The blood that flows perpetually through our veins and arteries performs numerous functions essential to our survival. Besides distributing oxygen, this vast circulatory system facilitates nutrient transport, deters infection and dispenses heat throughout our bodies. Since human blood has traditionally been considered to be an entirely sterile environment, comprising only blood-cells, platelets and plasma, the detection of microbes in blood was consistently interpreted as an indication of infection. However, although a contentious concept, evidence for the existence of a healthy human blood-microbiome is steadily accumulating. While the origins, identities and functions of these unanticipated micro-organisms remain to be elucidated, information on blood-borne microbial phylogeny is gradually increasing. Given recent advances in microbial-hematology, we review current literature concerning the composition and origin of the human blood-microbiome, focusing on bacteria and their role in the configuration of both the diseased and healthy human blood-microbiomes. Specifically, we explore the ways in which dysbiosis in the supposedly innocuous blood-borne bacterial microbiome may stimulate pathogenesis. In addition to exploring the relationship between blood-borne bacteria and the development of complex disorders, we also address the matter of contamination, citing the influence of contaminants on the interpretation of blood-derived microbial datasets and urging the routine analysis of laboratory controls to ascertain the taxonomic and metabolic characteristics of environmentally-derived contaminant-taxa. The human microbiome comprises a vast corpus of bacterial, archaeal, viral and fungal microbial taxa. While most of these micro-organisms are commensal, many are mutualistic and some are pathogenic. Regardless of whether their presence is beneficial, inconsequential or detrimental, our lives are inextricably linked to the microbes with which we share our bodies. In fact, despite being 1,000 times smaller than human cells, bacteria comprise ~2% of the adult human body mass (1.5 kg), roughly equivalent in size to the human brain or liver  and the advent of whole metagenome shotgun sequencing (WMGS) as techniques to study microbial genetic material present in different human body-sites  and the sequencing of PCR-amplified 16S rRNA and gyrB genes confirmed the presence of bacterial DNA in the blood of healthy individuals. Correspondingly, Moriyama et al.  treatment and cellular energy measurements  in the blood of healthy humans. The presence of archaeal DNA is generally not reported   in healthy individuals . As far back as 2000, Gresham et al. , the HBM appears to be dominated by the phyla Proteobacteria followed by Actinobacteria, Firmicutes, and Bacteroidetes , processing (using kits and storage), and generating (sequencing protocols and techniques) both healthy and diseased blood microbial data. This could be achieved by studying the blood microbiome together with that of the skin around the puncture site from where the blood was obtained, as well as characterizing microbial DNA derived from potential microorganisms present in the needle, vacutainers, reagents, and other consumable items used during sampling.i.e., vertical) origin. Samples from the skin-, oral- and Intestinal-microbiomes should be analyzed at the same time as blood samples to gain insight into the potential origin of these microbes. Finally, in order to ascertain the potential roles and functions linked to bacteria and other microorganisms in the human blood, studies on the HBM based on WMGS are undoubtedly essential. The increasing recognition of the existence of a healthy HBM stimulates novel and diverse research avenues, some of which may well turn out to be of considerable clinical significance.We furthermore encourage researchers to investigate this unique microbiome, as it promises to hold the potential to facilitate both the diagnosis and improved understanding of the onset of numerous human diseases. It is still unclear whether the putative healthy HBM comprises a core set of bacterial taxa, or a dynamic and adaptive group of micro-organisms. We recommend performing studies that also take into consideration the “time” element in the HBM. Most of the literature reviewed here deals with snapshots of blood bacterial communities, potentially overlooking important changes of the HBM across time. In addition, the influence of age, geography and socio-economic status  on healthy HBM composition remains ambiguous. With reference to the location of micro-organisms in the human blood-circulatory system, it is conceivable that bacteria either reside inside, or adhere to blood cells or in the plasma fraction of the blood. Moreover, with regards to the origin of the healthy HBM, it appears that blood-borne micro-organisms, particularly bacteria, may well originate from various body-sites and that many might have a maternal (DC initiated and conceived the review, investigated the literature, and wrote the review. RR contributed to the writing and critical revision of the manuscript. DAC contributed to revision of the manuscript. MP directed and contributed to the review design, investigated the literature, and contributed to the critical revision of the manuscript.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.


Drosophila, the odorant receptors (ORs) and the ionotropic receptors (IRs). Although both receptor families share the role of being the first chemosensors in the insect olfactory system, they show distinct evolutionary origins and several distinct structural and functional characteristics. While ORs are seven-transmembrane-domain receptor proteins, IRs are related to the ionotropic glutamate receptor (iGluR) family. Both types of receptors are expressed on the olfactory sensory neurons (OSNs) of the main olfactory organ, the antenna, but they are housed in different types of sensilla, IRs in coeloconic sensilla and ORs in basiconic and trichoid sensilla. More importantly, from the functional point of view, they display different odorant specificity profiles. Research advances in the last decade have improved our understanding of the molecular basis, evolution and functional roles of these two families, but there are still controversies and unsolved key questions that remain to be answered. Here, we present an updated review on the advances of the genetic basis, evolution, structure, functional response and regulation of both types of chemosensory receptors. We use a comparative approach to highlight the similarities and differences among them. Moreover, we will discuss major open questions in the field of olfactory reception in insects. A comprehensive analysis of the structural and functional convergence and divergence of both types of receptors will help in elucidating the molecular basis of the function and regulation of chemoreception in insects.Most insect species rely on the detection of olfactory cues for critical behaviors for the survival of the species, e.g., finding food, suitable mates and appropriate egg-laying sites. Although insects show a diverse array of molecular receptors dedicated to the detection of sensory cues, two main types of molecular receptors have been described as responsible for olfactory reception in  Drosophila resembles the organizational principles of the olfactory system of vertebrates despite being simpler  ORs of vertebrates  it is possible that ORs may be mixed ionotropic-metabotropic receptors , which is activated by the inositol 1,4,5-inositol triphosphate/diacyl glycerol (IP3/DAG) signal transduction cascade   Wicher, . These t Wicher, , then tw Wicher, . In the  Wicher, . In some Wicher, .Drosophila ORs, site-directed mutagenesis, resonance energy transfer and structural modeling efforts -related family of receptors, termed the IRs  signal transducer smoothened (Smo)   family member, as a determinant of the Or67d pheromone-sensing neuron development   and comparison with other large families, it has been demonstrated that chemosensory receptors do not show high rates of adaptive divergence between species but show genome-wide signals of recent selection within D. melanogaster , copy number variants (CNVs) and small insertions and deletions (indels), of chemosensory families from various Drosophila  over the olfactory metabotropic transduction of pheromones  , hissing cockroaches (Gromphadorhina portentosa), locusts (Schistocerca americana), honey bees (Apis mellifera) and moths (Manduca sexta), showing latencies as short as 2 ms  using IR25a and IR76b mutants, the requirement of IR25a and IR76b in fatty acid detection was shown (Ahn et al., Although little is known about fatty acid detection in insects, it has recently been linked to the IR family of chemoreceptors in Drosophila, it has been shown, by an optogenetic approach, that overconsumption of sugar could be avoided by activation of a circuit that inhibits sucrose feeding depending on IR60b (Joseph et al., A healthy metabolism requires the control of sugar consumption. In Drosophila by IR56d-expressing taste neurons together with IR25a and IR76b co-receptors (Sánchez-Alcañiz et al., Carbonation, a non-nutritious product of microbial fermentation, has been shown to be detected in In conclusion, different cell-specific IR subunit combinations seem to be the basis for different taste qualities.Although not the topic of this review, it should be mentioned that very recently, it was discovered that the IR family of receptors, unlike the OR family, covers other sensory modalities beyond chemosensation, such as hygrosensation (Enjin et al., Drosophila (see reviews by Wilson, Despite the recent increase in knowledge about the main receptor families in olfaction in For example, finding the ligands for orphan receptors in both chemosensory families will shed light on the different modalities that they subserve. Additionally, experiments identifying the transduction mechanisms used by the two types of chemoreceptors will also help understanding the exquisite sensitivity and specificity of these receptors. Also, accurate X-ray crystallographic structures will help in solving some of these standing issues such as the exact composition of olfactory receptor heteromers or their ligand binding sites.Drosophila, these two families of receptors are also present in larvae. However, few studies focus on larval chemosensory modalities. Twenty-five members of the OR family are expressed in the dorsal organ, the olfactory receptor organ in larvae (Fishilevich et al., Although this review has been focused mainly on adult Importantly, much evidence of both expression and functional roles is coming from the use of Gal-4 lines, which are a extremely useful tool in the field but also show some caveats. Surely, the generation of more knockout mutants for the different receptors will answer some of the controversies caused by the caveats of using RNAi knockdown strategies.Drosophila will shed light on the function and evolution of both the OR and IR families (Arguello and Benton, Another question that will be addressed in the future is that the most ecologically relevant ligands may not have been found yet. Olfactory receptors are considered narrowly or broadly tuned based on analysis of ligands that may not be relevant at all for the fly (Bohbot and Pitts, In this review, we have focused on the main peripheral chemosensory systems at the receptor level Table , but theAll the authors listed made substantial contributions to this review. CG-D, FM and EA drafted the initial manuscript. All authors critically read, corrected and approved the final version of the manuscript.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.


APOE, FOXO3A and 5q33.3 loci) have been successfully replicated in different ethnic groups and the emerging picture is complex. For instance, it is an understatement to think that long-lived people harbor only favorable variants, completely avoiding risk alleles for major age-related diseases; indeed, there is evidence that many disease alleles are present in long-lived people. It is more probable that the longevity phenotype is the result of a particular combination of pro-longevity variants and risk alleles for pathologies, likely interacting in networks in a sex- and age-specific way. Finally, characteristics of aging are extremely heterogeneous, even among long-lived individuals, due to the complex interaction among genetic factors, environment, lifestyle, culture and resiliency. Population and study specificity, lack of statistical power for such a rather rare phenotype and missing heritability represent further hard obstacles to overcome in genotype–phenotype association studies. Thus, many challenges remain to be addressed in the search for the genetic components of human longevity. In this Special Issue we included five original articles and two reviews covering different areas in the field of human longevity, to help the reader take stock of the situation and point to future perspectives of the field.Human average life expectancy in developed countries has increased dramatically in the last century, a phenomenon which is potentially accompanied by a significant rise in multi-morbidity and frailty among older individuals. Nevertheless, some individuals appear someway resistant to causes of death, such as cancer and heart disease, compared with the rest of the population, and are able to reach very old ages in good clinical conditions, while others are not. Thus, during the last two decades we have witnessed an increase in the number of studies on biological and molecular factors associated with the variation in healthy aging and longevity. Several lines of evidence support the genetic basis of longevity: from the species-specific maximum lifespan to the genetically determined premature aging syndromes. Studies in human twins, that aimed to distinguish the genetic from the environmental component, highlighted a heritability of life span close to 25%. In centenarian’s families, the offspring of long-lived individuals not only exhibit a survival advantage compared to their peers, but also have a lower incidence of age-related diseases. On the other hand, population studies found that genetic factors influence longevity in age- and sex-specific ways, with a most pronounced effect at advanced age and possibly in men compared to women. All this evidence indicates that a genetic influence on longevity exists, laying the foundation for the search for the genetic components of extreme long life. Consequently, over the past three decades, there has been a surge in genetic research, due in part to advances in molecular technologies, starting as studies of single genetic variants in candidate genes and pathways, moving on to array-based genome-wide association studies (GWAS) and subsequently to next generation sequencing (NGS). However, despite a plethora of studies, only few variants  directly deal with the search for the genetic component of longevity and healthy aging. The response to external injuries is the leitmotif unifying all three association studies.TERT variability, showing that shorter LTL at baseline may predict a shorter lifespan [As it is known, internal and external stresses disrupt telomere homeostasis. The contribution by Scarabino and co-workers confirms the genetic determination of leukocyte telomere length (LTL) by lifespan . FurtherIPMK (Inositol Polyphosphate Multikinase), a potential moonlighting protein performing multiple functions in pathways affecting the aging process, from nutrient-sensing to oxidative stress and telomere maintenance [CYP2B6, CYP3A5, COMT and ABCC2 genes, following either linear or non-linear trajectories with respect to the chance of becoming long-lived. Such findings underline once again that SNPs associated with longevity might behave either as pro-longevity or killing variants but also as deleterious variants neutralized by the protective effect of pro-longevity genes (buffered variant), an important aspect to take into account in disentangling the genetic contributors to human longevity.The papers by De Rango et al. and Crocco et al. highlight the complexity of longevity, a highly dynamic phenotype influenced by internal and external stresses, that makes the identification of genes robustly associated with it very challenging. De Rango’s paper investigated for the first time the contribution to the longevity phenotype of the genetic variability of ntenance . This pantenance . They foFinally, the paper by Revelas and co-workers contributes to the open debate of whether extreme longevity is coupled to risks for major diseases . The autOverall, we believe these papers, highlighting different facets and the complexity of the studies on the genetics of human longevity, may help to understand the path research has taken in the field up to now, and to explore some possible interventions, taking advantage of the pathways highlighted and of the perspectives they are unveiling for the future.So, where should the longevity genetic field go from here? Despite decades of genetic studies, the variants consistently identified as associated to human longevity explain only a small part of the estimated heritability. For facing the challenge of ‘missing heritability’, new and innovative approaches are needed. First of all, more studies are needed to elucidate the effect of rare variants with larger effect sizes, not captured by standard GWAS. The opportunity of high-throughput methodologies like NGS, together with large multi-center collaborative studies of extremely long-lived cohorts, can contribute to pave the way for untangling the networks involved in human longevity. In this sense, the study of epistatic effects of different genetic markers in gene-set and pathway-enrichment analyses, as well as the integration of several layers of biological variation  in polygenic risk scores could further help too. The application of a more functional genomic approach like the collection of whole-exome sequencing, genome-wide epigenetic, cell-specific transcriptomic data and the integration of all these layers of genomic information can help to disentangle the determinants of lifespan. Finally, the collection of life-long environmental and lifestyle variables known to influence an individual’s health (like microbiome and nutrition), can significantly improve our chance to untangle the intricate interplay between genes and environment in determining the longevity phenotype.


Birds and pterosaurs have pneumatic bones, a feature likely related to their flight capabilities but whose evolution and origin is still poorly understood. Pneumatic foramina are present on the external surface of the bone and are reliable indicators of post-cranial skeletal pneumatization present in Pterosauria, Eusauropoda, and Neotheropoda. Here, we carried out a qualitative analysis of the position, size and number of pneumatic foramina of the cervical and thoracic/dorsal vertebrae of pterosaurs and birds, as they have the potential to challenge hypotheses about the emergence and evolution of the respiratory trait in these groups. We also discussed differences between pneumatic and vascular foramina for identification purposes. Besides phylogenetic representativeness, the pterosaur taxonomic sampling considered the preservation of specimens and, for birds, their life habit, as this relates to the level of pneumatization. Pneumatic foramina on the lateral faces of the centrum of the mid-cervical vertebrae of pterosaurs and birds differ in position and size, and those adjacent to the neural canal additionally differ in number. The avian posterior cervical vertebrae show a higher number of pneumatic foramina in comparison to their mid-cervicals, while the opposite is true for pterosaurs, suggesting differences in the cervical air sac of these clades. Pneumatic foramina were found at the base of the transverse processes of the notarial vertebrae of birds, while they were absent from some of the pterosaurs analyzed here, revealing the presence of a pneumatic hiatus in the vertebral column that might be explained due to the distance of this structure to the cervical air sac. These findings indicate that, although the overall skeletal pneumatization of pterosaurs and birds present deep homologies, some pneumatic features occurred convergently because variation in the number of pneumatic foramina along the vertebral column is related to the position of the air sacs in pterosaurs and birds and/or the habit of each species. There is an evident reduction of the pneumatic foramina in birds that have aquatic foraging and an increase in the ones which perform static soaring. Although we did not find any external anatomical difference between pneumatic and vascular foramina, we observed that vascular foramina occur at specific sites and thus identification on the basis of location is reliable. Nowadays birds are the only extant tetrapods with post-cranial skeletal pneumatization, a condition correlated to the presence of the air sacs that are part of their respiratory system . The intGiven the presence of pneumatization in non-avian dinosaurs and pterosaurs, two different hypotheses about the origin of bone pneumatization are discussed: it might have arisen once in a non-ornithodiran archosaur ancestor, or it might have appeared two times independently in the evolutionary history of Ornithodira –8.Here, we compare the distribution, shape and size of pneumatic foramina present in the vertebral skeleton of pterosaurs and extant birds in order to test the before-mentioned hypotheses, taking into account the known life habit of each species, because this is a factor that influences the presence of pneumatization . We alsoAnhanguera piscator replica MN 5023-V; Tropeognathus cf. mesembrinus, MN 6594-V) and Tapejaridae, including Tapejarinae (AMNH 24445) and Thalassodrominae , as well as two undetermined Tapejaridae (MN 6588-V and MN 6511-V) [The analyzed pterosaurs come from the Romualdo Formation, in the Araripe Basin in northeastern Brazil. The three-dimensional preservation of the specimens recovered in this lithostratigraphic unit allows a more precise visualization of the cortical foramina that are probably connected to internal pneumatic cavities. The vertebrae belong to members of the families Anhangueridae ( 6511-V) . One lasRhea americana, MNA 766), Tinamiformes , Anseriformes , Galliformes , Procellariiformes , Pelecaniformes , Suliformes , Cathartiformes , Strigiformes , Falconiformes , and Psittaciformes . Rheiformes and Tinamiformes were selected because they represent basal clades of Neornithes, and Galliformes and Anseriformes represent basal clades of Neognathae [The studied vertebrae of Neornithes comprised living species of the taxa Rheiformes  and trunk  vertebrae were considered, because sacral and caudal vertebrae are normally not pneumatized in pterosaurs. Pterosaur vertebrae were termed following Bennett : ‘mid-ced Witmer  and term species . ‘Posterertebrae . The ‘noConcerning the pneumatic foramina in cervical vertebrae, we follow the nomenclature proposed by Vila Nova et al. , in whicOn the free dorsal/thoracic vertebrae and the notarium, we consider the term ‘lateral pneumatic foramina’ for all those found on the lateral surface of the centrum. We frequently observed structures, in cranial and/or caudal view, which are referred to as ‘pneumatic foramina present at the base of transverse processes’, because they, unlike the adjacent foramina seen in cervical vertebrae, lie in a more lateral position, considerably closer to the transverse processes, even though they are commonly close to the neural canal due to their size . We haveWe follow the nomenclature established by Wilson et al.  in regarMost foramina observed in this clade had a connection to the intertrabecular cavities and were thus identified as pneumatic. However, in some of them it was not possible to observe the connection between the cortex and the intertrabecular cavities, due to preservation issues or the presence of sediment. Because they were located in the same places as other pneumatic foramina, they were considered as such.The mid-cervical vertebrae of all observed specimens had at least one pneumatic foramen on the right and left sides of the lateral surface of the vertebral centrum. Independent of the different sizes of the vertebrae, which varied between taxa, these foramina had a long oval outline. Although there is usually only one lateral pneumatic foramen, some vertebrae had two, which were located exclusively on the cervical vertebrae V, VI and VII, as is observed in the anhanguerids MN 5023-V and MN 6594-V and in the tapejarids MN 6566-V, MN 6504-V, and MN 6511-V . In a siAnhanguera piscator, were found only laterally to the neural canal along the cervical series. Additionally, in all analyzed tapejarids, the pair of pneumatic foramina laterally adjacent to the neural canal was accompanied by a foramen dorsal to the neural canal. One case of asymmetry in the distribution of the foramina was observed: the specimen MN 6511-V (Tapejaridae sp.) showed two foramina on the right side of the neural canal, each one of different sizes, while there was only one foramen on the left of the canal. We interpret this as an individual variation.Pneumatic foramina adjacent to the neural canal were observed in cranial and/or caudal view in all vertebrae whose preservation allowed viewing . The forOn the posterior cervical vertebrae, we observed that the pneumatic foramina on the lateral faces of the centra were reduced, and even absent in some specimens. It is important to consider that these vertebrae also have shortened vertebral centra in relation to the mid-cervicals, being morphologically very similar to the first dorsal vertebrae . PosteriOne unusually placed pneumatic foramen was observed in the posterior cervical vertebrae of the thalassodromine MN 6566-V described by Buchmann et al. , and is Anhanguera piscator (MN 5023-V). These foramina had a circular outline and were always smaller than the neural canal. No external evidence of pneumatization was found on the centra of the posterior cervical vertebrae of the analyzed species.We observed adjacent pneumatic foramina located laterally to the neural canal in anterior view, and on the bases of the transverse processes in posterior view, in the posterior cervical vertebrae of Tropeognathus cf. mesembrinus (MN 6594-V) and a specimen of Tapejaridae (6588-V) have preserved notaria, but only in the second one pneumatic foramina at the base of transverse processes were found.In dsungaripteroid pterosaurs, the notarium forms in osteologically mature specimens . In thisAnhanguera piscator) and one unidentified dorsal vertebra of the specimen MN 6508-V (Pterosauria sp.) showed pneumatic foramina located at the base of the transverse processes  had a drastic reduction in their number: it only showed one small oval pneumatic foramen at the base of the transverse processes on the right and left sides of the thoracic vertebrae. We also note that its vertebrae show extremely laterally compressed centra.foramen transversarium in cervical vertebrae, as in Rhea americana and Ara ararauna, and associated with the ventral base of the transverse processes in thoracic vertebrae, as in Ardea alba and Ara chloropterus.Besides pneumatic foramina, most birds also possess foramina that are strictly vascular. They are frequently smaller and more oval than pneumatic ones, although size is not diagnostic. Vascular foramina are usually located laterally to the Rhea americana, MNA 766, and Tinamus solitarius, AZ 138 and AZ 139), and has a long and craniocaudally oval outline  and Ardea alba (MN 51321), although this foramen has been observed in other Galliformes  and Pelecaniformes .All analyzed birds have at least one circular craniolateral pneumatic foramen on the right and left lateral faces of the mid-cervical vertebrae, close to the ersarium . The absCairina moschata (AZ 763), Milvago chimachima (MNA 4305 and MNA 5086), and Amazona amazonica (AZ 066)  .Fregata magnificens (MNA 1977), with an additional foramen on each side, and in Diomedea chlororhynchus (MNA 1793), with an additional foramen only on the left side, resulting in an asymmetrical arrangement in this case  did not present pneumatic foramina dorsally adjacent to the neural canal, suggesting a probable character loss. The absence of the foramina in this position was also observed in other species, such as Rhea americana (MNA 766), Pipile jacutinga (AZ 767 and MNA 7664), Procellaria aequinoctiallis (MNA 8353), and Catharthes aura (AZ 578 and AZ 580).On the mid-cervical vertebrae of many species of Neornithes, we noticed a presence of a pair of small pneumatic foramina dorsal to the neural canal in caudal view. Both tend to show a circular or oval outline . The amohis case . Three sPenelope superciliaris (MNA 751 and MNA 2058) and in the analyzed Pelecaniformes, represented by species of Ardeidae . An exception are two pneumatic foramina that border the dorsal margin of the neural canal of the posterior cervical vertebrae of Egretta caerulea when seen in caudal aspect , Falco sparverius , and Ara macao (MNA 001), species which had only the lateral foramen, like Cairina moschata (AZ 763), Pipile jacutinga (AZ 767 and MNA 7664), Fregata magnificens (MNA 1977), Catharthes aura (AZ 578 and AZ 580), Milvago chimachima (MNA 4305 and MNA 5086), and Caracara plancus (MNA 4902 and MNA 4589), species which presented foramina in both positions, such as Sula leucogaster (MNA 766), Procellaria aequinoctiallis (MNA 8353), Pterodroma sp. (AZ 1196), Asio clamator (AZ 1190), and Ara chloropterus (AZ 762), and species with no foramina on the vertebral centrum, like the Paleognathae Rhea americana (MNA 776) and Tinamus solitarius   .Catharthes aura, for example, the foramina showed a reduced size in relation to the pneumatic foramina on the craniolateral surface of centrum, while they were considerably bigger in Procellaria aequinoctiallis  and Psittaciformes  , and are also present in Suliformes (USP 040) . These fUSP 040) .Pterodroma sp.) both are deep and without any association with pneumatic foramina; in MNA 009 (Ara ararauna), which only presents the prezygapophyseal parapodiapophyseal fossae, they are shallower and associated with pneumatic foramina on their interior; in MNA 8353  we observed pneumatic fossae on the lateral faces of the centra, which also are pierced by small oval pneumatic foramina on their bottom  [Baryonyx (NHMUK R9951) and Majungasaurus creatissimus (UA 8678) [Lilisternus [Variations in the pneumatic structures present on the cortical surface of the mid-cervical vertebrae were observed between Pterosauria and Neornithes. The pneumatic foramina observed on the lateral faces of the centra of mid-cervical vertebrae of all pterosaurs analyzed here are considered a synapomorphy for the Ornithocheiroidea , althougK R1034) , suggestK R1034) . The varK R1034) –29. ForaUA 8678) , 30. Theisternus , althougisternus . When coisternus , 30, 31.Raeticodactylus filisurensis (BNM 14524) [Austriadraco dallavechiai (BSP 1994 I 51) [Sericipterus wucaiwanensis (IVPP V14725) [Rapetosaurus krausei and Majungasaurus crenatissimus (UA 8678) [Pneumatic foramina on the lateral surfaces of the neural arch of mid-cervical vertebrae were described only in non-pterodactyloid pterosaurs, such as M 14524) , Austria94 I 51) , 32, and V14725) . Among t V14725) , 31, indUA 8678) , 34, whiSericipterus wucaiwanensis (IVPP V14725)[Majungasaurus crenatissimus (UA 8678) [The pneumatic foramina adjacent to the neural canal of the mid-cervical vertebrae of pterosaurs are present but not restricted to the clade Dsungaripteroidea , being aP V14725). The preP V14725), 36 and P V14725). At leasUA 8678) .Azhdarcho lancicollis, a vestigial lateral pneumatic foramen was recognized on the specimen ZIN PH 137/44, an eighth cervical vertebra, but was absent on specimens ZIN PH 131/44, ZIN PH 144/44, ZIN PH 147/44, ZIN PH 138/44, interpreted, respectively, as third, fourth, sixth and seventh cervical vertebrae [In the posterior cervical vertebrae, we find a tendency to size reduction and loss of foramina located on the lateral sides of the centra of several pterosaur clades , 37, 38,ertebrae . In contertebrae , 30. AddPneumatic foramina on the lateral surfaces of the neural arch of the posterior cervical vertebrae are reduced in size or absent in Pterosauria, but were frequently found in the Paleognathae, Anseriformes, Suliformes, Procellariiformes, and Falconiformes, in a position equivalent to that of the postzygapophyseal centrodiapophyseal fossae of non-avian Neotheropoda and Sauropodomorpha , 30, 39.Santanadactylus brasilensis and in Anhanguera sp. [Tropeognathus cf. mesembrinus (MN 6594-V), was also described in the literature [Some of the species of pterosaurs and birds we analyzed here presented a notarium. There are records in the literature of the presence of pneumatic foramina on the bases of transverse processes in the notarium of pterosaurs, as in a specimen tentatively referred to uera sp. , 40, 41.terature , 35, revterature –44. The terature . Howeverterature  describeterature , 46. We Majungasaurus crenatissimus (UA 8678) [Plateosaurus and Camarasaurus supremus [Differing from what was observed in the notarium, the free dorsal vertebrae of pterosaurs presented pneumatic foramina on the margins of the bases of the transverse processes, which were already reported in the Pteranodontoidea , 19, 29 UA 8678) , 5, 30. supremus . The widRaeticodactylus filisurensis [Dimorphodon macronyx [Torvosaurus (BYU 2008) [Despite the absence of pneumatic foramina on the lateral faces of the centra of the free dorsal vertebrae in the pterosaurs analyzed here, they have been previously observed in non-pterodactyloids, such as in the third dorsal vertebra of the holotype of surensis , and in macronyx . These pYU 2008) , also haSericipterus wucaiwanensis (IVPP V14725) [Rhamphorhynchus muensteri (MGUH 1891.783), these fossae are associated with pneumatic foramina [Pterodroma sp. (AZ 1196). The pneumatic fossae of birds and pterosaurs resemble, in position and appearance, the prezygapophyseal parapodiapophyseal fossae and postzygapophyseal centrodiapophyseal fossae present in the dorsal vertebrae of sauropodomorphs, exhibiting a concavity delimited by vertebral laminae [Despite the few records of pneumatic fossae in this work, in the literature, the presence of prezygapohyseal parapodiapophyseal fossae and of postzygapophyseal centrodiapophyseal fossae in pterosaurs has previously been observed on the dorsal vertebrae of Rhamphorhynchidae. These fossae had no connection with the internal cavities in the holotype of the rhamphorhynchid  V14725) . In Rhamforamina  similar  laminae , thus suPneumatic foramina present in unusual locations in certain clades, such as near the neural spine in the cervical vertebrae VIII in thalassodromine pterosaurs and in the posterior cervical vertebrae of Falconiformes, and unusually absent, such as on the lateral faces of the posterior cervical vertebrae in the Ardeidae and on the lateral faces of the mid-cervical vertebrae in the Ctenochasmatidae and the Azhdarchidae, can be interpreted as independent gains or losses in these clades , 26.Majungasaurus, Spinostropheus, Allosaurus, Monolophosaurus, and Sinraptor [Variations on the size of pneumatic foramina are observed along the vertebral column of the same individual, with the largest foramina generally closer to the air sac or lung that invades the bone , 5, 30. inraptor , 30. ThePhalacrocorax brasilianus  did not show any evidence of pneumatization in the axial postcranial skeleton, which may be related to its aquatic foraging habit, since less dense bones interfere with submersion, as is reported for penguins, loons and diving ducks [Procellariiformes, Suliformes, and Falconiformes have more numerous and larger pneumatic foramina than the other analyzed birds. These birds perform static soaring –18, an eng ducks , and/or arteria vertebralis. They can be present as a single foramen, as in Diomedea chlororhynchus (MNA 1793) and Sula leucogaster (MNA 7665), or subdivided into several smaller ones, without a standard shape, as in Rhea americana (MNA 766) and Ara ararauna (AZUSP 040)  and Ara chloropterus (AZ 762)  . They cadorsalis . We obse(AZ 762) . HoweverThe differences between positions, sizes, shapes and quantity of the pneumatic features present on the cortex of the cervical and dorsal/thoracic vertebrae of pterosaurs and birds suggests probable convergences in the development of the pneumatic foramina presented in the vertebral column of ornithodirans, either due to biomechanical influences or by the ecological habits performed.In both pterosaurs and birds, we observed presence and absence of pneumatic foramina in unusual places in certain groups, which were interpreted as independent acquisitions and losses in these groups, corroborating previous observations . These vThe variation in the size and/or quantity of pneumatic foramina along presacral vertebrae, which decreased from the mid-cervical to the posterior cervical vertebrae of Pterosauria, and exhibited the opposite trend in Neotheropoda, is interpreted here as a possible variation of the cervical air sac between these two clades, which is probably more robust cranially in pterosaurs. In addition, the pneumatic hiatus associated with to the apneumatic notarium observed in some pterosaurs is probably due to the distance of this structure from the cervical air sac, which is probably the one responsible for the formation of pneumatic diverticula in this area of the vertebral column.Although it was not possible to differenciate externally pneumatic and vascular foramina, we observed that the position of vascular foramina is probably the best feature to differenciate both, since their location is less variable.


Aesthetic experience concerns the appreciation of aesthetic objects and the resulting pleasure. Such pleasure is not derived from the utilitarian properties of the objects but linked to the intrinsic qualities of the aesthetic objects themselves. Hence, the aesthetic pleasure is disinterested . AesthetAesthetic experiences are offered by multiple contexts, . Several psychological perspectives considered aesthetic experience as a rewarding process and suggested a link between aesthetic experience and pleasure . Recent In this vein, neuroimaging studies highlighted that immediate emotional responses to artwork and low-intensity enduring changes in affective states cf. . MoreoveHere, we review evidence showing that arts promote well-being across several domains, and discuss the neural underpinnings of aesthetic experience, emotional processing, pleasure, and reward. In particular, we assess the idea that a common physiological mechanism underlies aesthetic processing in multiple places for experiencing art. Implications for therapeutic and educational uses of art are discussed.Benefits associated with aesthetic processing have been demonstrated in different settings, ranging from reproductions of paintings shown in laboratories to real art contexts such as museums.In the following sections, we present a review of the main research branches on art in which a beneficial effect on health has been shown.Several studies show benefits of art museums as settings for therapy . These bResearch has been conducted to identify the elements of the museum setting that facilitate the treatment goals, including psychological, social, environmental aspects . Museum Using psychophysiological measures, studies find visits to art museums decrease stress, which could promote health and well-being . Clow anSeveral studies have been conducted on the efficacy of art-based interventions in professional education, demonstrating a growing interest for this field, and posing challenging opportunities for the traditional learning methods that shape the current teaching practice . Art-basIn learning through art, the learner approaches a subject matter by creating art, responding to art, or performing artistic works not by studying art as a theoretical discipline . This arOne might wonder how the emotional experience elicited by the appreciation of diverse forms of art enables individuals to feel better and learn quickly and effectively, and whether the boosting effect of art on these different domains forms a basis of a common cognitive or affective mechanism. Here, we suggest that the processing of aesthetic artwork relies on the activity of reward-related brain areas, resulting in positive emotions and pleasure that, modulating affective state, increase the individual predisposition to cognitive activities such as learning.The studies reviewed so far demonstrated that the aesthetic value of artwork and their use in educational programs may affect psychological and physiological states, thus promoting well-being and enhancing learning. However, as we stated above, the mechanisms underlying the relationship between art and well-being are still unclear, probably due to the fact that the determinants of the aesthetic experience and its relationship with emotion processing and pleasure are still unresolved.Here, we review some neuroimaging evidence detailing the neural underpinnings of the relationship between aesthetic experience and activation of emotional states in the beholder, to provide a more comprehensive understanding of the aesthetic experience and how it provokes aesthetic emotion and pleasure in the beholder. Moreover, we relate these findings to influential models of aesthetic processing.From a psychological point of view, it has been suggested that the cognitive processing of art produces affective and often positive and pleasing aesthetic experiences. According to the information-processing stage model of aesthetic processing by On the other hand, various theories of emotion have been influential in describing the paradoxical enjoyment of negative emotions in art . SeveralThe pleasurable effect of negative emotions in art reception has been extensively investigated in the field of music . AccordiIn line with the conceptual frameworks offered by music research, it may be hypothesized that pleasure in visual art reception relies upon (1) emotional contagion with the valence conveyed by the artwork; (2) appraising a negative emotional stimulus as a fictional rather than realistic; (3) regulating emotion accordingly; (4) enjoying aesthetic experience and performing aesthetic judgment. If aesthetically pleasing, such an experience can be defined rewarding. The dynamic interaction of these and other factors for producing pleasurable aesthetic experience has been broadly described in theories of aesthetic processing e.g., . Providivia features that represent actions and emotions  can arouse a powerful aesthetic emotion. This could improve perceived well-being .Unfortunately, as far as we know, there are only a few studies that explore the neural correlates associated with cognitive- or affective-based accounts of the aesthetic experience and their relation to the use of art for promoting individual well-being. Furthermore, most empirical investigations of the relationship between art and well-being do not consider objective measures of stress, such as skin conductance, heart rate variability, or respiration rate. Further, any conclusion about a relationship between art appreciation and well-being is hampered by the use of quite different subjective measures of well-being, such as interviews and questionnaires. Nowadays, we know from the literature that the pleasure associated with aesthetic processing may be modulated by emotional responses of the beholder to the artwork, or may be function of the successful cognitive mastery of the aesthetic stimulus , or may Aesthetic experience, in many settings, may promote well-being. Neuroaesthetics research suggests that aesthetic pleasure is derived by the interaction between emotion processing that involves reward-related areas in the brain and top-down processes derived from the relationship of the beholder with the cultural artifact. The self-rewarding nature of aesthetic experience may influence the beholder’s affective state, possibly improving well-being. However, there still are many questions that future research should address to clarify the determinants of aesthetic pleasure and their relationship to health. First, the impact of aesthetic emotion on measured well-being has been assessed through subjective ratings using interviews or questionnaires, scarcely considering more objective indices recorded through psychophysiological measures. Moreover, it remains unclear whether proper use of art to improve well-being should emphasize the empathetic responses to the artwork or the possibility for the beholder to master the meaning of the artwork itself. Future research should consider these issues in developing art-based programs in healthcare and education.SM conceived the idea, reviewed the literature, and wrote the draft of the manuscript. SF reviewed the literature and wrote the draft of the manuscript. VB collaborated to the idea with SM, reviewed the literature on educational applications and supervised the manuscript writing.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.


Conducting polymers (CPs) have received much attention in both fundamental and practical studies because they have electrical and electrochemical properties similar to those of both traditional semiconductors and metals. CPs possess excellent characteristics such as mild synthesis and processing conditions, chemical and structural diversity, tunable conductivity, and structural flexibility. Advances in nanotechnology have allowed the fabrication of versatile CP nanomaterials with improved performance for various applications including electronics, optoelectronics, sensors, and energy devices. The aim of this review is to explore the conductivity mechanisms and electrical and electrochemical properties of CPs and to discuss the factors that significantly affect these properties. The size and morphology of the materials are also discussed as key parameters that affect their major properties. Finally, the latest trends in research on electrochemical capacitors and sensors are introduced through an in-depth discussion of the most remarkable studies reported since 2003. A. A3 gas x) through covalent binding. When the enzyme recognizes glucose, it induces a change in the source-drain current. This FET sensor showed high sensitivity in detecting glucose at the concentration range 0.5–20 mM. A similar concept has also been applied to detecting different kinds of biomolecules such as proteins [A rise in the number of people affected by disease has prompted researchers to develop suitable devices for the fast and accurate diagnosis of biological target species. A number of researchers have used CPs and CP composites for biosensor applications because of their high compatibility with biological systems. Yoon et al.  developeproteins ,156, horproteins , tastantproteins  and odorproteins .−1, but also showed high sensitivity, selectivity, and reproducibility in response. Recently, Hui et al. [Three-electrode electrochemical configuration is also a common strategy for the fabrication of CP-based biosensors. A gold/PANI nanocomposite was reported for the electrochemical detection of prostate-specific antigen (PSA) . The hybi et al.  developen-type and p-type, to induce an insulator-to-metal transition in the CPs. CPs offer many advantages in applications as in electrochemical capacitors, actuators, and sensors. However, the major disadvantages of CPs when used in these applications include their inferior long-term stability. Furthermore, the conductivity of CPs still has much room for improvement. The conductivity of CPs is lower than that of metals, and this has limited their use in specific applications such as transistors and memory devices. It is believed that overcoming these existing problems will make CPs strong candidates for a diverse range of future applications.We have described the electrical and electrochemical studies of CPs that have become important over the last few decades. Understanding these properties is of key importance in the development of CPs for application in various fields. The theoretical modeling of the transport properties of CPs is still challenging due to the extreme complexity of the polymeric conjugated systems. However, continuous study of the origins of conductivity and knowledge of doping reactions have made it possible to control the kinetics of the charge-transfer reactions. Electrons are either added to or extracted from the delocalized π-bonded polymer backbone, leading to the formation of charge carriers such as polarons, bipolarons, and solitons. The mobility of these carriers is affected by a variety of factors, such as the dopant, temperature, and inherent structure. The conductivity can be improved successfully by electrochemical doping, both


Lessons learned when adapting the evidence-based global family planning training resource package included the need to: (1) engage key nursing and midwifery educators for buy-in; (2) update the technical skills of educators in contraceptive technology and competency-based training methods; and (3) adapt to the local context including condensing the global content for the time-limited preservice education context. Lessons learned when adapting the evidence-based global family planning training resource package included the need to: (1) engage key nursing and midwifery educators for buy-in; (2) update the technical skills of educators in contraceptive technology and competency-based training methods; and (3) adapt to the local context including condensing the global content for the time-limited preservice education context. Tanzania and Uganda have high total fertility and maternal mortality rates, and low contraceptive prevalence rates. High-quality preservice family planning education for nurses and midwives can improve the quality of sexual and reproductive health care, thereby improving health outcomes.In 2015, we worked with relevant stakeholders in Tanzania and Uganda through a series of surveys, assessments, and workshops to adapt modules of the Training Resource Package for Family Planning (TRP), an evidence-based global resource, to improve the quality of preservice family planning education for nurses and midwives. With support, a wide range of stakeholders, including policy makers, program managers, educators from nursing and midwifery training institutions, and representatives from professional associations, identified relevant TRP modules and adapted them to each country's context to inform and develop their own lesson plans in accordance with national policies, guidelines, and standardized preservice education templates.Important lessons from the adaptation process include the following: (1) engage relevant ministries of health and education, professional associations, and regulatory councils at each step of the process to increase the acceptability and utility of the TRP; (2) use a context-specific process for adaptation of the TRP, as not one process will fit the needs of all countries; and (3) include nursing and midwifery educators in the adaptation process to create an established pool of trainers who can then cascade the TRP to other educators in their respective schools. Overall, participants in both countries expressed challenges with incorporating competency-based teaching methods into their curricula because they were unfamiliar with such approaches themselves and with reducing the extensive TRP content to fit within the time constraints for preservice education.Adaptation of an evidence-based global family planning training resource in Tanzania and Uganda resulted in substantive changes to the curricula of the reproductive health preservice course unit that will support nurses and midwives to provide quality, rights-based family planning services. For example, a 2013 survey of 70 nursing and midwifery professionals across East, Central, and Southern Africa revealed that many of the nursing and midwifery schools in these countries have limited materials and technical expertise.We worked with relevant stakeholders in Tanzania and Uganda to improve the quality of preservice family planning education for nurses and midwives. Stakeholders included the East, Central, and Southern Africa Community (ECSA), an organization focused on improving health across this region, as well as the Tanzanian and Ugandan governments. We focused on nurses and midwives because they comprise the majority of the professional health workforce.We worked with relevant stakeholders in Tanzania and Uganda to improve the quality of preservice family planning education for nurses and midwives.To enhance the quality of preservice family planning education for these cadres of health workers, we adapted the Training Resource Package for Family Planning (TRP)—a comprehensive set of family planning curricula—to meet the needs of the 2 countries, and supported use of the adapted curricula during preservice education .https://www.fptraining.org/. The TRP uses the most up-to-date technical information to inform the content of its modules.Developed in 2012 by the U.S. Agency for International Development (USAID), the United Nations Population Fund (UNFPA), and the World Health Organization (WHO), along with a number of sexual and reproductive health organizations, the TRP contains 13 modules, each outlining a particular topic. The topics include information on contraceptive methods, comprehensive contraceptive counseling, information on side effects, and youth-friendly contraceptive services . Each moBOXBenefits of Family PlanningCombined Oral ContraceptivesCondoms – FemaleCondoms – MaleContraceptive ImplantsEmergency Contraceptive PillsEmergency Contraceptive Pills Training for PharmacistsFamily Planning CounselingIntrauterine DevicesLactational Amenorrhea MethodProgestin-Only Injectable Contraceptives (Injectables)Standard Days MethodWHO's Family Planning Guidance Documents and Job AidsOur process, results, and lessons learned are further detailed in the following sections.In September 2014, ECSA held its 5th quadrennial general meeting in Harare, Zimbabwe, which was attended by representatives of ECSACON (ECSA's nursing- and midwifery-focused arm), development partners, nursing and midwifery educators, regulatory councils, professional associations, service delivery managers, providers, and students. The theme of this meeting was increasing access to quality nursing and midwifery care across ECSA's member states .In the days preceding the meeting, we hosted a half-day workshop with educators from ECSA member states during which we disseminated the TRP, demonstrated its use for in-service training, and discussed adaptation for preservice education. Participants at this half-day preconference workshop included tutors from preservice education of nurses and midwives, representatives of regulatory councils and professional associations, students, service delivery managers from ECSA and nonmember countries Botswana, Namibia, and South Africa. We demonstrated how to access the TRP online and oriented them on how it is structured. Participants reviewed the TRP counseling module and engaged in role-play exercises to demonstrate the module.After the workshop, between December 2014 and July 2015, participants applied their learning, sharing the TRP with colleagues, reviewing curricula and training methods, and identifying ways to strengthen family planning training at individual institutions. Some of the participants attempted to use the TRP as is and faced challenges with reducing the time and aligning the content with what is expected in preservice education. As a result, it was recommended to hold workshops in specific countries to align the TRP with their preservice education standards and to develop lesson plans.Participants from Lesotho, South Africa, Tanzania, Uganda, and Zimbabwe asked that we support them to implement the TRP to strengthen family planning training in their countries. The participants also suggested that in any future trainings, we upload the TRP content to flash drives so that participants can access the modules even when there is no Internet.Core Competencies in Adolescent Health and Development for Primary Care Providers,After the workshop, we developed a questionnaire based on WHO's A survey of nurses and midwives from ECSA countries revealed that countries dedicated insufficient time to comprehensive family planning training and that training focuses on provision of short-acting methods.Once we selected Tanzania and Uganda, in May 2015, we conducted a more detailed review of preservice family planning education in these 2 countries. We held discussions with ECSA and representatives of nursing and midwifery councils and preservice education program managers in Tanzania and Uganda, and we conducted a desk review of preservice curricula and resources. The desk review consisted of relevant websites including those of Johns Hopkins University's Knowledge for Health Project, Human Resources for Health's Global Research Center, the Global Health Workforce Alliance, implementing partners known to have supported preservice education programs, and ECSA and ECSACON. Following the online search, country-specific documents were requested from Tanzania and Uganda, such as scopes of practice, curricula for in-service and preservice education, and family planning and reproductive health policies and guidelines.Across both countries, nursing and midwifery councils accredit and regulate nursing and midwifery schools and determine curricula content. Content must then be approved by committees, which are typically composed of policy makers, educators, regulatory councils, content experts, and professional associations. Curricula must be reviewed and updated every 5 years, or as needed, based on changes in policies and new technology. Approved curricula are disseminated to the schools. Individual educators then design each training session using a standard template provided by the preservice training units of the Ministry of Health or Ministry of Education, leading to lack of standardization of the content taught, time allocated to teach family planning, and training methodology used. Both countries allocate 40 hours to the entire reproductive health module, which includes family planning, sexually transmitted infections, HIV/AIDS, adolescent and youth sexual and reproductive health, and postabortion care.While training standards in Tanzania and Uganda specify that curricula have to be competency-based, educators in both countries tend to have limited experience in use of such training methods. However, both countries cited that educators have limited experience in use of competency-based training methods and tend to depend instead on lectures. The Tanzanian preservice family planning curriculum was updated in 2015, but Uganda's curriculum had not been revised for many years, some parts for more than a decade.While training standards in Tanzania and Uganda specify that curricula have to be competency-based, educators in both countries tend to have limited experience in use of such training methods.WHO's 2015 Medical Eligibility Criteria (MEC)and the USAID High Impact Practices.In 2015, we organized 2 workshops in each country. During these initial workshops, we disseminated the TRP, oriented participants on its structure, and distributed The first planning workshop in each country was 3 days long and attended by key stakeholders, including preservice education policy makers, family planning program managers, educators from nursing and midwifery training institutions, and representatives from professional associations and curricula-development committees. The aim of this initial workshop was to disseminate the TRP, foster country ownership, and identify gaps in preservice family planning education curricula that could be addressed through application of the TRP. Participants arrived at recommendations for improvement of preservice family planning education based on the country's policies and standards, including family planning knowledge, attitudes, and skills to be developed during preservice education and job expectations post-graduation. At the initial workshop, participants then developed the design and schedule for the subsequent 5-day adaptation workshop; for example, they decided which TRP modules should be used to demonstrate adaptation of the TRP. The demonstration modules were selected based on what content the educators felt they and their students needed to know more about, and what content needed an update within the curricula. Participants also developed the schedule and methodology for the adaptation process.The second 5-day workshop in each country focused on adapting the selected TRP modules. The workshop included a broader set of participants to adapt the TRP: educators from public- and accredited private-sector schools for nursing and midwifery, regulatory councils, professional associations, in-service family planning trainers, and service providers from practicum training sites. Those who attended the 3-day planning workshop served as co-facilitators during the 5-day workshop. We documented our lessons learned for other program implementers to use when adapting the TRP to local contexts.In both countries, we established an online community of practice to allow participants to interact with one another and support continued learning. However, we recognized that the limited Internet connectivity in Uganda would somewhat hamper these participants' ability to engage.We held the Tanzanian 3-day planning workshop in July 2015. Participants decided on the following objectives for the 5-day adaptation workshop: (1) adapt the TRP to validate the newly updated family planning content; (2) demonstrate how to adapt the TRP using the counseling module as an example; (3) update the knowledge and skills of participants on family planning; (4) conduct contraceptive technology update; (5) demonstrate use of competency-based training methods; and (6) develop session plans for the updated curricula family planning module.The Ministry of Health and Social Welfare led the 5-day workshop, and 33 participants attended including educators from the nursing and midwifery training institutions, universities, and local ECSACON country representatives. We showed participants how to access and use the TRP online and on flash drives. In-service family planning master trainers from the Ministry of Health presented sessions on the WHO's MEC and the TRP's counseling module, demonstrating use of competency-based training methods. Participants then adapted the existing curricula according to the TRP content. We demonstrated how the trainers could adapt the TRP to inform their own lesson planning. To allow participants to practice, we divided them into groups of 3 and randomly assigned them a topic . Participants in the adaptation workshop used the TRP to develop lesson plans in accordance with national policies and guidelines in the standard templates. Once developed, participants presented their lessons to others and received feedback. Some adopted the session training materials and PowerPoint presentations directly from the TRP. Others removed slides that they felt were too advanced for their students. Still others made small modifications to the TRP based on their knowledge of students' needs and what is already taught through preservice education . All participants used the TRP to create comprehensive lesson plans. Participants reported that they would have liked to have had more time to develop lessons plans and practice session delivery for the whole family planning module.Participants in the adaptation workshop used the TRP to develop lesson plans in accordance with national policies and guidelines in the standard templates.In Tanzania, all participants had a laptop, so while we provided flash drives with the TRP modules included, participants primarily accessed the TRP online. Participants expressed satisfaction with the TRP itself, especially its ease of use and the many resources included with it. Overall, they preferred didactic teaching methods, rather than practicum methods. They suggested that in future sessions, facilitators might spend more time demonstrating role-play and other competency-based methods before they asked participants to do so. Lastly, they found adaptation a challenge, reporting difficulty reducing the exhaustive TRP content to fit within the allotted 20 hours of time for family planning training.Since the workshops, the National Pre-Service Education Coordinating Unit of the Ministry of Health and the Nurses Council aligned competencies in the diploma nursing and midwifery curricula with global standards, using the TRP as a benchmark. The National Council for Technical Education (NACTE) approved the updated curricula. The Coordinating Unit then organized workshops to orient 139 educators representing training institutions for nurses and midwives in Tanzania.Trainers' Reference Guide for Family Planning Pre-service Education for Nurses and Midwives, further described below.In December 2015, we hosted the 3-day planning workshop and subsequent 5-day adaptation workshop in Uganda. Based on feedback from participants, we then added a third, 2-week workshop where participants from the 5-day workshop reconvened to develop lesson plans that used Uganda's standardized template for preservice education. During the workshops, these lesson plans, along with supporting materials, were compiled into a During the 3-day workshop, participants decided on the following objectives for the 5-day workshop: (1) review the TRP to ensure its alignment with national policy; (2) update the preservice modules on contraceptive technology and competency-based training methods; and (3) standardize lesson planning.Thirty-two representatives of nursing and midwifery schools, the Uganda Nursing and Midwives Council, the Uganda Nursing and Midwifery Examination Board, and ECSA attended the 5-day workshop in Uganda. The Nursing and Midwifery Examination Board, which is under the Ministry of Education, was included instead of the Ministry of Health (as in Tanzania), because in Uganda the Ministry of Education has jurisdiction over preservice education. Participants spent the first few days of the Ugandan workshops reviewing the nursing and midwifery curricula to determine content gaps and needs. As the curricula had not been reviewed since 2003, participants viewed the TRP workshops as a useful opportunity to undertake a thorough curricula review of the entire reproductive health course unit's family planning objectives and content. We disseminated the TRP online and on flash drives. Based on the recommendations from the 3-day workshop to use modules with content that would be new to most of the participants, we demonstrated use of the TRP using modules on emergency contraceptive pills and the Standard Days Method. The use of role-plays and demonstration enhanced participants' learning and reinforced competency-based training methods.In small groups, participants cross-checked existing preservice curricula against the reproductive health unit family planning standards, in-service training curricula, and TRP content. During this examination, they revised learning objectives and updated knowledge, skills, and attitudes to be developed during preservice education. They then developed the content outline, resulting in a much more comprehensive curriculum .Once the objectives of the family planning curricula and content on knowledge, skills, and attitudes were established, the small groups allocated time to each objective, developing lesson plans and incorporating competency-based training methods. In Uganda, few participants had computers, so hard copies of the TRP modules were printed and distributed to participants to use in their small groups.Participants provided valuable feedback. Some workshop participants found it challenging to adapt the extensive TRP content into the limited time allotted for preservice education. They suggested that we develop more explicit guidance on how to use the TRP to develop preservice education lesson plans. Disseminating the TRP to all schools in the country would be helpful, participants said, in standardizing training content.Some workshop participants found it challenging to adapt the extensive TRP content into the limited time allotted for preservice education.Trainers' Reference Guide for Family Planning Pre-service Education for Nurses and Midwives. The Reference Guide includes a series of predesigned lesson plans that used Uganda's standardized template for preservice education; PowerPoint presentations; handouts; and knowledge evaluation questions that could be adapted to develop pre-post knowledge tests, quizzes, and skills assessment checklists. The Reference Guide includes guidelines for practicum training, development of practicum training sites, and preparation for practicum training. The Trainers Reference Guide is aligned with the TRP, and cognizant of the time allocated to family planning classroom teaching. Based on recommendations from the 2015 workshops, participants were given more time to practice delivering their sessions. They “pretested” lesson plans and used feedback from facilitators and peers to inform revisions. They added some lesson plans that are not part of the TRP, such as the family planning/reproductive health policies, guidelines, and strategies, and sessions on “family planning concepts” and permanent methods for enhanced knowledge on country context and links between family planning and maternal, newborn, and child health.In July 2016, a follow-up, 2-week workshop was organized in Uganda to adapt the TRP to develop lesson plans. During this workshop, we worked with participants to adapt the TRP to develop the Since developing the lesson plans, the National Curriculum Development Committee and educators who had attended the TRP workshops reviewed the certificate and diploma nursing and midwifery preservice education curricula, enabling the TRP to be a reference document. The lesson plans and Reference Guide have been disseminated to training institutions. The training institutions conducted workshops with other educators who had not attended the TRP workshops facilitating use of the lesson plans. The educators found the lessons plans to have sufficient content for preservice education.Adaptation of the TRP in Tanzania and Uganda resulted in substantive changes to the curricula of the reproductive health course unit that will support nurses and midwives to provide quality, rights-based family planning . In bothAdaptation of the TRP in Tanzania and Uganda resulted in substantive changes to the curricula of the reproductive health course unit that will support nurses and midwives to provide quality, rights-based family planning.Six months post-workshop, we checked in with workshop participants to assess whether the TRP was still in use. In Tanzania, educators who had attended the workshop were using the TRP and had shared the TRP and adapted lesson plans with their colleagues. The Ministry of Health had used the TRP as a reference to develop curricula for the country's newly formed community health worker cadre. Workshop participants were using the TRP resources and tools  in their classrooms; however, they reiterated the challenge of reducing content from the TRP to fit within the allotted amount of time for preservice family planning training. Because the Uganda lesson plans were developed within the time allotted to the reproductive health course unit, educators in Uganda did not experience the same challenge as those in Tanzania. However, they did express challenges in using competency-based training due to large class sizes and lack of adequate training resources, such as anatomic models. In Uganda, participants reported using the TRP and again affirmed the value of the Trainers' Reference Guide, which helped to standardize learning and contained lesson plans. Although educators who attended the workshops shared the TRP and adapted lessons with their colleagues, we do not know how the resources are being used by schools that were not part of the workshops.Basic foundational concepts for family planning service delivery, such as the meaning of healthy timing and spacing of pregnancy and rights-based family planningStrategies for deconstructing myths and misconceptions about family planningRecordkeeping and health data management including data use to improve family planning service deliveryGender and adolescent and youth sexual and reproductive healthOur workshops revealed some areas for improvement within the TRP. Any future revisions may consider adding content to address the following topics in greater depth:The process of reviewing the TRP with key stakeholders is a learning opportunity. Implementers of preservice education programs should organize content review workshops with educators and representatives of relevant ministries, allowing sufficient time for participants to identify and discuss in detail differences in their existing curricula and the TRP.Similarly, productive relationships with ministries of health and education, regulatory councils, and professional associations in each country proved invaluable for rolling out the TRP adaptations. Inclusion of nursing and midwifery educators in the adaptation process helped to create an established pool of trainers who could then cascade the TRP to other educators.Adaptation of the TRP for preservice education should be context specific. Adaptation is a complex process and not one process will fit the needs of all countries. It is therefore important for stakeholders involved in the adaptation process to have a thorough understanding of country context.Our experience reinforced our belief that high-quality preservice education is a crucially important element of building nurses' and midwives' foundational skills. But educators are only as good as the tools and resources available to them. It is critically important that educators' skills in contraceptive technology and competency-based training methods be routinely updated and they are equipped with technically accurate textbooks.Through the process of adapting the TRP, we learned several lessons that may be useful to other implementers as they implement the TRP in their own countries.Inclusion of nursing and midwifery educators in the adaptation process helped to create an established pool of trainers who could then cascade the TRP to other educators.Strengthening preservice family planning education for nurses and midwives can improve health outcomes for women, newborns, infants, and children. Quality preservice family planning education for nurses and midwives is therefore a “best buy” for countries seeking to reduce maternal, newborn, and child mortalities and morbidities. A global analysis conducted by UNFPA in 2014 concluded that midwives, when educated to international standards, have the competencies to deliver 87% of the 46 essential reproductive, maternal, and newborn health services needed by women and newborns.How-To Guide for adaptation of the TRP to improve preservice family planning education (see https://www.e2aproject.org/publication/guide-adaptation-training-resource-package-family-planning-improve-pre-service-education/). The How-To Guide contains detailed steps elaborating the adaptation process, including examples of adapted training modules. The second is a package of modules—one for preservice family planning education and one for preservice education on adolescent and youth sexual and reproductive health and gender . Adolescent and youth sexual and reproductive health is taught in its own standalone unit in both countries, but the broader curricula do not discuss age biases in family planning service provision. The modules contain lesson plans and supporting materials that can be used by educators to teach essential family planning, adolescent and youth sexual and reproductive health, and gender competencies to nurses and midwives during the time allotted for preservice family planning education.Based on our experience in Tanzania and Uganda, we developed 2 useful tools for those interested in replicating the process described in this article. The first is a


The brainstem reticular formation (RF) represents the archaic core of those pathways connecting the spinal cord and the encephalon. It subserves autonomic, motor, sensory, behavioral, cognitive, and mood-related functions. Its activity extensively modulates cortical excitability, both in physiological conditions  and in disease . Such a wide variety of effects arises from the long course and profuse axonal branching of isodendritic reticular neurons, which allows the neuronal message to travel toward the entire cerebral cortex and downstream to the spinal cord. On the other hand, the isodendritic architecture featuring a monoplanar branching allows most RF neurons to cover roughly half of the brainstem and to be impinged by ascending and descending pathways. In parallel, such a generalized influence on CNS activity occurs in combination with highly focused tasks, such as those involved in the coordination of gaze.Venkatraman et al.). This brainstem region was explored in pioneer electrophysiological studies carried out by Moruzzi and Magoun . This paper, while confirming classic morphological studies on the isodendritic core of the RF . Consistently, this issue includes an original investigation on how proprioceptive trigeminal afferents may affect attention and arousal via a tight neuroanatomical interaction between the proprioceptive trigeminal mesencephalic nucleus and the LC . The specific role of LC in sustaining cognitive functions is substantiated by its diffuse branching . Glial cells are also critical for releasing cytokines and chemokines messengers with both proinflammatory and neuroprotective actions. This may lead to an endogenous neuroprotective effect mediated by P27R receptors, as demonstrated by Lim et al.The high connectivity of reticular nuclei may explain why a variety of different sensory information  may impact cognitive functions through ascending reticular neurons, pertaining to the catecholamine nucleus Locus Coeruleus (LC) .Within this framework, Gambardella et al.). Frequently, the onset of PD, instead of consisting of motor disturbances, coincides with autonomic alterations and pain. In this regard, the role of the RF in driving painful stimuli, and controlling pain-related circuitries, was reviewed by Martins and Tavares. These authors centered brainstem pain control in a reticular loop, which includes the periaqueductal gray, the rostro-ventro-medial medulla and the ventro-lateral medulla (Martins and Tavares).For instance, specific patterns of neuronal loss affecting catecholamine-containing reticular nuclei may produce a constellation of phenotypes in Parkinson Disease (PD). In fact, depending on which reticular nucleus is affected, a variety of both motor and non-motor  symptoms, may occur. This mostly applies to non-motor symptoms, which appear to underlie different PD subtypes, each one owing a specific pattern of brainstem involvement  with midbrain DA neurons might be crucial for the hyperlocomotion induced by amphetamines .The key role of the brainstem RF in mediating those activities relevant to species survival, such as pain and reward, sets the ground for these brain regions as preferential targets for drugs of abuse as reported by Wang et al.).So far, the RF has been viewed mainly as an archaic collection of ascending and descending systems, and interconnected nuclei, which play only a rough and ancestral role in interlacing various CNS areas. Nevertheless, specific nuclei of the RF act as premotor centers, involved in the fine-tuning of the gaze, both along the vertical and horizontal plane. This latter function was investigated by Wang et al. who defined the central mesencephalic reticular formation as a conduit for the collicular saccadic signals in the horizontal gaze (All these features are covered by specific contributions of the research topic, which offers an updated view to define the anatomical correlates of the multiple and interconnected roles played by the brainstem reticular formation in health and disease.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


We analyzed the performance and throughput of currently available analytical techniques for quantifying body burden and cell internalization/distribution of silver nanoparticles (Ag NPs). Our review of Ag NP biological fate data shows that most of the evidence gathered for Ag NPs body burden actually points to total Ag and not only Ag NPs. On the other hand, Ag NPs were found inside the cells and tissues of some organisms, but comprehensive explanation of the mechanism(s) of NP entry and/or in situ formation is usually lacking. In many cases, the methods used to detect NPs inside the cells could not discriminate between ions and particles. There is currently no single technique that would discriminate between the metals species, and at the same time enable localization and quantification of NPs down to the cellular level. This paper serves as an orientation towards selection of the appropriate method for studying the fate of Ag NPs in line with their properties and the specific question to be addressed in the study. Guidance is given for method selection for quantification of NP uptake, biodistribution, precise tissue and cell localization, bioaccumulation, food chain transfer and modeling studies regarding the optimum combination of methods and key factors to consider. Wh. Wh35]. P-ICP-MS  may in tP-ICP-MS  is alreaIn the case of NPs with high dissolution potential, such as Ag NPs, it is important that the method is able to discriminate between ions (and their ligand complexes) and particles. The techniques with this ability are synchrotron-based techniques (XANES) , hypersphigh, medium and low. We estimate that AAS, ICP-MS, SP-ICP-MS and AF4-ICP-MS have the highest throughput (and accessibility). Medium throughput methods are those enabling tissue/cellular microscopy and spectroscopy, while the lowest throughput is offered by synchrotron-based techniques  the time input for sample preparation, (ii) thevtime input for data acquisition and processing, as well as (iii) the method accessibility. The time demand for sample preparation was evaluated based on the number and duration of required steps, such as drying, freezing, chemical fixation, acid digestion and processing . Methods with the highest time input needed for sample preparation are TEM-EDX, FIB-SEM-EDX and spectroscopy-based techniques  B. Methodchniques B.We designed an interpretation scheme for biological fate data of metal-based NPs with dissolution potential, illustrated using the literature data for Ag NPs . The aim+, Ag NPs-Ag+ complexes and/or Ag+-organic and inorganic ligand complexes associated with, or internalized by the organism [The most commonly applied techniques to study Ag NP body burden are AAS, ICP-MS, and radioactive labelling . These torganism . Recentlorganism . The latorganism ,41. Thes+ may lead to false positive results on cellular internalization of Ag NPs. In this case the size, shape, composition and density of re-precipitated particles compared to the engineered ones differ and should be compared by TEM and X-ray diffraction analyses. Secondary formation of Ag particles in vivo could be confirmed by exposure of organisms to ionic Ag only.A number of different techniques have previously been applied to study the cellular internalization and body distribution of Ag NPs . The majTetrahymena thermophila [Ochromonas danica [Nereis diversicolor, Ag NPs were found in the endocytic pits and endosomes of gut epithelia [D. rerio embryo via transport through the chorionic channels, which was confirmed by real-time in vivo imaging using scanning near-field optical microscopy [C. elegans Ag NPs were found predominantly around the uterine tissue which implies the entrance of NPs through an external opening, i.e., the vulva [Daphnia magna embryos, because the brood chamber is open and in direct contact with the ambient water [On the other hand, synchrotron-based techniques (XANES), hyperspectral imaging, SP- and AF4-ICP-MS and TOF-SIMS enable the differentiation between ions and particles and are therefore appropriate to study NP distribution. Using these approaches, Ag NP distribution inside organisms was reported in some organisms ,42,43,44rmophila , in alvermophila , and in s danica . In the pithelia . Ag NPs pithelia ,32,43,48pithelia  and planpithelia  cells hahe vulva ,22. In phe vulva . Geislerhe vulva  found Agnt water .Taken together, these considerations imply that further studies on NPs body distribution and cellular internalization should consider two important aspects in terms of interpretation of the obtained data. Firstly, there are serious limitations in terms of techniques utilized to confirm internalization of intact NPs and to discriminate between ions and particles, as well as to discriminate between the uptake of intact NPs from the external environment and the secondary precipitation of particles from internalized ions. The latter could be effectively resolved by employing the corresponding metal salt control. Even more important is the fact that physiological interpretation of observed NP internalization is rarely provided which may result in an inaccurate understanding of the obtained data. For example, endocytosis, in particular phagocytosis, is a common physiological mechanism used by many protists or cells of multicellular organisms to acquire nutrients or to serve as an important defense mechanisms against infection by microorganisms  and the process of removing cellular debris . The presence of NPs in certain types of cells  therefore does not necessarily point to potential hazard for the organism, but may suggest the organism is capable of processing the NPs for removal or degradation. Also, there is no convincing evidence that the internalized NPs, especially those with high dissolution potential, bioaccumulate inside the body of organisms after passing through the epithelial barriers. Analogously, the fact that NPs were found inside the organism after passage through an opening to the external environment  does not necessarily mean that they have passed the epithelial barriers and accumulated in the body.+, Ag NPs and Ag NPs-Ag+ complexes rather than confirming uptake of intact Ag NPs by organisms commonly used in environmental studies. Although not included in our analysis due to the sheer volume of publications, it is reasonable to assume that the same conclusion would be drawn from data gathered on other test systems including bacteria, yeast, protozoa and mammalian cell lines, and for other soluble or partially soluble NPs. On the other hand, many reports of internalized Ag NPs that were supported by appropriate methodology were lacking a comprehensive explanation of the possible uptake/translocation route(s). Two important aspects to be included in the interpretation of biological fate of Ag and other metal and metal oxide NP data are: limitations of the techniques utilized, the physiological background or mechanism of the observed NPs internalization, and the discussion of uptake should be grounded in consideration of the potential implications for bioaccumulation and/or hazard.Our review shows that most of the evidence gathered to date for Ag NP body burden actually assesses total Ag, including AgThis review categorizes methods for studying the biological fate of NPs, according to their performance in terms of answering questions regarding body burden and body distribution as well as throughput and accessibility of the analytical methodology. Although we focused our review on Ag NPs, being among the most studied NPs, our conclusions regarding appropriate method selection according to the scientific aim in question can be readily applied to other metal and metal oxide NPs. There is currently no single technique that would discriminate between ions and NPs (including discriminating between the engineered variants and secondary particles precipitated in situ by organisms), and at the same time enable localization and quantification of NPs up to cellular level. We suggest that for each NP investigated, the selection of technique should be based on the characteristics of the NPs , be driven by the aim of the study and further users of data/results , and include complementary methods capable of quantification and visualization of localization as well as appropriate control experiments to rule out confounding factors. For example, in the case of bioaccumulation, food chain transfer and modeling studies, high throughput analyses are of vital importance. Data should be robust enough to be further used by experts with non-biological backgrounds. For precise tissue and cell localization, high resolution imaging and discrimination among ions and particles (engineered versus in situ precipitated) are of prime importance. These data have to be interpreted by experts with knowledge on the specific organism’s anatomy, physiology and particular feeding strategy as well as the ecology of the investigated species.At the present state of knowledge it appears that the biological fate of metal and metal oxide NPs could partly be the same as the fate of other metal species  and partly that of microorganisms, cell debris, and other foreign bodies entering the organism , although the relative proportions will depend on the specifics of the NP preparation and its ratio of ions to NPs under the specific environmental conditions. However, there are also particle-specific characteristics of NPs which need to be taken into consideration when their biological fate is studied. Among them are the adsorption to organism body surfaces in particular, and the transformation of NPs within the organism body following uptake. In the future, some new methodological approaches might complete our understanding of NP biological fates. For example, analysis of gene expression may indicate processes in the cell facilitating metal ion precipitation or synthesis of in situ particles. With subsequent gene knock-out of the identified processes, it might be possible to better determine the origin of intracellular NPs.


The claustrum (CLA) is a subcortical structure, present only in mammals, whose function remains uncertain. Previously, using resting-state functional magnetic resonance imaging (rs-fMRI) in awake head-fixed rats, we found evidence that the CLA is part of the rodent homolog of the default mode network (DMN; Smith et al.,  The claustrum (CLA) is a subcortical structure whose precise function remains unknown, but has been implicated in various mechanisms involved in directing attention Mathur, , saliencWe recently used neuroanatomical tracing in conjunction with resting-state functional magnetic resonance imaging (rs-fMRI) in awake, head-fixed rats to investigate the relationship between the structural and functional connections of the rodent CLA  activity related to each function, as measured by fMRI see . One sucTwo more ICNs are thought to operate in opposition to the DMN: the SN and the executive control network ECN; . In contr2 = 0.89) with visual analog scores (VAS) from a pre-scan anxiety assay  may arise, at least partly, from the CLA , must be interpreted cautiously  guided tracing of the BLA  and other experiments identifying the frontal eye fields in cingulate cortex of rats (Awh et al., redators .via IT-type neurons, but not actual motor output, which is conveyed by the PT-type neurons (Shepherd, When attention needs to be managed across multiple threats in opposing spatial fields , we hypoThe connectivity between the CLA and Cg cortex is of particular interest with regards to salience processing. The human SN is defined as coupling between the dACC and a region that appears to encompass the CLA. In addition to our seed-based analysis that corroborates this functional connection in rodents, we have demonstrated anatomical and functional connections from the CLA to Cg cortex. The CLA to Cg cortex circuit seems well-suited to guide salience processing owing to the limbic inputs received by the CLA from the BLA, which are relayed to Cg cortex: a motor output region shown to guide eye movements (Smith and Alloway, Major connectivity networks such as the DMN and SN can have aberrant connectivity patterns that are thought to underlie a host of neurological and psychiatric disorders Menon, . For exaBased on our recent studies, we propose that the CLA plays a role in SN-related neuropsychiatric disorders (Kalaitzakis et al., Future neuroimaging and optogenetic behavioral studies are necessary to demonstrate what role, if any, the CLA plays in emotional processing, particularly with regard to anxiety. Nevertheless, this endeavor represents a new frontier for the treatment of affective neuropsychiatric disorders using deep brain stimulation or drugs that target the CLA. In fact, kappa opioid receptors, which are abundantly expressed in the CLA, may serve a therapeutic role in treating depression (Stiefel et al., Our results demonstrate a role for the CLA in both the DMN and the SN. Furthermore, these findings expand the view of the CLA from being only a sensorimotor structure (Smith and Alloway, This study was carried out in accordance with the guidelines and approval of the Institutional Review Board at the University of Massachusetts and Penn State University.JS and GW wrote the first draft of the manuscript, constructed figures, and performed anatomy experiments and analysis. JS, GW, NZ and KA made significant revisions to multiple drafts of the manuscript. ZL, YL and NZ performed rs-fMRI experiments and analysis. NZ and KA obtained funding and supervised all data collection and analysis. All authors approve this submission and take responsibility for all information contained in this article.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.