The homeostasis of the central nervous system (CNS) is ensured by the endothelial, epithelial, mesothelial and glial brain barriers, which strictly control the passage of molecules, solutes and immune cells. While the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) have been extensively investigated, less is known about the epithelial and mesothelial arachnoid barrier and the glia limitans. Here, we summarize current knowledge of the cellular composition of the brain barriers with a specific focus on describing the molecular constituents of their junctional complexes. We propose that the brain barriers maintain CNS immune privilege by dividing the CNS into compartments that differ with regard to their role in immune surveillance of the CNS. We close by providing a brief overview on experimental tools allowing for reliable in vivo visualization of the brain barriers and their junctional complexes and thus the respective CNS compartments. The brain barriers established by the endothelial blood-brain barrier (BBB), the epithelial blood-cerebrospinal fluid barrier (BCSFB), the meningeal brain barriers and the blood spinal cord barrier are essential for maintaining central nervous system (CNS) homeostasis . While tDevelopment of the CNS vasculature begins with the process of vasculogenesis, where angioblasts, which originate from the mesoderm, move to the head region and form the primary perineural vascular plexus around the developing brain. The proliferating neuroectodermal tissue is invaded by vascular sprouts from the primary perineural plexus establishing the brain vasculature by a process called angiogenesis. The unique barrier properties of the brain endothelial cells are not intrinsic and are rather induced by a process referred to as barriergenesis by the continuous crosstalk with the developing neuroectodermal tissue ,3,4,5.The ChP develops from different locations along the dorsal axis of the neural tube, with the hindbrain ChP of the fourth ventricle being the first to be formed, once the neural tube is closed . While tThe meningeal barriers comprise three layers: the dura mater, the arachnoid mater and the pia mater, and cover the brain and the spinal cord from its earliest stages of development, establishing a protective covering of the CNS in the adult. Our current understanding of the development of the meninges has not much advanced beyond identifying the origins of pial, arachnoid and dural fibroblasts in the frontal brain from neural crest, and in the midbrain, hindbrain and spinal cord from mesoderm . DespiteTaken together, junctional complexes established between cells of the brain barriers might not only differ between the different barriers but also within one respective barrier along the rostral-caudal axis. Here, we summarize the current knowledge of the brain barriers, with a particular focus on the molecular components of their junctional complexes. Additionally, we suggest that visualization of brain barriers junctional complexes provides useful landmarks for in vivo imaging of immune surveillance of the CNS.The brain vasculature is a big and complex network composed of arteries and arterioles, capillaries, venules and veins, which allows for the vital distribution of nutrients and oxygen to the CNS at the level of the microvasculature, namely arterioles, capillaries and post-capillary venules . Brain mSLC2A1) is also highly enriched in the BBB endothelium, allowing for glucose delivery to the CNS .terminus ,104,105.terminus ,105.Finally, claudin-12 is an atypical member of the claudin family because it does not have a PDZ-binding motif . PromineTaken together, reports describing junctional localization of claudin-1, claudin-3, and claudin-12 proteins in brain endothelial cells may be inaccurate due to not well-defined cross-reactivities and thus false-positive staining of claudin antibodies. Cross-reactivity of the anti-claudin antibodies may be due to the high degree of conservation and a common amino acid sequence within some members of the claudin family, which could potentiate a cross-reactivity of anti-claudin antibodies . FurtherThus, at present, claudin-5 is the only claudin confirmed to be expressed at the mRNA and protein level in brain endothelial cells, to be localized to BBB TJs and to play a major role in BBB TJs integrity. Advanced transcriptome profiling studies have identified additional claudins, e.g., claudin-25/CLDND1 to be expressed in brain microvascular endothelial cells, with their exact function to be determined ,105.The TAMP family comprises the proteins occludin, tricellulin and MARVELD3 ,108,109,+ T cells into the CNS but is not involved in the α4β1-integrin-mediated migration of CD4+ T cells across the BBB [The third group of proteins found in BBB TJs are the members of the JAM family, JAM-A, JAM-B and JAM-C. These proteins are immunoglobulin (Ig)-like molecules composed of a single transmembrane domain, two extracellular Ig domains and a PDZ-binding motif in their C-terminus that allows for an interaction with cytoplasmic proteins linking the JAMs to the actin cytoskeleton ,122. JAM the BBB ,133,134. the BBB ,135.The claudins, occludin and the JAMs expressed in TJs connect to the cytoskeleton via the interaction with intracellular scaffolding proteins, which form the TJ plaque. The membrane-associated guanylate kinase (MAGUK) proteins represent the major subgroup of scaffolding proteins at the TJs ,137. TheAdherens junctions (Ajs) have a role distinct from TJs. Prior to TJ formation, cell-cell contacts are established by AJs, which is a prerequisite for TJ maturation and maintenance. Thus, AJs are generally required for TJ formation, and a continuous crosstalk between AJs and TJs is necessary for the organization and preservation of the junctional complexes (reviewed in Reference ). VE-cadAdditional molecules are found to be localized at the endothelial cell-cell junctions, outside of the organized AJs and TJs. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a transmembrane protein that belongs to the Ig superfamily. It is highly concentrated and restricted to the endothelial junctions but is found outside the organized TJs and AJs complexes ,160. HowAlthough a large number of molecules localized at the cell-to-cell contacts of the BBB within TJ, AJs and beyond has thus been described, their dynamic interplay and regulation during CNS homeostasis and how their expression and localization changes during neurological disorders when BBB function is impaired remains to be investigated in more detail. In addition, species differences in the expression of molecular constituents of the BBB junctional complexes have been described ,167,168,The development and maintenance of the CNS is assured by the cerebrospinal fluid (CSF), found in the spinal and brain subarachnoid space, cisterns, sulci and in the cerebral ventricles ,170. Imp+ in the proximal tubule of the kidney suggesting a role for claudin-2 in paracellular transport of small cations across the BCSFB [In accordance to the endothelial BBB, TJs and AJs are present in the BCSFB epithelium, ensuring not only the barrier function but also the apicobasal polarity of the BCSFB . Claudinhe BCSFB . Intereshe BCSFB ,91. Accohe BCSFB . If any he BCSFB ,179. In he BCSFB  which wahe BCSFB . Thus, sLike any other epithelial barrier, the BCSFB epithelial cells establish AJs, where the transmembrane E-cadherin binds to the catenin complex, which anchors the AJs to the epithelial actin cytoskeleton allowing to control adhesive interactions between the BCSFB epithelial cells ,181.A schematic representation of the junctional complexes at the ChP BCSFB can be found in The meninges constitute three layers, the dura mater, the arachnoid mater and pia mater, which sheath the brain and spinal cord  have allowed for unprecedented observations of immune cell entry into the CNS during immune surveillance and neuroinflammation. We have proposed that the brain barriers play a vital role in separating compartments in the CNS that provide different access to the immune cells. Advanced imaging has also allowed for precise localization of cellular junctional molecules ,204 in vThere are few major approaches allowing for visualization of junctional complexes in brain barriers. First, transgenic mouse models, where a fluorescent reporter is expressed under the promoter of the junctional protein expressed in the respective cell, can be employed. In this case, the soluble fluorescent protein will be visible in the whole volume of the cells expressing the corresponding junctional protein. For example, claudin-5, which has been identified as a major constituent of the TJs of CNS endothelial cells, was used to create the Tg (Cldn5-GFP) Cbet/U reporter mouse line , where gAlternatively, knock-in mouse models can be employed where the fluorescent protein is fused to the respective junctional molecule. This approach enables visualization of the subcellular localization of the respective junctional molecule in the cells via the fluorescent tag. For example, the VE-cadherin-GFP knock-in mouse , expressSimilarly, the E-Cadherin-CFP knock-in mouse expressing the fluorescent protein monomer Cyan (mCFP) fused to the C-terminus of E-cadherin enables visualization of the AJs of epithelial cells . This moIn vivo imaging of BBB TJ complexes in the mouse has already been successfully reported . To this11 fragment of only 16 amino acids formed of a single β-strand in case of the GFP1–10D7/11M3 OPT split protein [11 fused to the target protein.Another shortcoming of the fusion protein models is the potential impact of the relatively big fluorescent proteins on the appropriate localization of junctional molecules and their interaction with intracellular scaffolding proteins. This issue can be addressed by employing self-complementing split fluorescent proteins ,209, whe protein . The GFPAdditional fluorescent reporter mouse models can be obtained with the versatile aid of the Cre/loxP system. For example, by crossing the Ai14 line , where tVisualization of the BCSFB can be achieved by employing the FOXJ1-GFP reporter mouse or FOXJ1 Cre models , specifiTo distinguish capillary and post-capillary segments of the CNS vascular tree, differential visualization of smooth muscle cells and pericytes can be used. For example, the expression of Cre and eGFP under the promoter of smooth muscle myosin heavy chain was developed to visualize the vascular and non-vascular smooth muscle cells . VisualiFinally, in peripheral vascular beds visualization of junctional components has been achieved by intravenous injection of fluorescently tagged non-function blocking antibodies targeting, e.g., PECAM-1 to label endothelial cell junctions in vivo . This apAt the surface of the brain or spinal cord assignment of a vascular structure to a specific layer within the meninges or a compartment in the CNS can be facilitated by making use of the second harmonic generation (SHG) signal  obtainedVarious small molecule tracers, such as TRITC and FITC Dextran, nanoparticles can be employed to visualize the luminal compartment of blood or lymphatic vessels. In neuroinflammation, imaging junctional complexes combined with the injection of vascular fluorescent tracers may allow to image BBB junctional leakiness. By injecting tracers into the ventricles or into the cisterna magna, distribution of the fluorescent tracers has been successfully applied to visualize paravascular CSF drainage pathways along subarachnoid vessels  allowingDespite decades of research, there are still a lot of unresolved questions concerning the precise molecular composition and interaction of TJs and AJs of the brain barriers, which have hampered the development of therapeutic strategies targeting the brain barriers in neuroinflammatory disorders. Regarding the BBB, it has been recently confirmed by several reports that claudin-1 and claudin-3 are absent from BBB endothelial cells and thus, claudin-5 is the only critical TJ claudin proven to date to contribute for the mouse BBB function . HoweverStructural and cellular junctions of meningeal barriers are less studied as compared to the BBB and the BCSFB. Meningeal barriers from the front and posterior region of the nervous system have been described to develop from different origins and therefore will have a different molecular make-up of their respective junctional complexes which may lead to regional differences in meningeal barrier functions.A first set of available tools for in vivo visualization of the junctional components of the different brain barriers allows to study the dynamic interactions of immune cells with the brain barriers during health and neuroinflammation. Identifying the junctional components of the endothelial, epithelial, meningeal and glial brain barriers will set the stage for developing further mouse models, which will allow for in vivo imaging of the brain barriers. With these tools, one can thus explore their function in maintaining CNS immune surveillance and the impact of brain barrier disruption in neuroinflammation in vivo.


To demonstrate the role of cadaveric hands-on training model on surgical skills and confidence levels of surgeons during transobturator tape (TOT) surgery.A pre-test and post-test evaluation to measure skills during the practice of the steps of TOT surgery was performed on a total of 15 postgraduate urologists and gynecologists during a urogynecologic cadaveric dissection course. The course was shaped with regard to theoretical lessons, full pelvic cadaveric dissection and TOT surgery on cadavers.Good handling of the TOT needle, identifying the right place for groin incision, adequate size of groin incision, identifying the right place for incision at the anterior vagina, dissection of bladder pillars from the vagina, identifying the right place at the vaginal foramina for TOT needle exit, and good placement of mesh were reviewed. The post-test scores were statistically significant for all parameters and also for self confidence level (p<0.001).Cadaveric workshops are important landmarks of surgical education to improve surgical skills, and gain experience and confidence. Many residents and young surgeons attend to cadaveric dissection courses, which are in association with surgical anatomy and procedures, because detailed anatomic knowledge is essential for accurate practice in surgical procedures ,2. DespiPostgraduate courses highly focus on improving anatomic knowledge in surgical procedures, and physicians need a good knowledge of anatomy to manage patients intra-operatively and post-operatively. Cadaveric workshops have a beneficial status on the learning points of surgical procedures without any stress of the operation room .Stress urinary incontinence (SUI) is highly common among pre and postmenopausal women, the prevalence is between 4% and 35% , and theThis study aimed to analyse the effect of a cadaveric dissection course on the surgical competence of participants for TOT procedure with a pre/post-test.In total, 15 postgraduate physicians, urologists and gynaecologists, attended this urogynecologic cadaveric dissection course to gain adequate knowledge in pelvic reconstructive surgery and urogynecology. The faculty consists two urologists, one gynaecologist, and one anatomist. After theoretical lessons, a detailed pelvic dissection was performed with two fresh-frozen female cadavers and afterwards a pre/post-test, which measured 8 parameters. The points scale was from 0 (no success) to 5 (very good) and 3 physicians evaluated all participants. First, participants tried to perform a TOT procedure, then mentors showed the tips and tricks of the TOT procedure with step-by-step anatomic landmarks, the participants repeated the procedure and were evaluated for each described intervention (flowchart). One of the check points after the test the measurement of the confidence level of the participants for TOT procedures.Statistical analyses were performed using the SPSS software version 21. Visual and analytical methods were used to investigate whether the variables were normally distributed. Median and minimum-maximum values were used to present non-normally distributed variables. The Wilcoxon test was used to compare the change in scores between the pre-test and post-test analysis. A p value of less than 0.05 was considered to show a statistically significant result.A 16-F Foley catheter was placed in the bladder, clamped, and tracked to localise the position of the urethra neck. Then, the clitoris was palpated and the parallel location at the genitofemoral fold was used for the groin incision, 0.5-1 cm in length. A mid-urethral vaginal incision 1-1.5 cm in length was performed and, with sharp and blunt dissections, the pubo-cervico-vaginal fascia was opened cranio-laterally until the superposed obturator foramina. After the bladder was removed from the operation field, a TOT needle was inserted from the groin incision (out-in technique) and two clicks was felt while getting into the lateral field of vagina. The pathway of the needle after the skin is the obturator externus muscle, obturator membrane, obturator internus muscle, endopelvic fascia, and vaginal incision. Afterwards, the needle was directed to the vaginal incision to exit. This intervention was performed bilaterally; the tape was laid in front of the urethra with a distance of a Metzenbaum scissor.Parameters that were identified to measure the ability for TOT procedure were: good handling of the TOT needle, identifying the right place for the groin incision, adequate size of groin incision, identifying the right place for incision at the anterior vagina, dissection of the bladder pillars from the vagina, identifying the right place at the vaginal foramina for TOT needle exit, and good placement of mesh. At the end, the participants self-evaluated their confidence level as per their situation before the demonstration by mentors and after the course. All parameters were detected as statistically significant (p<0.05) .TOT procedures are very commonly performed by gynaecologists and urologists to increase the quality of life of women due to SUI. It has quite low rates of complications and discomfort after surgery with a desirable improvement in symptoms ,10. DespThis study has an important corner, which is in conjunction with urogynecologic practice and anatomic cadaveric studies. It showed that proper surgical education at the cadaveric workshops improved the surgical skills for all steps of a procedure, with increased confidence levels for the operation room.Surgical training needs a good knowledge of anatomy and clinical consideration for better surgery. In general, there are no formal post-graduate training centres in surgery. However, surgical anatomy education programs should be based on the need of physicians and course directors should plan the flow-chart of the training module according to deficits in surgical anatomy. Additionally, observation and one-to one dissection with mentors, and performing procedures under their guidance increases the basic and advanced anatomic knowledge of participants with clinical concordance .A literature search revealed many anatomic cadaveric studies in the field of urogynecology. Smajda et al.  exploredThis urogynecologic cadaveric dissection course aimed to make surgical procedures easier with cadaveric dissections. Detailed pelvic anatomy with self-practice of participants aimed to increase the topographic view of pelvic and nearby anatomic landmarks during pelvic and urogynecologic surgeries. For surgical approaches, many physicians find it beneficial to attend cadaveric workshops to improve surgical skills in an atmosphere of less stress. This course was planned to be held 3 times per year with 15 participants to improve skills in pelvic reconstructive and urogynecologic surgery.All the steps of TOT procedure were analysed by mentors and mistakes in the practice of surgery were solved with self-practice. During these steps, close anatomic landmarks were discussed and identified with dissection of the pelvis, perineum, and obturator space. The axis of the trocar needle must be accurate and that needs a good level of experience because the path of the trocar needle is blind and it passes many tissues and fascia around the vessel and nerve structures. Otherwise, the obturator artery, obturator vein or obturator nerve be injured on the lateral pelvic wall, additionally the bladder may also be damaged. Movement of TOT needles were analysed and viewed from the abdominal incision to check probable complications by all participants; the proximity of obturator nerve and obturator foramina, the position of bladder, the location of vagina and endopelvic fascia were evaluated during vaginal dissection and TOT needle insertion . At the All anatomic steps during TOT surgery , place oAs a conclusion, cadaveric workshops improve basic and advanced anatomic knowledge during the steps of transobturator tape procedure with improved skills and increased confidence status.


While the positive effects of environmental enrichment (EE) applied after weaning, in adulthood, during aging, or even in the presence of brain damage have been widely described, the transgenerational effects of pre-reproductive EE have been less examined. And yet, this issue is remarkable given that parental environmental experience may imprint offspring’s phenotype over generations through many epigenetic processes. Interactions between individual and environment take place lifelong even before conception. In fact, the environment pre-reproductively experienced by the mother and/or the father exerts a substantial impact on neural development and motor and cognitive performances of the offspring, even if not directly exposed to social, cognitive, physical and/or motor enrichment. Furthermore, pre-reproductive parental enrichment exerts a transgenerational impact on coping response to stress as well as on the social behavior of the offspring. Among the effects of pre-reproductive parental EE, a potentiation of the maternal care and a decrease in global methylation levels in the frontal cortex and hippocampus of the progeny have been described. Finally, pre-reproductive EE modifies different pathways of neuromodulation in the brain of the offspring . The present review highlights the importance of pre-reproductive parental enrichment in altering the performances not only of animals directly experiencing it, but also of their progeny, thus opening the way to new hypotheses on the inheritance mechanisms of behavioral traits. Organisms adapt their physiology and behavior in response to environmental modifications. The influence of environmental experiences during lifespan is confined not only to the neurobehavioral profiles of the directly exposed individual, but it can also be evident in the next generations . GrowingIt is well-known that pre-natal/early post-natal as well as ancestral experience of environmental insults  may result in modifications of the phenotype later in life . In the Environmental enrichment is an experimental paradigm apt to potentiate social, cognitive, and sensorimotor stimulations experienced by animals . By incrEnvironmental enrichment as well as exercise may improve learning and memory, and enable neuroplasticity processes involving increased neurogenesis, possibly via neurotrophin-mediated mechanisms . NotablyIn the last years, attention has been paid to the transfer of proactive effects of enriching experiences from parents to the progeny, and the number of researches on this issue is significantly increased over time . By usinThe effects of parental enrichment across generations have been mainly investigated by exposing mothers to different kinds of environmental stimulations during gestation and/or lactation .A rising literature demonstrates the role of maternal , 2018 orTo our knowledge, the literature on the effects of early EE by parents in humans is still scarce, and principally deals with fetuses’ and newborns’ stimulation. In fact, besides an accumulating evidence on the beneficial consequences of tactile  and auditory stimulation  on the neurobehavioral development of preterm infants  or the eIn the following paragraphs, we will take into account the impact of pre-reproductive parental exposure to enriching experiences on maternal behavior and on physical, motor, cognitive and emotional features of the offspring in rodents. Furthermore, based on our studies and the few others present in literature, we will focus on the changes in a key neurotrophic factor, such as the brain-derived neurotrophic factor (BDNF), and in the oxytocinergic system following pre-reproductive parental housing in highly stimulating environments. Whenever possible, comparisons with the effects of parental enrichment during gestational and/or lactation periods  will be also considered. Namely, in Table constellation” of behaviors of preparation for the arrival of the newborns as well as their nurturing and protection  and arched-back nursing (ABN) during the third lactation week .Mother rats enriched before and during gestation show heightened LG behavior and reduced pup nursing .Maternal enrichment during pre-reproductive, gestational and lactation phases induces an increase in ABN, but a reduction in the time spent in the nest and nursing .There are also studies in which parental enrichment does not affect  or even Taken together these data, even somewhat contrasting, seem to account for a modulatory effect of environmental conditions on maternal care that reflects the different EE protocols used or the timing of enrichment exposure.The more effective EE protocols for increasing maternal care are the ones providing at least social, cognitive and physical stimulations during pregnancy and lactation . MoreoveA variable conserved across dams housed in EE is the reduction in the time/frequency in contact with the nest. It may be attributed to the increased physical space of the enriched cage, when the EE protocol is applied at least during gestation and lactation , given tpost-partum care in comparison to standard-reared females, and their offspring show increased frequency of nursing  in the hypothalamus of dams  in the study by As evident, the implications of environmental factors on the complex relationship among paternal, maternal and offspring phenotypes still represent a crucial challenge in the study of the mechanisms driving paternal effects.A proper assessment of the transgenerational effects of parental experiences on offspring developmental trajectories can be achieved by examining the maturation of motor behaviors in rodents. This represents a useful tool to carefully assess early post-natal neurodevelopment, because the appearance of sensorimotor reflexes and motor skills typically follows a definite timing during the first 3 weeks after birth .Unfortunately, few studies paid specific attention to the transgenerational effects of EE on offspring’s motor development and even fewer ones used the pre-reproductive EE paradigm, although it shows the possibility to distinguish pre- from post-natal effects of EE, thus allowing the study of Lamarckian inheritance.To this aim, we performed a series of studies to examine the hypothesis that pre-reproductive maternal EE could affect progeny’s phenotype, by rearing female Wistar rats in an enriched environment from weaning until mating , 2018. TThis pre-reproductive maternal EE did not affect litter characteristics, such as litter size and male/female ratio, thus suggesting that this experience does not influence the reproductive ability or the pregnancy of female rats , 2017. IWe found that pre-reproductive maternal enrichment affects offspring’s body weight only at birth, with enriched mothers’ offspring weight less than controls , 2017, sOverall, it seems that parental environmental experiences affect the body weight of the progeny. Namely, enriched mothers influence progeny’s body weight only at birth, in a way ambiguously linked to different EE conditions, whereas enriched fathers influence offspring’s body weight at adulthood. Thus, it is possible to speculate that both maternal and paternal influences are driven by pre-natal metabolic programming, even if we cannot exclude that a specific maternal behavior in the pre-weaning phase may account for the particular fitness outcome observed in the progeny of enriched fathers .The pre-reproductive maternal EE alters the motor development of the progeny, leading to an earlier acquisition of those abilities that require complex sequencing and coordination of the motor output . FurtherThe open field test (OF) is one of the most frequently used method to assay locomotor behavior in rodents and is sensitive to EE-induced exploratory modifications. The analysis of the effects of pre-reproductive parental enrichment on explorative activity of the progeny provided conflicting results. In fact, increased OF activity levels have been reported in young male offspring of pre-reproductively enriched fathers , while aIn conclusion, the exposure of rodents to EE during the pre-reproductive phase significantly shapes the neurodevelopment of progeny, by accelerating the acquisition of motor abilities . No signIn rodents the effects of parental enrichment on the offspring’s cognitive performances and anxiety levels have been primarily investigated by exposing mothers to complex stimulations during pregnancy and/or lactation, and by using different kinds of behavioral tasks and biochemical correlates. Namely, the exposure of mothers to different kinds of EE paradigms during pregnancy facilitates learning and memory abilities , and incMaternal exposure to EE during pregnancy and lactation improves spatial memory performances in the Morris water maze, reduces anxiety, and prevents the hippocampal tissue loss after neonatal hypoxia ischemia .Also the exposure of mothers to exercise only paradigms during pregnancy  or durinThe maternal exposure to EE before and during gestation affects offspring’s development trajectories by modifying cognitive and emotional outcomes in a sex-specific manner with improved learning ability only in females and increased anxiety mainly in males .Lastly, data resulting from post-natal social enrichment obtained by communal nesting are variable and still difficult to reconcile across studies. For example, a study by The few studies available on the effects of pre-conceptional parental enrichment on the cognition and emotional response of the offspring generally indicate an amelioration of memory performances and a reduced anxiety. In fact, Low LG female offspring housed under enriched conditions display increased LG behavior and higher levels of exploration in comparison to standard housed low LG females . These vIn addition, communal nesting is able to increase maternal care and reduce anxiety across generations .As for pre-reproductive paternal enrichment experiences, the existing studies mainly addressed the emotional responses of the offspring. Namely, a very recent study by In a genetic rat model of absence epilepsy, early enrichment of fathers from weaning to breeding induces anti-epileptogenic and anxiolytic effects that were heritable across generations . These fIn conclusion, it seems that pre-reproductive paternal housing conditions which include an overt motor enrichment by running-wheel presence are able to induce anxiolytic effects.With regard to indirect effects of parental enriching experiences on the pups’ behavioral trajectories through the modulation of maternal care, the few data currently available are still little explicative, since maternal behavior has not been systematically investigated. However, it can be noted that when an enhancement in maternal care is evident following pre-reproductive maternal enriching experiences, the offspring’s phenotype can be characterized by enhanced maternal care and/or reduced anxiety  or by imAs for paternal enrichment, it seems that a clear relationship between maternal care and offspring’s behavior is still not well-definite. In fact, studies reporting behavioral modifications in the offspring of not-enriched females mated with males enriched during the pre-reproductive period fail to report maternal care modifications, thus suggesting that the progeny could be differently influenced by the pre-reproductive paternal experiences according to the different environmental manipulations used , regardless maternal investment . InteresSocial interactions are essential for survival and proper neural and behavioral development. After weaning, playful interactions with peers allow the acquisition of social and cognitive competence. Social play behavior is a highly rewarding activity in humans and animals, as it can instill a sense of well-being and pleasure, motivates approach behaviors toward a specific social stimulus, and finally elicits associative learning in order to attribute salience to socially related cues . InteresAs for pre-reproductive maternal EE, two studies demonstrated that it reduces social interaction in males, but not in females either in adult and adolescent offspring . SimilarNevertheless, pre-reproductive maternal EE does not induce any difference in sociability as assessed in the three-chamber sociability test , in whicEnvironmental enrichment has been considered as prevention/intervention strategy to counteract the negative consequences of stress .The ability to cope with environmental challenges is essential to be successfully adapted. To face acute and chronic stressors, the sympathetic system and the hypothalamic-pituitary-adrenal (HPA) axis guarantee the recruitment of necessary resources, the inhibition of non-necessary ones, and fine feedback mechanisms for homeostasis. Glucocorticoids (GCs) secreted from adrenal glands act peripherally and in the brain through their binding with mineralcorticoid (MR) and glucocorticoid (GR) receptors. GR are recruited when GCs levels are elevated, as in a stressful condition, and are involved in negative feedback on HPA axis .Genetic background  and pareParents have a pivotal role in programming the offspring’s HPA axis functioning as widely demonstrated in rodents , and morParental stress has been associated with offspring’s greater risk of psychopathology , higher Danio rerio)  .In rats long-lasting moderate maternal EE during pregnancy, and together with pups during lactation until weaning, modifies the response of the female offspring to a chronic stress . WhereasMoreover, the positive effects of EE on stress response are reported in different strains of mice. Remarkably, the enhancement of stimulations provided by EE produces changes that propagate across generations influencing descendant’s future responses to chronic and acute stress  and promPre-reproductive EE can modify the effect of social isolation, in rats. The offspring born to standardly reared mothers and subjected to social isolation from weaning to adolescence show greater GR expression in amygdala compared to not-isolated controls, whereas socially isolated offspring born to pre-reproductively enriched mothers do not show any difference in GR expression when compared to the respective not-isolated controls. Furthermore, a blunted amygdaloid c-Fos immunoreactivity in response to the FST is evident in the offspring of pre-reproductively enriched mothers in comparison to the offspring of standardly reared dams .Recently, the transgenerational effects of pre-conceptional paternal EE on despair behaviors and neuroendocrine phenotypes of the adult offspring in mice has been investigated . EE effeEnvironmental enrichment and stress indeed activate similar circuitries and involve HPA axis activity and changes in GR expression and functionality .Several theoretical models have been proposed to account for the beneficial or contrasting effects of EE on stress response. Considering conflicting transgenerational EE effects, the degree of fitness between the environmental challenges experienced by the offspring and the environmental demands for which parents and grandparents have equipped future generations to cope with has to be keep in mind .Finally, transgenerational inheritance could interact with ingrained individual differences to determine the phenotypic scenarios “for better or for worse” .Neurotrophins constitute a protein family whose components show analogous structure and exert an essential action on the development and function of the neurons. They regulate cell proliferation and differentiation, growth and readjustment of axons and dendrites, and plastic changes involved in synaptic function . In partRecently, it has been evaluated if the known EE effects on BDNF-mediated brain plasticity could be transgenerationally transmitted. Namely, some research has been specifically devoted to study the effects of the pre-reproductive exposure of mothers to EE on the offspring’s BDNF brain levels . InteresIn line with our results, in a different model of maternal EE, pups born to mothers enriched during pregnancy and lactation do not show variations in cortical or hippocampal BDNF levels few days after birth . In addiOn the whole, the available evidence supports the influence of the maternal enrichment during pre-reproductive and post-reproductive periods on the pup brain BDNF-mediated neuroplasticity, regardless of the specific characteristics of the enrichment paradigm.The neuropeptide oxytocin is widely implicated in the social behavior of mammalian by modulating maternal care, infant behavior, social bonding, agonistic behavior and social recognition . It is spost-partum care and of oxytocin receptor density in the lateral septum across generations  literature is the diversity of paradigms as for timing of exposure and enhanced stimulations used. Nevertheless, a common pattern emerges in findings. As for dams, the pre-reproductive enrichment tendentially results in increased maternal care, brain BDNF and oxytocinergic levels. As for offspring, the pre-reproductive parental enrichment appears to accelerate the acquisition of complex motor abilities, potentiate cognitive performances and coping skills, reduce anxiety, increase brain BDNF levels and modulate social behavior and oxytocinergic system in a sex-dependent manner.On the whole, the available evidence supports an influence of pre-reproductive parental enrichment on mother’s and pups’ brain and behavior. However, given the diversity in the different parental enrichment paradigms, systematic analyses on the selective exposure to EE  in the different pre-reproductive and post-reproductive periods are required to provide exhaustive evidence on brain and behavior changes induced in pups by parental experiences.It is interesting to speculate on the mechanisms of inheritance involved in the transmission of the environmental influence from parents to the offspring. The inheritance across generations may involve epigenetic modifications in the germline or can be passed to the offspring through maternal care during early post-natal life . Non-genFinally, the study of the inter- and transgenerational origin of age- or stress-related diseases offers the opportunity to identify predictive or diagnostic biomarkers crucial to develop new interventions according to a tailored clinical approach that claims healthy aging and mental health in today’s population and future generations.DC and LP coordinated the work, wrote, and edited the article. EB, FG, PC, and DL contributed in writing the 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.


Over the previous decades, numerous studies focused on how oceanic islands have contributed to determine the phylogenetic relationships and times of origin and diversification of different endemic lineages. The Macaronesian Islands , harbour biotas with exceptionally high levels of endemism. Within the region, the vascular plants and reptiles constitute two of the most important radiations. In this study we compare relevant published phylogenetic data and diversification rates retrieved within Cabo Verde endemic lineages and discuss the importance of choosing appropriate phylogeny-based methods to investigate diversification dynamics on islands. From this selective literature-based review, we summarize the software packages used in Macaronesian studies and discuss their adequacy considering the published data to obtain well-supported phylogenies in the target groups. We further debate the importance of Next Generation Sequencing (NGS), to investigate the evolutionary processes of diversification in the Macaronesian Islands. Analysis of genomic data provides phylogenetic resolution for rapidly evolving species radiations, suggesting a great potential to improve the phylogenetic signal and divergence time estimates in insular lineages. The most important Macaronesian reptile radiations provide good case-studies to compare classical phylogenetic methods with new tools, such as phylogenomics, revealing a high value for research on this hotspot area. During the last two decades, one of the fields that took the most benefit of the explosive growth of bioinformatic tools was phylogenetics, which uses molecular data to establish evolutionary relationships among species ,2. PreseAn outstanding area for studies of evolution and speciation is the Macaronesian Region  [Gonosperminae (Asteraceae) [Bencomia (Rosaceae) [Pericallis (Asteraceae) [Lactuca (Asteraceae) [Cistus (Cistaceae) [Azorina, Campanula, Trachelium, Lobelia, Musschia, and Wahlenbergia (Campanulaceae) [Gallotia (Lacertidae) [Hemidactylus (Gekkonidae) [The number of bioinformatics tools available today represents a huge framework for each question that arises, presenting several alternative solutions. For phylogenetic analysis, this can prove to be an extraordinary time, as new knowledge, powerful software and hardware frameworks, are continuously improved ,12. Threulaceae) ; Gonospeeraceae) ; Bencomiosaceae) ; Pericaleraceae) ). More reraceae) ; Cistus staceae) ; Azorinaulaceae) ; and endertidae) ; Hemidackonidae) .When working with ML methods that were made popular by Fisher in the early 20th century , the objPrograms such as PAUP*   and MEGALimonium, Plumbaginaceae [To specifically perform ML and BI analysis with Macaronesian reptiles  and planginaceae  and on Cginaceae .Bayesian approaches also allow obtaining divergence time estimations ,58, an aDue to the complexity of the divergence time estimation methods, the choice of the software to perform these analyses is paramount. The most popular software used in this type of analysis is BEAST  and the Globularia amygdalifolia (Plantaginaceae), Cynanchum daltonii (Apocynaceae) and Umbilicus schmidtii (Crassulaceae) [Ammi, Angelica lignescens, Azorina vidalii, Euphorbia stygiana, Pericallis malvifolia) [Lactuca [Other used approaches in Macaronesian phylogenetic inference studies, have been the estimation of phylogenetic networks, to test the existence of cryptic events  and often relate to intraspecific genealogies in the given molecular data set. The phylogenetic networks were used to produce complete community-level phylogenies and can be implemented in several software packages like TCS  and Spliulaceae) ) and of vifolia)  and of b[Lactuca . These m[Lactuca , which h[Lactuca , where tIslands can be broadly classified as one of two types: continental or oceanic. Continental islands are fragments of a larger landmass, whereas oceanic islands result from volcanoes rising above the water on or near a mid-ocean ridge. Particularly, oceanic islands, as is the case of the Macaronesian Islands, were the focus of studies that contributed to the understanding of speciation and the origins of biodiversity . The terDifferent patterns of colonization within the Macaronesian Region have been an ongoing subject of debate. Some of the islands within this region are near the mainland, with the closest being Fuerteventura in the Canary Islands, which distances only ca. 95 km west of mainland Africa A. This pCampanula [Diplotaxis [Echium [Lotus [Chalcides; Chioninia; Gallotia; Hemidactylus; Tarentola and Teira) occur within the Macaronesian Region  revealed that the tree is largely unresolved, and the clades within the tree show very weak support (55–69%). Therefore, particularly for plants, there is a need to perform more studies to understand the evolutionary patterns and dating analyses [Even with all challenges considered, reptiles are still one of the most well-studied groups within the Macaronesian region, and their evolutionary patterns appear to be clearer and more comprehensible . Plants analyses ,91.As stated above, phylogenetic methods used to date to establish the relationships among insular lineages were mostly based on single locus or a small number of loci  and often the resulting trees included unresolved clades and were thus unable to fully reflect the speciation events within the Macaronesian Region.Considering (i) the inherent drawbacks of the targeted molecular marker-based techniques, namely, the high effort required for finding a considerable number of informative loci; (ii) their little resolving power at low taxonomic levels, which ignores many population-level effects, and (iii) increasingly affordable sequencing costs resulting from the continuous advancement of next-generation sequencing (NGS) technologies, phylogenomics emerges as an promising alternative tool for studying evolutionary relationships based on comparative analysis of genome-scale data .Laurus azorica (Seub.) Franco, [Phylogenomics can be seen as the phylogenetics branch which specializes in inferring species relationships based on whole genomes (WGS) or genome-partition approaches and is starting to be applied in evolutionary and ecological studies on Macaronesian islands ,94. The  Franco, ) show so Franco, ), restri Franco, ,99,100.  Franco, . Even th Franco, .Another consideration when performing RAD-Seq analyses is the type of assembly to be used–de novo or referenced-based. As complete genomes are still scarcely available for most of the Macaronesian lineages, the selection of the method is usually limited, and the most viable solution is a de novo assembly . The useA scenario of rapid plant and reptile radiations is found among most of the Macaronesian lineages, with molecular differences not being correctly depicted by traditional molecular markers and phylogenetic analyses, mostly due to low accumulation of differences in the genomic regions screened between different species in different islands or even within species at intra-islands levels/different habitats ,105. MacTolpis [Micromeria [In regard to insular evolutionary studies, RAD-Seq has proven to be very effective to contribute to clarify the relationships within closely related taxa, as well as providing a good alternative to study groups where Sanger sequencing failed to provide accurate results ,111. In Tolpis  and Micrcromeria . Finally, other phylogenomics methods can be used to address the current shortcomings of relatively deep phylogenetic divergences  to shallow-scale divergences are based target enrichment to sequence protein-coding exons ,115 or aThe interdisciplinary field of bioinformatics was born from the need to develop and employ methodologies to study large amounts of biological data, namely proteins and DNA. Many scientific areas benefited greatly from the introduction of specialized and powerful tools that improved the implementation of databases, data analysis and biological interpretations. For evolutionary study fields such as phylogenetics, the knowledge jump has been substantial since the introduction of bioinformatic methods in their analysis. Making use of highly advanced software packages it is now possible to perform high-throughput DNA sequencing, and thoroughly analyse this data with several methods of phylogenetic inference, to obtain informative and well supported phylogenies. Moreover, the introduction of NGS was one of the biggest achievements in the biology field in the last couple of decades. Several biological fields were revolutionized and some only reached their true potential when high-throughput sequencing methods were finally widely available at a relatively low cost. For phylogenetic sciences, this was an revolutionary occurrence, as it was finally possible to sequence, store and analyse complete genomes, and in doing so, reconstruct more comprehensive phylogenies.The power of phylogenetic sciences has been crucial as tools in the development and consolidation of knowledge of insular native/endemic species in Macaronesia, clarifying their classifications, understanding genetic patterns and evolutionary processes that occurred on the islands and/or archipelagos. The combined data from phylogeny and divergence time estimations are able to clarify diversification hypothesis and assign probable colonization events, and can even help discover new species, providing useful information for the field of conservation biology in insular environments. Phylogenomics are at a turning point and it is highly likely that in the next few years, new understanding on several issues regarding species diversification and adaptation will be instigated by the application of new methodologies to natural laboratories such as the Macaronesian Islands.


Despite decades of research and major efforts, malignant brain tumors remain among the deadliest of all cancers. Recently, an increasing number of psychiatric drugs has been proven to possess suppressing activities against brain tumors, and rapid progress has been made in understanding the potential mechanisms of action of these drugs. In particular, the traditional mood stabilizer valproic acid, the widely used antidepressants fluoxetine and escitalopram oxalate, and the atypical psychiatric drug aripiprazole have demonstrated promise for application in brain tumor treatment strategies through multiple lines of laboratory, preclinical, and clinical evidence. The unexpected discovery of the anticancer properties of these drugs has ignited interest in the repurposing of other psychiatric drugs to combat brain cancer. In this review, we synthesize recent progress in understanding the potential molecular mechanisms underlying the brain cancer–killing activities of representative psychiatric drugs. We also identify key limitations in the repurposing of these medications that must be overcome to enhance our ability to successfully prevent and treat brain cancer, especially in the most vulnerable groups of patients, such as children and adolescents, pregnant women, and those with unfavorable genetic variants. Moreover, we propose perspectives that may guide future research and provide long-awaited new hope to patients with brain cancer and their families. Brain cancer is among the most lethal of all cancers. A large fraction of adult patients with glioblastoma multiforme (GBM), also known as glioblastoma, the most common and aggressive primary brain tumor, dies within 2 years of GBM diagnosis . The treOver the past decades, psychiatric medications have been shown to be clinically effective in the treatment of a broad range of mental illnesses, and they have been targets for drug repurposing to increase cost effectiveness. Existing psychiatric drugs have several advantages, such as free penetration of the BBB and thorough study in clinical settings . BesidesRapid progress has also been made recently in the understanding of mechanisms potentially underlying the brain cancer–killing activity of certain psychiatric drugs. However, limitations of the repurposing of these drugs must be overcome to enhance our ability to successfully prevent and treat brain cancer, especially in the most vulnerable groups of patients, such as children, adolescents, pregnant women, and those with unfavorable genetic variants. In the present review, we summarize recent progress in understanding of the brain cancer–killing activity of representative psychiatric drugs and potential underlying mechanisms. Additionally, we identify potential obstacles that must be overcome to enhance our ability to successfully prevent and treat brain cancer and propose perspectives for future research.Several epidemiological studies have shown that the incidence of cancer is significantly lower in patients with schizophrenia than in the general population . AdditioResearch teams throughout the world have extensively explored the molecular mechanisms underlying the anticancer activities of certain psychiatric medications, which may help to explain the reduced cancer incidence in patients taking these drugs and guide their repurposing for use against GBM and other forms of brain cancer. The characteristics of the representative psychiatric drugs with anticancer activity and potential mechanisms of action in the brain that are considered in this review are summarized in The dopamine (DA) receptor pathway is a well-recognized neurotransmitter in the brain with critical roles in multiple important brain processes  and behaviors in humans. Abnormal DA levels have been found in patients with psychiatric disorders and neurological diseases . DA exerEpigenetic dysregulations, apart from genetic aberrations, are well-known drivers of the development and progression of malignant tumors, including GBMs . The mecSeveral studies have demonstrated the beneficial effects of VPA in patients with brain tumors . Retrospin vitro and in vivo studies support the results of clinical studies , potassium (Kv), and calcium (Cav) channels; and reduce N-methyl-D-aspartic–mediated responses, through which VPA modulates the excitation of neurons. The anti-GBM effects of VPA observed in several  studies , but the studies . Clozapi studies . Followi studies . Moreove studies . CLC-4 h studies . Thus, mSrc, also known as cellular-Src, is a proto-oncogene tyrosine kinase and an important regulator of intracellular signal-transduction pathways. Src pathway activation is tightly regulated under normal conditions; its perturbation or excessive activity has been observed frequently in patients with many forms of malignancy, and constant Src activation in malignant tissues has been shown to promote proliferation, invasion, metastasis, and angiogenesis . The inhStudies of the role of the Wnt/β-catenin signaling pathway in the context of brain tumors have generated conflicting results. Several studies have shown that the overexpression of β-catenin reduces the proliferation and migration of glioma cells and the expression of stem cell markers, and others have shown that the enhancement of Wnt expression stimulates the self-renewal and proliferation of brain tumor cells . Rampazzvia the induction of autophagy. Notably, escitalopram oxalate’s induction of autophagy in GBM8401 cells in culture and animal models suggests the potential of repurposing this drug for the treatment of GBM  is the most thoroughly studied single-nucleotide polymorphism associated with a wide range of psychotic disorders (DRD2 gene and consists of a single nucleotide change (C/T), with the two alleles referred to as A2 (C) and A1 (T)  . Howeverd A1 (T)  alter thThe surprising findings regarding the antitumor effects of psychiatric drugs in the brain are of clinical importance. In particular, these psychiatric drugs hold potential for repurposing for the treatment of patients with brain cancers, such as GBM. The successful treatment of GBM with current conventional therapies is very challenging for the following main reasons: GBM cells are highly resistant to these therapies; the brain tissues, especially those of children and adolescents, are highly susceptible to damage by these therapies; and the brain has very limited self-repair capacity. As the clinical characteristics of these psychiatric drugs, such as safety and toxicity, have been studied extensively, they are likely to bypass lengthy preclinical studies. Furthermore, unlike the majority (90%) of existing drugs, these psychiatric drugs can successfully penetrate the BBB. Thus, these psychiatric medications with anticancer activities offer new hope for patients with brain cancer. However, greater understanding of the mechanisms of action of their anticancer effects is needed. Multicenter studies of psychiatric drug repurposing for the treatment of primary and metastatic brain cancer in vulnerable groups, such as children, adolescents, and pregnant women, are needed.CZ, ZX, and WH designed the idea; FJ, XL, and HT acquied the references; WZ, MC, CL, CC, and WW made a draft for this work; CZ, ZX, and WH performed the final version of this work.This work was supported by grants from the National Natural Science Foundation of China (81871052 to CZ), the Key Projects of Natural Science Foundation of Tianjin, China (17JCZDJC35700 to CZ), the Tianjin Health Bureau Foundation (2014KR02 to CZ), the Tianjin Anding Hospital Research Award 300000 yuan for CZ, and the Wenzhou Science and Technology Project (ZS2017011 to XL).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.


Maladaptive habits are behavioral patterns of inflexible behavior that reflect poor self-control and occur despite their harmful consequences. Those behaviors lead to undesirable outcomes in a broad range of a person's life domains. It is important to note that maladaptive habits, while prevalent, are not always a symptom of a disorder. Maladaptive habits develop in both psychiatric/neurological disorders and non-clinical samples; and reflect context-specific failure of self-control. There are many explanations concerning the etiology, pathogenesis, and consequences of maladaptive habits. Yet, more research is needed for understanding neurobiological and psychological mechanisms that support maladaptive habit formation and maintenance.The study of maladaptive habits is particularly relevant since online tempting behaviors have never been so readily available and easy to engage in. Specifically, the current easy access to computers, tablets, and smartphones allows people to get repeated and continuous rewards by just pressing a button . Moreover, the high volume of cues  makes it increasingly hard to resist the temptation of engaging in such activities.In the light of today's high-availability of ready-to-consume rewards, the aim of this Research Topic was to integrate contributions concerning the understanding, assessment, etiology, and consequences of different types of maladaptive habits. The collection includes ten articles addressing such topics.Pompeia et al.). In this paper, the authors found that the psychometric properties of the Brazilian Short Version of UPPS-P support the use of a single and general impulsivity score for this scale.Considering the assessment of impulsive behavior, this Research Topic includes a study presenting the adaptation of assessment tools such as the short version of UPPS-P to a Brazilian population  pertaining to traffic accidents in youth, and proposed strategies for preventing them. Jelihovschi et al. examined the relationship between cognitive impulsivity and decision making in professionals and students from the management field. They found that cognitive impulsiveness, related to immediatism impair complex decision-making assessed by the Cognitive Reflection Test. Together, these two studies stress the relevance of considering tempting and impulsive behavior within non-clinical samples.Two articles addressed the issues of impulsivity and maladaptive behaviors in non-clinical samples. Lima et al.), attention deficits and hyperactivity disorder , hoarding behavior , excessive smartphone use , and feeding and eating disorders . In these articles, the authors present several patterns of hampered impulse control in individuals presenting mental health traits, suffering from mental disorders, including among their relatives. Taken together, these studies support the relationship between habits, impulsiveness, and inhibitory control deficits within a broad range of maladaptive outcomes.Within the frame of mental disorders, the consequence of impulsivity and maladaptive habits were presented and discussed in relation to suicidal behavior . In this paper, the authors address the association between a genetic polymorphism related to the DRD2 expression (polymorphism C957T) and the memory for the reward stimulus related to addictive behavior.Finally, the neurobiological perspective of understanding the etiology and maintenance of maladaptive habits has been adopted by a study examining the relationship between dopaminergic system and reward memory in addiction (Overall, the diversity of issues and knowledge fields that contributed to this topic provides unequivocal evidences that maladaptive habits are a pervasive pattern of behavior with undesirable outcomes in a large variety of settings. To a broader extent, this Research Topic offers a relevant framework for further understanding the multifactorial basis of maladaptive habits and its relationship with adverse outcomes across the lifespan and life domains, in both clinical and non-clinical samples. We hence call for more research on this topic.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.


The mutualistic, commensal, and parasitic microorganisms that reside in the rumen and lower gastrointestinal tract of cattle and other ruminants exert enormous influence over animal physiology and performance. Because these microbial communities are critical for host nutrient utilization and contribute to the metabolic capacity of the rumen, past research has aimed to define host-microbe symbioses in cattle by examining the rumen and lower gut microbiomes with respect to production phenotypes, such as feed efficiency. The mutualistic, commensal, and parasitic microorganisms that reside in the rumen and lower gastrointestinal tract of cattle and other ruminants exert enormous influence over animal physiology and performance. Because these microbial communities are critical for host nutrient utilization and contribute to the metabolic capacity of the rumen, past research has aimed to define host-microbe symbioses in cattle by examining the rumen and lower gut microbiomes with respect to production phenotypes, such as feed efficiency. However, as the field of bovine gut microbial ecology progresses, multidisciplinary approaches must be employed, combining host genomics and other omics-based techniques to understand the complex host-microbe network. In this perspective, I discuss the direction of the field of bovine gut microbial ecology with regard to feed efficiency and explore how the grand challenge of such research will be to maintain host-efficient gut microbiomes in cattle production through manipulations of genome-microbiome interactions. To address food security challenges, we must continue to actively seek and develop novel tools and resources to produce animal proteins in an economically, environmentally, and socially acceptable manner that meets the demands of an increasing population . In beefFirmicutes taxa were observed in the group of steers with greater ADG and less ADFI , indicating that increased feed efficiency is correlated with more abundant Firmicutes populations. This association was intriguing because the Firmicutes-to-Bacteroidetes ratio is often implicated in human obesity research  were enriched in feed-efficient steers , demonstrates that host genetics is important in determining the composition of the gut microbiome of mice and humans . The fie


Sepsis-associated encephalopathy (SAE) is commonly complicated by septic conditions, and is responsible for increased mortality and poor outcomes in septic patients. Uncontrolled neuroinflammation and ischemic injury are major contributors to brain dysfunction, which arises from intractable immune malfunction and the collapse of neuroendocrine immune networks, such as the cholinergic anti-inflammatory pathway, hypothalamic-pituitary-adrenal axis, and sympathetic nervous system. Dysfunction in these neuromodulatory mechanisms compromised by SAE jeopardizes systemic immune responses, including those of neutrophils, macrophages/monocytes, dendritic cells, and T lymphocytes, which ultimately results in a vicious cycle between brain injury and a progressively aberrant immune response. Deep insight into the crosstalk between SAE and peripheral immunity is of great importance in extending the knowledge of the pathogenesis and development of sepsis-induced immunosuppression, as well as in exploring its effective remedies. Sepsis is one of the major threats to the survival and prognosis of patients in intensive care units (ICUs) but lacks specific and effective treatments. According to the new definition of sepsis 3.0, life-threatening organ dysfunction caused by an aberrant immune response to infection is responsible for the pathogenesis and progression of sepsis . The intAs we know, dysfunction of the central nervous system is responsible for the collapse of the peripheral immune system because of its central role in multiple types of neuroendocrine immune networks, including the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic and parasympathetic nervous systems. The inflammatory signals reach the different brain regions mainly through two disparate ways: the humoral and neural pathways, which involve a compromised blood–brain barrier (BBB) and activation of afferent fibers of the vagus nerve, respectively . The braMultiple factors are reportedly involved in the pathogenesis of SAE Fig. , includiInflammatory signals can reach different brain regions in both neural- and humoral- dependent manners after initiation of septic syndrome. For example, visceral inflammation can be detected by the vagus nerve via terminal cytokine receptors, which further stimulates brain cholinergic nuclei and acts as negative feedback for the inflammatory response by releasing acetylcholine (Ach) at the terminal efferent nerve . Other mNeuroinflammation is critically involved in the pathogenesis of SAE, as uncontrolled inflammation is a primary indicator of septic conditions. Neuroinflammation is responsible for dysfunction and massive apoptosis of brain cells, including microglial cells, neurons, and endothelial cells . Both peReduction in brain perfusion plays an important role in the development of SAE, and it can directly result in the abnormal cellular metabolism and oxidative stress . CerebraThe dysregulated immune response is considered as a major cause for the development of sepsis and is a critical contributor in the pathogenesis of SAE. The influence of brain dysfunction on the systemic immune response is also a noteworthy issue for the progression of sepsis, as it plays an integral role in the neuroimmune networks Table . In thisThe functional status and differential phases of monocytes or macrophages reveal the responsive capacity of the innate immune system, which serves as the main repository for multiple types of inflammatory mediators. Several studies have documented that monocytes and macrophages are either hyporesponsive or hyperactive during sepsis and have dysregulated cytokine production, resulting in compromised survival of septic animals –65. InflThe function and polarization of monocytes are compromised by different types of brain dysfunction. For instance, nosocomial infection is commonly complicated and regarded as a major cause of mortality secondary to severe TBI, which brings about outward signs of systemic immune depression , 68. TheHowever, the specific mechanism underlying the unresponsiveness and apoptosis of monocytes and macrophages in acute TBI remains elusive. In physiological conditions, the central nervous system manipulates systemic inflammatory signals and immunomodulation mainly through the following three pathways: HPA axis, CAP, and sympathetic nervous system. In brain injury, however, these mechanisms act as feedback for the loss or dysfunction of central nuclei and elicit outward signs of hyperactivation, which are responsible for the abnormal response of the immune system. It has been documented that the activity of the HPA axis is enhanced after brain injury, and it relates to marked immunosuppression by increasing the production of corticosteroid . A similNeutrophils are an active participant in the innate immune response and are mobilized early after infection. Reprograming of neutrophils is regarded as one of the major features of sepsis-induced immune dysfunction, as evidenced by abnormal accumulation and inefficient chemotaxis and bactericidal effects in sepsis . RestoriPeripheral neutrophils suffer from disorder and dysregulated apoptosis after exposure to different brain injuries. For example, in the early stage of TBI, peripheral neutrophils have remarkable infiltration in the central nervous system, which is deemed a protective behavior by eliminating damaged neurons and improving tissue repair . NeverthDendritic cells (DCs) are essential in maintaining homeostasis of the immune response, serving as a bridge connecting innate immunity to the adaptive immune system. Functional integrity and activity of DCs are closely related to the survival and prognosis of septic patients, while dysfunction of DCs has been identified as one of the major contributors to sepsis-induced immunosuppression and accounts for increased mortality and poor outcomes , 78. DCsIt has been documented that the function and activity of DCs are affected by different types of brain dysfunction. In patients with severe head injury, for example, the host response to common antigens showed significant suppression, along with a marked increase in opportunistic infections . The numT lymphocytes are classically representatives of the adaptive immune system, which are important for the prognosis of septic complications, and their impaired activity and aberrant differentiation are noteworthy in sepsis . This in+ T cells play a crucial role in maintaining the integrity of the CAP, which is rendered incapable of potent anti-inflammatory effects either through splenectomy or depletion of CD4+ T cells [The recruitment of T cells occurs during the early stages of TBI, which controls damage and tissue repair through the release of anti-inflammatory cytokines . However T cells . Thus, a T cells . Periphe T cells , 97. Mit T cells .The enhanced activity of the HPA axis and the production of glucocorticoids are theoretically responsible for the abnormal function and polarization of peripheral T cells that have anti-inflammatory capacities, which might act as feedback for the loss of effective brain nuclei , 99. RegThe aberrant responses of multiple neuroendocrine immune networks are reportedly responsible for the disorders in host immunity that are secondary to brain injury. The central nervous system plays an essential role in maintaining the functional integrity of the immune system, as the command center of various neuromodulatory pathways and has close connections to peripheral organs. Thus, clarifying the specific mechanism with regard to both neurologically dependent and independent pathways is of great importance for timely recognition and prompt interference in the vicious cycle between SAE and intractable immunosuppression.CAP represents an important branch of the parasympathetic nervous system and serves as an effective therapeutic target for various inflammatory diseases. It mainly constitutes the following three parts: brain for signal integration, processing and transition, efferent vagus nerve for signal transmission, and cellular α7nAchR for initiating intracellular machinery . Periphe2 (PGE2) levels and disrupting the stromal cell-derived factor-1 (SDF-1) axis after enhancing β-AR activation [Activation of sympathetic nervous system (SNS) represents the “fight or flight” response to external threats. It is essential for inflammatory control and modulation of the immune response by releasing norepinephrine (NE) at terminal nerves, which is a pivotal part in the inflammatory reflex. The SNS shows critical involvement in the development and response of immune cells. For example, failure of intact SNS signaling impairs the steady-state circadian rhythmicity of hematopoietic stem cells and disturbs their mobilization . Basic βtivation . Other stivation . In sepstivation . HoweverHPA axis belongs to the neurological branch of stress system that is programmed for maintaining functional homeostasis. The hypothalamus and brain stem constitute a central component of the HPA axis and induce a cascade of endocrine hormones, including corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and glucocorticoid . The levl-selectin activity [Glucocorticoids reportedly enable the anti-inflammatory effects of the HPA axis by either directly forming complex with transcription factors, e.g., NF-κB, to interfere its binding with proinflammatory genes, or promoting expression of anti-inflammatory proteins, such as annexin A1, mitogen-activated protein kinase phosphatase-1 (MKP-1), and glucocorticoid-induced leucine zipper protein (GILZ) . Reductiactivity . The disactivity . Aberranactivity . The coractivity . Though activity . It has activity . In addiactivity . Therefoactivity . In addiThe interplay between host immune depression and development of SAE is responsible for the deteriorating outcome of sepsis due to uncontrolled neuroinflammation and disorders of systemic immunity Fig. . The infNonetheless, specific mechanisms of both SAE and its impacts on peripheral immune response have not yet been established, even though dysfunction of the brain has been identified, which impairs immune homeostasis and worsens outcomes in septic cases. Moreover, the specific point at which the initiation of SAE becomes a vicious cycle of immunosuppression must be clarified for timely recognition and prompt treatment. Therefore, the interplay between the pathogenesis of SAE and the abnormal response of immune cells is noteworthy and can be used for constructing a predictive algorithm after confluence analysis, given the participation of multiple immune effectors. In addition, exploration of a set would be beneficial for preventing such a vicious cycle.SAE is commonly yet severely complicated by sepsis but is prone to be neglected in clinical practice. Brain damage plays a critical role in the survival and prognosis of septic patients, which should be recognized as not only a compromised organ but also an essential participant in impaired immunomodulation secondary to sepsis because brain is the command center for multiple types of neuroendocrine immune networks, such as CAP, HPA axis, and sympathetic nervous system. The impaired activity of these neuromodulatory mechanisms reportedly contributes to decreased counts and abnormal responses of peripheral immune cells, including neutrophils, macrophages/monocytes, DCs, and T lymphocytes, which might drive brain injury into a vicious cycle of sepsis-induced immunosuppression as a result of uncontrolled neuroinflammation in the wake of progressively aberrant immunity. Therefore, it may be an efficient strategy for the sepsis-induced immunosuppressive state to block the vicious cycle between SAE and peripheral immune dissonance.


Diabetologia, Kusmartseva et al (10.1007/s00125-017-4494-x) provide further evidence to support the use of organ donor pancreases for the study of human diabetes. They show that length of terminal hospitalisation of organ donors prior to death does not seem to influence the frequency of inflammatory cells infiltrating the pancreas and the replication of beta cells. These findings are reassuring, demonstrating the reliability of this precious and valuable resource for human islet cells research.Direct in vivo assessment of pancreatic islet-cells for the study of the pathophysiology of diabetes in humans is hampered by anatomical and technological hurdles. To date, most of the information that has been generated is derived from histological studies performed on pancreatic tissue from autopsy, surgery, in vivo biopsy or organ donation. Each approach has its advantages and disadvantages (as summarised in this commentary); however, in this edition of  Insulin-secreting beta cells, sophisticated and unique endocrine cells, comprise the majority of cells in the pancreatic islets. Their failure, owing to the interplay of genetic, epigenetic and environmental factors, is key to the onset and progression of type 1 and type 2 diabetes . HoweverFor over a century since the discovery of the pancreatic islets in the rabbit by Paul Langerhans , microscMore recently, pancreatic surgical samples obtained after removal of portions of the gland  have been used in research. This approach has been generally applied for the comparison of islets and beta cells from those with and without type 2 diabetes, and also to study beta cell traits in individuals with insulin resistance –17. ObviOn a few occasions, pancreatic tissue samples have been obtained by laparoscopic-assisted biopsy procedures in individuals with recent-onset type 1 diabetes –23. ThisMany researchers have started to use whole pancreases (or part of them) retrieved from brain-dead multi-organ donors. The advantages of using such a resource include the transplantation-grade quality of the tissue, better representation of the demographics of the general population, and possible availability of additional tissues, such as spleen and pancreatic/non-pancreatic lymph nodes . Indeed,www.jdrfnpod.org), which was started in 2007 by JDRF in the USA, has reported use of organ donor pancreases in their research. Similarly, the more recent programme INNODIA  has also adopted use of these samples. However, there are some concerns regarding the use of organ donor pancreases. For instance, the longer the interval between organ removal and tissue processing for clinical or research purposes (CIT), the worse the outcome of pancreas transplant or islet isolation [For the reasons mentioned above, the use of organ donor pancreases in research is being extensively implemented by single research groups and also, very importantly, by dedicated networks. For example, the Network for Pancreatic Organ donors with Diabetes .Importantly, however, in this edition of va et al  report tr issues , which iKusmartseva et al also found that length of hospitalisation prior to organ donation did not significantly influence beta cell replication, as assessed by evaluating the presence of the marker Ki67 in insulin positive cells. Cells positive for both insulin and Ki67 staining have been previously observed in surgical pancreatic samples  and someIn conclusion, whilst each source of pancreatic tissue from humans has advantages and disadvantages (summarised in Table


In a flow-focusing microdroplet generator, by changing the flow rates of the two immiscible fluids, production speed can be increased from tens to thousands of droplets per second. However, because of the nonlinearity of the flow-focusing microdroplet generator, the production speed of droplets is difficult to quantitatively study for the typical flow-focusing geometry. In this paper, we demonstrate an efficient method that can precisely predict the droplet production speed for a wide range of fluid flow rates. While monodisperse droplets are formed in the flow-focusing microchannel, droplet spacing as a function of time was measured experimentally. We discovered that droplet spacing changes periodically with time during each process of droplet generation. By comparing the frequency of droplet spacing fluctuations with the droplet production speed, precise predictions of droplet production speed can be obtained for different flow conditions in the flow-focusing microdroplet generator.  The design of microdroplet generators requires that a wide range of droplet production speeds can be tuned for different experiments. In particular, controlling and predicting the production speed of droplets is of significant importance for droplet-based microfluidic systems ,2,3,4 anAccording to the literature, in a flow-focusing microdroplet generator, the principle of droplet formation has been studied both theoretically and experimentally ,14,15. FIn this paper, we provide an efficient method to precisely predict droplet production speed for a wide range of fluid flow rates in a flow-focusing microdroplet generator. Different viscosities of silicone oil were chosen for experimental measurements of droplet production speed. The nonlinear relationship between droplet production speed and flow-rate ratio was tested experimentally, and the effects of fluid viscosity on this nonlinear relationship of droplet formation are discussed. While monodisperse droplets are generated in a flow-focusing microchannel, it can be observed that droplet spacing changes periodically with time during each process of droplet generation. Most importantly, by increasing the droplet production speed from approximately 10 to 100 per second, the comparison between the frequency of droplet spacing fluctuations and the production speed of droplets was obtained experimentally. As a result, from experimental measurements of the periodic fluctuations of droplet spacing, the prediction precision of the production speeds of droplets can be quantitatively studied for various flow conditions in the flow-focusing microdroplet generator.A flow-focusing microdroplet generator was designed for measuring the periodic fluctuations of droplet spacing as monodisperse droplets were produced. Meanwhile, the frequency of droplet spacing fluctuations was quantified experimentally, which could be used to precisely predict the production speed of droplets. Here, The commercial syringe pump  was used to supply the flow rates of the continuous and disperse phases. While monodisperse droplets were formed in the flow-focusing microdroplet generator, the production rates of droplets could be verified by changing the flow rates of the two phases. In addition, a high-speed camera  was chosen to take images of the droplet generation, and the speed of image acquisition of droplet formation was specified as 1000 fps. Using the method of image processing, the image of the droplet generation was analyzed by the image processing toolbox of the Matlab software . By calculating the pixels of the droplet spacing along the microchannel, the time-varying droplet spacing could be measured experimentally, while monodisperse droplets were produced within the microchannel. For our experiments of droplet formation, the production rates of droplets spanned from approximately 10 to 100 per second; therefore, the sampling frequency of the droplet spacing acquisition was much higher than the production speed of the droplets. Consequently, the periodic fluctuations of droplet spacing could be easily captured, while monodisperse droplets were produced in the flow-focusing microdroplet generator. For the flow-focusing microdroplet generator, three different viscosities of the silicone oil (Particularly, while monodisperse droplets were produced in the microchannel, the relationship between droplet production speed, From the experimental results, it can be observed that the droplet production speed varies nonlinearly with the flow-rate ratio for different viscosities of the fluids. More importantly, since all of the experiments of monodisperse droplet production were conducted at low capillary numbers (eglected . ConsequFor the flow-focusing microdroplet generator, a wide range of droplet production speeds can be obtained by changing the flow-rate ratio of the two phases. Droplet spacing as a function of time was tested experimentally for the different production speeds of the droplets. Here, Based on the experimental measurements, we note that droplet spacing,From In a flow-focusing microdroplet generator, we demonstrate an efficient method that can precisely predict the droplet production speed for a wide range of fluid flow rates. While monodisperse droplets were being formed in the flow-focusing microchannel, we discovered that the droplet production speed varies nonlinearly with the flow-rate ratio, and especially for a fixed flow-rate ratio, the droplet spacing changes periodically with time during each process of droplet generation. Most importantly, as the droplet production speed varies from approximately 10 to 100 per second, the frequency of the droplet spacing fluctuations coincides with the production speed of droplets. As a result, from experimental measurements of the periodic fluctuations of the droplet spacing, it is validated that the production speed of droplets can be precisely predicted for various flow conditions within the flow-focusing microdroplet generator.


During recent years, the significant influence of mitochondria on osteoarthritis (OA), the most common joint disease, has been consistently demonstrated. Not only mitochondrial dysfunction but also mitochondrial genetic polymorphisms, specifically the mitochondrial DNA haplogroups, have been shown to have an important influence on different OA-related features, including the prevalence, severity, incidence, and progression of the disease. This influence could probably be mediated by the role of mitochondria in the regulation of different processes involved in the pathogenesis of OA, such as energy production, the generation of reactive oxygen and nitrogen species, apoptosis, and inflammation. The regulation of these processes is at least partially controlled by the bi-directional communication between the nucleus and mitochondria, which permits the regulation of adaptation to a wide range of stressors and the maintenance of cellular homeostasis. This bi-directional communication consists of an “anterograde regulation” by which the nucleus regulates mitochondrial biogenesis and activity and a “retrograde regulation” by which both mitochondria and mitochondrial genetic variation exert a regulatory signaling control over the nuclear epigenome, which leads to the modulation of nuclear genes. Throughout this mini review, we will describe the evidence that demonstrates the profound influence of the mitochondrial genetic background in the pathogenesis of OA, as well as its influence on the nuclear DNA methylome of the only cell type present in the articular cartilage, the chondrocyte. This evidence leads to serious consideration of the mitochondrion as an important therapeutic target in OA. Osteoarthritis (OA) is the most common chronic progressive disorder that involves movable joints, occurring in 10-20% of the population over 50 years of age; the incidence of OA is estimated to double within the next 30 years . The patThe nature of OA is heterogeneous, because a combination of factors such as occupation, age, gender, body mass index, and genetics have a profound influence on its pathogenesis. As a consequence of this, several different phenotypes characterize this disease, including inflammatory, aging-related, metabolic, pain, and post-traumatic phenotypes . In agreBecause articular chondrocytes are highly glycolytic cells that obtain their energy mainly from anaerobic glucose metabolism, the role of mitochondria in the pathogenesis of OA was not studied in depth until early 2000. However, a large number of studies have demonstrated the profound influence of mitochondria in the pathogenesis of OA.Terkeltaub and co-workers published a full review describing how mitochondrial impairment of chondrocytes is an important mediator of the establishment of OA . SpecifiMitophagy is a form of autophagy, a process that involves the removal of damaged macromolecules and organelles to regulate cell homeostasis ; specifiMitochondria are considered unique organelles because they contain their own maternally inherited DNA, the mtDNA, containing 2 rRNAs, 22 tRNAs, and 13 essential mitochondrial protein-coding genes. The mutation rate of this circular molecule is higher than that of the nuclear DNA, mainly due to i) its proximity to the main source of ROS production, ii) the lack of an efficient repair system, and iii) the higher replication rate of mtDNA . As a coIn this sense, OA is not an exception. During recent years, different studies have shown clinical associations between specific mtDNA haplogroups and different OA-related features, including the prevalence, progression, and incidence of the disease Table 1.Different studies have shown significant associations between specific mtDNA haplogroups and the prevalence of OA. Specifically, mtDNA variants belonging to the European cluster JT have been associated with a lower risk of knee and hip OA in a cohort of Spanish patients . mtDNA hA plausible cause for the lack of replication among different case-control studies, even when nuclear DNA polymorphisms are analyzed, could be the unavailability of knee (or hip) radiographs for most population-based controls. This is not a minor issue, since up to 50% of people without joint symptoms may develop radiographic changes comparable to OA . In thisThe use of well-characterized prospective cohorts of patients permits rigorous studies to analyze the influence of mtDNA haplogroups on the rate of progression and incidence of OA over time. mtDNA variants within mitochondrial cluster JT were associated with lower rates of radiographic knee OA progression in different world populations, including Spain, the USA, and the Netherlands. Specifically, in a Spanish cohort, mtDNA cluster JT was associated with a lower rate of radiographic progression in terms of Kellgren and Lawrence grade , and eveIn terms of disease incidence, a meta-analysis including 3217 individuals from the OAI and CHECK cohorts concluded that, compared with the most common Caucasian haplogroup H, subjects with mtDNA haplogroup J show a lower rate of incident knee OA over an eight-year period . This stIn an effort to detect structural changes in an early stage of the disease, to monitor disease progression, or even to assess therapeutic responses with more sensitivity and reliability, molecular biomarkers have been developed in OA . In a seIn terms of imaging biomarkers, the identification and quantification of early bone marrow lesions (BMLs) has great relevance for assessing symptomatic progression and radiographic worsening over time in patients with OA . In thisIn summary, in Caucasian populations, it seems that haplogroups belonging to mitochondrial cluster JT are protective against OA and have even been associated with increased longevity in some European populations . HoweverIt is well known that bi-directional communication exists between the nucleus and mitochondria with the aim of maintaining cellular homeostasis and regulating adaptation to a broad range of stressors . This coBased on the above findings, and taking into account the reported associations between common mtDNA variants and different physiological and pathological phenotypes , it can In terms of animal models, interesting work in conplastic mice (mice with a constant nuclear background but different mtDNA genomes) showed profound differences in health longevity between conplastic strains. The level of divergence between the two strains, equivalent to that between human African and Eurasian mtDNAs, showed different behavior in terms of mitochondrial proteostasis, reactive oxygen generation, obesity, and insulin signaling as well as in cell-senescence-related parameters such as telomere shortening and mitochondrial dysfunction . Most of3) by S-adenosyl-methionine (SAM) to a cytosine that lies at 5´ of guanine (CpG site) to give rise to methylated cytosine. When this process occurs in high-density CpG regions of promoters, gene silencing occurs; in contrast, when methylation occurs in gene bodies, it leads to increased gene expression  suppressing mitochondrial oxidative damage and restoring extracellular matrix homeostasis , ii) actFB and IR-P contributed equally to the design and coordination of the study; both conceived the study and participated in its design. AD-S and PR-L contributed to some of the findings described in the manuscript and helped to draft the final version of the manuscript.This work is supported by grants from Fondo de Investigación Sanitaria  integrated in the National Plan for Scientific Program, Development and Technological Innovation 2013–2016 and funded by the ISCIII-General Subdirection of Assessment and Promotion of Research-European Regional Development Fund (FEDER) “A way of making Europe”. IR-P is supported by Contrato Miguel Servet-II Fondo de Investigación Sanitaria (CPII17/00026).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.