for elderly care. These countries face an additional burden of accelerated population aging and a lack of institutional support to meet the needs of older individuals . ChangesEvidence from the literature shows that migration can affect the health of those left behind both positively and negatively. With additional money coming from remittances, people have easier access to health services, can buy expensive medicine, and eat better quality food . Thus, mOn the other hand, some research notes that out-migration of young adults has severe negative consequences for ageing parents, namely, loneliness, isolation, and the loss of basic instrumental and economic support . A studyAlthough the separation of families due to migration might have serious implications on the health status of elderly left behind and their health care-seeking behavior, the possible health effects on elderly left behind in Thailand remain unclear. This study aims to explore the impact of migration of adult children on the health of the elderly left behind and on their health care-seeking behavior. In-depth examination of these issues will provide policy makers and program planners with needed information and will raise public attention to this public health issue. The findings of this study will help fill the gap in the literature and provide the understanding necessary for advocacy and for the design of appropriate interventions related to migration in Thailand.This paper uses data from a cross-sectional survey conducted in 2007 among the elderly in Thailand. This nationally representative survey was conducted by the National Statistical Office (NSO) of Thailand. The NSO has conducted three nationally representative household surveys of older persons, in 1994, 2002, and 2007 -26 to fuThe survey is based on a probability sample of persons aged 50 years old or older who were usual residents of their own or family members' households. A stratified two-stage sampling procedure was employed to collect the information. The primary sampling units were blocks for municipal areas and villages for non-municipal areas. The secondary sampling units were households, using random sampling from the list of all enumerated households in each block or village selected. The number of households selected was 15 households per block in municipal areas and 12 households in non-municipal areas. In total, 56,002 persons were interviewed. However, the analysis for this paper is confined to those who were aged 60 years or above and who had at least one child  . A structured questionnaire was used by trained interviewers to interview selected subjects at the household level. This study was approved by the ethics committee of the NSO of Thailand.This study examines several aspects of the health of elderly parents. These include symptoms of poor mental health, self-assessed health status, chronic diseases, illnesses, and treatment-seeking behavior.Items measuring symptoms of poor mental health experienced by elderly persons during the month preceding the survey were: i) feeling stress, ii) feeling moody, iii) feeling hopeless, iv) feeling useless, v) feeling down/unhappy, and vi) feeling lonely. One composite indicator, "symptoms of poor mental health," was developed from all the above-listed symptoms (Cronbach's α = 0.86), resulting in 4 categories: had no symptoms of poor mental health, had 1 symptom, had 2 symptoms, and had 3 or more symptoms. For the logistic regression, two further categories were created: "no symptoms poor mental health" and "at least one symptom of poor mental health".Respondents in the survey were asked, "How has your health been in the past 7 days?" The question had five response categories . The authors categorized the response categories into two groups: "good," which included "very good" and "good," and "poor," which included "fair," "bad," and "very bad."Individuals were asked about the presence of hypertension, heart disease, diabetes, cancer, stroke, and paralysis. One composite indicator, "chronic disease condition," was developed from all the chronic diseases, resulting in 4 categories: had no chronic disease, had 1 chronic disease, had 2 chronic diseases, and had 3 or more chronic diseases. For the logistic regression, two more categories were created: "no chronic disease" and "at least one chronic disease."The study explored whether the elderly had symptoms of an illness (self-reported) during the five years preceding the survey. This variable was split into two categories: "experienced symptoms" and "did not experience symptoms."The study explored whether the elderly who experienced symptoms of an illness during the five years preceding the survey sought treatment for the most recent illness. This variable was categorized into two categories: "sought treatment" and "did not seek treatment."In this study, we have defined an out-migrant child as one living outside their parents' province. We have used migration of adult children as a main independent variable. This variable was split into two categories: "0 no migrant child" and "1 at least one migrant child."The other demographic and socioeconomic variables were used as control variables in this study. Many previous studies have shown that demographic and socioeconomic variables have strong effects on health and health care-seeking behavior. Hence we needed to control for their effect to determine the independent effect of migration. Demographic variables included age , sex , place of residence , and number of respondent's children. The economic variable was the average total income per year . Social variables included education  and living arrangements .Analysis for this study was confined to those who were aged 60 years or above and who had at least one child  . Univariate and multivariate analysis were used to analyze the data. Initially, univariate or descriptive analysis was used to describe the respondents' socio-demographic characteristics. Then, after controlling for the other socio-demographic and economic variables, multivariate analysis in the form of logistic regression was used to identify whether migration of adult children affected the elderly's likelihood of experiencing symptoms of diseases, self-assessed health, experience of chronic diseases, illnesses, and treatment-seeking behavior.More than half of the sampled respondents were aged 60-69 while about one out of seven were aged 80 or above, and nearly three out of five were female. The large majority had only an elementary-level education. Respondents had an average of 4 children. About three in five respondents were from rural areas. Half reported that their household income per year was about 30,000 baht or less. Only about one in ten elderly lived alone, and two-thirds had at least one migrant child. About four in five elderly (79%) who had at least one migrant child had received money from their child(ren) during the 12 months preceding the survey (Table Table It is encouraging to note that an overwhelming majority of elderly who felt sick (88%) had sought treatment for their most recent illness. Interestingly, a higher proportion of the elderly who had migrant child(ren) had sought treatment for the last illness compared to those whose children did not migrate Table .Logistic regression was used to assess the net effect of the migration of children on the health of the elderly left behind and their treatment-seeking behavior after controlling for the other control variables in the model.Separate logistic regressions were run for 5 dependent variables. The odds ratio is presented in Table Migration of children was found to have a strong association with poor mental health symptoms among the elderly left behind. Those elderly who had at least one migrant child were more likely  to have poor mental health symptoms than those whose children had not migrated. On the other hand, no significant association was observed with physical health, such as experience of chronic disease, self-rated health status, or experience of symptoms of illness during the previous 5 years.Furthermore, the results show that the migration of children is a significant predictor of treatment-seeking behavior among the elderly left behind. For instance, those elderly who had at least one migrant child were more likely  to seek treatment for their most recent illness than were those who had no migrant children  have at least one out-migrant child and that an overwhelming majority (79%) received money from their migrant children.Out-migration of adult children was highly associated with poor mental health symptoms among the elderly left behind. It is likely that out-migration of children reduces opportunities for face-to-face interaction. This finding is similar to other studies ,23 that Our study found that the migration status of children was not associated with the physical health of the elderly left behind . It should be noted that this may be because adult children are less likely to migrate when a parent is ill . Howevera, we also found that those who had received remittances from their children were more likely  to seek treatment than those who had no migrant children. This could be due to the fact that the benefits of remittances offset the potential negative effect of migrant children living apart from their parents. The financial support provided by migrant children may operate primarily through amelioration of financial hardship of their parents. Studies have also found that most elderly Thai parents are getting financial support from their children and that this contributes positively to their material well-being [An earlier national survey in Thailand also found that 77% of Thai elderly reported receiving financial support from children during the previous year . Furtherll-being ,30. Manyll-being  and to ell-being . The othll-being  that helll-being . Yet anoThere are some limitations in the interpretation of the results of this study. Because of the survey's cross-sectional design, all of the factors analyzed in the study were measured at a single point in time. Thus, the analysis can only provide evidence of statistical association between those items and the health status of the elderly left behind and health care-seeking behavior; it cannot show a cause-effect relationship. Besides, we have to be careful while comparing the results of this study with those of other research studies that have used pre-existing validated questionnaires to measure mental and physical health.Our study provides novel evidence on an issue of special importance to countries affected by heavy out-migration of adult children, an issue that has received little attention. Findings here show that out-migration of adult children is highly associated with poor mental health but is not associated with the physical health of elderly left behind. Out-migration of children is also highly associated with greater utilization of health facilities. Thus, in order to decrease morbidity among the elderly as well as to maintain and enhance the well-being of families, programs should focus on alleviating the symptoms of poor mental health among the elderly left behind and aim to reduce the differences in utilization of health care-seeking behavior among elderly with children present in the community and elderly left behind.a. We were unable to keep both 'remittances' and 'out migration of children' in the same model as it is highly correlated. Therefore, we run separately to see the association between 'remittance' and 'treatment seeking' variables after controlling for other demographic and socio-economic variables.The authors declare that they have no competing interests.RA analyzed, interpreted the data, and drafted the manuscript. AJ and AC commented and provided suggestions on the analysis and interpretation. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/143/prepub


The unusual case of dense in dente in mandibular molar area with extra oral sinus in a 30-year-old female is presented. The chief complaint of the patient was wound formation and pus discharge from the right side of lower jaw for many years. Clinical examination revealed extra oral sinus and mild swelling in vestibular region opposite the right mandibular molars. On radiographical examination, right mandibular third molar had bulbous root associated with periapical radiolucency. It appeared that there was a tooth within a tooth and the invagination extended nearly to the root apex. A clinical diagnosis of dense in dente Type III was confirmed by stereomicroscopy of ground section of the tooth. Extra oral sinus healed after extraction of the mandibular third molar within a month. Dens in dente, also known as dens invaginatus or dilated composite odontome is a rare developmental tooth anomly characterized by invagination of the enamel organ into the dental papilla. The frequency of dens in dente is more common in permanent maxillary lateral Incisors. In the A 30-year-old female patient reported in dental OPD of Sir Sunder Lal Hospital, Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi with a chief complaint of wound formation and pus discharge from the right side of lower jaw for many years. The general health of the patient was normal and her medical history revealed no significant problem. Extraoral examination revealed facial sinus with pus discharge from the body of the mandible . On intrDens in dente is a rare developmental tooth anomaly characterized by invagination of the enamel organ into the dental papilla that begins at the crown and often extends to the root even before the calcification of the dental tissues. Dens in dente is more common in permanent maxillary lateral incisors 0.25-5.1%), but


Escherichia coli, has been the method of choice in most studies. These studies have greatly enlarged our understanding on the prevalence and distribution of AMR and associated risk factors. Recent studies using  metagenomics, however, highlighted the unappreciated diversity of AMR genes in the human microbiome and identified genes that had not been described previously. Next to metagenomics, more targeted approaches such as polymerase chain reaction for detection and quantification of AMR genes within a population are promising, in particular for large-scale epidemiological screening. Here we present an overview of the indigenous microbiota as a reservoir of AMR genes, the current knowledge on this “resistome” and the recent and upcoming advances in the molecular diagnostic approaches to unravel this reservoir.The gut microbiota is amongst the most densely populated microbial ecosystem on earth. While the microbiome exerts numerous health beneficial functions, the high density of micro-organisms within this ecosystem also facilitates horizontal transfer of antimicrobial resistance (AMR) genes to potential pathogenic bacteria. Over the past decades antibiotic susceptibility testing of specific indicator bacteria from the microbiome, such as  Antimicrobial resistance (AMR) is worldwide one of the most important public health threats that we face currently. AMR reduces clinical efficacy and increases treatment costs. Furthermore, AMR jeopardizes the achievements of modern medicine, since the success of interventions such as organ transplantation, cancer chemotherapy, and major surgery depends on effective antimicrobial agents for prevention and treatment of  infections. With a lack of novel antibiotics in the pipeline, the conservation of existing ones is crucial.While the majority of studies on the epidemiology of antibiotic susceptibility have been focused on clinical isolates, the human microbiota warrants special attention as perhaps the most accessible reservoir of resistance genes due to the high likelihood of contact and genetic exchange with potential pathogens. Here we present a non-comprehensive overview of the indigenous microbiota as a reservoir of AMR genes, the current knowledge on this “resistome” and the recent and upcoming advances in the molecular diagnostic approaches to unravel this reservoir.The human gut microbiota has long been recognized to contribute to health and disease by influencing gut maturation, host nutrition and pathogen resistance . More reThe establishment of the indigenous microbiota starts as soon as the amniotic membranes rupture and subsequently involves a succession of bacterial populations waxing and waning as the diet changes and the host develops.The bacteria colonizing the infant gut during the first days of life originate mainly from the mother’s birth canal, the living environment and from handling by other individuals. . VaginalOther factors that can influence the neonatal intestinal microbiota composition are: the environment during birth, prematurity, hygiene measures, and the type of infant feeding . The newThe majority of indigenous microbes cannot be cultivated, and it’s only since the recent application of sequencing techniques that the human microbiota has been described in all complexity including the unculturable and rare taxa. Bacteroides, Prevotella, and Ruminococcus, respectively  project, identified a staggering number of some 3.3 million different bacterial genes among the studied individuals, which is 150-fold more than in our own human genome. .Next to the apparent beneficial effects on human health, the human microbiota warrants special attention as perhaps the most accessible reservoir of antibiotic resistance given the high density of micro-organisms within this ecosystem. The gastrointestinal tract is an open system, which every day encounters a myriad of bacterial acquisitions originating from the environment  was based on the clinical relevance of these organisms; i.e., whether they were implicated in causing infections, but also on the cultivability of these organisms rather than for example those who represented the majority of the microbiome. Cultivability has been of major importance because even today, every method to assess antibiotic susceptibility (rather than antibiotic resistance) involves culturing the micro-organism .E. coli or enterococci as indicator micro-organisms and they have gained invaluable insights regarding: (i) geographic differences in AMS prevalence and changes in prevalence over time  of the gut microbiota have used (selective) culturing and isolation of specific micro-organisms, followed by culture-based antibiotic susceptibility testing to obtain their results. Most of these studies focused on ver time ; (ii) thver time ,b; and. ver time . Additiofections . Finallyegatives , but alsegatives . These lIn a follow-up study on the samples from a remote Indian community with minimal antibiotic exposure, Metagenomics, refers to the study of metagenomes, genetic material recovered directly from environmental samples. It is the genomic analysis  applied to all the micro-organisms of a microbial ecosystem without previous identification . Three dThe accumulating evidence, showing that resistance genes are transferred within an ecosystem and across species, has highlighted the potential of application of easy-to-use PCR-based metagenomics. Numerous PCR methods are currently available for the detection of resistance genes or gene families. Furthermore, when real-time PCR is used to perform such analysis, (semi-)quantitative results can be generated and the relative abundance of resistance genes can be determined. This approach has been used to study the emergence of resistance over time in soil and water environments . Knapp eE. coli). Next, these transformants are screened for the expression of resistance genes by growing them on antibiotic containing media at concentrations where the wild-type host strain is susceptible, where after antibiotic resistant clones are being sequenced , and the subsequent expression into heterologous hosts . On the other hand the foreign gene may as well interact in novel ways with the cellular machinery of the surrogate host resulting in false positives . The multidrug resistance efflux pump and fluoroquinolone resistance were also the predominant RATC functional groups in the cattle rumen and chicken cecum libraries, whereas the fluoroquinolone resistance was less abundant in the human stool library.Over six percent (6.44%) of the sequences of the buffalo metagenomic library  and overAs plasmids are important mediators of HGT of AMR genes, a more focussed sequencing of the plasmid metagenome (plasmidome/metamobilome) has also been applied .Although with traditional metagenomic sequencing every single extra-chromosomal element within an environment can be sequenced (as long as the sequencing depth is sufficient), the proportion of chromosomal and extra-chromosomal genomes will always remain hugely in favor of chromosomes resulting in masses of redundant data when one is merely interested in the extra-chromosomal metagenome  The reseAs described above, many resistance genes identified in studies applying functional metagenomics have low similarity to known genes. In sequence-based metagenomics, sequences with low similarity to known reference sequences cannot be easily identified, in other words this approach is generally limited to identifying genes that are already known . The annIn addition, sequence-based metagenomics does not provide any information on the expression of the identified genes. On the other hand, sequence-based metagenomics provides a wealth of information not only on AMR genes, but on the entire gene content thereby enabling the identification of the community composition and metabolic profile. In particular these metagenomic data sets enable to look at which members of the bacterial community are carrying particular functional genes.Previous research on marker gut microbes has provided a wealth of information on amongst others AMS of these strains in different hosts, over time and at different geographic locations. In the current era of molecular techniques, there is still a place for culture-based studies as it is a necessity to assess antibiotic susceptibility. Yet, targeted, functional, or sequence-based metagenomics are required to gain more insight in the potential of the human gut microbioma as an AMR reservoir. These techniques have their own strengths and limitations and application of these techniques in population-based studies is only starting to emerge. Within the coming years, new metagenomics-based studies are likely to reveal many novel insights into this subject.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Avian influenza virus reveals persistent and recurrent outbreaks in North American wild waterfowl, and exhibits major outbreaks at 2–8 years intervals in duck populations. The standard susceptible-infected- recovered (SIR) framework, which includes seasonal migration and reproduction, but lacks environmental transmission, is unable to reproduce the multi-periodic patterns of avian influenza epidemics. In this paper, we argue that a fully stochastic theory based on environmental transmission provides a simple, plausible explanation for the phenomenon of multi-year periodic outbreaks of avian flu. Our theory predicts complex fluctuations with a dominant period of 2 to 8 years which essentially depends on the intensity of environmental transmission. A wavelet analysis of the observed data supports this prediction. Furthermore, using master equations and van Kampen system-size expansion techniques, we provide an analytical expression for the spectrum of stochastic fluctuations, revealing how the outbreak period varies with the environmental transmission. Understanding the dynamics of infectious diseases in humans has become a increasing focus in public health science Multi-year periodicity in epidemics is widely observed in time series from many cities with greatly varying climatic and demographic conditions et alRecently, a method, the so-called van Kampen's system-size expansion, which is based on a simple individual-based mathematical formulation of stochastic dynamics, has been applied to investigate stochastic population dynamics Here, we estimate the outbreaks period of avian influenza in North Americas with a wavelet method, which reveals 4–8 year periodicity from empirical data. To explain this, we first develop a fully stochastic two species' avian influenza model (host and virus) with two routes of transmission: environmental indirect transmission and direct transmission through contact between individuals within the wild bird population. Then, we provide a prediction for the dominant period of disease oscillations by analytically calculating the power spectral density from a stochastic Fokker-Planck equation. From a geographically  and temporally restricted data, the model gives general insights into long-term patterns of disease dynamics in wild bird populations. Some conclusions may also apply to other infectious diseases characterized by two transmission routes. Our analysis sheds new light on the importance of environmental transmission for avian influenza outbreaks and persistence. Our results show that, in principle, it is possible to reduce the frequency and intensity of the outbreaks of avian influenza by controlling the environmental route of transmission.In avian influenza, susceptible hosts are not only infected by direct contact with infected individuals with avian influenza viruses (AIV), but also by virus particles that persist in the aquatic environment. AIV are transmitted via the fecaloid route of the host and subsequent drinking or filtering of water while feeding To better understand the effects of demographic stochasticity and virus persistence in the environment on epidemic outbreaks and extinction, we describe virus population dynamics and environmental transmission using an explicit stochastic host-pathogen model that assumes global mixing, i.e, random contact between individuals. Hence the epidemiological dynamics of the host falls within the susceptible-infected-recovered (SIR) framework Although births and deaths are intrinsically distinct events, we assume, for simplicity, that host birth and death rates have the same value where Our individual-based stochastic model fully integrates the abundance of virus particles in the environment into the SIR framework. Virus particles All the transitions of the host and the virus associated with their corresponding rates are illustrated graphically in The basic ingredients of our new framework are the susceptible The transition probability per unit time from state InfectionDeath/BirthRecoverywhere So far we have formulated a fully stochastic host-parasite model with both direct and indirect environmental transmission, assuming well-mixed conditions. Given the specified analytical formulations for transition probabilities Using van Kampen's system-size expansion of the stochastic dynamics, as discussed in section A1 of where where It is simple to verify that these equations have a trivial fixed point and a unique non-trivial fixed point where It is important to investigate whether the existence of a stable fixed point in the deterministic system generates oscillations and multi-year periodicity in the corresponding stochastic system. In order to investigate this and describe the stochastic fluctuations of the system by an analytical method, the higher-order terms should be included in the van Kampen system-size expansion where The Langevin equations (6) describe the temporal evolution of the normalized fluctuations of variables around the equilibrium state. By Fourier transformation of these equations, we are able to analytically calculate the power spectral densities (PSD) that correspond to the normalized fluctuations, independent of community size The complete derivation of these PSDs and detailed descriptions about the way the functions Unlike Fourier analysis, wavelet analysis is well suited for the study of signals whose spectra change with time. This time–frequency analysis provides information on the different frequencies (i.e. the periodic components) as time progresses If we denote the time-series as et alIn the definition, parameters Previous studies over 15 years from 1976 to 1990 established a cyclic pattern of occurrence of influenza A viruses in wild ducks The data on aquatic wild birds revealed a clear periodicity in the outbreaks of avian influenza in agreement with literature Direct comparison of the deterministic and stochastic simulations reveals that demographic noise and environmental transmission can induce rich multi-period patterns, corresponding to deterministically damped oscillations see . Our anaThe original PSD formula (7) further allows us to examine how the period of the epidemic outbreak varies with changes of the environmental transmission rate We characterize the region of the parameter space that allows for disease persistence both in the deterministic model Click here for additional data file.


Objective: To investigate the morphological and structural changes of oral mucosa under the influence of alcohol.Methods: Sixty male and female specimens  who died of chronic alcoholism were selected in this study. The specimens (5-7 mm) were sliced by the morphological-histological detection method, and stained by the HE and Spielmeyer (myelin staining) protocols respectively. Then five immune peroxidase chemical reaction tests were performed.Results: 10% of the tissue sections had epithelial hyperplasia points with hyperkeratosis and acanthosis. 90% of the sections had epithelial atrophy points with different degrees of damage, and had moderate infiltration of lymphocytes-macrophages in the basal oral mucosa simultaneously. For the tissue sections of patients who died of cardiovascular diseases with a history of alcoholism, about a half showed that extensive necrotic points were observed in different parts of oral mucosa, accompanied by a secondary infection. Approximately 15% of the sections had more dense and homogeneous necrotic tissues with microbial colonization, and the necrotic focus of 5% of the sections was located above the epithelial tissue, which was not distinctively different from other tissues. 48% of the sections were subjected to small nerve bundles with jeopardized deep oral mucosa, accompanied by necrosis of neuron axon and its myelin membrane.Conclusion: The findings of this study show that drinking alcohol over an extended time may lead to carcinogenic changes in oral mucosa. Besides, alcohol is able to decompose the lipid composition of the outer epithelial membrane of mucosal tissue, which augments the "susceptibility" of oral mucosa to other carcinogens. In addition, ethanol can also act on the large and small salivary secreting glands in oral cavity to increase saliva secretion and viscosity. The detailed function of alcohol is still undefined hitherto. Meanwhile, the dangers of drinking alcohol to human body as well as the influence on oral mucosa remain unknown.1,2From the viewpoint of epidemiology, chronic alcoholism is an important factor in the development of oral and its mucosal diseases. Many studies have shown that ethanol also plays a key role in the progress of cancerous lesions of oral mucosa. However, the specific pathological process remains partially unclear, which may be attributed to that ethanol Sixty male and female specimens  who mainly died of chronic alcoholism and alcoholic cirrhosis were selected in this study, of which the ages ranged between 41 and 74 years old . The samThe tissue with the size of 1×3 cm was taken from the oral mucosa as the specimen. The position was selected in the junction region between the attached mucosa of upper and lower alveolus and buccal mucosa and lip mucosa. All specimens were immersed in 10% formalin solution, and then sent to the Pathological Testing Laboratory of St. Petersburg Medical University, during which all specimens were not polluted and undermined. After being labeled, all specimens were being added with paraffin and prepared into 5-7 mm thick specimen slices, then stained with HE , and the Van-Gieson method (VG), Mallory method (phosphotungstic acid hematoxylin) and Spielmeyer method (myelin staining) were used respectively. Then various immune peroxidase chemical reaction tests were performed to determine the presence of leukocytes. The reactions include: (1) reaction with common leukocyte antigen (LCA); (2) reaction between T-lymphocyte and B-lymphocyte ; (3) reaction with T-helper cell (CD-4); (4) reaction with T-suppressing cell (CD-8); (5) reaction with T-natural killer cell (CD-56).The imaging characteristics under the electron microscope are shown in . About 1About 90% of the sections had epithelial atrophy points with different degrees of damage, and meanwhile had moderate infiltration of lymphocytes-macrophages in the basal oral mucosa . Muscle The oral mucosa in the tissue sections of patients who died of cardiovascular diseases with a history of alcohol abuse changed similarly to the oral mucosa of those who did not have a long history of alcoholism. Nevertheless, about half of the sections showed that extensive necrotic points were observed in different parts of oral mucosa, accompanied by a secondary infection, trace amount of inflammatory reaction, as well as extension along the outer periphery of focus. It is worth noting that the necrosis was structurally featured in stratification and pagination .About 15% of the sections were of more dense and homogeneous necrotic tissue with microbial colonization and a small amount of filamented neutrophils and macrophages .The necrIn 48% of the sections were found damaged small nerve bundles in deep oral mucosa, accompanied by necrosis in neuron axon and its myelin membrane . Notably3Fiorettiet al4 found that alcoholic beverages manly accounted for the oral and pharyngeal cancers in non-smoking patients. There are several pathogenesis for ethanol acting on the oral mucosa. Active oxidation may directly undermined DNA. The invasive ability of carcinogen surrounding oral mucosa is increased, which relies on the enhanced solubility of the carcinogen or increased mucosal permeability.5 The carcinogenic effect of ethanol commonly occurs in case of daily ethanol intake higher than 45 ml.6Preliminary discussion was conducted herein concerning the impact of alcohol on oral mucosa. Alcohol can destroy the lipid composition that the protective layer of oral mucosa covers acanthosis granules, and disrupt the normal order of epithelial lipid molecules, resulting in a gap between epithelial cells and increasing oral mucosal permeability. In other words, alcohol opens a pathway for deep soft tissues. Meanwhile, previous researches have confirmed that the most important risk factors in the development of oral cancer are smoking and alcohol.7Homann et al7 demonstrated increased salivary acetaldehyde levels even after ingesting moderate amounts of ethanol, allowing significant acetaldehyde accumulation in oral tissues during chronic ethanol consumption and may explain some of the cytologic anomalies in the oral mucosa of non-smoker alcoholics. Several researchers also reported some anomalies, including increased nuclear area,8 epithelial atrophy due to decreased basal cellular size,7 dysplastic changes with keratosis and increased number of mitotic figures.9 However, in the present study, the nucleus/cytoplasm ratio was not determined by the cytomorphometry. The exfoliative cytology of established oral cancers exhibited polyploid DNA profiles and reduced cytoplasmic area.10 Since polyploidy DNA profiles were discerned only in established oral cancer results, more subtle changes in DNA and nuclear morphologic characteristics may be observed in clinically normal mucosa, and may be just as crucial for detecting future cancer in susceptible patients as polyploidy is for detecting cancer.11Acetaldehyde, the first metabolite of ethanol, has the largest carcinogenic potency. Acetaldehyde has been verified to be highly toxic, mutagenic and carcinogenic in different cell cultures and animal models.12 Regardless, the total amount of ethanol and the duration of ethanol consumption may be more important than the type or composition of the alcoholic beverage consumed.13,14The presence of contaminants, such as polycyclic aromatic hydrocarbons and nitrosamines, is a significant carcinogenic factor in alcoholic beverages.All the above experimental studies have proven that the stratified structure of oral mucosa was damaged in the presence of alcohol, thus resulting in a gap between epithelial cells, increasing the permeability of oral mucosa, and facilitating deep penetration of carcinogen. Alcohol evidently promotes the development of oral cancer.According to the histopathological characteristics of the specimen sections obtained from 60 patients, we found that oral mucosa was altered under the stimulation of alcohol. A half of the specimen sections could be described as necrosis, especially the lesion in the basal oral mucosa was far more dramatic. The extent of necrosis, however, was not exactly the same. A small number of sections were subjected to only small necrotic points with a small amount of hyperkeratosis and acanthosis, and no inflammatory reaction was found. While the other half were of necrotic points that were rendered as "stratification". Muscle fibers underwent atrophy deep in the submucosa in part of specimen sections, as well as the necrosis of neuron axon and myelin membrane. In summary, the stratified structures of oral mucosa were susceptible to different degrees of injury in the presence of alcohol. The epithelial layer of oral mucosa was manifested as epithelial hyperplasia points with hyperkeratosis and acanthosis, as well as part of necrosis. Not only necrotic points were found in the basal oral mucosa and submucosa, but also atrophy muscle fiber atrophy was observed in deep submucosa with injury in the "neurological regulatory system".FL: Designed the protocol and prepared the final manuscript.WLL: Clinical data collection and experiments.


The clinical outcome of hemiarthroplasty for proximal humeral fractures is not satisfactory. Secondary fragment dislocation may prevent bone integration; the primary stability by a fixation technique is therefore needed to accomplish tuberosity healing. Present technical comparison of surgical fixation techniques reveals the state-of-the-art approach and highlights promising techniques for enhanced stability.A classification of available fixation techniques for three- and four part fractures was done. The placement of sutures and cables was described on the basis of anatomical landmarks such as the rotator cuff tendon insertions, the bicipital groove and the surgical neck. Groups with similar properties were categorized.Materials used for fragment fixation include heavy braided sutures and/or metallic cables, which are passed through drilling holes in the bone fragments. The classification resulted in four distinct groups: A: both tuberosities and shaft are fixed together by one suture, B: single tuberosities are independently connected to the shaft and among each other, C: metallic cables are used in addition to the sutures and D: the fragments are connected by short stitches, close to the fragment borderlines.A plurality of techniques for the reconstruction of a fractured proximal humerus is found. The categorisation into similar strategies provides a broad overview of present techniques and supports a further development of optimized techniques. Prospective studies are necessary to correlate the technique with the clinical outcome. Hemiarthroplasty represents an established treatment method for three or four-part fractured proximal humeri. Pain relief is often achieved by this surgical intervention, but the functional result is less predictable ,2. ConseThe first operation of a shoulder replacement was performed 1893 by Dr. Péan . Horse hPublished fixation techniques were often tested in a biomechanical test to analyse strength and stability. In prior studies, different in-vitro loading profiles were applied such as load-to-failure testing of fixation techniques ,28. In oA summary of existing fixation techniques may support the identification of further advantageous techniques. By comparing the most frequent techniques, promising features and innovative procedures may be combined. Existing publications focus primarily on one specific technique; it is therefore of interest to have a direct comparison. Classifying the different techniques in distinct groups supports a schematic innovation process to develop novel techniques. The aim of this investigation is therefore the analysis of existing fixation techniques for proximal humeral four-part fractures for hemiarthroplasty.A review of the different fixation techniques in the literature was carried out focusing on proximal humeral four-part fractures. Suture and wire placement based on illustrations from literature Figure , left wa- the number of strands connecting the humeral shaft to the greater tuberosity;- the number of strands connecting the humeral shaft to the lesser tuberosity;- the number of strands connecting the greater and lesser tuberosity to each other;- the number of strands connecting the greater and lesser tuberosity to the shaft;- the design of the middle parts of corresponding prosthesis including- the number of holes and fins in the proximal shaft- the qualitative prosthesis shapeA uniform terminology of suture placement was defined that corresponds to the frequently used conventions in literature: the strands oriented parallel/collinear to the shaft axis were defined as longitudinal, leading from proximal-to-distal. Circular strands were perpendicular to the longitudinal axis of the humerus, placed circumferentially around the cuff. Transverse sutures represented a placement through the prosthesis. Diagonal sutures were guided from the GT-LT fragment to the anterior-posterior diaphysis of the shaft. The use of dotted lines represented transosseous sutures. Blue lines represented a tuberosity connection to the shaft, and green lines represented interfragmentary connections between the LT and the GT. Cerclages around the GT and LT, guided through the prosthesis, are shown as magenta, and metallic braided cables as black. Sutures interconnecting all three fragments like GT, LT and shaft are shown as red.Several investigations have applied the figure-of-eight technique by interconnecting all three fragments such as the shaft, the GT and the LT -34. DineSimilar to the previous technique of Dines et al, the technique of Frankle et al uses the same prosthesis type ,22. BothIn contrast to the previously discussed fixation techniques, both tuberosities may be fixed individually to the shaft by separate figure-of-eight tension bands . The UniFirst, the circular sutures connecting the tuberosity fragments are tied, then the strands to the shaft are tightened. A suture is placed in a predrilled GT hole and a second one in the LT hole. A cerclage suture is passed through the SSC tendon, around the GT and the LT and ends at the ISP and TM tendon insertion. A suture in the humeral shaft, medial to the bicipital groove, pulls the distal end of the lesser tuberosity back down to the shaft. Additional sutures in the middle of the prosthesis are used for further reduction.Reuther et al. use the Affinis fracture prosthesis (Mathys Ltd) . To achiIn the technique of Hertel et al, fixation consists only of metallic cables, without using sutures . This meA tension-band technique using braided polyester sutures has been used for biomechanical testing  using thSimilar fixation technique has also been applied to the humerus reconstruction without using an implant . Such coStability was investigated in a biomechanical test for three different fixation techniques by Abu Rajab et al . The monMetallic wires are also used for a figure-of-eight tension band technique . The antRecent hemiarthroplasty treatments are using a modified prosthesis' middle part. Schittko et al propose a middle part with multiple holes for an unconstrained placement of the tuberosities using the Ortra prosthesis . A furthBased on the analysis in the review, four groups of different fixation techniques are built. Each group summarises therefore a similar strategy of fixation:Group A: Tuberosities and shaft are connected by one single suture. In group A, Dines et al and Frankle et al use a figure-of-eight tension band over the entire surface of the rotator cuff to connect all three fragments such as the humeral shaft, the GT and the LT.Group B: The single tuberosities are independently connected to the shaft and among each other: In group B, Voigt et al and Gerber et al use the figure-of-eight tension band to connect only single fragments independently to the stem, without involving all three fragments. Voigt et al, Boileau et al and Reuther et al place several sutures ranging from the SSC to the ISP tendon insertion and additionally apply a tension-band technique between the LT and the GT in a horizontal orientation.Group C: Metallic cables are used in addition to the sutures: Reuther et al, De Wilde et al and Hertel et al use metallic cables either applied alone or in combination with sutures. The cables are often placed circumferentially around the shaft humeral shaft.Group D: Short suture loops are used to connect adjacent fragments together. The suture loops are placed close to their fragment borderlines.Recently, the fixation technique of proximal humeral four-part fractures is often discussed in literature. The number of acquired references demonstrates the high relevance in fixing humeral proximal four-part fractures.Generally, securing tuberosity fragments against medial displacement is done by horizontal sutures, circumferentially around the cuff. The sutures are passed around the prosthesis and the humeral long bone in form of a closed-loop. A similar placement is done for the metallic cables. No anchor in the bone is therefore needed, since two suture/cable ends are fixed together by knots.Placement of stable sutures along the humeral axis on the bone surface - connecting the shaft to single tuberosities - seems to be more challenging: Drilling holes are needed in the shaft and in the fragments as anchor points. The effect of cutting-out of sutures through the bone has to be expected. Due to the fact that proximal fragment displacement is seen clinically, the scenario of a cutting effect has to be assumed. Further studies discuss a fixation of sutures at the proximal humeral shaft without using drilling holes To be able to strengthen proximal-to-distal tuberosity-to-shaft connections, a placement of a cable along the humeral axis would be of interest. Connecting two circumferential oriented cables  by another cable presumably leads to enhanced stiffness  Figure .Further prospective studies are necessary to correlate specific techniques with the clinical outcome. A standardised, biomechanical testing strategy according to physiological loads is needed to evaluate the strength of such techniques.LT: Lesser Tuberosity; GT: Greater Tuberosity; SSP: Supraspinatus; ISP: Infraspinatus; SSC: Subscapularis; TM: Teres Minor.The authors declare that they have no competing interests.No fees or funding was received from a commercial partner.DB as the main author was responsible for the preparation of the manuscript.The technical analysis of existing fixation techniques was provided by BN, RM and SL. ES is the head of the department and approved the strategic background of present publication. All authors read and approved the final manuscript.


Principal Components Analysis showed that the Xavante Indians are concentrated in the Amerindian axis near other populations of known Amerindian ancestry such as Karitiana, Pima, Surui and Maya and a low degree of genetic admixture was observed. This is consistent with the historical records of bottlenecks experience and cultural isolation. By calculating pair-wise Fst statistics we characterized the genetic differentiation between Xavante Indians and representative populations of the HapMap and from HGDP-CEPH project. We found that the genetic differentiation between Xavante Indians and populations of Ameridian, Asian, European, and African ancestry increased progressively. Our results indicate that the Xavante is a population that remained genetically isolated over the past decades and can offer advantages for genome-wide mapping studies of inherited disorders.Characterization of population genetic variation and structure can be used as tools for research in human genetics and population isolates are of great interest. The aim of the present study was to characterize the genetic structure of Xavante Indians and compare it with other populations. The Xavante, an indigenous population living in Brazilian Central Plateau, is one of the largest native groups in Brazil. A subset of 53 unrelated subjects was selected from the initial sample of 300 Xavante Indians. Using 86,197 markers, Xavante were compared with all populations of HapMap Phase III and HGDP-CEPH projects and with a Southeast Brazilian population sample to establish its population structure Knowledge of genetic diversity patterns of human populations provides important insights of their evolutionary history and is useful in genetic mapping studies of complex diseases and their component traits th century in the Brazilian Central Plateau and this period was marked by epidemics, armed conflicts and forced labor imposed by the Portuguese colonial government. In the middle of 19th century, in an attempt to escape mistreatment, they moved westward to their present habitat and remained relatively isolated until the early 20th century. In the 1940s Brazilian government decided to stimulate settlements in its central region, considered a sparsely populated area, aiming to promote greater integration of this area with the rest of the country. As a result of this political decision the Xavante groups were forced to deal with the settlers and the post-contact period was characterized by epidemics and conflicts that resulted in a severe reduction of their population. By the end of the 1950s, the Xavante were reduced to small patches of settlements. However, in the last decades with the demarcation of their lands, health programs and establishment of peaceful contacts with non-Indians they experienced an increase in their population. Importantly, despite of the interaction with the outsiders, the Xavante maintain their own complex social organization and cultural values that were preserved over the years Xavante is an indigenous population living in Mato Grosso state, Central Brazil. They comprise approximately 10,000 individuals, one of the largest indigenous groups in Brazil, and are Jê-speaking people The aim of the present study was to characterize the genetic structure in Xavante Indians providing valuable baseline data for future genetic studies and to compare it with a Southeast Brazilian population A cross-sectional study was conducted in the Sangradouro Reservation, Mato Grosso state, Brazil. The initial sample of Xavante Indians was comprised of 300 individuals and blood samples were collected from each subject. Genomic DNA was extracted from peripheral blood leukocytes using a commercial kit .http://pngu.mgh.harvard.edu/~purcell/plink/) confirmed the presence of related individuals in this sample. Genetic data were used for obtaining maximum likelihood estimates of relationship among pairs of individuals using the ML-Relate software http://www.montana.edu/kalinowski/Software/MLRelate.htm). This approach uses simulation to determine which relationships are consistent with the empirical genotype data, comparing putative relationships with possible alternatives. Only main pairs of individuals, such as parent-offspring, full-siblings, and half-siblings, are identified by the software. The ML-Relate software was unable to estimate the relationships using the complete panel of SNPs , therefore we selected a set of approximately 10% independent markers (730 SNPs) to perform this analysis. The selection of unrelated individuals was conducted in several steps. The individual with the greatest number of relationships was identified and removed from the Xavante sample. Next, the relatedness was recalculated, generating other relationship structures. Again, the individual with the higher number of relatedness was removed and the relatedness was recalculated. This process was repeatedly made until we had only an unrelated sample. We identified 53 unrelated Xavante Indians in our sample.Individuals were genotyped in 731,442 SNPs using Human Omni Express Bead Chip plataform . Genome-wide pair-wise identity-by-descent (IBD) estimated using the PLINK package Genotype data from the HapMap project (Phase 3) The Indian leaders and the study participants were informed about the purposes of this study and gave their consent. The majority of the population gave their written consent, for the ones who were illiterate (14%), fingerprint impressions were used to document their approval. A Xavante health agent worked as an interpreter when necessary. This study was approved by Ethics Committee of Escola Paulista de Medicina, Universidade Federal de São Paulo and Brazilian National Ethics Commision (CONEP).Principal component analysis (PCA) was applied to genotype data to infer continuous axes of genetic variation using the SmartPCA program of the Eigensoft package st estimates were also computed by SmartPCA program to characterize the genetic differentiation between Xavante Indians and the populations of the final dataset.Pair-wise Fhttp://evolution.genetics.washington.edu/phylip.html) and visualized with HyperTree http://kinase.com/tools/HyperTree.html).Next, we investigate the genome-wide genetic distance among individuals of all populations using a distance matrix computed by the PLINK package We knew there was a familial structure in the Xavante Indians sample. Genealogical relationships can be represented mathematically as probabilities that individuals share zero, one, or two alleles identical-by-descent. All 731.442 markers were used to estimate the genome-wide pair-wise identity-by-descent using the PLINK package, confirming the presence of related individuals in our sample, as shown in The ML-Relate software was used to select unrelated individuals. Estimates of relatedness and relationship of main related pairs  are computed by maximum likelihood approach. After the removing process, a subset of 53 unrelated Xavante Indians were selected for the genetic population structure analysis. In The group of 53 Xavante Indians was merged with HapMap, HGDP-CEPH, and São Paulo databases and the set of markers genotyped in all datasets was determined. The merged dataset is composed of 2,363 individuals  genotyped in 86,197 markers.. Although the São Paulo sample has been removed from our final dataset, we plotted the first three eigenvectors including it to illustrate the ancestry of the general Brazilian population, as shown in Ten continuous axes of genetic variation (eigenvectors) were computed using the Eigensoft package. Principal component analyses (PCA) using different subsets of populations were firstly performed to investigate which populations were important for determine the genetic structure of the Xavante Indians. These datasets were composed by Xavante Indians and, respectively: (a) only Amerindians samples; (b) Amerindians and Asians samples; (c) Amerindians, Asians and São Paulo samples; (d) the complete dataset. Three-dimensional plots were provided to all analyses, considering the first three principal components. Comparing plots of each subset of populations, we decided to remove São Paulo and GHI samples from the final dataset for PCA, since these two populations do not enhance the determination of the eigenvectors of the Xavante sample. Thus the final dataset for PCA was composed by Xavante Indians and all populations of HAPMAP and HGDP projects, excluding only São Paulo and GHI samples. A principal component analysis was then performed using this final datasetWe also studied the genetic similarity at the individual level from a genetic distance matrix obtained by calculating the complementary values of the genome-wide average proportion of alleles identical-by-state shared among pairs of individuals from all studied samples. The results of a neighbour-joining tree analysis are shown in st estimates were also computed by the SmartPCA program of the Eigensoft package for all populations of our final dataset with more than 6 sampled individuals. The genetic differentiation between Xavante Indians and representative populations of the European, Asian, African, and Amerindian ancestry are shown in Pair-wise FIn this study we compared the genetic variation of 2,363 individuals  genotyped in 86,197 markers. Our results showed that the Xavante population has a low level of genetic admixture. This is consistent with the historical records of bottlenecks experience, cultural isolation with subsequent reduced gene flow. Despite using different methodologies, which difficult the comparison, our data are consistent with previous studies that have shown low level of admixture in the Xavante population suggesting that no significant changes in this villagés gene pool has occurred over last decades Genetic studies of isolated populations have been subject of interest since they may help to map genes underlying simple monogenic, as well as, complex diseases. In isolated populations, monogenic disorders are less likely to show non-allelic heterogeneity Principal component analyses demonstrate that the Xavante population is a distinct ethnic group more closely related to individuals of Amerindian ancestry and genetically distinct from other HapMap and HGDP populations and from São Paulo individuals.st statistics, we found that the genetic differentiation between the Xavante population and representative populations of Amerindian, Asian, European and African ancestry increased progressively. These results are consistent with the Americas history of peopling that suggests a main colonization event from Siberia. The migration of humans from Eurasia to the Americas took place via Bering Strait and spread throughout North, Central and South Americas, diversifying into several culturally distinct native populations By calculating pairwise FThe findings from this study add to our understanding of genomic variation across the South American native populations and confirm that the Xavante is a closed Indian population that can provide a unique opportunity for genome-wide mapping studies of inherited disorders.


Drosophila, the Hedgehog signaling pathway controls a wide range of developmental processes and is implicated in a variety of cancers. The success of a screen for chemical modulators of this pathway, published in 2002, opened a new chapter in the quest to translate the results of basic developmental biology research into therapeutic applications. Small molecule pathway agonists are now used to program stem cells, whilst antagonists are proving effective as anti-cancer therapies.First discovered in  Journal of Biology, Jeff Porter and colleagues described their use of a cell-based assay to identify and characterize small molecules that modulate the activity of the Hedgehog signaling pathway [hedgehog (hh) gene that had originally been studied in Drosophila. A burgeoning body of evidence supported the conviction that manipulation of Hh signaling could have applications in both regenerative medicine and cancer therapy. The Frank-Kamenetsky et al. paper [In November 2002, in what was only the second research article to be published by the  pathway . In the l. paper , along wThe cloning of Sonic Hedgehog (Shh), one of three vertebrate Hh orthologues, in 1993  led quicin vitro and in vivo' had been filed at the end of 1993 [Drosophila geneticists to a potential target for pharmaceutical manipulation. And since pharmaceutical companies prefer the lower production costs and longer shelf life of small molecules compared to biologics, the search began for chemical modulators of its activity.Such a use of Shh had in fact been anticipated with its original discovery and a patent application relating to its exploitation 'to generate and/or maintain an array of different vertebrate tissue both  of 1993 . This paDrosophila had identified the multipass-transmembrane protein Patched as being the receptor for Hh [The early studies of Hh signaling in r for Hh  and the r for Hh ,11. GPCRr for Hh . So the r for Hh .in vitro assays of Hh pathway activation had been based either on the differentiation of explanted embryonic tissues or on alkaline phosphatase activity in 10T1/2 cells, an assay that took over 5 days to complete [Drosophila of the cryptically named Cubitus Interruptus protein, a member of the Gli family of transcription factors, had revealed its role in mediating the transcriptional response to Hh through binding to enhancers upstream of ptc, itself a Hh target gene [et al. paper, however, was the identification of Hh-Ag1.1, the first example of a Hh-pathway agonist.Encouraged by this finding, Porter and colleagues at Ontogeny (which in 2000 merged with two other companies to become Curis Inc.) set out to discover novel chemical modulators of Hh. Up to this point, complete . The keyget gene , a findiin vivo, they also mimicked the cellular responses elicited by Hh proteins, acting as potent mitogens on primary rat cerebellar neurons and inducing specific neuronal progenitor subtypes in neural plate cultures. This recapitulation of Hh neural patterning activity together with the ability to rescue Shh mutant embryos by oral gavage of pregnant mothers established the in vivo utility of the Hh-Ag molecules. A set of epistasis tests suggested that both agonists and antagonists (cyclopamine and CUR-61414) act at the same level downstream of the Hh receptor Patched, but upstream of the intracellular modulator of Gli activity, Protein Kinase A (PKA). Moreover, the inability of agonists to rescue the mouse Smo mutant phenotype pointed to the Smo protein itself being their target. Formal proof of this came through use of radiolabeled agonist in Smo protein binding and competition assays, which at the same time proved that the antagonists also act by binding Smo directly  were identified with enhanced potency, reduced toxicity and/or improved stability. Not only was the strong transcriptional activation evoked by these agonists reproducible et al. paper. In the August 2002 edition of Cell, Jessell and colleagues [The first reported application of SAG in directing differentiation of embryonic stem (ES) cells actually pre-empted publication of the Frank-Kamenetsky lleagues  reportedlleagues  (Figure in vitro [The use of purmorphamine to generate oligodendrocytes from ES cells provided the first demonstration that Hh agonists can induce glial as well as neuronal differentiation in vitro . Excitinin vitro . Both puin vitro . In anotin vitro . Togethein vitro model of ALS, purmorphamine was shown to be cytoprotective against oxidative stress [As well as its role in neural patterning and survival, the Hh pathway functions in a variety of other developmental processes. The ability of purmorphamine to direct mesenchymal cells to an osteogenic fate has been shown to occur via induction of the master regulator of bone formation, Runx2, and there has already been an attempt to load purmorphamine onto artificial bone adhesives to promote fracture repair . In mouse stress . TogetheJournal of Biology paper was followed up in early 2003 with a report of the first evidence for therapeutic efficacy of such a molecule. Two in vitro BCC culture systems were established by the Curis team, in both of which CUR61414 was found to elicit complete regression of the lesions [Ptc1/+; p53/p53 mice, following treatment with HhAntag-691, another antagonist from the Curis screen [Clinical application of Hh pathway antagonists Figure , by cont lesions . The fols screen . Within s screen , whilst s screen . These ps screen  and in eWhile many challenges remain, not least emergence of mutant forms of Smo that are resistant to vismodegib, the prospects for the use of Hh antagonists as therapies for a range of cancers look promising. By any measure, the development of a novel anti-cancer drug as well as reagents promoting the directed differentiation of human stem cells represents an impressive output from a single screen!http://www.biomedcentral.com/bmcbiol/series/tenthanniversary.This article is part of the BMC Biology tenth anniversary series. Other articles in this series can be found at


A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase) that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion) of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber) required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass . Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic cleavage of the cellulose chains  Bioethanol derived from the bioconversion of lignocellulosic feedstocks continues to attract global interest as a potentially environmentally compatible alternative to current petroleum-based transportation fuels. However, considerable technical improvements are still needed before efficient and economically feasible lignocellulosic biomass-based bioethanol processes can be commercialized. One of the major limitations of this process is the consistently high cost of the enzymes involved in the conversion of the cellulose component into fermentable sugars . This isIn a typical batch enzyme-based process, cellulose conversion-time experiments are characterized by a three-phase curve Figure . This usPrevious technoeconomic modeling has shown that the long hydrolysis time associated with achieving complete cellulose saccharification adds significantly to the operating costs of the enzymatic hydrolysis step and, consequently, to those of the overall biomass to ethanol bioconversion process . RecentlVarious substrate- and enzyme-related factors have been suggested to explain the slowdown in the rate of hydrolysis and, in many cases, the incomplete hydrolysis of cellulosic materials. Although there is still considerable debate about the contribution of each of these factors, it has been suggested that the accessible surface area of the cellulose is one of the most important factors influencing the rate and extent of enzymatic hydrolysis of lignocellulosic substrates -9. This One of the major barriers faced by cellulase enzymes during lignocellulose hydrolysis is their limited access to much of the cellulose, which is buried within the highly ordered and tightly packed fibrillar architecture of the cellulose microfibrils . Celluloet al. [Earlier work by Grethlein  showed aet al. , using fet al. . In eithAlthough previous studies have highlighted the importance of cellulose accessibility during enzymatic hydrolysis -20, the 2)-catalyzed steam and ethanol organosolv (EO) pretreatment at previously determined conditions [In the present study, a range of lignocellulosic feedstocks  were subjected to sulfur dioxide  derived from Aspergillus niger - were used in the enzymatic hydrolysis experiments. Protein concentrations were determined using the modified ninhydrin method [Two commercial preparations  - a cellulase cocktail  derived from n method . Bovine Representatives of agricultural residues (corn stover and corn fiber), softwood (douglas fir and beetle-killed lodgepole pine) and hardwood (hybrid poplar) were used in this study.2-catalyzed steam and/or EO pretreatment as described previously [Lignocellulosic feedstocks were pretreated by SOeviously . Most ofeviously , corn fieviously , douglaseviously , and EO-eviously  and popleviously ) to obtaThe chemical composition of the pretreated materials was determined according to a standard method , as previously described . Monosac4 full factorial design experiment with three central points. The dependent variable was glucan conversion, expressed as percentage, and the independent variables were the cellulase (Celluclast 1.5 L) and β-glucosidase (Novozym 188) loadings, hydrolysis time, and solids loading. The range and the levels of these variables are given in Table Optimization of minimum protein loadings required for efficient glucose release from a broad range of pretreated substrates was performed according to a central composite design in the form of a 2To describe and predict the effect precisely and quantitatively, the hydrolysis data for each of the pretreated materials was fitted using a second-order polynomial model and Statistica software .®, Yang-Chung, Seoul, Korea) at 50°C. The conditions for cellulase and β-glucosidase loadings, hydrolysis time, and solids loadings were determined according to the statistical design of experiments  were mechanically shaken in an orbital shaker incubator  adsorption was used as an indication of the surface area of a particular substrate available for protein binding. Protein adsorption isotherms were established by varying the amounts of protein (cellulase + β-glucosidase) added to the different pretreated substrates (2 mg/mL) in sodium acetate buffer . The cellulase:β-glucosidase ratios were obtained by assessing the minimum protein loading required for efficient hydrolysis. Free protein was determined by measuring the amount of protein in the supernatant after incubation at 4°C and 150 rpm for 1 hour to reach equilibrium. Bound protein was calculated as the difference between free protein and the total protein initially added to the reaction medium. The protein content was determined using the ninhydrin assay . The expads is the concentration of adsorbed protein (mg of protein/mg of substrate), Pmax is the maximal adsorbed protein (mg of protein/mg of substrate) and Kp is the equilibrium constant (mL/mg of protein).in which P is the concentration of unadsorbed protein (mg of protein/mL), Pet al. [The external surface area of the cellulosic-rich substrates measured as the average fiber length of the pretreated substrates was determined using a high-resolution fiber quality analyzer (FQA)  in accordance with the procedure described by Robertson et al. . Brieflyet al. [et al. [Simons' stain (SS), a staining technique used in the pulp and paper industry to examine changes in the physical structure of pulp fibers under the microscope, and adapted for evaluating the pore structure  of cellulosic materials , was peret al. . Pontami [et al. .It has been predicted that a diverse range of plant biomass will be needed to satisfy the projected demands for second-generation bioethanol . As woulThe minimum protein loading for efficient hydrolysis was initially optimized before any correlation was made, to account for the possibility that previous predictions of the hydrolyzability of pretreated lignocellulosics based on either low or high protein levels might not be as meaningful or as accurate as predicted. For example, predictions based on low protein loadings might only include saccharification of the so-called 'easy/accessible' cellulose, and thus factors that control the digestibility of cellulose at high levels of conversion might not have been assessed. By contrast, experiments carried out at high protein loadings might, by saturating the substrate with enzymes, mask important factors limiting efficient hydrolysis.The enzymatic digestibility of the seven pretreated samples using varying protein and solids loadings and hydrolysis times was assessed by monitoring the amount of glucose released Table , and theP ≤ 0.05, or values near to this, were included in the models. The validity of the models was evaluated as a function of their respective coefficients of determination (R2). The value of the correlation coefficient provides a measure of variability in the observed response values that can be explained by the experimental factors and their interactions . The models computed for the R2 value ranged between 0.91 and 0.96  were carried out to obtain mathematical models Table  that betT. reesei, consists mainly of cellobiohydrolases and endoglucanases [in situ β-glucosidase activity, this T. reesei cellulase system is commonly supplemented with an excess of β-glucosidase to avoid any end-product inhibition caused by the accumulation of cellobiose, which would mask the actual minimum cellulase requirement at higher levels. It has also been reported that synergism between cellulase enzymes decreases at high cellulase concentrations (around saturation levels) [The commercial cellulase cocktail (Celluclast 1.5 L) derived from the filamentous fungus ucanases -40. Owin levels) . Thus, tIn this work, we defined effective hydrolysis as at least 70% of the original cellulose in the pretreated lignocellulosic materials being hydrolyzed to glucose. With this percentage conversion as a target, a meaningful assessment of cellulose saccharification could be made before the typical, significant slowdown in hydrolysis rate took place Figure . As mentThe effect of cellulase and β-glucosidase loading on cellulose saccharification yields for most pretreated substrates was significantly affected by cellulase and β-glucosidase loadings in the linear term, and less significantly affected by their interaction with each other, and by their interaction with solids loading and hydrolysis time Table . UnexpecThe significance of the quadratic coefficients of cellulase loading for SPCF, SPDF and OPLPP, and of β-glucosidase loading for SPCS, SPCF, SPLP, SPDF, OPCF, OPLPP, indicate that cellulose saccharification yields increase with protein loading up to a certain level Table . Beyond The mathematical models Table  obtainedThe reliability of the equations was also assessed by comparing the experimental values of the responses at the centre point conditions, an average of three independent experiments Table . All of It was apparent that the minimum protein requirement ranged between 18 and 63 mg protein per gram of glucan. The minimum protein requirement increased as follows: OPCF < SPCF < OPLP < OPP < SPCS < SPDF < SPLP. It was observed that for the same feedstock , EO pretreatment generally resulted in substrates that required less protein to achieve efficient hydrolysis than did steam pretreatment. Within the group of feedstocks pretreated by the same process, the pretreated agricultural residues (corn stover and corn fiber) required lower protein loading per gram of glucan to achieve high glucan conversion than did the pretreated forest biomass . This confirmed that the nature of the lignocellulosic feedstock plays an important role in determining the amount of protein required for effective hydrolysis. This was not unexpected, as the plant cell-wall architecture and molecular structure, which are the primary lignocellulosic factors contributing to biomass recalcitrance, are likely to be different in woody biomass and herbaceous plant-derived biomass. For instance, softwoods have a more rigid structure and a higher lignin content, and are therefore expected to display more resistance towards deconstruction  than the less structurally recalcitrant biomass derived from herbaceous plants.To achieve efficient bioconversion of cellulose to ethanol, it is desirable that the hydrolyzate obtained after enzymatic hydrolysis contains a sufficiently high concentration of fermentable sugars to result in a high ethanol concentration. To obtain this high sugar concentration, hydrolysis should be carried out at high solids loading. Raising the solids loading in the enzymatic hydrolysis step is crucial to minimizing subsequent distillation costs, and it is also expected to decrease process cost by lowering the reactor size and minimizing water requirements ,43. It hRegardless of the pretreatment process used, the effect of solids loading was highly significant for all of the pretreated agricultural residues  Table . This neThe linear effect of hydrolysis time was significant for all of the pretreated substrates, with the exception of the SPCF sample Table . This laThe hydrolysis yields obtained with minimum protein loading for the steam pretreated wood substrates (SPDF and SPLP) did not seem to be affected by increasing the hydrolysis time from 24 hours to 72 hours, and the hydrolysis yields for the steam-pretreated agricultural residues (SPCF and SPCS) were only slightly affected Figure . The greWhen the effect of solids loading on the hydrolysis efficiency of the pretreated materials at the optimized minimum protein loading was assessed Figure , it was R2 > 0.9790) was obtained. An assessment of the Langmuir adsorption isotherm revealed significant differences for protein adsorption onto the different pretreated lignocellulosic materials. The maximum adsorption capacity (Pmax) of proteins onto pretreated materials ranged from 11.0 to 89.3 mg/g substrate and increased as follows: steam-pretreated samples: SPLP < SPDF < SPCS < SPCF; then EO-pretreated samples: OPP < OPLP < OPCF , two distinct curves were observed  of the substrates to bind protein.The high correlation values, For feedstocks pretreated by the same pretreatment technology, it was observed that the higher the capacity of the substrate to adsorb proteins, the lower the amount of protein required to attain efficient hydrolysis. This suggests that the more available the surface area of the cellulose-rich material for the proteins to bind to, the lower the protein-loading requirement for efficient cellulose saccharification. We next wanted to confirm the role that the available surface area of cellulose might have on the minimum protein loading required for efficient hydrolysis of lignocellulosic substrates.2-catalyzed steam-pretreated eucalyptus chips, the substrate accessibility to the cellulases could not be readily predicted from the differences in their cellulose DP or crystallinity, but these substrate characteristics did indicate the likely mode of action of the enzymes [It has been suggested that the cellulose surface area accessible to the cellulase enzymes is one of the most important factors determining the ease of hydrolysis of cellulosic materials, and it is also affected by several substrate characteristics. These features include distribution of particle size, pore volume, degree of crystallinity and degree of polymerization (DP) ,37,46,51 enzymes . From th enzymes .In this study, we assessed the influence of the exterior surface area of the cellulosic-rich materials, determined by fiber dimension/length, on the minimum protein requirement for effective enzymatic digestibility of pretreated lignocellulosic substrates, using a FQA, which is an automated particle size analyzer. We found that the minimum protein requirement for efficient hydrolysis had no correlation with the average initial particle size Figure . SeveralIt is known that cellulose microfibrils are porous substrates, and their overall accessible surface area is expected to be a combination of exterior and interior surface area . One method that we have adapted is the SS technique, which measures a combination of both the interior and exterior surface area of the exposed/accessible cellulose ,35. SS iThe overall available surface area of cellulose increased with the increasing glucan content of the pretreated substrates, with the exception of SPCF, as evidenced by the linear correlation observed between the glucan content and the DO:DB ratio Figure . This coet al. [It has been reported that the internal surface area of cellulose is much larger than the external surface area . Therefoet al.  supportset al. ,55.Previous work has suggested that the limited available surface area of cellulose is a key factor that necessitates the need for relatively high enzyme dosages to attain effective cellulose hydrolysis. However, the majority of these studies used highly digestible, purified cellulosic substrates such as filter paper, Solka floc or Avicel. In the present study, we used a broad range of more realistic heterogeneous, lignocellulosic feedstocks pretreated by promising technologies under more representative conditions. Regardless of significant differences in the origin, structure and chemical composition of the feedstocks and the pretreatment process used, it appears that the minimum protein loading required for efficient hydrolysis of pretreated lignocellulosic substrates has no direct relationship with only the external surface area of the cellulose-rich materials. However, protein loading did appear to be strongly influenced by the overall enzyme accessibility, as determined by the SS technique, which as well as measuring the external cellulose surface area, also takes into account the porosity/topology of the available cellulose.A strong linear relationship between cellulose accessibility and the minimum amount of protein required to achieve effective hydrolysis was apparent, at least with the enzyme cocktail used in this study. As regards the enzymatic mechanism, these results suggest that some of the cellulase components may initially penetrate into areas of the cellulose, particularly the amorphous regions that are large enough to accommodate cellulase enzymes, disrupting/fragmenting the cellulose fibers before significant hydrolysis of cellulose takes place.per se but rather the limited accessibility of the enzymes to the cellulose chains within the substrate matrix.The fact that the more available/exposed cellulose in the pretreated lignocellulosic structure required lower protein levels per gram of glucan to attain high digestion rates suggests that the rate-limiting step during hydrolysis may not be the actual catalytic cleavage of the cellulose chains The authors declare that they have no competing interests.VA planned and carried out the experiments, analyzed the results and wrote the paper. JNS participated in the design of the study, helped analyzing the results and contribute to the draft of the manuscript. All authors read and approved the final manuscript.