Instituto Butantan

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

We present a draft sequence of the genome of Aedes aegypti, the primary vector for yellow fever and dengue fever, which at approximately 1376 million base pairs is about 5 times the size of the genome of the malaria vector Anopheles gambiae. Nearly 50% of the Ae. aegypti genome consists of transposable elements. These contribute to a factor of approximately 4 to 6 increase in average gene length and in sizes of intergenic regions relative to An. gambiae and Drosophila melanogaster. Nonetheless, chromosomal synteny is generally maintained among all three insects, although conservation of orthologous gene order is higher (by a factor of approximately 2) between the mosquito species than between either of them and the fruit fly. An increase in genes encoding odorant binding, cytochrome P450, and cuticle domains relative to An. gambiae suggests that members of these protein families underpin some of the biological differences between the two mosquito species.

Our ability to correlate biological evolution with climate change, geological evolution, and other historical patterns is essential to understanding the processes that shape biodiversity. Combining data from the fossil record with molecular phylogenetics represents an exciting synthetic approach to this challenge. The first molecular divergence dating analysis (Zuckerkandl and Pauling 1962) was based on a measure of the amino acid differences in the hemoglobin molecule, with replacement rates established (calibrated) using paleontological age estimates from textbooks (e.g., Dodson 1960). Since that time, the amount of molecular sequence data has increased dramatically, affording ever-greater opportunities to apply molecular divergence approaches to fundamental problems in evolutionary biology. To capitalize on these opportunities, increasingly sophisticated divergence dating methods have been, and continue to be, developed. In contrast, comparatively, little attention has been devoted to critically assessing the paleontological and associated geological data used in divergence dating analyses. The lack of rigorous protocols for assigning calibrations based on fossils raises serious questions about the credibility of divergence dating results (e.g., Shaul and Graur 2002; Brochu et al. 2004; Graur and Martin 2004; Hedges and Kumar 2004; Reisz and Müller 2004a, 2004b; Theodor 2004; van Tuinen and Hadly 2004a, 2004b; van Tuinen et al. 2004; Benton and Donoghue 2007; Donoghue and Benton 2007; Parham and Irmis 2008; Ksepka 2009; Benton et al. 2009; Heads 2011). The assertion that incorrect calibrations will negatively influence divergence dating studies is not controversial. Attempts to identify incorrect calibrations through the use of a posteriori methods are available (e.g., Near and Sanderson 2004; Near et al. 2005; Rutschmann et al. 2007; Marshall 2008; Pyron 2010; Dornburg et al. 2011). We do not deny that a posteriori methods are a useful means of evaluating calibrations, but there can be no substitute for a priori assessment of the veracity of paleontological data. Incorrect calibrations, those based upon fossils that are phylogenetically misplaced or assigned incorrect ages, clearly introduce error into an analysis. Consequently, thorough and explicit justification of both phylogenetic and chronologic age assessments is necessary for all fossils used for calibration. Such explicit justifications will help to ensure that divergence dating studies are based on the best available data. Unfortunately, the majority of previously published calibrations lack explicit explanations and justifications of the age and phylogenetic position of the key fossils. In the absence of explicit justifications, it is difficult to distinguish between correct and incorrect calibrations, and it becomes difficult to reevaluate previous claims in light of new data. Paleontology is a dynamic science, with new data and perspectives constantly emerging as a result of new discoveries (see Kimura 2010 for a recent case where the age of the earliest known record of a clade was more than doubled). Calibrations based upon the best available evidence at a given time can become inappropriate as the discovery of new specimens, new phylogenetic analyses, and ongoing stratigraphic and geochronologic revisions refine our understanding of the fossil record. Our primary goals in this paper are to establish the best practices for justifying fossils used for the temporal calibration of molecular phylogenies. Our examples derive mainly, but not exclusively, from the vertebrate fossil record. We hope that our recommendations will lead to more credible calibrations and, as a result, more reliable divergence dates throughout the tree of life. A secondary goal is to help the community (researchers, editors, and reviewers) who might be unfamiliar with fossils to understand and overcome the challenges associated with using paleontological data. In order to accomplish these goals, we present a specimen-based protocol for selecting and documenting relevant fossils and discuss future directions for evaluating and utilizing phylogenetic and temporal data from the fossil record. We likewise encourage biologists relying on nonfossil calibrations for molecular divergence estimates (e.g., ages of island or mountain range formations, continental drift, and biomarkers) to develop their own set of rigorous guidelines so that their calibrations may also be evaluated in a systematic way. Most studies use a Bayesian framework for estimating divergence dates with probability curves between a minimum and a maximum bound to represent calibrations (time priors) (Thorne et al. 1998; Drummond et al. 2006; Yang 2006; Yang and Rannala 2006). An appropriately constructed fossil calibration uses the oldest assigned fossil of a taxon as the basis for its minimum age and then constructs these other parameters around it (Benton and Donoghue 2007; Donoghue and Benton 2007). One key to improving the use of paleontological data is recognizing that this first step can be tied explicitly to one or a small set of museum specimens, creating a readily auditable chain of evidence. To minimize error and maximize clarity, all calibration data should be derived explicitly from specific fossil specimens. If links between calibration data and specimens cannot be made, then there are serious questions about the validity of the proposed time priors. In this respect, the fossil specimens used for calibrations represent a standard, much in the same way that a holotype specimen (or type series) is a taxonomic standard. In both cases, these specimens provide a necessary reference point for future inquiries. The explicit reporting of specimen data is just as crucial to the scientific integrity of a fossil calibration study as is making genetic sequences publicly available or reporting analytical methods. Thus, it is worthwhile to compile, reiterate, and expand on the caveats from previous studies that pertain to the construction and reporting of fossil calibrations (e.g., Graur and Martin 2004; Hedges and Kumar 2004; van Tuinen and Hadly 2004a, 2004b; Benton and Donoghue 2007; Donoghue and Benton 2007; Gandolfo et al. 2008; Parham and Irmis 2008; Benton et al. 2009; Ksepka 2009; Sanders et al. 2010) while providing a simple and explicit protocol (in checklist form) to address them. The checklist can be divided into justifying phylogenetic position and justifying age and In cases, the data to calibrations are in a but to be In to derived from is explicitly as for a rigorous and explicit approach is for justifying the use of paleontological and geological data for divergence The can be used to develop new calibrations and as a checklist for and justifying previously published calibrations based on fossils. If all are then a calibration can be of that all the relevant and data should be of specimens to the taxon should be An of the or an phylogenetic analysis that the should be on the of and molecular data should be The and stratigraphic the best of from the should be to a published age and of age should be a fossil used for calibration be based on a specimen that all the that it to be assigned to a taxonomic are from specimens, are to be from a divergence dating studies that use paleontological data for calibrations on from phylogenetic that are based on of specimens to a taxon In cases, the basis for a taxonomic can be as as documenting that the specimen was from the same or where other specimens previously Consequently, are a in et al. Parham fossil are not it is necessary to the and of specimens. may be to specimens from to a taxon there are or through phylogenetic analysis et al. 2004; et al. 2009; In where previously cannot be it is necessary to the calibration to a of specimens (e.g., and Parham or the from the calibration. Incorrect phylogenetic of fossil calibrations can introduce into divergence estimates Brochu van Tuinen and Hedges 2004; et al. dating studies on the paleontological for calibration but of the oldest of a have been in a phylogenetic analysis. Gandolfo et al. in incorrect and taxonomic to inappropriate fossil is a for that are a fossil in a taxon than the data can in the (e.g., and 2005; et al. 2010; Sanders et al. The that may use the same taxon to to biological the and may be the fossil record of is we the use of an approach to and phylogenetically specimens that are relevant for paleontological guidelines can also be to fossils (e.g., in the case that their are and evidence for the of a based on explicit and et al. 2008; et al. 2011). fossils are have phylogenetic the analytical on paleontological it is that evidence the taxonomic of relevant be explicitly A is the of to the oldest geological record of a based upon evidence. can be on provide evidence to are of or with specimens, it can be difficult to distinguish the fossil to the or the of the clade that it is used to the earliest will the of the of the and so assigning fossils to the or the of a clade of evolution that is not fossil specimens of may not be as lack one or more of the as a of or secondary Donoghue and 2009; et al. is for that are on the basis of molecular evidence but for is known (e.g., or et al. is also to in of that evolution their In those cases, the that might be of in the time of divergence from the may be difficult to the to the phylogenetic of specimen used for is not to a paper that the taxon or the of used in the phylogenetic of fossils it to The phylogenetic position of a fossil taxon can be specimens are a thorough of the paleontological is to that the recent study is claims about the oldest of a may as new data and are A of this is the case of the oldest the are fossils that to be the clade of of the of do not the In more recent analyses, have been the tree et al. and are to be on the of and where no evidence about a minimum for in phylogenetic position from about the of than from in study or discovery of specimens. specimens, new of specimens, and phylogenetic lead to revisions in the phylogenetic of fossils. as the may and stratigraphic associated with fossil specimens, but relevant phylogenetic justifying the taxonomic of these specimens is rates of in and to be as as and the of taxonomic in as the to our specimen-based are useful for the oldest specimens to a given is necessary to the phylogenetic position of a specimen for calibration. In the best cases, fossil specimens that to be assigned to a with In these assigning fossils to is of the tree the fossil will the and as a calibration for all in it is In other cases, the position of a fossil is and is on the of a specific analysis. In to the position of a taxon given (see between of and molecular phylogenetic is a that has been (Benton et al. 2009; et al. 2010; et al. from and molecular can fossil calibrations in In cases, the of a fossil may become to about it can be used to If data of the of also may be to of evolution, the of fossils in a tree et al. 2005; et al. and molecular are in the phylogenetic position of a fossil cannot be to a a that a fossil taxon is justification for a fossil calibration. A fossil with can be assigned to a specific with of the the fossil will the and as a calibration for all it is phylogenetic from data can the position of fossil In the a fossil is to be to and the that the fossil A molecular study with a and making the of the fossil If the fossil is to then it If the fossil is to then it is a calibration for just one to can the of and of fossils. In the a fossil is in the to A molecular analysis the of the and In a the for the clade are in a way and so using the fossil to clade be problems of and molecular can be (e.g., Brochu and 2008; and 2008; Ksepka or through the use of a in and, the phylogenetic position of known from fossils (e.g., et al. and Parham 2006). those approaches and explicitly to the data from fossil specimens with the of molecular about the of molecular data. methods do not so a approach to based on or is In cases, it may be that the and molecular data are so that a evidence a molecular approach are for the position of an given the phylogenetic position of use of the oldest fossil specimens to has a probability of error into the analysis (see et al. et al. We using to divergence dating analyses. are to analysis is the of specimens used for calibrations be The with a fossil can be to a specific in a stratigraphic but on the data might be to a in a stratigraphic or a or or a specimens, those more than or those derived from the lack stratigraphic and data and so have for calibration fossil in can be assigned to its and to a stratigraphic that In the best cases, calibration data will be based upon fossils with and stratigraphic that can be assigned to a in a The with a fossil can be a stratigraphic framework will have a on estimates of its and in light of in revisions of and in (e.g., formations, and are the key used to correlate and the sequence in a have (e.g., and explicitly and fossil taxon has and geological that provide a basis for its The given is for on the can be a useful minimum calibration for specimens of are known and the of is the oldest specimens. is from the in the it is from the the it is from is of a stratigraphic for the the can be in the the can be assigned to the and a the of this and is to represent in the stratigraphic where et al. the is to on the basis of ages and methods based on the et al. specimen a minimum age of are of in of or represent of Most do not represent of may be might represent of with of the time range at do the between with geochronologic of the of a fossil to a a of the age of the fossil that can then be used to establish a age as is not a stratigraphic is or with the of new and new or and can lead to of the present at a (e.g., and The dynamic of the of for fossil specimens in order to the of stratigraphic and upon divergence dating calibrations and, divergence time dating ages, but do not use or The age of a fossil is the of for the geochronologic data for dates can be difficult to establish for a and much and so provide a more framework for reporting fossil The of fossil to ages a chain of through on the basis of geological and paleontological evidence (e.g., van Tuinen and Hadly Benton et al. 2009; 2011). for the majority of calibrations, this is not the used in are not The age of a fossil is not it is established through than through dating at the in the fossil was age for a fossil specimen is the best and can be through dating methods have dating an order of in the as a result of new of and methods (e.g., et al. 2004; 2006; et al. and ages that to (e.g., et al. of this ongoing it is to the basis upon the age is If the chain of is the of revisions will be its our for justifying the age of a calibration point is that the of from paleontological studies should reference or published that ages (e.g., and et al. et al. 2004; 2010; and on are constantly and can become these it for to A of this step in the protocol the of the age from the geological a minimum age the age of the fossil should be used the of the relevant time than the of a in the a fossil the of the to it is the age from an will the minimum from the age of it is to that the minimum age is one a and is to not on its the age of the minimum age should the age of the fossil the error associated with the geochronologic age Tuinen et al. 2004; Donoghue and Benton 2007; Benton and Donoghue 2007; Benton et al. age should be as a The assigning based on the age of the fossil has been (e.g., van Tuinen et al. 2004; Benton and Donoghue 2007; Donoghue and Benton 2007). may to use in of or but The of a minimum age that the estimates for a fossil should be The justification for the might to a but paleontological data are established and that to introduce error into the analysis. In cases, of or the age of a fossil may not be a stratigraphic in cases, it is to much more and dates than are given a stratigraphic data may not be available in the the fossil specimens used for calibrations, and so it is necessary to evidence from fossil may not be to those data more than molecular but the specimen and ages in a may the the of dates In to the of the specimen-based we that about the of that or Such of calibrations in (e.g., Benton and Donoghue 2007; et al. 2007; Benton et al. it for to the justification the relevant and We should that through and analysis that the calibrations be or In order to the evolution of justifications, we that (or of should become a of calibration The justification of the phylogenetic position and age of a fossil is an first step to a in a divergence dating analysis. In to can be assigned time may not have this step the data from the fossil the minimum bound of a calibration The maximum bound and the of the are also based on the fossil but in a much more probability of the oldest known The of these other parameters from a protocol for them. the maximum is established as than all the oldest to a time the and for the of the are but no are the maximum an approach that into and phylogenetic has been proposed (e.g., Reisz and Müller Müller and Reisz 2005; Benton and Donoghue 2007; Donoghue and Benton 2007; Benton et al. approach is and from the fossil established Marshall who use this approach should provide justifying their so that can and, the of Benton and Donoghue and and the maximum should be and Most studies use a Bayesian framework for estimating divergence dates with probability curves between minimum and maximum In may be of the fossil but there is no way to parameters and and of little more than A of recent studies that these parameters are not et al. The of these are et al. 2010; et al. and et al. the that a is to that have a on results et al. et al. is a of molecular divergence dating The of methods for estimating maximum and probability curves should be a (see In order to the of our specimen-based we apply it to used calibrations in the vertebrate of the tree of the and the from A of the the of the paleontological data for these The of the specimen-based protocol to these results in new We also provide examples of our calibration as as maximum the the approach of Benton and Donoghue of ages for point calibrations, The data for this can be in The of Paleontology and holotype of et al. that the of (in the clade a of it in the clade also et al. a previously proposed calibration point for et al. can be in it the processes of the at the for the is the and the of the of the an is it a of the that is to the of the the of the the a is present at the of the of the for of of into the are on the and it the of of the et al. 2011). The of et al. is with molecular of that a (e.g., and 1998; Hedges and and et al. et al. 2005; et al. 2007; et al. 2009; et al. et al. et al. molecular data have for a clade and and et al. et al. or a clade and for these an where are the taxon to a is et al. et 2005; et al. The majority of recent molecular a et al. 2005; et al. 2007; et al. 2009; et al. 2009; et al. and of as or not the oldest calibration point or the phylogenetic of of the vertebrate fossils that are to be to other in and 2005; et al. is relevant is that the an and that is in age and and to other and 2007; and the also the taxon and a taxon in throughout et al. and 2005; and The data that these the and The is with ages, so the of the is to et al. The and is one of the fundamental calibrations for vertebrate studies (e.g., et al. 2007; et al. it as an calibration for both and molecular (e.g., et al. 2002; and 2006; 2007; and 2010) and is relevant to in (Benton and Donoghue 2007). used a secondary calibration for this has been and Martin 2004; Müller and Reisz on the to have not been used for calibration the earliest fossils from the and Reisz 2005; Benton and Donoghue 2007). Müller and Reisz proposed an age of for this based on the of the the of the is using recent age data et al. is phylogenetically as a of the (e.g., and 2006; 2007; and 2007; et al. 2011). its age is is from the of the in this has been to the using vertebrate and and this is with et al. and age data and Unfortunately, vertebrate is in (e.g., et al. 2009; Irmis et al. so the age of is not an age for this it a minimum age of divergence for the of et al. studies have this calibration (e.g., et al. Benton and Donoghue proposed an age of for this based on the of the from the of is a for the it has been in a phylogenetic and there is evidence that it is a of phylogenetically the and et al. from the of a that is the oldest of the of phylogenetically this fossil is no than also problems in the age of is vertebrate (see et al. et al. from the but these the same problems as other (see and are no than from the of was first as a of and with this phylogenetic of the specimen et al. that it to the age of a minimum for the for the maximum age for is difficult recent fossil discoveries have the age of divergence for this fossil evidence that the earliest based on the that a of have to the et al. but this not the that of will be to have a the of the with present in and et al. 2011). this (e.g., the the with We that the age of the oldest of the but to the a for a maximum it is the oldest fossil and all fossils. is clearly as a and and is to the and The is ages to that we do not of the is we that a maximum for be studies inappropriate calibrations that have error into divergence dating (e.g., Graur and Martin 2004; Gandolfo et al. 2008; Ksepka 2009; Sanders et al. In order to the of the specimen-based protocol can identify inappropriate calibrations, we examples and In the the published minimum age cannot be with specimen-based evidence so we a much minimum age in the the published minimum age cannot be with specimen evidence. In we cannot identify specimen that will all the of the protocol for that and so that future do not is not that these be but then it not be the fossil data calibrations is clearly to data into analysis. The checklist is an first step to other incorrect calibrations and more reliable time priors. calibrations are more to be of will molecular divergence dates more and provide in A specimen-based protocol will attention on between the fossil record and published calibration making it for to identify and correct and refine calibrations as new data to the reporting of data in (e.g., our is a crucial first In to providing this we identify as the for more methods for selecting parameters of time maximum and probability and the associated with from In both cases, can be in our checklist protocol will help identify the oldest fossil of a that can a time with an minimum fossils the time of the represent (e.g., Marshall Benton and Benton and Donoghue 2007). The probability of the oldest fossil is the other Bayesian calibration parameters to these parameters estimates of that to the of a et al. for of fossil and to provide of in the fossil The amount of to rigorous paleontological for is To studies have of fossil with of record at small taxonomic or Benton et al. 2004; and 2007; and for the of time priors. approach was to Bayesian and 2010; and 2010) on dates that be as Bayesian for divergence dating but we do not of studies that have this of time based on the temporal of and then used as time for divergence dating et al. 2011). The and of these and other methods to time parameters should be a for the divergence dating the of will be the of relevant data. genetic sequences is not the but the will more from in order to and the data from the fossil record. A the problems we address is the associated with from the first step of the specimen-based specimen and justifying is a for a molecular a for their Such challenges can be through or a study that has the are not but also introduce and these data be of time or at more The step is to that the and of paleontological calibration data to the way that molecular sequence data are on The is an for this as the based on Benton and Donoghue We can a of that et al. that is to other of biological data as the and the of We encourage and to a more providing data that the in and to to provide these data to their have to calibration data for divergence If paleontological data can be to their position in this it will result in more and the to to be explicitly with molecular will encourage the of phylogenetic for of data and for future (e.g., differences in rates of and molecular evolution, between and The recommendations to explicitly ages will and with The for more to maximum dates should the of methods for the fossil record. on the fossils will all the of to the we can a new community of to develop a more and rigorous approach to the study of evolution and the of life. can be in the data was the and of the of the of and a and The and and from the is the to the was through an from the of and are for their and was at a in in The of at the of is for this of with and the fossil record. We for to use the of from the for

Mirror-image allodynia is a mysterious phenomenon that occurs in association with many clinical pain syndromes. Allodynia refers to pain in response to light touch/pressure stimuli, which normally are perceived as innocuous. Mirror-image allodynia arises from the healthy body region contralateral to the actual site of trauma/inflammation. Virtually nothing is known about the mechanisms underlying such pain. A recently developed animal model of inflammatory neuropathy reliably produces mirror-image allodynia, thus allowing this pain phenomenon to be analyzed. In this sciatic inflammatory neuropathy (SIN) model, decreased response threshold to tactile stimuli (mechanical allodynia) develops in rats after microinjection of immune activators around one healthy sciatic nerve at mid-thigh level. Low level immune activation produces unilateral allodynia ipsilateral to the site of sciatic inflammation; more intense immune activation produces bilateral (ipsilateral + mirror image) allodynia. The present studies demonstrate that both ipsilateral and mirror-image SIN-induced allodynias are (1) reversed by intrathecal (peri-spinal) delivery of fluorocitrate, a glial metabolic inhibitor; (2) prevented and reversed by intrathecal CNI-1493, an inhibitor of p38 mitogen-activated kinases implicated in proinflammatory cytokine production and signaling; and (3) prevented or reversed by intrathecal proinflammatory cytokine antagonists specific for interleukin-1, tumor necrosis factor, or interleukin-6. Reversal of ipsilateral and mirror-image allodynias was rapid and complete even when SIN was maintained constantly for 2 weeks before proinflammatory cytokine antagonist administration. These results provide the first evidence that ipsilateral and mirror-image inflammatory neuropathy pain are created both acutely and chronically through glial and proinflammatory cytokine actions.

Typical and atypical enteropathogenic Escherichia coli (EPEC) strains differ in several characteristics. Typical EPEC, a leading cause of infantile diarrhea in developing countries, is rare in industrialized countries, where atypical EPEC seems to be a more important cause of diarrhea. For typical EPEC, the only reservoir is humans; for atypical EPEC, both animals and humans can be reservoirs. Typical and atypical EPEC also differ in genetic characteristics, serotypes, and virulence properties. Atypical EPEC is more closely related to Shiga toxin-producing E. coli (STEC), and like STEC these strains appear to be emerging pathogens.

Melatonin is an old and ubiquitous molecule in nature showing multiple mechanisms of action and functions in practically every living organism. In mammals, pineal melatonin functions as a hormone and a chronobiotic, playing a major role in the regulation of the circadian temporal internal order. The anti-obesogen and the weight-reducing effects of melatonin depend on several mechanisms and actions. Experimental evidence demonstrates that melatonin is necessary for the proper synthesis, secretion, and action of insulin. Melatonin acts by regulating GLUT4 expression and/or triggering, via its G-protein-coupled membrane receptors, the phosphorylation of the insulin receptor and its intracellular substrates mobilizing the insulin-signaling pathway. Melatonin is a powerful chronobiotic being responsible, in part, by the daily distribution of metabolic processes so that the activity/feeding phase of the day is associated with high insulin sensitivity, and the rest/fasting is synchronized to the insulin-resistant metabolic phase of the day. Furthermore, melatonin is responsible for the establishment of an adequate energy balance mainly by regulating energy flow to and from the stores and directly regulating the energy expenditure through the activation of brown adipose tissue and participating in the browning process of white adipose tissue. The reduction in melatonin production, as during aging, shift-work or illuminated environments during the night, induces insulin resistance, glucose intolerance, sleep disturbance, and metabolic circadian disorganization characterizing a state of chronodisruption leading to obesity. The available evidence supports the suggestion that melatonin replacement therapy might contribute to restore a more healthy state of the organism.

We present a phylogenetic analysis of spiders using a dataset of 932 spider species, representing 115 families (only the family Synaphridae is unrepresented), 700 known genera, and additional representatives of 26 unidentified or undescribed genera. Eleven genera of the orders Amblypygi, Palpigradi, Schizomida and Uropygi are included as outgroups. The dataset includes six markers from the mitochondrial (12S, 16S, COI) and nuclear (histone H3, 18S, 28S) genomes, and was analysed by multiple methods, including constrained analyses using a highly supported backbone tree from transcriptomic data. We recover most of the higher-level structure of the spider tree with good support, including Mesothelae, Opisthothelae, Mygalomorphae and Araneomorphae. Several of our analyses recover Hypochilidae and Filistatidae as sister groups, as suggested by previous transcriptomic analyses. The Synspermiata are robustly supported, and the families Trogloraptoridae and Caponiidae are found as sister to the Dysderoidea. Our results support the Lost Tracheae clade, including Pholcidae, Tetrablemmidae, Diguetidae, Plectreuridae and the family Pacullidae (restored status) separate from Tetrablemmidae. The Scytodoidea include Ochyroceratidae along with Sicariidae, Scytodidae, Drymusidae and Periegopidae; our results are inconclusive about the separation of these last two families. We did not recover monophyletic Austrochiloidea and Leptonetidae, but our data suggest that both groups are more closely related to the Cylindrical Gland Spigot clade rather than to Synspermiata. Palpimanoidea is not recovered by our analyses, but also not strongly contradicted. We find support for Entelegynae and Oecobioidea (Oecobiidae plus Hersiliidae), and ambiguous placement of cribellate orb-weavers, compatible with their non-monophyly. Nicodamoidea (Nicodamidae plus Megadictynidae) and Araneoidea composition and relationships are consistent with recent analyses. We did not obtain resolution for the titanoecoids (Titanoecidae and Phyxelididae), but the Retrolateral Tibial Apophysis clade is well supported. Penestomidae, and probably Homalonychidae, are part of Zodarioidea, although the latter family was set apart by recent transcriptomic analyses. Our data support a large group that we call the marronoid clade (including the families Amaurobiidae, Desidae, Dictynidae, Hahniidae, Stiphidiidae, Agelenidae and Toxopidae). The circumscription of most marronoid families is redefined here. Amaurobiidae include the Amaurobiinae and provisionally Macrobuninae. We transfer Malenellinae (Malenella, from Anyphaenidae), Chummidae (Chumma) (new syn.) and Tasmarubriinae (Tasmarubrius, Tasmabrochus and Teeatta, from Amphinectidae) to Macrobuninae. Cybaeidae are redefined to include Calymmaria, Cryphoeca, Ethobuella and Willisius (transferred from Hahniidae), and Blabomma and Yorima (transferred from Dictynidae). Cycloctenidae are redefined to include Orepukia (transferred from Agelenidae) and Pakeha and Paravoca (transferred from Amaurobiidae). Desidae are redefined to include five subfamilies: Amphinectinae, with Amphinecta, Mamoea, Maniho, Paramamoea and Rangitata (transferred from Amphinectidae); Ischaleinae, with Bakala and Manjala (transferred from Amaurobiidae) and Ischalea (transferred from Stiphidiidae); Metaltellinae, with Austmusia, Buyina, Calacadia, Cunnawarra, Jalkaraburra, Keera, Magua, Metaltella, Penaoola and Quemusia; Porteriinae (new rank), with Baiami, Cambridgea, Corasoides and Nanocambridgea (transferred from Stiphidiidae); and Desinae, with Desis, and provisionally Poaka (transferred from Amaurobiidae) and Barahna (transferred from Stiphidiidae). Argyroneta is transferred from Cybaeidae to Dictynidae. Cicurina is transferred from Dictynidae to Hahniidae. The genera Neoramia (from Agelenidae) and Aorangia, Marplesia and Neolana (from Amphinectidae) are transferred to Stiphidiidae. The family Toxopidae (restored status) includes two subfamilies: Myroinae, with Gasparia, Gohia, Hulua, Neomyro, Myro, Ommatauxesis and Otagoa (transferred from Desidae); and Toxopinae, with Midgee and Jamara, formerly Midgeeinae, new syn. (transferred from Amaurobiidae) and Hapona, Laestrygones, Lamina, Toxops and Toxopsoides (transferred from Desidae). We obtain a monophyletic Oval Calamistrum clade and Dionycha; Sparassidae, however, are not dionychans, but probably the sister group of those two clades. The composition of the Oval Calamistrum clade is confirmed (including Zoropsidae, Udubidae, Ctenidae, Oxyopidae, Senoculidae, Pisauridae, Trechaleidae, Lycosidae, Psechridae and Thomisidae), affirming previous findings on the uncertain relationships of the "ctenids" Ancylometes and Cupiennius, although a core group of Ctenidae are well supported. Our data were ambiguous as to the monophyly of Oxyopidae. In Dionycha, we found a first split of core Prodidomidae, excluding the Australian Molycriinae, which fall distantly from core prodidomids, among gnaphosoids. The rest of the dionychans form two main groups, Dionycha part A and part B. The former includes much of the Oblique Median Tapetum clade (Trochanteriidae, Gnaphosidae, Gallieniellidae, Phrurolithidae, Trachelidae, Gnaphosidae, Ammoxenidae, Lamponidae and the Molycriinae), and also Anyphaenidae and Clubionidae. Orthobula is transferred from Phrurolithidae to Trachelidae. Our data did not allow for complete resolution for the gnaphosoid families. Dionycha part B includes the families Salticidae, Eutichuridae, Miturgidae, Philodromidae, Viridasiidae, Selenopidae, Corinnidae and Xenoctenidae (new fam., including Xenoctenus, Paravulsor and Odo, transferred from Miturgidae, as well as Incasoctenus from Ctenidae). We confirm the inclusion of Zora (formerly Zoridae) within Miturgidae.

We report the isolation of a population of immature dental pulp stem cells (IDPSC), which express embryonic stem cell markers Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 as well as several other mesenchymal stem cell markers during at least 25 passages while maintaining the normal karyotype and the rate of expansion characteristic of stem cells. The expression of these markers was maintained in subclones obtained from these cells. Moreover, in vitrothese cells can be induced to undergo uniform differentiation into smooth and skeletal muscles, neurons, cartilage, and bone under chemically defined culture conditions. After in vivo transplantation of these cells into immunocompromised mice, they showed dense engraftment in various tissues. The relative ease of recovery and the expression profiles of various markers justify further exploration of IDPSC for clinical therapy.

The study of topological insulators has generally involved search of materials that have this property as an innate quality, distinct from normal insulators. Here we focus on the possibility of converting a normal insulator into a topological one by application of an external electric field that shifts different bands by different energies and induces a specific band inversion, which leads to a topological state. Phosphorene is a two-dimensional (2D) material that can be isolated through mechanical exfoliation from layered black phosphorus, but unlike graphene and silicene, single-layer phosphorene has a large band gap (1.5-2.2 eV). Thus, it was unsuspected to exhibit band inversion and the ensuing topological insulator behavior. Using first-principles calculations with applied perpendicular electric field F⊥ on few-layer phosphorene we predict a continuous transition from the normal insulator to a topological insulator and eventually to a metal as a function of F⊥. The tuning of topological behavior with electric field would lead to spin-separated, gapless edge states, that is, quantum spin Hall effect. This finding opens the possibility of converting normal insulating materials into topological ones via electric field and making a multifunctional "field effect topological transistor" that could manipulate simultaneously both spin and charge carrier. We use our results to formulate some design principles for looking for other 2D materials that could have such an electrical-induced topological transition.

As more data are generated from proteome and transcriptome analyses of snake venoms, we are gaining an appreciation of the complexity of the venoms and, to some degree, the various sources of such complexity. However, our knowledge is still far from complete. The translation of genetic information from the snake genome to the transcriptome and ultimately the proteome is only beginning to be appreciated, and will require significantly more investigation of the snake venom genomic structure prior to a complete understanding of the genesis of venom composition. Venom complexity, however, is derived not only from the venom genomic structure but also from transcriptome generation and translation and, perhaps most importantly, post-translation modification of the nascent venom proteome. In this review, we examine the snake venom metalloproteinases, some of the predominant components in viperid venoms, with regard to possible synthesis and post-translational mechanisms that contribute to venom complexity. The aim of this review is to highlight the state of our knowledge on snake venom metalloproteinase post-translational processing and to suggest testable hypotheses regarding the cellular mechanisms associated with snake venom metalloproteinase complexity in venoms.

BACKGROUND AND OBJECTIVES: In dentistry, low-power lasers have been used in the treatment of dentin hypersensitivity, gingivitis, periodontitis, and different forms of oral ulcers. This in vitro study focuses on the biostimulation of NIH-3T3 fibroblasts by a low-power Ga-As-pulsed laser. STUDY DESIGN/MATERIALS AND METHODS: We have studied cell growth and procollagen synthesis of cultured fibroblasts submitted to low-power laser irradiation with energy densities varying from 3 to 5 J/cm(2) over a period of 1-6 days. The light source was a 120 mW Ga-As diode laser (lambda = 904 nm). Growth curves and procollagen immunoprecipitation were obtained. RESULTS: Irradiation of 3 and 4 J/cm(2) increased the cell numbers about threefold to sixfold comparing to control cultures. However, this effect was restricted to a small range of energy densities since 5 J/cm(2) had no effect on cell growth. The energy density of 3 J/cm(2) remarkably increased cell growth, with no effect on procollagen synthesis, as demonstrated by the immunoprecipitation analysis. CONCLUSIONS: Our results showed that a particular laser irradiation stimulates fibroblast proliferation, without impairing procollagen synthesis.

In one of his final essays, statesman and former United Nations secretary general Kofi Annan said, ‘Snakebite is the most important tropical disease you’ve never heard of’ [1]. Mr. Annan firmly believed that victims of snakebite envenoming should be recognised and afforded greater efforts at improved prevention, treatment, and rehabilitation. During the last years of his life, he advocated strongly for the World Health Organisation (WHO) and the global community to give greater priority to this disease of poverty and its victims.

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.

Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

We confirmed the ability of the triterpenoid betulin to protect against neurotoxicity caused by Bothrops jararacussu snake venom in vitro in mouse isolated phrenic nerve-diaphragm (PND) preparations and examined its capability of in vivo protection using the rat external popliteal/sciatic nerve-tibialis anterior (EPSTA) preparation. Venom caused complete, irreversible blockade in PND (40 μg/mL), but only partial blockade (~30%) in EPSTA (3.6 mg/kg, i.m.) after 120 min. In PND, preincubation of venom with commercial bothropic antivenom (CBA) attenuated the venom-induced blockade, and, in EPSTA, CBA given i.v. 15 min after venom also attenuated the blockade (by ~70% in both preparations). Preincubation of venom with betulin (200 μg/mL) markedly attenuated the venom-induced blockade in PND; similarly, a single dose of betulin (20 mg, i.p., 15 min after venom) virtually abolished the venom-induced decrease in contractility. Plasma creatine kinase activity was significantly elevated 120 min after venom injection in the EPSTA but was attenuated by CBA and betulin. These results indicate that betulin given i.p. has a similar efficacy as CBA given i.v. in attenuating the neuromuscular effects of B. jararacussu venom in vivo and could be a useful complementary measure to antivenom therapy for treating snakebite.

Studies of the peopling of the Americas have focused on the timing and number of initial migrations. Less attention has been paid to the subsequent spread of people within the Americas. We sequenced 15 ancient human genomes spanning from Alaska to Patagonia; six are ≥10,000 years old (up to ~18× coverage). All are most closely related to Native Americans, including those from an Ancient Beringian individual and two morphologically distinct "Paleoamericans." We found evidence of rapid dispersal and early diversification that included previously unknown groups as people moved south. This resulted in multiple independent, geographically uneven migrations, including one that provides clues of a Late Pleistocene Australasian genetic signal, as well as a later Mesoamerican-related expansion. These led to complex and dynamic population histories from North to South America.

Abstract Aim The knowledge of biodiversity facets such as species composition, distribution and ecological niche is fundamental for the construction of biogeographic hypotheses and conservation strategies. However, the knowledge on these facets is affected by major shortfalls, which are even more pronounced in the tropics. This study aims to evaluate the effect of sampling bias and variation in collection effort on Linnean, Wallacean and Hutchinsonian shortfalls and diversity measures as species richness, endemism and beta‐diversity. Location Brazil. Methods We have built a database with over 1.5 million records of arthropods, vertebrates and angiosperms of Brazil, based on specimens deposited in scientific collections and on the taxonomic literature. We used null models to test the collection bias regarding the proximity to access routes. We also tested the influence of sampling effort on diversity measures by regression models. To investigate the Wallacean shortfall, we modelled the geographic distribution of over 4000 species and compared their observed distribution with models. To quantify the Hutchinsonian shortfall, we used environmental Euclidean distance of the records to identify regions with poorly sampled environmental conditions. To estimate the Linnean shortfall, we measured the similarity of species composition between regions close to and far from access routes. Results We demonstrated that despite the differences in sampling effort, the strong collection bias affects all taxonomic groups equally, generating a pattern of spatially biased sampling effort. This collection pattern contributes greatly to the biodiversity knowledge shortfalls, which directly affects the knowledge on the distribution patterns of diversity. Main conclusions The knowledge on species richness, species composition and endemism in the Brazilian biodiversity is strongly biased spatially. Despite differences in sampling effort for each taxonomic group, roadside bias affected them equally. Species composition similarity decreased with the distance from access routes, suggesting collection surveys at sites far from roads could increase the probability of sampling new geographic records or new species.

Since 2005, the Brazilian Ornithological Records Committee (CBRO) has published updated checklists of Brazilian birds almost every year. Herein, we present a completely new and annotated version of our checklist. For the first time, we list all bird subspecies known from Brazil that are currently accepted by at least one key ornithological reference work. The inclusion of the subspecies should be seen as a synthesis, and not as a taxonomic endorsement. As such, we include in the new checklist 1919 avian species, 910 of which are treated as polytypic in reference works (2042 subspecies), totaling 3051 taxa at the species and subspecies level. We anticipate that several of the subspecies included in our list may be subject to future taxonomic upgrades to species status, while others will probably be shown to be invalid in the light of future taxonomic studies. The results highlight Brazil as a megadiverse country and reinforce the need for proper enforcement of political tools, laws and international commitments assumed by the country to preserve its biodiversity.

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.