Latinos are exposed to adverse psychosocial factors that impact their health outcomes. Given the heterogeneity and rapid growth of this population, there is an urgent need to understand the mechanisms through which psychosocial factors impact substance abuse and anxiety between immigrant and U.S. born Latino adults. The present study employs a multi-group path analysis using Mplus 7.2 to examine generational differences in the paths between affiliation culture, years of formal education, contact with important people, and length of full-time employment to substance abuse and anxiety in immigrant and U.S. born Latino adults who completed substance abuse treatment. A total of 131 participants  in recovery from substance abuse completed self-report measures. Results from the multi-group path analysis suggest that being more affiliated to the U.S. culture is associated with substance abuse, whereas years of formal education and longer full-time employment is associated with reduced anxiety in the immigrant group. Conversely, frequent contact with important people and affiliation to the U.S. culture are associated with fewer years of substance abuse, whereas longer full-time employment is associated with substance abuse in the U.S. born group. Anxiety and substance abuse was correlated only in the U.S. born group. The implications of these findings are discussed. With a population exceeding 51 million, Latinos are the largest and fastest growing minority in the United States ,3. NatioThe sparse research on access and substance abuse treatment utilization among Latinos shows mixed results –9. AlthoThe co-occurrence between substance abuse and anxiety on Latinos who complete SAT deserves consideration. A review of the literature shows that anxiety disorders were present in nearly 75% of individuals with substance use disorders (SUDs), partially supporting the self-medication pathway ,25. The Currently, Latino immigrants comprise 36% of the total Latino population and most of them are middle age age 35 and over) ,14. Alth and overSubstance abuse literature has documented existing disparities in substance abuse within Latinos based on level of acculturation, country of origin, years of residence in the U.S., and generational status ,48. StudThe acquisition of U.S. cultural norms related to acculturation has been linked to mental health problems and substance abuse among Latinos ,35,52. HFewer years of formal education and poor employment history have been found to increase the likelihood of relapse ,63. A reThe collectivistic orientation of the Latino culture emphasizes the importance of social networks for their well-being . HoweverThe dearth of research on clinical populations who completed SAT remains significant for Latinos . Researcage = 36.3; SD ± 10.5), 113 were males (86.3%) and 18 females (13.7%). Nearly half of the participants immigrated from Mexico, Puerto Rico, and other Central American countries (48.1%), with a mean length of stay of 19.2 years (SD ± 13.71) in the U.S. The majority of the participants had alcohol and substance abuse treatment previously (n = 124), while for seven participants it was their first time in treatment. For sociodemographic characteristics see Participants for this study were part of a larger NIH-funded study that examined community-based recovery homes for Latinos in recovery from substance abuse . A totalRecruitment of participants took place from fall 2009 to spring 2012 for a larger NIH-funded study aimed to examine recovery homes for Latinos in recovery from substance abuse ,75. A caAfter completing the interview, participants received $30 as compensation for their participation.A 24-item demographic questionnaire was used to collect participants’ age, gender, place of birth, country of origin, and treatment setting.Participants were asked to report their place of birth and were assigned either to the immigrant or U.S. born groups. Puerto Ricans who were born on the island were placed in the immigrant group. We acknowledge that Puerto Ricans are U.S. citizens by birth. However, given the fact Puerto Rico endorses traditional cultural norms similar to those of other Latin American countries, we determine to place Puerto Ricans born in the island with other Latino immigrants.th Edition, assesses problems during the individual’s lifetime and during the 30 days prior to the interview in seven areas: alcohol use, drug use, illegal activity, interpersonal and family relations, medical problems, employment, and psychiatric problems [th Edition of the ASI has been used with Latino participants in several large studies and yielded valid data [The Addiction Severity Index (ASI), 5problems . A sampllid data ,22,77. Tlid data . CorrelaThe Bidimensional Acculturation Scale for Hispanics (BAS) is a 24-item, 4-point Likert-type (1 = low or not well to 4 = high or very well) self-report measure of English and Spanish use as a proxy for acculturation . Three sThe Important People and Activities Inventory (IPA) examines the impact of social network by asking participants questions about their relationships with significant people . Particinever to 3 = often) measure that evaluates symptomatology in adults associated with traumatic experiences [The Trauma Symptom Checklist 40 (TSC-40) is a 40-item, 4-point Likert-type , with a mean age of 36.15 years. Means, standard deviations and correlations for all study variables are presented in Independent-samples t-tests were conducted to compare the impact of the aforementioned psychosocial factors on immigrant and U.S. born Latinos. Results from the t-tests revealed that U.S. born Latinos were more affiliated to the U.S. Culture, t (129) = 9.18, p < 0.001; completed more years of formal education, t (129) = 3.26, p < 0.001; and had more contact with important people, t (129) = 2.83, p = < 0.01, than their immigrant counterparts. In contrast, there was a significant difference in age between the immigrant  and U.S. born Latino  groups, t (129) = 3.09, p < 0.01. Conversely, there was no significant difference between the immigrant and U.S. born groups in terms of substance abuse, anxiety, and employment pattern.A multiple-group analysis was conducted to compare immigrant and U.S. born Latinos on the impact of psychosocial factors on substance abuse and anxiety using the Mplus computer software, version 7 . Maximum2 = 6.61, df = 3, p = 0.08, CFI = 0.92, TLI = 0.47, RMSEA = 0.13, RMSEA 90% C.I. = 0.00 – 0.27, SRMR = 0.03). Next, recurrent nonsignificant pathways were constrained to zero  to improve model fit indices. The constrained multiple-group model yielded an adequate fit to the data as indicated by the values on the following fit indices . Next, a model with all paths constrained to equal was estimated . The model fit for the fully constrained model was significantly worse than both, the model with freely estimated paths and the trimmed model. To test for group difference, the chi-square from the trimmed model was compared to the chi-square from a model with paths constrained to equal. The two models were significantly different , suggesting group differences . Similarly, years of formal education  and full-time employment pattern  were significantly negatively associated with anxiety. In other words, for each 1 standard deviation (SD) increase in affiliation to the U.S. culture, there is a 0.28 SD increase in years of substance use. Conversely, for each 1 standard deviation (SD) increase in years of formal education and length of full-time employment, there is a −0.38 SD and −0.37 SD decrease in anxiety rates. Substance abuse lifetime and anxiety were not related .Conversely, in the U.S. born group, average contact with important people , and affiliation to the U.S. culture  were negatively associated with substance use lifetime. Conversely, full-time employment pattern was significantly positively associated with substance use lifetime . Substance use lifetime and anxiety were significantly positively correlated . In other words, for each 1 standard deviation (SD) increase in average contact with important people and affiliation to the U.S. culture, there is a −0.24 SD and a −0.22 decrease in years of substance use. Conversely, for each 1 standard deviation (SD) increase in length of full-time employment, there is a 0.22 SD increase in years of substance abuse  and 3. OThe purpose of the present study was to examine whether generational differences moderate the association between psychosocial factors and anxiety on a sample of Latinos who completed SAT. Results from the multi-group analysis revealed significant differences between the two groups, relative to the outcomes of interest. Specifically, in the immigrant group, after controlling for age, being more affiliated to the U.S. mainstream culture was associated with more years of substance use. Conversely, contact with important people, years of formal education, and length of full-time employment were not related to years of substance use. Results for the U.S. born group indicate that, after controlling for age, longer full-time employment was associated with more years of substance use, whereas contact with important people and affiliation to the U.S. culture were associated with fewer years of substance use. Years of formal education were not related to substance abuse.With regards to anxiety, in the immigrant group and after controlling for age, more years of formal education and longer full-time employment were associated with reduced anxiety. Conversely, affiliation to the U.S. culture and contact with important people were not related to anxiety. In the U.S. born group, none of the pathways was related to anxiety. Lastly, substance abuse was positively correlated with anxiety in the U.S. born group, but not in the immigrant group.These findings illustrate substantial differences between the immigrant and U.S. born group in the direction and strength of the paths from psychosocial factors to substance abuse and from psychosocial factors to anxiety. Although some research have focused on disparities during early stages of treatment and outpatient SAT, the present study advances previous research on substance abuse on Latinos by examining generational differences on a sample of Latinos who completed SAT ,73,85. OOur results showed that affiliation to the U.S. culture was associated with more years of substance use among immigrants, but it was not related to anxiety. Although Latino immigrants, particularly middle-age adults experience greater difficulty to adopt social norms and values of the U.S. culture, those who live in cultural enclaves, or communities with high concentration of immigrants, may be less exposed to discrimination and receive support from community members ,58,88. AThe result indicating that more affiliation with U.S. mainstream culture was associated with fewer years of substance use seems to contradict previous studies conducted on community samples of U.S. born Latinos ,48. is uOur results suggest that more years of formal education and longer full-time employment reduce anxiety in the immigrant group. These findings shed light on the extent to which the above factors may protect Latino immigrants from anxiety. It is plausible that Latino immigrants with more years of formal education are more likely to learn the English language and U.S. cultural norms, facilitating the acculturation process. In addition, for most Latino immigrants who moved to the U.S. looking for better jobs, being gainfully employed may elicit a sense of success. Given the co-occurrence between anxiety and substance abuse, recovery oriented services should be tailored to the needs of the population. Specifically, securing employment and stable housing have been reported as an important recovery goal among Latinos working in their recovery . In a stThe findings for the U.S. born group indicating that frequent contact with important people was associated with fewer years of substance use is consistent with the social control theory . Strong Lastly, the path from longer full-time employment to years of substance abuse seems to be in function of being affiliated to the U.S. culture and having more years of formal education. More important, the results indicating that anxiety was correlated with substance abuse in the U.S. born group, but not in the immigrant group suggest unique sources of anxiety that affect this group. Support for the link between anxiety and substance abuse can be found in studies conducted on community samples ,96. HoweContrary to our hypothesis, the same path was not correlated in the immigrant group. It is plausible that Latinos may underreport anxiety symptoms due to artifactual level explanations . Severaln = 30, 47.6% of the participants assigned to the immigrant group) into this group. Although Puerto Ricans may contend with discrimination, low educational attainment, and have difficulties acculturating to the U.S. mainstream culture, their status as U.S. citizens afford them access to resources that non-U.S. citizens do not have. is may have driven the high affiliation to the U.S. mainstream culture in tandem with the length of residence in the U.S. Further studies are needed to explore the impact of psychosocial factors within Latino immigrants. Another limitation is that the low number of Latino women in the dataset did not allow for an examination of gender differences within each group. Lastly, most participants were from Puerto Rican and Mexican background (in both immigrant and U.S. born groups), which may limit generalization of findings to other Latino subcultures.There were several limitations, including a small sample that did not allow for exploration of other psychosocial factors . The cross-sectional nature of the present study does not allow for causal inferences. Participants on the present study completed SAT in multiple settings, which may reduce generalizability to similar populations. In addition, in an attempt to test the proposed model on Latinos who grew up in Latin American countries, we included Puerto Ricans who were born in the Island . More important, to develop effective substance use prevention programs for individuals who completed SAT, research is needed to understand the underlying processes through which psychosocial factors impact clinical samples of Latinos to inform ways to intervene. Specifically, there is the need for more studies to understand the culture of reception wherein Latinos are immersed, how symptoms manifest and the responses to prevention among this ethnic group in order to culturally informed prevention programs . By the


Janelle R. Thompson was not included as an author in the published article. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way.The original article has been updated.CN wrote the manuscript and TK and TB provided clinical wastewater samples. CN, T-HL, LH, and HC conducted sampling and performed the experiments. CN, MT, and BT analyzed datasets. JT provided computational resources and supervision for data analysis. CN, MT, BT, and KG conceived and designed the experiments.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.


Studies focusing on a joint evaluation of both Working Memory (WM) and Math Ability (MA) in autism are far from abundant in literature, possibly due to inadequate methodological approaches and reported inconsistencies between results obtained in each separate field of research, resulting in contradictory conclusions. The specific aim of this case report is therefore evaluating and integrating results on these two cognitive abilities in a child with autism spectrum disorder without intellectual impairment. Our data on an autistic 10-year-old child (M.N.) show that the levels of functional (active vs. passive), rather than structural , data manipulation are quite relevant in the way the child scored differently in the various tasks. Furthermore, M.N. generally displayed average to good ability levels in math calculation, except for oral multiplication, and division activities. By way of conclusion, data are discussed in terms of strengths and weaknesses in relation to special learning trajectories in education and the relevant achievements. According to Baddeley's model  in children with autism still accounts for a limited share of studies in the literature, although investigations carried out on typical development clearly show a significant correlation between these two variables  with Typical Development (T.D.). Evaluation was carried out at the end of fourth grade. Parents (both mothers and fathers) were asked to sign an informed consent to (1) authorize their children's participation in the study and (2) authorize the publication of the research report.The following tests were carried out: Digit Span Forwards task  = 3.67, p = 0.002], scoring a 99.92 percentile (95% CI: 99.36–100.00). In the Listening Span test M.N.'s performance was within the average . M.N.'s score was approximately 55.98 percentile (95% CI: 38.71–72.42), with respect to the T.D. distribution.M.N.'s memory abilities were evaluated through tasks testing WM-related componential and manipulation levels using Digit Span Forward, Listening Span, Visual Pattern Test, and Counting Span Test. M.N.'s performance can be described as follows: compared to the typical development (T.D.) group, M.N. was better at remembering numbers in the Digit Span Forward test , and a 99.77 percentile (95% CI: 98.44–100%) in the box test , with respect to the T.D. group. M.N. obtained an average score with respect to the control group in the Counting Span Task  as shown in Table The visuo-spatial sketchpad was carried out via the Visual Pattern test, whose indexes yielded significant results: M.N. scored a 99.87 percentile (95%: CI 99.04–100%) in the matrix test [The MA profile of M.N. was outlined using the AC-MT tool , independently of WM components involved. On the other hand, when tested on complex manipulation tasks , M.N. scores do not differ from those achieved by T.D. children, independently from the type of data to be processed. Results thus obtained suggest that the level of data manipulation implicated in the tasks resolution, rather than the type of component, seems to determine the degree of difficulty for M.N. This result seems to corroborate data underlining the importance of elaboration level when evaluating WM in autism, while partially invalidating claims concerning the role of the type  of material proposed .In conclusion, M.N.'s MA seems to be closely related to his WM skills. We are aware of the fact that functioning of children with autism spectrum disorder without intellectual impairment may largely depend on the age at which the ability is assessed (Titeca et al., SP designed the study, drafted a first version of work and provided a final approval of the manuscript. MP supervised a critical revision of the work and proposed the final approval of the version to be published.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.


Echinococcus and Echinococcosis, Part A. Volume 95, Advances in Parasitology 95. Academic Press; 2017. 525 pages, ISBN 978-0-12-8114711 (hardcover); 9780128114728 (eBook).Thompson RCA, Deplazes P, Lymbery AJ, Editors.  Echinococcus. The life-cycle is completed through a predator-prey interaction. The parasite matures in the small intestine of domesticated and/or wild carnivores. This book may be described as the second edition of “Echinococcus and Hydatid Disease” edited by RCA Thompson and AJ Lymbery, published in 1995.Echinococcosis is the most lethal chronic hepatic disease caused by accidental ingestion of eggs of the genus Echinococcus were recognized as rigid independent species. Among them, E. granulosus and E. multilocularis are of public health importance, since they cause cystic echinococcosis (CE) (hepatic abscess) and alveolar echinococcosis (AE) , respectively.Approximately two decades ago, only four species of E. granulosus (sensulato) (s.l.) consists of five independent species: E. granulosus (sensu stricto) (s.s.), E. equinus, E. ortleppi, E. canadensis and E. felidis. Among these five species in E. granulosus (s.l.), E. granulosus (s.s.) (G1) and E. canadensis (G6/7) are the major pathogens for human CE. Furthermore, a new species, E. shiquicus, a sister species of E. multilocularis, was described from the Tibetan plateau, China in 2005, and E. felidis from African lions, a sister species of E. granulosus (s.s.) (G1), was resurrected as an independent species from E. granulosus felidis in Africa in 2008. Therefore, it is really timely for us to have an updated review book on these topics.Recent molecular studies have revealed that E. felidis should not be rare. In Asia, mainly in the Tibetan plateau, China, E. shiquicus may also be confirmed from humans.People involved in the control of echinococcosis are expected to do any surveillance based on molecular identification of the pathogens. In Africa, it is easy to expect that human cases with The definitive hosts, either domesticated or wild carnivores, have been contaminating our living environment with eggs. Accidental human infection is mainly caused by human invasion into the wild animal territories, but also by wild animals such as red foxes which also often invade our living environment in big cities in developed countries.Echinococcus. The authors are well known experts in each topic. Chapter 1 by Eckert and Thompson is on “Historical aspects of Echinococcosis” and is highly informative. It mainly focuses on the pioneer researchers and their scientific contribution with their memorial photos. It is interesting to learn what the first senior author, Eckert, has done for the establishment of WHO informal working group on echinococcosis etc. over 60 years’ lifework. If it had included the two new species, E. shiquicus and E. felidis, or the most recent debate on E. canadensis, it would be perfect.This book with six chapters is an updated overview on the highly pathogenic parasites in the genus Echinococcus” and “Phylogenetic Pattern, Evolutionary Processes and Species Delimitation in the Genus Echinococcus”, respectively. Although they recently stressed that E. canadensis should be divided into three species, i.e. E. intermedius, E. borealis and E. canadensis , ChapteE. granulosus (s.l.) through endogenous budding, and the reverse topology of laminated layer in mother vs daughter cyst is resolved. But the laminated layer might be differentiated from undifferentiated stem cells in the germinal layer in the mother cyst.From Chapters 1–4, I still have no clear idea on the mechanism as to how the laminated layer emerges in daughter cysts in Echinococcus Species” is informative with excellent figures showing the life-cycle of each species.Chapter 5 by Romig et al. on “Ecology and Life Cycle Patterns of E. multilocularis and E. granulosus (s.l.) and highly informative in general. It clearly indicates the historical evidence for the distribution of different genotypes of E. multilocularis and different species of E. granulosus (s.l.) on a global scale. It is easy to understand that Echinococcus parasites have been traveling the world. It is the time for field and laboratory parasitologists to join together using molecular tools.Chapter 6 by Deplazes et al. on “Global Distribution of Alveolar and Cystic Echinococcosis” is focused on the distribution of It is easy to believe that the living environment of humans is becoming more complicated than we expected before reading this book. Is it possible for us to eradicate echinococcosis? Or should we be patient to live together with those pathogens, but challenge for sustainable education and practice for prevention of echinococcosis? If an innovative metacestocidal drug is developed, its benefit would be enormous!I dedicate this book review to the late Professor the Lord EJL Soulsby who passed away on 12 May 2017.


Passiflora from samples containing glands in the petiole and foliar blade fixed in 50% solution of formaldehyde-ethanol-acetic acid and stored in a 70% ethanol solution. For anatomical analyses, part of the material was embedded in Paraplast, longitudinally sectioned and double stained with safranin and astra blue. Scanning electron microscopy analysis was also carried out. To analyze the presence of sugars in the secretion of foliar glands, a glucose strip test was used. Based on the results of morphological, anatomical and glucose strip tests, the foliar secretory glands in Passiflora can be grouped into two categories: Type I glands, defined as nectaries, can be elevated or flattened, and can have a sugar content high enough to be detected by the glucose strip test analysis. Type II glands are elevated and did not show a positive reaction to the glucose strip test. From an anatomical viewpoint, glands characterized as extrafloral nectaries show a multistratified secretory epidermis, typically followed by two flat layers of nectariferous parenchyma with dense content. Internal to these layers, vascular bundles are immersed in the subsecretory parenchyma and terminate in phloem cells. On the other hand, type II glands show a single layer of elongated secretory epidermal cells. Internal to this single layer, parenchyma and vascular tissue with both phloem and xylem elements can be observed. The analyzed species show a wide diversity of gland shape and distribution, and the combined analysis of morphology, anatomy and preliminary tests for the presence of glucose in the exudate in different Passiflora subgenera suggests the occurrence of two categories of glands: nectaries and resin glands.Extrafloral glands in Passifloraceae species have aroused the interest of many researchers because of their wide morphological diversity. The present work analyzed the foliar glands on 34 species of  Passiflora L. is the most representative genus in the Passifloraceae s.s. family, comprising more than 500 species [Astrophea (DC.) Mast., Deidamioides (Harms) Killip, Decaloba (DC.) Rchb., Passiflora [Tetrapathea (DC) P.S. Green, the latter restricted to Oceania [Admired for the beautiful flowers and their edible fruits, the Passifloraceae Juss. Ex. Rouseel family is the object of study in different areas.  species –4. Accorssiflora , and Tet Oceania .Passiflora [The occurrence of extrafloral nectaries (EFN) in Passifloraceae species is very common, and their presence, as well as their shape, has been widely used as a diagnostic characteristic for species or species groups within the genus ssiflora –10. EFN ssiflora .Nectaries are specialized structures that secrete a sugary substance known as nectar , 12, whiPassiflora species. They can appear as scars or protrusions, and they can also resemble small barrels, trumpets or spoons [The petiolar EFN are one of the main characteristics used to identify r spoons . Laminarr spoons .Passiflora and Adenia Forssk. species can also have glandular spots at the abaxial surface, which have an external appearance of rounded brown spots. When observed in cross-section, the glandular spots show many layers of elongated epidermal cells at the glandular region, which are followed by tissue with several crystal formations. The author also suggests that the petiolar glands, which are frequently observed in Passiflora, are modifications of these glandular spots.According to Solereder , the leaPassiflora species. In this study, the author points out that petiolar nectaries show a variety of sizes and morphologies, usually distinguishable by the organ in which they are found. Thus, they fit the classification of elevated nectaries proposed by Zimmermann [In an extensive work about the species of Passifloraceae in the American continent, Killip  describemmermann . On the mmermann  classifimmermann .Passiflora, these nectaries could be described as vascularized, according to Elias [Passiflora is quite similar to that of species already reported. Therefore, considering that various authors have used different nomenclature, three different and specialized regions are usually recognized in these types of nectaries: secretory epidermis, where the nectar is liberated to the exterior and where stomata and trichomes may be either present or absent; secretory parenchyma that produces or stores the nectar, and is composed of layers of small cells with a dense content, located immediately under the epidermis; and subsecretory parenchyma, which has large and fewer juxtaposed cells [Based on EFN studies already carried out in to Elias . From aned cells , 18, 19.Dysosmia DC. section [Passiflora, that shows a characteristic absence of petiolar glands, being replaced with “gland-tipped hairs” [Passiflora foetida L., Roth [et al. [Passiflora foetida shows a variability in its gland morphology and physiology that can be regarded as a transition from a true EFN to lipophilic secretory glands, thus naming them as resin glands.Besides the presence of nectaries, the occurrence of a glandular type referred to as “gland-tipped hairs”, “sticky glands” and “sticky hairs” are also reported , 5, 8. T section , from sud hairs” , 8. StudL., Roth  describe [et al.  were notPassiflora has not been conducted. To better understand the diversity of secretory glands, the current study was aimed at comparative morphological and anatomical analyses associated with the presence of glucose in the exudate, as determined by the glucose strip test. We described two categories of secretory glands and discuss the use of terms regarding the different shapes.Detailed investigation to elucidate the morphological and anatomical structures of foliar glands in Astrophea (DC.) Mast. (1 species), Deidamioides (Harms) Killip (2 species), Decaloba (DC.) Rchb. (5 species) and Passiflora (26 species) , from a greenhouse cultivation at the State University of Santa Cruz , and from the greenhouse of a private collector. One species was obtained from its natural environment (Thirty-four species were selected from the subgenera species) , and speironment . The vouironment .et al. [In order to structurally characterize the glands, samples were analyzed through scanning electron microscopy (SEM) and the shape definition was based on the descriptions for solid shapes by Radford et al.  and the et al. . Sampleset al. , dehydraet al. . The anaFor the anatomical analysis, samples of petiolar and laminar glands were fixed in FAA 50 for 24 hours  and embePassiflora sublanceolata (Killip) MacDougal cultivated in a Passiflora active germplasm bank at UESC (BAG-Passifloras).In order to analyze the presence of sugars in the secretion of foliar glands, the glucose strip test was used (Inlab Diagnóstica–Alamar Tecno Científica Ltda.) for the species cultivated at the Institute of Biosciences, University of São Paulo. The same method was used for the species Passiflora, glands were found in the petioles (petiolar glands) in pairs or dispersed over the petiole at the margin of the leaf blade , on the abaxial face of the leaf blade and/or dispersed on both sides of the foliar blade (laminar glands). In the same species, glands could be seen in more than one region, and the most common combination observed was the presence of petiolar and marginal glands on the same leaf  and P. racemosa.The secretory parenchyma does not show druses or vascular endings in most species. Nevertheless, vascular endings formed only by phloem occur in inner layers of the secretory parenchyma in osa Figs  and 7B. P. watsoniana is particularly short. Extension of the subsecretory parenchyma in petiolar glands is particularly notable in P. ligularis (The innermost region of the glands is formed by non-secreting parenchyma (subsecretory), vascular bundles and idioblasts with druses Figs  and 8. Tigularis . In glanigularis .P. gardneri vascular endings are composed of both phloem and xylem cells  petiolar nectaries, when present, and leaf blades with nectaries in the shape of spots. Passiflora has two to six petiolar nectaries (rarely absent or more than six), and in the foliar blades nectaries may be either absent or present  [Tetrapathea has petiolar nectaries, up to two per petiole when present, in ovoid, sub-sessile or crateriform shapes. When present, it has up to eight laminar nectaries that are ovoid and, in some cases, inserted in pairs between the median and primary veins at the base of the leaf blade, or even dispersed along the major veins [al ones) , 2. Tetror veins .Passiflora, many species displaying both petiolar and laminar glands in the same specimen. As mentioned in the description of the subgenus, the laminar glands observed in the present study were marginal, with the exception of the glands of the abaxial surface of leaf blade of P. coccinea. Still, Silva et al. [Passiflora, P. glandulosa Cav. In the subgenus Decaloba, the analyzed species have only one type of gland, either petiolar or dorso-laminar, except for P. ferruginea, which has petiolar glands and ocellus glands on abaxial surface of leaf blade. Among the species belonging to the subgenus Deidamioides, P. deidamioides show features in common with those described for the group, with two petiolar nectaries, in this case, nectaries are also present in the petiolule. However, for P. contracta, besides the petiolar nectaries, nectaries in the abaxial surface of leaf blade are also present. Unlike the description for the group, P. haematostigma, from the subgenus Astrophea, has nectaries on the foliar blade in addition to petiolar nectaries.Among the analyzed species, we found considerable morphological diversity in the glands of the subgenus a et al.  reportedPassiflora, we can denominate such pattern I glands as elevated nectaries ("Hochnektarien") according to the classification of Zimmermann [Passiflora species, other authors, such as Durkee [et al. [Piriqueta and Turnera (genera currently within Passifloraceae) Gonzales & Ocantos [Among species that show glands defined as EFN and verified through chemical tests for the presence of total carbohydrates , 28–30, mmermann . In descs Durkee  and Silv Ocantos  classifiPassiflora contracta, P. ferruginea, P. misera and P. organensis, two of which have glands exclusively on the abaxial surface of the leaf blade (P. misera and P. organensis), while the other two have petiolar glands that fall under morphological pattern I. The marginal glands included in this morphological type are present in only two of the analyzed species, P. galbana and P. odontophylla. Since this morphological pattern is also representative among the EFN of Passiflora, we classified these glands as flattened nectaries ("Flachnektarien") according to Zimmermann [et al. [Piriquetta Aubl. and Turnera L.  present on abaxial surface of leaf blade and some marginal glands among the flattened nectaries.In pattern II, the glands that occur on abaxial surface and margin of leaf blade have a circular shape forming an ocellus, and the projection in relation to the leaf tissue is not evident to the naked eye. In this group, we mention mmermann . Durkee mmermann  mentionemmermann  to Zimmemmermann  classifi [et al.  classifi [et al.  classifi [et al.  also plaThe nomenclature of the different glandular shapes varies among authors. The description of the glands for some species is often mentioned only as sessile or stipitate, or as concave or convex , 28, 30.et al. [Passiflora species. When analyzing EFN in several species of Piriquetta and Turnera , Gonzales & Ocantos [Passiflora.In this work, we are using nomenclature for gland shape based on the solid form descriptions found in Radford et al.  and Harret al. . This ap Ocantos  divide tPassiflora species [Anatomically, the EFN show small variations of the same pattern among the various studied species. All extrafloral glands framed as nectaries are vascularized nectaries according to the definition of Elias . Anatomi species –30, 32,  species  named thPassiflora species, the secretory parenchyma contains isodiametric cells that are larger than those of the epidermis, usually more vacuolated with a dense cytoplasm and abundant calcium oxalate crystals [et al. [In crystals , 18, 28. [et al.  report t [et al.  verifiedPassiflora and Malesherbia (currently Passifloraceae). In the genus Passiflora the author reported that these "trichomes" are restricted to some species like Passiflora clathrata Mast., P. foetida, P. lepidota Mast. and P. villosa. Solereder [In addition to the presence of EFN, as described by several authors in Passiforaceae, Solereder  reportedolereder  also repDysosmia (DC.) Killip and Dysosmioides Killip (subgenus Passiflora) have a petiole without a "true gland", although they often have gland-tipped hairs. On the other hand, in an extensive work on EFN in Passifloraceae, Cusset [P. foetida as glandular pseudo-hairs ("pseudo-poils glanduleux"). When assessing glandular development in this same species, Roth [Killip  highligh, Cusset , named tes, Roth  concludePassiflora foetida cytologically and chemically, Durkee et al. [in situ. The authors conclude that the glands present in P. foetida may represent a transition from true EFN to lipid-secreting glands, subsequently denominating them as resin glands.When analyzing e et al.  treated et al. [The glands described in the present work as type II are small and elongated, with a cylindrical stipe, which can, after only a cursory analysis, be confused with trichomes, having an anatomical structure similar to that of glands described by Roth  and Durket al. . This glet al. , 34, 35.et al.  has showPassiflora foetida already described as having resin glands, P. arida, P. sublanceolata and P. villosa studied in the present work have secretory glands with very similar morphological and anatomical characteristics. Also, they did not show a positive reaction to the glucose strip test, suggesting that they are structurally similar to the resin gland in Passiflora foetida.Apart from Dysosmia DC. is characterized by the presence of gland-tipped hairs, and P. foetida is coated by small sticky glands and sticky hairs [Dysosmia species may be related to the presence of resin glands, a probable evolutionary novelty. However, this hypothesis needs to be verified in a phylogenetic context. A similar situation was reported by Conceição et al. [Chamaecrista (Leguminosae). In the Bignonieae (Bignoniaceae) tribe, Nogueira et al. [The section ky hairs , 8. The o et al. , who vera et al.  verifiedPassiflora. The combined morphological and anatomical analyses, together with preliminary tests for the presence of glucose in the exudate of different Passiflora subgenera, suggests the occurrence of two categories of glands, resin glands and nectaries, the last one with a wide morphological diversity. The next challenges will involve determining the nectary histochemistry and ultrastructure, as well as the chemical nature of the exudate. Once the hypothesis of resin glands has been corroborated only in species of the Dysosmia section, we can think of a new interpretation of the evolution of the secretory glands in the group.In summary, this work reflects the initial stage of a much more extensive study of extrafloral secretory glands in S1 FigA-C Petiolar glands in P. actinia, P. ambigua and P. coccinea, respectively. D Gland of P. coccinea on abaxial surface of leaf blade. E-F Glands of P. contracta on petiole (E) and on abaxial surface of leaf blade (F). G-H Glands of P. deidamioides on petiole (G) and petiolule (H). I-J Glands of P. edmundoi on petiole (I) and at the margin of leaf blade (J). K-L Glands of P. eichleriana on petiole (K) and at the margin of leaf blade (L).(TIF)Click here for additional data file.S2 FigA-B Glands of P. elegans on petiole (A) and at the margin of leaf blade (B). C-D. Glands of P. ferruginea on petiole (C) and on abaxial surface of leaf blade (D). E-F. Glands of P. galbana on petiole (E) and at the margin of leaf blade (F). G. Gland of P. gardneri at the margin of leaf blade. H-I. Glands of P. haematostigma on petiole (H) and at the margin of leaf blade (I). J-K. Glands of P. incarnata on petiole (J) and at the margin of leaf blade (K). L. Gland of P. kermesina on petiole.(TIF)Click here for additional data file.S3 FigA. Gland of P. kermesina at the margin of leaf blade. B-D. Petiolar gland of P. laurifolia, P. maliformis and P. ligularis, respectively. E-F Glands of P. miersii at the margin of leaf blade (E) and petiole (F). G. Gland of P. misera on the abaxial surface of leaf blade. H. Petiolar gland of P. morifolia. I-J Glands of P. odontophylla on petiole (I) and at the margin of leaf blade (J).(TIF)Click here for additional data file.S4 FigA. Gland of P. organensis on abaxial surface of leaf blade. B. Petiolar gland of P. racemosa. C-D. Glands of P. serratodigitata on petiole (C) and at the margin of leaf blade (D). E. Petiolar gland of P. setacea. F-G. Glands of P. sidifolia on petiole (F) and at the margin of leaf blade (G). H. Petiolar gland of P. suberosa. I-J. Glands of P. subrotunda on petiole (I) and at the margin of leaf blade. K Gland of P. umblicata at the margin of leaf blade. L. Petiolar gland of P. watsoniana.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


This report reviews the European, National, and Regional catalogues of protected species, focusing specifically on the Orchidaceae family to determine which species seem to be well-protected and where they are protected. Moreover, this examination highlights which species appear to be underprotected and therefore need to be included in some catalogues of protection or be catalogued under some category of protection. The national and regional catalogues that should be implemented are shown, as well as what species should be included within them. This report should be a helpful guideline for environmental policies about orchid's conservation in Spain, at least at the regional and national level. Around 76% of the Spanish orchid flora are listed with any figure of protection or included in any red list, either nationally (about 12–17%) or regionally (72%). The family Orchidaceae is widely represented in Europe, with 35 known genera, of which 25 are present in the Iberian Peninsula—26 including the Canary Islands—where the representation of this family is one of the most extensive ones, at least on an extratropical, equivalent level or only slightly lower than in other countries of the Mediterranean region. Taxonomic and floristic studies of this family in the Iberian Peninsula have been increasing slowly, though in a fragmented way over time. The first published quotes regarding the Orchidaceae family date back to 1861 , but it  Serapias , wh, wh86],  Corallorhiza trifida Châtel., Epipogium aphyllum Sw., O. robusta, and Orchis spitzelii Saut. ex. W. D. J. Koch , according to the 2010 Red List of Spanish Vascular Flora: J. Koch  86].. CoralloC. trifida. In the 2000 Red List of Threatened Vascular Flora of Spain, this species was listed as Endangered (EN), having gone to be classified as Critically Endangered (CR) in the 2008 Red List of Threatened Vascular Flora of Spain . H.. H.H. mence 2004  and it ince 2004 , 82 and nce 2004 ; therefo D. cantabrica, Epipactis phyllanthes G.E. Sm., O. canariensis, Serapias nurrica Corrias and S. occidentalis.The vulnerable orchid species include D. cantabrica, O. canariensis and S. occidentalis. The fourth one is O. robusta (CR), which was discussed above.There are sixteen species included in the Red List of Spanish Vascular Flora  86], an, an86], O. robusta, D. cantabrica, and S. occidentalis—most likely are in the Red List and no other catalogue because they have recently been described as separate species . If. IfD. caIn Tables 2 D. sulphurea. In addition, Gennaria diphylla (Link) Parl. and Gymnadenia conopsea (L.) R. Br. in W. T. Aiton, both listed as VU in the Red List of Andalusia, should be included as well in the RC of Andalusia [The Red List of Andalusia . Andalusndalusia . C. trifida and C. calceolus    [86].In the case of Asturias  Table 5Table 5,  S. nurrica is notable, as it is also included in the 2010 Red List of Spanish Vascular Flora (VU)    86]. Th. Th S. nred (CR)  [86]. D. sulphurea and N. gabasiana are listed as CR, and the other species listed as Rare in the RC of the Basque Country  are catalogued in the Red List of the Basque Country as VU, as well as Orchis italic Por in Lam., which is still listed as VU. The seventh species, Himantoglossum hircinum (L.) Spreng., is catalogued as Special Interest in the RC of the Basque Country whereas in the Red List of the Basque Country it is catalogued as Near Threatened. The Red List of the Basque Country adds to these seven species five more: E. phyllanthes, which is listed as CR in the Basque Country and as VU at the national level according to the 2010 Red List of Spanish Vascular Flora ( Barlia robertiana (R. J. Loisel) Greuter and Orchis cazorlensis Lacaita, both listed as CR; Ophrys aveyronensis (J. J. Wood) P. Delforge in P. Delforge & D. Tyteca as VU; and Orchis papilionacea L., which is classified as DD ; Ba; Ba D. sed as DD . It woul H. metlesicsianum (included as B. metlesicsiana. in the category Extinction Danger) .. H. metl Epipactis palustris (L.) Crantz; however, it seems clear that E. phyllanthes should be included as well, as it is included in the 2010 Red List of Spanish Vascular Flora as a vulnerable species (VU) (The RC of Cantabria  Table 1 includes G. odoratissima (DD), D. sulphurea (LC), and N. gabasiana (LC) ( O. spitzelii is not included and perhaps it should be due to the few populations of this species in this region [ana (LC)  86]; ho; ho G. os region ; this sp C. trifida, E. aphyllum, and O. spitzelii) or Endangered  at the national level, all of them catalogued as in Danger of Extinction according to the RC of Catalonia; only G. odoratissima (DD) is not threatened according to the Spanish Red List, classified as Vulnerable according to the RC of Catalonia. Therefore, and considering that it is one of the most recent catalogues, the regional catalogue of Catalonia seems to be very up to date. It should be noted that it is the only regional catalogue to include O. spitzelii, an endangered species according to the 2010 Red List of Spanish Vascular Flora  (ora (LC)  86]. Th. Th D. iD. insularis and S. perez-chiscanoi; in the case of the latter, catalogued for Extremadura as Endangered and however listed nationally as Near Threatened (NT) according to the 2010 Red List of Spanish Vascular Flora . It should be listed as VU according to the 2010 Red List of Spanish Vascular Flora , it should at least include D. cantabrica, catalogued nationally as VU according to the 2010 Red List of Spanish Vascular Flora  according to the 2010 Red List of Spanish Vascular Flora  Rich and Neottia nidus-avis (L.) Rich. The list of possible species to be included is long, with very specific or apparently extinct species in this autonomous community, as evidenced by recent studies .. Orchis After conducting a joint analysis of the different regional catalogues, the following observations can be made. Orchis, with 28 inclusions of different species in the regional catalogues, which precisely shows the greater number of species at the national level (21 species) (R2 = 0.79) (species)  76]. In. In Orch = 0.79) . Neottia, single grazing (Neottia nidus-avis (L.) Rich), and Spiranthes, with two species , which are included in six and nine regional catalogues, respectively.At the opposite end, there are the genera Neottia and Spiranthes, as they are only represented by one and two species, respectively.In a complementary manner, in  Orchis, which remains standing with 16 species included in various regional catalogues, the other genera are represented through six to nine species in the different regional catalogues. This drop in the number of species is because several species are repeated in several catalogues, but there are really not so many different species, especially in the case of Orchis and Epipactis, featuring 28 and 15 appointments in different regional catalogues . Likewise, if the number of times that a genus is included in any regional catalogue is correlated with the number of species of that genus included in regional catalogues, the correlation is quite high , as expected.For talogues  correspotalogues . The cor S. aestivalis and N. nidus-avis, found in seven and six regional catalogues, respectively. None of the six most cited species in regional catalogues (four to seven cites) are included in the 2010 Red List of Spanish Vascular Flora [ S. aestivalis is included in the CNEA [Similarly, ar Flora , which ithe CNEA .R2 = 0.1; figure not shown); nevertheless, it is quite obvious for some regional catalogues that seem obsolete, as in the cases of Madrid [ E. aphyllum in Aragón, La Rioja, and Navarre, or multiple species that appear necessary to include in the catalogue of Madrid according to De la Torre & Gamarra [As evident in f Madrid , Asturiaf Madrid , Navarref Madrid , La Riojf Madrid , and Basf Madrid , with an Gamarra , which i Gamarra , the Can Gamarra , Cantabr Gamarra , and Gal Gamarra .R2 = 0.38; figure not shown), amounting to R2 = 0.57 if the curious case of Andalusia mentioned above is not taken into account (figure not shown). Although this regional catalogue dates from 2012, it is suggested to include at least four more species based on the Red List of the Vascular Flora of Andalusia [However, if the correlation is made between the antiquity of the catalogue and the number of species suggested to include, in this case, an acceptable correlation is obtained  and the mountainous regions of the Centre-North  or South , where on average there are more than 16 genera of orchids, as opposed to other regions with a low diversity of orchids  . C. calceolus, which is the species that is best protected at a legal level, and S. aestivalis.There are no orchid species whose distribution area includes Spain that are included in the Berne Convention , and theOut of the 96 species of the Spanish orchid flora considered in this review, up to 73 species are included in any catalogue of protection; 16 are in the 2010 Red List of Spanish Vascular Flora (five of them not endangered) , and 69  H. metlesicsianum (Endangered) [ C. calceolus (Endangered) [ O. provincialis and S. aestivalis  [ C. calceolus (EN) and H. metlesicsianum (EN), which are already included [The National Catalogue of Threatened Species (CNEA) lists a total of four species:angered) , 82, C. angered) , 82, andincluded  , E. aphyllum (CR), O. spitzelii (CR), C. calceolus (EN), and G. odoratissima (DD).It is remarkable that the RC of Catalonia  is the oThe other fourteen regional catalogues would need to be modified to a greater or lesser extent, highlighting the RC of Madrid , which i E. aphyllum (CR) and perhaps O. spitzelii (CR) (its presence not confirmed); the RC of the Balearic Islands [ O. robusta (CR), a species endemic to this autonomous community; the RC of the Canary Islands [ O. canariensis (VU), a species endemic to this autonomous community; the RC of Extremadura [ S. occidentalis (VU), which is endemic to Extremadura; the RC of Galicia [ D. cantabrica, endemic to this autonomous community.Several regional catalogues should also be highlighted for their remarkable absences: The RC of Aragón  should b Islands  should b Islands  should iremadura  should i Galicia  should iOrchis spitzelli (CR): this species should be included in the regional catalogue of Castilla y León. Epipogium aphyllum (CR): this species should be incorporated into three regional catalogues: Aragón, La Rioja, and Navarre. Epipactis phyllanthes (VU): this species should be included in two regional catalogues: Cantabria and Basque Country. The main species that are listed in the 2010 Red List of Spanish Vascular Flora  and that


To the Editor: The Chernobyl Exclusion Zone (CEZ) surrounds the center of the 1986 Chernobyl nuclear power plant disaster. Preliminary study shows predominance of Dermacentor reticulatus ticks in the CEZ; ticks of other species, such as Ixodes ricinus, are surprisingly rare, even in habitats where they should be relatively common  in these ticks is higher in the CEZ than in other regions  by use of PCR with RpCS.409d and RpCS.1258n primers (http://www.ncbi.nlm.nih.gov/blast/download.shtml). All obtained sequences were submitted to GenBank (accession nos. KX056493 and KX056494).DNA was extracted by use of the ammonium hydroxide method (Rickettsia spp. was detected in 72.64% of ticks (R. raoultii isolated from D. marginatus ticks from China (GenBank accession nos. KU171018.1 and KT261764.1) and D. reticulatus ticks from Poland (KT277489) and Hungary (LC060714.1). In the CEZ, the predominant tick species is D. reticulatus; no D. marginatus ticks have been found in the CEZ (R. raoultii vector is D. reticulatus ticks. Infection with of ticks . A higheR. raoultii infection among D. reticulatus ticks (68.42%–74.07%) is significantly higher in the CEZ than in other regions. A previous study found prevalence of A. phagocytophilum infection in the CEZ to be high, mainly associated with Ixodes ticks (D. reticulatus ticks. The prevalence of Babesia canis infection, also vectored by this tick, was within the usual range (D. reticulatus ticks in the CEZ than in other region is not known and needs more study. The prevalence of these pathogens among mammals that inhabit the CEZ is also not known; the influence of radiation on pathogen level has not been studied. The nucleotide sequences of R. raoultii detected in ticks in the CEZ are identical to sequences originating from other regions and deposited in GenBank; the sequences of A. phagocytophilum and B. canis from the CEZ were also similar to those described elsewhere (R. raoultii infection prevalence is radiation, then radiation also influences the ticks—some morphologic abnormalities have been noted on D. reticulatus ticks collected from the CEZ (The prevalence of R. raoultii in D. reticulatus ticks in the CEZ. The structure of zoonotic foci in the CEZ seems to differ from that in other regions. Confirmation of this hypothesis needs follow-up study of tickborne pathogens in wild mammals that might serve as a source of infection for ticks in the CEZ. This study confirms presence of


Psittacosaurus sp. from the Chinese Jehol biota [Psittacosaurus was countershaded [Psittacosaurus sp. inhabited a closed habitat such as a forest with a relatively dense canopy.Countershading was one of the first proposed mechanisms of camouflage , 2. A daol biota , 17. Thiershaded  with a l  •Psittacosaurus suggest countershading camouflagePreserved pigments in the dinosaur •We predicted the optimal countershading camouflage for different light environments•The dinosaur’s patterns would have been cryptic in a forest, but not open, habitat•We can also infer that dinosaur predators used shape-from-shading cues to detect prey Psittacosaurus was countershaded and cryptic in a forested environment.Countershading camouflage uses a dark-to-light gradient from back to belly to counter the light-to-dark gradient created by illumination. The body appears flatter and less conspicuous. Vinther et al. use 3D reconstruction and radiance modeling to show that the dinosaur  The past years have witnessed an increased interest in the coloration of fossil animals. Studies of fossil melanosomes in particular have allowed reconstruction of the plumage patterns of theropod dinosaurs , 13, 15 Psittacosaurus sp. (SMF R 4970), from the Early Cretaceous Jehol Biota, with tail filaments and extremely well preserved skin preservation as a compressed film outlining the body, superimposed on the skeleton [Here we study a ceratopsian ornithischian, skeleton   imaging . The colDistinct scales are present on the specimen  and S4. Internal organs, such as the liver, may also contain melanin , 25. We There is a clear difference in pigmentation between the ventral surface on the lower belly and tail relative to the dorsum, which is more heavily pigmented . The traThe distribution of the pigmentation patterns in SMF R 4970 suggests color patterns congruent with camouflage through background matching and countershading, in addition to some putative disruptive coloration on the legs, coupled with strong facial pigmentation suggestive of a signaling function and some protective/strengthening melanization.Psittacosaurus SMF R 4970, carefully considering the volume and thickness of the body based on myoanatomical reconstructions and the preserved body outline (see the In order to interpret and further test the function of the observed color patterns, we produced a life-size model of  see the . We prod see the  and S2.  see the  and S17.The animal’s reconstructed patterns more closely match the predicted optimal countershading for diffuse illumination, as would be experienced in a “closed” light environment such as under a forest canopy . The graPsittacosaurus. It is, of course, possible that there was variation in color across the species’ range, and we would predict that this would mirror differences in the light environment.By comparing the observed distribution to that predicted for obliterating shape-from-shading cues under diffuse illumination, we infer that this small ceratopsian lived in a closed light environment such as under a forest canopy. Paleobotanical studies indicate that the lakes of the Jehol biota were surrounded by predominantly coniferous forest, with only a minor contribution from deciduous plants , 34, 35.Bristles on the tail  would haIt has been shown that countershading also correlates with positional behavior, as is observed in many species of caterpillar that live upside down and exhibit reverse countershading , 46. In More speculatively, we suggest that the predators of this species most likely used shape-from-shading cues in detecting prey, given that optimized countershading obliterates this information . The likPsittacosaurus sp. held at the Senckenberg Museum, SMF R 4970, was studied in the public exhibit (see the The well-preserved specimen of  see the . Small s see the .The preserved skeleton was measured and used to produce a 1:1 anatomical model of SMF R 4970. Cranial details were obtained from a 3D printed skull of a different specimen. Volume was added to the skeleton based on knowledge of major muscle groups and the preservation of integument outlines of the specimen. Decay has not lead to distortion of the integument relative to the skeleton, which suggests rapid burial after deposition and little prior rot to the animal before it sank to the bottom of the lake. Exposed scale patterns were projected onto the model and extrapolated between unexposed regions. Exposed color patterns were similarly carefully projected onto the model and extrapolated across unexposed parts based on similar patterns, assuming bilateral symmetry between left and right patterns as confirmed in certain parts (for more information, see the In order to predict the environment in which the observed countershading was optimal, we took a model, painted uniformly gray, and photographed it under different lighting conditions; we chose a sunny and a cloudy day and photographed the specimen in an open and a closed habitat. The model was cropped and the image inverted in order to produce the optimal countershading patterns under each circumstance in order to compare with the observed patterns in the fossil (for more information, see the J.V. conceived the study. J.V., R.N., M.P., T.G.K., and G.M. studied the color patterns and taphonomy. R.N. modeled and painted the dinosaur. S.L. provided additional 3D models and figures. E.R. and S.L. supervised the anatomical modeling. I.C.C. and J.V. did the calibrated photography. S.L. did the 3D photogrammetry. T.G.K. and M.P. performed the LSF imaging. I.C.C. did the visual modeling. J.V. and I.C.C. wrote the paper with comments from all authors.


G. pentaphyllum (Gynostemma pentaphyllum), a creeping herbaceous perennial with many important medicinal properties, is widely distributed in Asia. Gypenosides (triterpenoid saponins), the main effective components of G. pentaphyllum, are well studied. FPS (farnesyl pyrophosphate synthase), SS , and SE  are the main enzymes involved in the synthesis of triterpenoid saponins. Considering the important medicinal functions of G. pentaphyllum, it is necessary to investigate the transcriptomic information of G. pentaphyllum to facilitate future studies of transcriptional regulation. After sequencing G. pentaphyllum, we obtained 50,654,708 unigenes. Next, we used RPKM (reads per kilobases per million reads) to calculate expression of the unigenes and we performed comparison of our data to that contained in five common databases to annotate different aspects of the unigenes. Finally, we noticed that FPS, SS, and SE showed differential expression of enzymes in DESeq. Leaves showed the highest expression of FPS, SS, and SE relative to the other two tissues. Our research provides transcriptomic information of G. pentaphyllum in its natural environment and we found consistency in unigene expression, enzymes expression , and the distribution of gypenosides content in G. pentaphyllum. Our results will enable future related studies of G. pentaphyllum. Gynostemma pentaphyllum (Thunb.) Makino is a kind of creeping herbaceous perennial that is distributed in Asia. Gynostemma pentaphyllum (G. pentaphyllum) grows in many places of China, including Guangxi, Guangdong, Fujian, Guizhou, Yunnan, Hubei, Anhui, Hebei, Jiangsu, Henan, Shandong, Sichuan, and Shanxi and in Taiwan. G. pentaphyllum also grows in neighboring countries such as Bangladesh, India, Indonesia, Japan, Republic of Korea, and Malaysia  .–16.14–16mations) . G. pentaphyllum have various and vital applications in medicine and health [ G. pentaphyllum [ G. pentaphyllum relative to Panax ginseng (P. ginseng). It was interesting that G. pentaphyllum has such diversity in triterpenoids compared to other plants [ G. pentaphyllum related to the synthesis pathway of triterpenoids. However, this article had two important limitations. First, the samples used for sequencing only included two tissues (leaves and roots) and G. pentaphyllum that was sampled and sequenced was planted in water and not in its natural environment [ G. pentaphyllum exhibits great phenotypic diversity in different environments because of its strong adaptability [ G. pentaphyllum was determined. In 2015, Zhao et al. identified EST-SSR makers by analyzing the sequencing data of two species of Gynostemma (Cucurbitaceae) [ G. pentaphyllum were mixed up together to extract RNA for constructing cDNA to sequence. In other words, the sequencing data and related information in that article were a mixed result and these results could not be classified by tissues. Therefore, to address this deficiency of knowledge, we collected G. pentaphyllum in natural environment and sequenced its transcriptome separately by Illumina's NextSeq 500.Like the ginsenosides, gypenosides (triterpenoids) ind health . Howeveraphyllum –28. In or plants . This isr plants –37, becar plants –39. In 2ironment . This istability , 40–44. itaceae) . In thatG. pentaphyllum used for sequencing was planted in the Medicinal Plant Garden of Guangxi Traditional Chinese Medical University, Nanning City, Guangxi Autonomous Region, China. In July 2015, we harvested G. pentaphyllum after identification by Mr. Yilin Zhu . Fibrous roots, leaves, and stems were separately collected and cleaned and removed of impurities like soil  . Finally, the samples were saved in cryotubes and submerged in liquid nitrogen immediately. G. pentaphyllum was sent to Personalbio Company  for transcriptome sequencing using Next-Generation Sequencing (NGS) technology based on the sequencing platform of Illumina's NextSeq 500. First, the mRNA was cleaved into little segments after treatment with chemical reagents and high temperature. Next, the segments were used to construct a cDNA library that was sequenced by paired end (PE) reads.The plant tissue sample ofTrinity  professional software was used to assemble the RNA sequence . First,  G. pentaphyllum and the calculation method of RPKM is described below [p value < 0.05). The final result was displayed by Venn diagram.RPKM (reads per kilobases per million reads) was used to calculated unigene expression ofed below . Before ed below . The RPKed below . The expAfter categorization, unigenes were annotated for functions using five databases: NCBI nonredundant protein (NR) sequences, Gene Ontology (GO) , 51, Kyo30H52O3) as a standard sample to calculate the actual content of gypenosides in samples by the standard curve method.First, dried powder of the sample (about 15 mg) was mixed with 10 mL of extraction solvent (ethanol containing 5.0% pure water) and was processed by a continued supersonic treatment for 30 minutes. Second, the mixed solvent was evaporated to dryness using a rotary evaporator. The dry gypenosides were redissolved in 5.0 mL hot water (50°C). Third, this solution was applied to a chromatography column containing D101 macroporous resin and allowed to stand for 20 minutes. Fourth, pure water was used to rinse unbound material from the macroporous resin, while extraction solvent (ethanol containing 50% pure water) was used to remove the gypenosides form column. Fifth, the solvent of gypenosides was brought up to 5.0 mL and processed with a color reaction by vanillin. We then detected the absorbance of the solvent (after color reaction) by UV-Vis Spectrophotometer at a wavelength of 584 nm. Finally, we used Panaxadiol  for all transcriptome sequences in the NR database. The transcriptome sequence with the highest score in BLAST was saved and the transcriptome sequences with same gi number were categorized as coming from the same unigene. Finally, we obtained 50,654,708 unigenes with a mean length of 755 bp. The overview of sequences is presented in The mRNA ofWe used five databases, NR, GO, KEGG, eggNOG, and Swiss-Port, to annotate unigenes for functions . The overview of annotation is listed in β-AS (beta-amyrin synthase) in β-AS showed the highest expression in leaves and the lowest expression in fibrous roots. In the unigene expression of β-AS, the leaves showed almost 125 times higher expression than in the fibrous roots. The unigenes of FPS, SS, SE, and β-AS showed higher expression in the stems than in the fibrous roots. Based on this sequencing data, we detected the differential expression of unigenes using the software DESeq. We obtained the upregulated and downregulated unigenes in the pairwise comparison among the data from the fibrous roots, stems, and leaves  and unigenes with lower or higher expression were the minority of AUC . In the d leaves . We alsod leaves . Combini G. pentaphyllum. Leaves had the highest content (3.189%) of gypenosides of all samples (R2) of 0.996 in the standard curve indicates that our result was accurate and reliable .A UV-Vis Spectrophotometer was used to detect the content distribution of gypenosides in samples , and theBased on the KEGG Pathway, a more detailed result about the enzymes expression in the triterpenoids synthesis was obtained. We categorized related enzymes into three parts according to the synthesis pathway of the triterpenoids: (1) enzymes involved in the synthesis of IPP or DMAPP, (2) enzymes involved in the synthesis and cyclization of squalene, and (3) enzymes in the squalene functionalization reaction . We focuG. pentaphyllum is a creeping herbaceous perennial with medicinal properties used in traditional Chinese medicine. Triterpenoid saponins, the main effective components of G. pentaphyllum, have been widely studied [ studied , 23, 24. studied . We anal G. pentaphyllum. Since G. pentaphyllum has wide applications for health and medicine, it is essential to identify the secondary metabolites with various and vital medicinal functions.We found that the expressions of unigenes and enzymes were positively associated with the distribution of gypenosides content. Generally speaking, unigenes and enzyme expression  in the samples  determined to the distribution of gypenosides content. Higher expression of unigenes and enzymes (encoded by unigenes) caused the higher content of enzymes' production (gypenosides), and the lower expression of unigenes and enzymes caused less enzyme production. This consistent result could facilitate future studies of other secondary metabolites in G. pentaphyllum determined in our study showed high similarity to the transcriptome sequences related to bitterness in cucumber as reported previously [ G. pentaphyllum. The BLAST result was surprising, as all thirteen available sequences related to bitterness in that article showed a high degree of similarity to the specific unigene sequences of G. pentaphyllum. This high similarity predicted that genes related to the biosynthesis, regulation, and domestication of bitterness in cucumber may also be present in G. pentaphyllum. G. pentaphyllum also has two tastes (sweet and bitter) and this difference of taste may be caused as in cucumber. The taste of G. pentaphyllum from bitter to sweet predicts that G. pentaphyllum may change in response to domestication and contain a similar mutation. A further genetic exploration of the domestication of G. pentaphyllum may provide understanding of its changed taste.One interesting finding was the observed differences between general expression and individual expression of unigenes . A groupeviously . We down G. pentaphyllum using sequencing, we corrected the limitations of the previous study and enriched the analysis of triterpenoid synthesis of G. pentaphyllum. G. pentaphyllum used for analysis in our study was planted in a natural environment and three common kinds of plant tissues  were used to provide tissue samples for sequencing. Together with the sequencing, an exploration of the distribution of gypenosides content was performed to confirm the analysis of enzymes and unigenes. We found a positive association of unigene expression, enzyme expression, and the distribution of gypenosides. Our study will facilitate more genetic studies examining the regulation of transcription and the change of bitterness in G. pentaphyllum.Although this study was not the first report of the transcriptome information for G. pentaphyllum, we used RNA sequencing technology to sequence the transcriptome of G. pentaphyllum. We found a positive association of unigene expression, enzyme expression, and the distribution of gypenosides. Our results will enable future related studies of G. pentaphyllum.To provide more complete and high-quality transcriptional information of naturalG. pentaphyllum (Sample) 01. Description: This is the picture of intact G. pentaphyllum in our study.Figure S1. G. pentaphyllum in our study.Figure S2. G. pentaphyllum (Sample) 02. Description: This is the picture of intact G. pentaphyllum in our study.Figure S3. Leaves (Sample). Description: This is the picture of leaves sample of G. pentaphyllum in our study.Figure S4. Stems (Sample). Description: This is the picture of stems sample of G. pentaphyllum in our study.Figure S5. Fibrous Roots (Sample). Description: This is the picture of fibrous roots sample of Figure S6. The general result of annotation. Abbreviations: NR: Nonredundant protein sequences; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genome; eggNOG: Evolutionary genealogy of genes: Nonsupervised Orthologous Groups.Figure S7. The result of GO Slim. Abbreviation: GO Slim: Cut-down versions of the GO ontologies.Figure S8. The result of eggNOG annotation.Figure S9. The result of KEGG annotation.Figure S10. The standard curve of absorbance.


To the Editor,th month of maintenance therapy due to new onset of non-flushing catheter. The patient had no other complaints. On posterior anterior chest X-ray, the catheter was found to be disconnected from its reservoir  are widely used in these patients for administration of chemotherapeutic agents, parenteral nutrition, fluids, and blood products ,2. Fracteservoir . Echocareservoir . The embeservoir . Our cas


Helicobacter pylori is the most abundant bacterium in the gastric epithelium, and its presence has been associated with the risk of developing gastric cancer. As of 15 years ago, no other bacteria were associated with gastric epithelial colonization; but thanks to new methodologies, many other non-H. pylori bacteria have been identified. It is possible that non-H. pylori may have a significant role in the development of gastric cancer. Here, we discuss the specific role of H. pylori as a potential trigger for events that may be conducive to gastric cancer, and consider whether or not the rest of the gastric microbiota represent an additional risk in the development of this disease. Helicobacter pylori was the first bacterium whose presence was associated with increased risk of developing any type of cancer, in this case gastric cancer [H. pylori was, and continues to be, responsible for one of the most prevalent infections in humans globally [H. pylori eventually developed gastric cancer [H. pylori infected more than half of the world’s population [H. pylori prevalence that has been occurring in the last 100 years [H. pylori is at its peak [c cancer ,2. This globally . Howeverc cancer . H. pylopulation , but it pulation ,6. Furth00 years ,8. This its peak .H. pylori is responsible for a series of histopathological changes in the gastric mucosa that are recognized as major factors in the development of gastric cancer. This particular series of histopathological changes that lead to gastric cancer are collectively known as Correa’s model [H. pylori, and almost 15 years before the recognition of this organism as a risk factor for gastric cancer [H. pylori findings, with the progression from a normal gastric mucosa prior to infection followed by chronic superficial gastritis, atrophic gastritis, intestinal metaplasia, dyspepsia and finally gastric carcinoma [’s model . Correa’c cancer . Surprisarcinoma .H. pylori, many other organisms can be present, colonizing the stomach [H. pylori varies depending of the H. pylori status of the individual [H. pylori in the development of gastric cancer.Recent progress in the identification of bacteria that colonize different body sites of the human host since birth, collectively named microbiota, has provided detailed insights into the numerous organisms present in the gut and a variety of other body sites, including the oral cavity, skin, lungs, etc. . The hum stomach . The reldividual . As a reH. pylori. We also discuss which H. pylori traits contribute to increasing the risk of the development of gastric cancer and whether other members of the gastric microbiota possess similar capabilities.We discuss, in this review, the potential role of the human gastric microbiota in gastric carcinogenesis in the presence or absence of H. pylori infection and gastric cancer was derived from three independent epidemiological studies published in 1991. All three studies reported elevated odds ratios for the development of gastric cancer in subjects who had tested positive for H. pylori more than 2 decades before the diagnosis of gastric cancer, when compared with subjects without H. pylori infection [H. pylori as the most relevant risk factor in the development of gastric cancer [The first solid evidence of the association between nfection ,16,17. Tc cancer .H. pylori as a risk factor in gastric pathology was obtained from animal models, including piglets, dogs and monkeys. Their normal gastric mucosa was challenged with H. pylori, and the development of active and chronic superficial gastritis, and in some cases atrophic gastritis, was confirmed to be associated with H. pylori colonization, but not with gastric cancer [H. pylori inducing a progression from superficial gastritis to intestinal metaplasia  was with the use of the Mongolian gerbil model [H. pylori can lead to the development of gastric cancer [H. pylori challenges were associated with the development of gastric cancer [H. pylori and its role in gastric cancer.The second type of evidence that implicates c cancer . The firil model . Some yec cancer . More rec cancer . This moH. pylori in gastric carcinogenesis is the use of in vitro models. Most of these studies have been dedicated to investigating the role of the major virulence factors of H. pylori, including the cytotoxin-associated gene pathogenicity island (CagPAI). It was discovered that H. pylori strains expressing the CagPAI are more virulent, and are more frequently isolated from patients with severe clinical outcomes of the infection, including peptic ulcer disease and gastric cancer. The CagPAI is a major chromosomal insertion, encoding around 34 genes, that can be acquired by horizontal transfer [H. pylori [H. pylori CagA, vacuolating cytotoxin (VacA), or any of its other components. Assessment of mutagenic activity of these factors using methods such as the Ames test is needed to confirm the role of CagA as a true oncogenic protein. The Ames test is a biological assay to assess mutagenic potential of individual compounds using bacteria to test its mutagenic activity. An in vivo experiment in a transgenic mouse model demonstrated that expression of CagA predominantly in the stomach was associated with epithelial hyperplasia, gastric polyps and adenocarcinoma. Systemic expression of CagA was associated with leukocytosis, leukemia and B cell lymphomas [H. pylori and its components have no direct mutagenic effects in gastric epithelial cells, how does H. pylori affect the gastric epithelium in order for it to be considered a major risk factor in gastric cancer development?Another approach to documenting the role of transfer . The demtransfer . As a re. pylori . It is iymphomas . It is iH. pylori occurs early in life, and is maintained for decades or perhaps for the whole life of the colonized individual. The presence of H. pylori induces a superficial chronic gastritis influencing the balance between the rate of cellular loss and regeneration [H. pylori maintains a constant production of a cascade of cytokines, which attracts neutrophils that generate oxidative radicals that have the potential to damage the host DNA. Infection with H. pylori has been associated with a reduction in cell replication, and increase in apoptosis, autophagy induction, and endoplasmic reticulum and oxidative/nitrosative stress [H. pylori, there is a greater chance of acquiring potentially malignant characteristics, which may explain the relevance of this infection as a major risk factor in the development of gastric cancer [H. pylori infection and the development of gastric cancer.To answer this question, we need to revisit the Correa model. Colonization with neration . The chre stress . These ic cancer . This chc cancer . The proH. pylori and the host. The original study of El-Omar et al. [H. pylori-associated gastric cancer. This study provides strong evidence for the influence of host genetic factors and the possibility that the immune necrotic state is relevant for the development of gastric cancer. In addition, these results indicate that an increased inflammatory response, particularly linked with an over-production of pro-inflammatory cytokines and mediators in the Th1 pathway, increase the odds ratio of H. pylori-positive individuals developing gastric cancer [H. pylori strains expressing CagA and VacA, and simultaneously carrying cytokine polymorphisms associated with pro-inflammation. Here, the odds ratio for developing gastric cancer increases more than 40-fold [Pro-inflammation is a key player in the chronic interaction between r et al.  reportedc cancer . Several 40-fold .H. pylori, other bacteria also colonize the gastric mucosa [H. pylori microbiota in the process of gastric carcinogenesis? Particularly, what role do these microorganisms play once H. pylori has produced gastric atrophy and intestinal metaplasia, and is no longer capable of colonizing the affected gastric mucosa?With the improvement of sequencing technology, it has been demonstrated that in addition to c mucosa . An impoH. pylori infection [H. pylori sequences in biopsy samples of subjects who had been identified as H. pylori negative by most conventional methods [H. pylori by conventional methods, the sequence data demonstrated the relative abundances of Helicobacter to be <2.0%. The clinical relevance of these low-relative-abundance sequences of H. pylori remains unsolved. To assess the role of the gastric microbiota as a potential player in H. pylori-associated gastric cancer, and to determine the potential interactions between the gastric commensal bacteria and H. pylori, it is important to consider the relative abundance of these players, and what type of effect they could produce. In addition, we need to consider the presence of H. pylori and its impact on the gastric microbiota, as well as the selectivity that H. pylori has in colonizing the gastric epithelium, where any changes affecting the gastric mucosa may affect the ability of H. pylori to colonize the stomach.The recent development of methods for the analysis of 16S rRNA data has provided a clear picture of the bacterial communities present in the human host . Of partnfection . One int methods . These r methods . In the Another relevant point that needs to be considered is the occurrence of dysbiosis in the gastric community. Most currently available studies report changes in the gastric microbiota in patients with gastric cancer compared to those without . BecauseH. pylori in the stomach induces several histopathological changes in the gastric epithelium. In particular, the early pre-malignant changes involving the presence of intestinal metaplasia may precipitate the elimination of H. pylori from the human stomach  ,42, or cH. pylori gastric microbiota in the development of gastric cancer. However, in some regions of the world, despite the decline of H. pylori infections, increased incidence of gastric cancer has been reported [H. pylori, could be implicated in this age-specific increase in gastric cancer [There are no well-designed studies that implicate the participation of non-reported ,45. Intereported ,47,48. Tc cancer . There ac cancer ,47,48,49c cancer ,47,48.A possible explanation for the recent increase in gastric cancer in the USA is perhaps the large immigration of Hispanic populations that has occurred recently . HoweverH. pylori subjects [H. pylori negative and living in high-prevalence areas of gastric cancer and H. pylori infection for potential development of gastric cancer. Two independent studies have recently reported the benefits of antibiotic treatment in reducing the development of gastric cancer particularly in older individuals [A recent Japanese study suggested the assessment of aberrant DNA methylation in gastric tissue as a means to determine risk of gastric cancer . Hypermesubjects . Therefoividuals ,54. BothH. pylori has a major effect on the composition and relative abundance of the gastric microbiota. In the absence of H. pylori the gastric microbiota likely contributes to the perpetuation of the inflammatory stimuli. The role of the microbiota as inflammation stimuli is particularly important in patients previously colonized by H. pylori. The pre-malignant changes in the gastric epithelium may favor the conditions for bacteria other than H. pylori to induce the inflammatory process related to cancer development. The term “point of no return” in the cascade of events that lead to gastric cancer has been associated with patients with intestinal metaplasia and dysplasia, independent of H. pylori status, who are at the highest risk of developing gastric cancer [H. pylori continue on the pathway of gastric carcinogenesis.In conclusion, the published studies support the idea that the presence of c cancer . This ph


Grant 2014–37 (http://www.ec.js.edu.cn/) from Jiangsu Education Department was received by Affiliated Hospital of Stomatology, Nanjing Medical University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The publisher apologizes for the errors.There are errors in the Funding section. The correct funding information is as follows: Grant 81370025 (