Polycystic ovary syndrome (PCOS), one of the most common and complex endocrine disorders affecting up to 15 % of reproductive age women, is considered a predominantly hyperandrogenic syndrome according to the Androgen Excess Society. It is generally accepted that androgens determine the characteristic features of PCOS; in this context, a hyperactive androgen receptor (AR) at the levels of the GnRH pulse generator in the hypothalamus and at the granulosa cells in the ovary, skeletal muscle or adipocytes senses initially normal testosterone and dihydrotestosterone as biochemical hyperandrogenism and might be a crucial connection between the vicious circles of the PCOS pathogenesis.Polymorphism of the AR gene has been associated with different androgen pattern diseases. Several studies have demonstrated an association between AR with increased activity encoded by shorter CAG repeat polymorphism in the exon 1 of the AR gene and PCOS, although there are conflicting results in this field. The phenomenon is more complex because the AR activity is determined by the epigenetic effect of X chromosome inactivation (XCI). Moreover, we must evaluate the AR as a dynamic heterocomplex, with a large number of coactivators and corepressors that are essential to its function, thus mediating tissue-specific effects. In theory, any of these factors could modify the activity of AR, which likely explains the inconsistent results obtained when this activity was quantified by only the CAG polymorphism in PCOS. Although the aetiology remains unknown, some investigators consider that androgen excess is the sine qua non of PCOS [5], which is consistent with the theory that androgens determine the characteristic features of the syndrome, for example, oligo-anovulation and infertility, inhibition of follicle development with polyfollicular ovarian morphology, hirsutism, acne or androgenic alopecia [10]. Polycystic ovary syndrome (PCOS) is a complex endocrine disorder [17]. These data provide evidence that androgen excess is a key factor in the PCOS etiopathogeny.  Experiments conducted in animal models as well as equivalent natural situations in humans, i.e., pregnancy in PCOS or congenital adrenal hyperplasia, demonstrated that exposure to androgen excess during the foetal life and infancy determines the PCOS features in adult life, with LH hypersecretion, oligomenorrhoea, polyfollicular enlarged ovaries, insulin resistance, abdominal obesity, impaired insulin response to glucose and dyslipidemia [20]. In these adult women with PCOS, the blockade of androgen action by flutamide administration restored the sensitivity of GnRH to estradiol and progesterone, thus supporting the role of an increased androgenic activity mediated by the androgen receptor (AR) to reduce the progesterone negative feedback at the level of the GnRH pulse generator [21]. More specifically, experiments conducted in animal models showed that a special KNDy subpopulation of cells in the arcuate nucleus, which colocalise three important neuropeptides, kisspeptin, neurokinin b and dynorphin, express steroid receptors that include AR and connect with GnRH neurons in a network, are responsible for transporting the testosterone influence on GnRH neurons in the hypothalamic GnRH pulse generator [22].  In women with PCOS, the abnormality in the regulation of hypothalamic GnRH secretion, with persistently rapid GnRH pulsatility, pituitary synthesis of LH over that of FSH, and increased LH concentrations and LH/FSH ratios, has been explained, at least in part, by a decreased sensitivity of the GnRH pulse generator to the progesterone suppression [24], an increased androgenic activity could amplify insulin resistance as soon as testosterone in physiological concentrations enhanced the insulin-induced IRS-1 serine 636/639 phosphorylation in differentiated rat skeletal muscle myotubes [25]. AR is present in human addipose tissue, at the level of preadypocites and adipocytes [27] and Corbould demonstrated that testosterone induces a selective insulin resistance in cultured subcutaneous adipocytes of women, with action mediated by the classical AR, given that AR antagonists, cyproterone acetate and flutamide attenuated the effect of testosterone on the glucose uptake [28].  Although the insulin resistance of PCOS partially has a genetic substrate . In this condition, compensatory hyperinsulinaemia in synergistic action with LH will amplify all steroidogenesis and specifically testosterone synthesis at the level of responsive ovarian theca cells. The androgens, either circulatory or produced locally, impair the ability of primary follicles to grow normally, with a marked increase in the number of growing preantral and antral follicles, particularly in the population of growing and classic primary follicles, leading to the typical polycystic ovarian morphology [30]. Several studies have demonstrated that high-dose androgen treatment of transsexual women induced enlarged ovaries with increased numbers of cystic follicles and theca-interstitial hyperplasia, which meet the morphological criteria for PCOS [33]. Although the gonadotropins were suppressed in these subjects, their ovaries were not suppressed. These observations suggested that androgens per se might induce ovarian follicular and theca interstitial growth [7]. These data, correlated with experiments in non-human primates [7] and the evidence for expression of AR in the human ovary, at germinal epithelium, granulosa cells of antral follicles, corpus luteum, and thecal and stromal cells [34], indicated that the characteristic polycystic ovarian morphology, with enlarged ovaries and numerous small follicular cysts, might be the result of direct, receptor-mediated androgen activity.  Whereas the classic target tissues such as muscle and fat manifest insulin resistance in PCOS, the ovaries of these patients remain sensitive to insulin or perhaps hypersensitive to it  .  The AR, encoded by the AR gene located at Xq11-q12, is a nuclear transcription factor and member of the steroid receptor superfamily [42]. After ligand binding, AR adopts an active conformation that facilitates the dissociation of heat shock proteins, dimerisation, and binding to response elements in the promoters of responsive genes in the nucleus. The N-terminal activation domain accounts for more than 60% of the AR protein and has a potent transcriptional activator function (AF-1) at the level of androgen response elements [43]. The unbound AR is inactive in the cytoplasm as a large dynamic heterocomplex, together with heat shock proteins (Hsp70 and Hsp90) and their co-chaperones [44], partial androgen insensitivity syndrome (Reifenstein syndrome) [45] or prostate cancer [44].  So far, have been described different point mutations, deletions or insertions at the level of the AR gene that influence the activity of AR in different diseases, such as complete androgen insensitivity syndrome (CAIS) [49]; however, there is no uniform consensus [50].  Moreover, has been shown that the activity of AR is influenced by a genetic polymorphism in exon 1 of the AR gene. There are two polymorphic trinucleotide repeat fragments, CAGn and GGNn, in the first exon of the AR gene, encoding two polyglutamine and polyglycine tracts in the N-terminal activation domain. Although the functional consequences of variation in the length of the GGNn fragment are unclear, several in vitro studies have evidenced an inverse relationship between the length of the CAG polymorphic repeat  and the AR transcriptional activity [47], whereas the expansion of the CAG repeat length of over 40 is related to SBMA, a rare neuromuscular disorder characterised by spinal and bulbar muscular atrophy and is associated with androgen insensitivity, decreased virilisation, testicular atrophy, reduced sperm production, and infertility [51]. Expanded CAGn repeat in the AR gene modulates other male-specific phenotypes, such as prostate growth under androgen substitution in hypogonadal men [52] or different phenotypic features in untreated patients with Klinefelter syndrome [54].  In vivo studies support the majority of functional data, which demonstrate a negative correlation between the CAG repeat number and AR activity. Healthy men whose AR has a CAG repeat length of greater than 28 have an increased incidence of impaired spermatogenesis and infertility [59] or premature pubarche [60].  Conversely, shorter AR polyglutamine tracts and thus a more transcriptionally active AR have been associated with prostate cancer [CAG polymorphism of AR gene in PCOS69].  According to functional and in vivo data, CAG repeat polymorphism, encoding an AR with increased activity, might play a role in PCOS pathogenesis. However, several studies centred on the association between the CAG repeat polymorphism of the AR gene and PCOS have conflicting results [36]; this finding is consistent with that of another study that described all of the subjects with 15 or fewer CAG repeats as part of the PCOS group [37]. Azziz et al and Schuring et al reported significantly lower CAG repeats in patients with PCOS than in controls [69], a finding consistent with our preliminary data that demonstrated significantly shorter biallelic means of CAG repeats in patients with PCOS than in normal women [67].  Some authors observed that short androgen receptor CAGn alleles tended to appear more frequently in women with PCOS than in controls [70].  Mohlig et al demonstrated that in women with PCOS, the AR CAG repeat polymorphism has metabolic consequences by modifying the impact of testosterone on insulin resistance, which is consistent with prior known functional data. In a statistical model that described 42.5% of the HOMA-IR variation, the elevated testosterone increased the insulin resistance in carriers of short CAG lengths; this effect attenuated with increasing biallelic CAG length until it turned into the opposite at a CAG length of longer than 23 [68]; however, when compared PCOS groups constructed by androgen levels, one of these studies showed a trend for lower CAG biallelic means in the subgroup of women with PCOS and lower testosterone levels, which was consistent with a role for androgen hyperactivity in the development of the syndrome [61].  Other studies showed no difference in CAG repeat length between patients with PCOS and controls, regardless of the fact that both alleles were considered together or separately [62].  Contrary to the theory of an inverse relation between CAG repeat number and receptor activity, the study published by Hickey et al has shown that infertile women with PCOS exhibited a greater frequency of CAG alleles or biallelic means greater than 22 repeats compared with both the fertile controls and the general population [68] nor acne [68] was significantly associated with the CAG repeat polymorphism, whereas other studies found that short CAG repeat was associated with hirsutism and acne [71].  Regarding clinical hyperandrogenism of PCOS, several studies found that neither hirsutism  [69]. DNA methylation is an important epigenetic mechanism in X chromosome inactivation [72], which normally occurs in early embryogenesis as a random event. A nonrandom X inactivation, either due to environmental exposures or allelic differences, may contribute significantly to the expression of PCOS [38]. Experimentally induced animal models, in monkeys or sheep, showed that in utero hyperandrogenism exposure may lead to abnormalities of epigenetic reprogramming in foetal reproductive and metabolically active tissues and may produce an impact on the neuroendocrine development, gonadal differentiation and pancreatic organogenesis [16], which ultimately results in a PCOS phenotype in the adult life [16]. Prenatally androgenised female rhesus monkeys developed a PCOS-like phenotype as adults with increased visceral adipose tissue and basal serum insulin levels [73], conditions that correlated with different DNA methylation patterns in both infant and adult visceral adipose tissue [74]. An equivalent, naturally occurring situation with foetal exposure to the androgen excess occurs in pregnant women with PCOS [77].  In addition to the conflicting data on the association between genetically encoded CAG repeats and PCOS, we should consider the X chromosome inactivation (XCI) as a significant phenomenon present in females because the AR gene is located on the X chromosome; therefore, the AR activity is determined not only by the AR genotype but also by the epigenetic effect of XCI .  We hypothesise that a hyperandrogenemic environment could modify the process of methylation, including the AR expression in the active X chromosome, even when it appears in adult life, based on the data that demonstrated a loss of methylation of the LH receptor in the ovary of a mouse model with PCOS induced by the administration of dehydroepiandrosterone .  In this context, supplementary information can be derived by investigating the methylation pattern of AR’s entire sites near the polymorphic CAG repeats because all of those CpG sites may be involved in regulating AR expression in the active X chromosome [69]. One important study demonstrated that total methylation of peripheral blood leukocyte DNA is unaltered in patients with PCOS vs. matched controls [84].  b. We should consider that XCI can vary in different tissues. In all of the previous studies, XCI was generally tested by using blood samples, which does not appear to reflect the condition in androgen target tissues [85]. Future genetic and physiologic studies are required to identify the functional interactions between the CAG repeats and these AR coregulators [38]. 3. Finally, a large number of AR coactivators and corepressors have been identified as critical to the AR function. When they mediate tissue-specific androgen effects, a variation in the expression or function of these coregulators might contribute to the development of PCOS [Genetic data that demonstrate a hyperactive androgen receptor in PCOS are limited to the evaluation of the CAG repeat polymorphism of the AR gene and are inconsistent, thereby requiring more in vitro studies to identify functional AR polymorphisms that affect AR transactivity and different PCOS phenotypes. The important role that AR with increased activity plays in the PCOS pathogenesis is interesting in light of continuous efforts to understand this complex disease, which might represent an important connection between the multiple characteristic pathways, including the androgens, neuroendocrine and insulin axis.


Genomic concepts are based on the assumption that phenotypes arise from the expression of genetic variants. However, the presence of non-Mendelian inheritance patterns provides a direct challenge to this view and suggests an important role for alternative mechanisms of gene regulation and inheritance. Over the past few years, a highly complex and diverse network of noncoding RNAs has been discovered. Research in animal models has shown that RNAs can be inherited and that RNA methyltransferases can be important for the transmission and expression of modified phenotypes in the next generation. We discuss possible mechanisms of RNA-mediated inheritance and the role of these mechanisms for human health and disease. Our basic understanding of transgenerational inheritance relies on observations made by our ancestors generations ago. Gregor Mendel's 1860s pioneering studies of pea plant crosses and breeding represent the foundation of what is known about inheritance mechanisms to this day. By crossing pea plants with pure-breeding, i.e. homozygous, characteristics and later also heterozygotes in different combinations, Mendel was able to propose two basic principles, or laws, which apply to sexually reproducing, diploid organisms: The first is the law of segregation, which states that the two alleles of a given trait from each parent segregate into two gametes, which are then passed down to the offspring. This law was confirmed in later studies on meiosis and ensures genomic stability by maintaining ploidy while allowing genetic diversity through new and random combinations of alleles of the same gene. Mendel further observed that different alleles of a trait (such as pea colors) do not blend, but either dominate or recess, allowing only one defined trait to be expressed at a time. Mendel's second law, the law of independent assortment, states that separate traits  are inherited independently from one another. This principle allows a vast variety of combinations of traits and thus ensures genetic diversity among the offspring.As a result of decades of research in the framework of Mendelian genetics, we now know that DNA is the main carrier of genetic information from one generation to the next. Methods for genetic analysis have evolved considerably and now allow the deciphering of entire genomes. DNA microarrays and next-generation sequencing have made it possible to identify millions of genetic variants, such as single nucleotide polymorphisms and copy number variants, in thousands of individuals. Currently, large-scale genome-wide association studies (GWAS) are used to unravel highly complex genotype–phenotype relationships and represent a sophisticated conceptual development that is based on Mendelian genetics Genetic information is organized in higher order structures, which consist of DNA, proteins, and RNA Transgenerational epigenetic inheritance has been described in various systems and detailed reviews on this topic have been published recently Kit paramutation” which describes a stable modification of Kit gene expression. Kit encodes a tyrosine kinase receptor with roles in developmental processes including hematopoiesis, germ cell differentiation, and melanogenesis. A homozygous deletion of Kit is lethal, whereas heterozygotes carrying one allele inactivated by a LacZ insertion (tm1Alf/+Kit) show a white-tail phenotype +/+Kit progeny and in subsequent crosses with wild type partners  represents the most prominent modification of mammalian mRNAs. The availability of m6A-specific antibodies provided an excellent opportunity to identify methylated RNAs by immunoprecipitation and sequencing of mRNA fragments. Indeed, two recent studies have identified adenine methylation marks in several thousand mRNAs, with a distinct enrichment in the vicinity of stop codons and in internal exons Cytosine-5 methylation (m5C) represents another prominent modification of RNA, which can be detected at single-base resolution by bisulfite sequencing Additionally, the regulatory role of RNA methylation in gene expression has also been investigated through the functional characterization of the DNMT2 RNA methyltransferase. DNMT2 is closely related to the established DNA methyltransferases and key epigenetic regulatory enzymes DNMT1 and DNMT3. However, DNMT2 does not methylate DNA Dnmt2 gene Kit RNA fragments into wild-type fertilized eggs induced up-regulation and possibly methylation of the Kit transcript, these processes could not be observed in Dnmt2-deficient mice. Importantly, corresponding Kit genomic DNA sequences remained unmethylated in wild-type as well as in Dnmt2-deficient mice Finally, it has been shown that the inheritance of paramutant mouse phenotypes requires an intact Interestingly, several recent findings suggest that the mechanisms of RNA-mediated inheritance might be relevant for human health and disease . For exaFurthermore, RNA-mediated inheritance could also provide an explanation for the missing heritability problem of complex human diseases. The “missing heritability” phenomenon was defined after even extensive GWAS failed to identify major risk factors for complex diseases


Stimulation of sGC mediates physiologic responses including smooth muscle relaxation, inhibition of inflammation, and thrombosis. In pathophysiologic states, NO formation and bioavailability can be impaired by oxidative stress and that tolerance to NO donors develops with continuous use. Two classes of compounds have been developed that can directly activate sGC and increase cGMP formation in pathophysiologic conditions when NO formation and bioavailability are impaired or when NO tolerance has developed. In this report, we review current information on the pharmacology of heme-dependent stimulators and heme-independent activators of sGC in animal and in early clinical studies and the potential role these compounds may have in the management of cardiovascular disease.The heme-protein soluble guanylyl cyclase (sGC) is the intracellular receptor for nitric oxide (NO). sGC is a heterodimeric enzyme with  Guanylyl cyclase (GC) is an enzyme that catalyzes the formation of guanosine 3′,5′-monophosphate (cGMP) from guanosine triphosphate (GTP) and is found in tissues throughout the animal kingdom , 2. Solu3), sodium nitrite (NaNO2), hydroxylamine (NH2OH), nitroglycerin (C3H5N3O9), and sodium nitroprusside (Na2[Fe(CN)5NO]) were able to activate GC [Initial investigations into the role of NO were conducted showing that nitrogen-containing compounds such as sodium azide NaN, sodium ivate GC –23  and amyl nitrite have been used in the treatment of angina pectoris and heart failure for over 140 years , 55, theStudies in the literature provide evidence that vasorelaxant responses to GTN are mediated by the formation of NO or aclosely related S-nitroso molecule , 73, 74.Studies have shown that the bioactivation of GTN requires the presence of thiols or sulfhydryl-containing compounds and that NO or NO-like compounds are believed to be the biologically active species , 78, 79.The activation of sGC enhances the conversion of GTP to cGMP that mediates physiologic responses including smooth muscle relaxation and inhibition of platelet aggregation –18. HoweCompounds have been developed that can directly stimulate sGC and increase cGMP formation in pathophysiologic conditions when NO formation and bioavailability are impaired or when NO tolerance has developed –36 Figu. The pyrIn an intact chest rat model, administration of BAY 41-8543 under control or baseline tone conditions produced small decreases in pulmonary arterial pressure, larger dose-dependent decreases in systemic arterial pressure, and increases in cardiac output . HoweverThe effect of NOS inhibition with L-NAME on vasodilator responses to BAY 41-8543 was investigated in the intact chest rat model, and following administration of the NOS inhibitor, decreases in pulmonary and systemic arterial pressures in response to BAY 41-8543 were reduced when compared to responses in U46619-infused animals. Comparisons of responses to BAY 41-8543 at the same level of pulmonary arterial pressure indicate that decreases in pulmonary arterial pressure in response to the sGC stimulator are reduced by more than 50% in L-NAME-treated animals . These rThe role of BAY 41-8543 synergy with exogenous NO was examined in the intact chest model with the NO donor, sodium nitroprusside (SNP). Although separate administration of BAY 41-8543 and SNP produced significant decreases in pulmonary and systemic arterial pressures, coadministration of a small dose of the NO donor along with BAY 41-8543 produced decreases in pulmonary and systemic arterial pressures that were significantly greater than the sum of responses to either agent when administered alone . These rBAY 41-8543 and BAY 41-2272 were synthesized based upon analysis of the structure of YC-1 , 129. ThAlthough BAY 41-8543 had beneficial effects in experimental models of pulmonary hypertension, this agent does not have favorable pharmacokinetic properties and cannot be used in clinical trials . In contAlthough pulmonary vasodilator response to the sGC stimulator, BAY 41-2272, was not dependent on the formation of endogenous NO in the awake sheep model, the response strongly synergized with inhaled NO . TherefoP < 0.0001) and in patients with pulmonary arterial hypertension (PAH) . Moreover, similar improvements were also observed in patients on chronic bosentan therapy [The first sGC simulator to undergo clinical study was BAY 41-8543 . However therapy . The mos therapy .3+ instead of Fe2+) state, or when the heme group is lost [in vivo studies indicates that these NO-independent receptors exist and may become more abundant under pathological conditions associated with oxidative stress [The oxidation of the heme iron on sGC decreases the responsiveness of the enzyme to NO and promotes vasoconstriction . Recentl is lost , 138  induced with the stable endoperoxide analogue, U46619 , 128. InThe development of heart failure due to a number of etiologies is a common final stage in cardiovascular disease that is associated with high morbidity and mortality . StimulaAs sGC stimulators have been shown to stimulate sGC independently of NO donors, this class of agonists could have an important role in the acute hospital setting when a sudden increase in pulmonary vascular tone can occur during sympathetic overstimulation following major surgery that would be refractory to conventional NO-donor therapy, such as in patients who develop ARDS and are refractory to inhaled NO (iNO). The application of sGC stimulators would then make iNO therapy more effective in the treatment of acute pulmonary hypertension and possibly decease the incidence of associated right ventricular heart failure.In contrast to the benefits from sGC stimulators, the application of sGC activators may have an important role in the therapeutic management of chronic-diseased blood vessels with extended periods of efficacy such as seen in systemic hypertension, atherosclerosis, diabetes mellitus, angina, and heart failure. Moreover, the use of this class of drugs may reduce the development of oxidative stress from NO-donor therapy and may reduce the common side effects from NO-donor therapy such as headache that would improve patient compliance.Research has shown that heme-dependent drugs are effective in the treatment of cardiopulmonary disorders, but that their effects are diminished under pathological conditions associated with increased oxidative stress and the development of tolerance. In the past decade, the development of heme-independent compounds has been shown to higher affinities for the oxidized form of sGC, and they are being developed for the treatment of acute decompensated heart failure and pulmonary hypertension. These two classes of drugs, sGC stimulators and sGC activators, have been studied in animal studies. These compounds are now undergoing preliminary clinical trials and may be available for clinical use within the near future.


Although astrogliosis and microglia activation are characteristic features of multiple sclerosis (MS) and other central nervous system (CNS) lesions the exact functions of these events are not fully understood. Animal models help to understand the complex interplay between the different cell types of the CNS and uncover general mechanisms of damage and repair of myelin sheaths. The so called cuprizone model is a toxic model of demyelination in the CNS white and gray matter, which lacks an autoimmune component. Cuprizone induces apoptosis of mature oligodendrocytes that leads to a robust demyelination and profound activation of both astrocytes and microglia with regional heterogeneity between different white and gray matter regions. Although not suitable to study autoimmune mediated demyelination, this model is extremely helpful to elucidate basic cellular and molecular mechanisms during de- and particularly remyelination independently of interactions with peripheral immune cells. Phagocytosis and removal of damaged myelin seems to be one of the major roles of microglia in this model and it is well known that removal of myelin debris is a prerequisite of successful remyelination. Furthermore, microglia provide several signals that support remyelination. The role of astrocytes during de- and remyelination is not well defined. Both supportive and destructive functions have been suggested. Using the cuprizone model we could demonstrate that there is an important crosstalk between astrocytes and microglia. In this review we focus on the role of glial reactions and interaction in the cuprizone model. Advantages and limitations of as well as its potential therapeutic relevance for the human disease MS are critically discussed in comparison to other animal models. Demyelination of the central nervous system (CNS) is the hallmark of diseases like multiple sclerosis (MS). MS is generally considered to be an autoimmune disease, however, the causative agent of MS and a possible trigger of this disorder are still not well understood. The pathology of MS lesions is heterogeneous  cuprizone to different mouse strains Carlton, , howeverThe gender-related differences in de- and remyelination pattern were already mentioned by Ludwin who notified that Swiss female mice did not undergo demyelination Ludwin, . The demIn the last decade it has become accepted that MS lesions arise also within gray matter structures, particularly in the cortex  synchronously to cuprizone administration (0.5%) did not reduce the toxic effects Carlton, . When miIn the light of all these evidences it is obvious that cuprizone directly or indirectly impairs a variety of essential cell functions. However, mitochondria seem to be mainly affected by this toxin, leading to increased production of oxidative agents, and disturbance of energy metabolism of oligodendrocytes with subsequent cell death. Additionally, since cuprizone susceptibility depends on a variety of co-factors such as age, strain, genetic background, dose, and duration of exposure, the mode of action seems to be a complex mechanism requiring multiple steps and interplayers. Inflammatory metabolites of activated microglia/astrocytes may harm oligodendrocytes as well.During cuprizone-induced demyelination, oligodendrocytes start to undergo apoptosis after 3–7 days of cuprizone exposure  local and migrated OPC start to proliferate within lesions  newly generated oligodendrocytes progressively undergo apoptosis. Furthermore, also the pool of OPC becomes diminished  at the onset of vascularization of the CNS  and displayed a high ex vivo phagocytic activity accompanied by up-regulation of several phagocytic receptors such as Trem2b, FcγR II/III, CD36, and Tim-3  our group analyzed the composition of the microglial population and expression of a subset of phagocytic markers and some known pro- and anti-inflammatory cytokines in the cuprizone model , a known immune inhibitory molecule  infiltrated into the demyelinated CC of cuprizone-treated mice ] indicating a potential beneficial role of FABP7 expression in activated astrocytes for remyelination.It is well known that astrocytes produce different growth factors during cuprizone-induced demyelination, thereby promoting OPC proliferation by FGF-2 and platelet-derived growth factor alpha (PDGFα), or regulating oligodendrocyte differentiation and survival via ciliary neurotrophic factor (CNTF). Recently, the expression of the radial glia cell marker fatty acid binding protein 7 (FABP7) was found to be induced in activated astrocytes after cuprizone-induced acute demyelination whereas expression of this marker and the extent of astrogliosis was diminished after chronic demyelination , a family of zinc-dependent endopeptidases, are highly involved in the perpetuation of inflammation, myelin degradation, and remodeling of extracellular matrix but also proposed to modulate different physiological processes such as cellular development/growth, survival, apoptosis, and migration  and TNFR2(p75) , or TNF-β, belongs to the TNF-superfamily and acts through TNFR1, TNFR2, and lymphotoxin-β receptor (LtβR). In the cuprizone model, Lt-α is expressed primarily by astroglia  is highly expressed in bone but is also secreted by a variety of cell types including endothelial cells, activated macrophages, leukocytes, and T cells . Being a potent mitogen for OPC PDGFα is one of the prominent compounds of the culture medium of primary cortical OPC derived from new-borns or embryos as well as immortalized OPC in vitro  are acting via heterodimeric gp130/LIFRβ  receptor complex. Both molecules possess multiple functions in a variety of cell types including promotion of oligodendrogenesis, differentiation, myelin synthesis, and survival of oligodendrocytes  in modulating oligodendrogliogenesis and remyelination  cuprizone for 6 weeks to achieve complete demyelination. However, it was already described in the early work by the group of Matsushima and confirmed by others that complete demyelination of the CC occurs at week 5 of cuprizone feeding. Despite continued cuprizone exposure mature oligodendrocytes occur between week 5 and 6 and start subtle remyelination. When cuprizone is fed even longer these oligodendrocytes are damaged again and the myelin disappears after week 6. Thus, it should be kept in mind that remyelination does not strictly start with the end of cuprizone administration but all the necessary prerequisites are already made available before that. A recent study provided insights that even 2–3 weeks of cuprizone ingestion is sufficient to induce glial responses and de- and remyelination pattern similar to that described for 5–6 week treatment  male C57BL/6 mice are used in the cuprizone model.The dose of cuprizone needs to be adjusted in accordance to species, mouse strains, and age of animals. For young male C57BL/6 mice (8–10 weeks old) the cuprizone dose of 0.2% (w/w) is known to be well tolerated and reliable to induce a profound demyelination. When using other doses again the controls need to be well characterized since comparison to most published studies is not possible.Demyelination in the cuprizone model is region-dependent. Most studies have investigated the medial CC where complete demyelination occurs after 5 weeks of 0.2% (w/w) cuprizone administration. We therefore, recommend to stop cuprizone after 5 weeks. However, complete demyelination in the cortex or the hippocampus is reached after 6 weeks of cuprizone feeding and thus the duration of treatment needs to be adjusted according to the region to be investigated. The spatiotemporal pattern of cuprizone-induced de- and remyelination is brain region specific and may even vary within a structure like the CC. Therefore, it is of great importance to always investigate a similar bregma level in all animals.Remyelination is fast and robust after acute demyelination (cuprizone feeding for 5 weeks). Thus, in young mice differences may be picked up only early at 0.5 or 1 week after stopping cuprizone feeding since smaller effects may be compensated at later time points. After chronic demyelination (12 weeks) the remyelination capacity retains after withdrawal from the cuprizone diet but is strongly decreased.Different myelin proteins are temporally individually degraded and re-expressed. Knowledge of this pattern is required to plan and interpret the experiments.In order to generate sustainable conclusions it is important to investigate several time points and chose them carefully depending on the question to be answered.Models using additional inhibitors of proliferation like rapamycin in order to better synchronize remyelination are not well characterized. Such substances also affect microglia and astrocytes the reaction of which has not been characterized so far. Thus, the system is even more artificial and the interpretation of results may be impossible because beside the oligodendrocyte damage there is interference with all other proliferating cells.The cuprizone model is a well established and investigated paradigm to study de- and remyelination in rodents. It seems that the regeneration program initiated after the toxic insult via cuprizone proceeds in a highly controlled manner running sequences of tightly orchestrated events on molecular and cellular levels beginning with an activation of microglia and astrocytes as a response to the oligodendrocyte insult within the first 3 weeks of cuprizone feeding followed by a massive proliferation of all glial cells and myelin clearance between the third and fifth week, and onset of remyelination around week 5.5 Figures . There iAlthough well characterized the cuprizone model lacks the autoimmune component of MS. Thus, it possesses only a limited spectrum of MS-relevant mechanisms in particular in lesion induction. However, it is useful to study T cells-independent de- and especially remyelination as well as basic myelin repair mechanisms, interaction of glial cells and their communications with axons. Since no animal model covers the complex pathomechanisms of human MS, the various animal models should be applied selectively or complementary to investigate MS-relevant processes and develop putative regenerative and protective treatments complementing immunomodulating therapies.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.


Arabidopsis, the loss-of-function mutants of ACAULIS5 (ACL5) gene, encoding thermospermine synthase, exhibit a severe dwarf phenotype, suggesting that thermospermine acts as a regulator of stem elongation [Plant polyamines are preferentially detected in actively growing tissues and have been implicated in growth and developmental processes such as embryogenesis, floral developmental, fruit ripening, senescence and stress responses . Recentlongation ,3. HowevACL5-like sequences in Populus trichocarpa genome allowed us to identify three putative ACL5 orthologous genes. Based on the degree of sequence similarity, we have selected one of them, PtACL5, to generate transgenic plants bearing the constructs for overexpression and silencing of this gene in poplar.A search for PtACL5 in overexpression transgenic lines have been found to be correlated to higher thermospermine content in leaves and young stems, but not to a higher level of other polyamines, suggesting that PtACL5 encodes a thermospermine synthase in poplar, and it is most probably an ortholog of ACL5 in poplar. Interestingly, these plants display altered and arrested shoot development in the early stages following p35S::PtACL5 transformation, as well as severe dwarfism. Anatomical changes associated to the lack of elongation include arrested development of the root system and no elongation of the stem from the first internode onwards. Because Arabidopsisacl5 loss-of-function mutants show accelerated vessel cell death and ACL5 expression is confined to xylem vessel elements [High expression levels of elements , we furtArabidopsis, in our transgenic poplar the high levels resulting from overexpression of thermospermine synthase gene seem to overcome any turn-over that might be occurring of the excess thermospermine being produced. Currently we are pursuing the spatial localization of the ACL5 transcript in poplar plants through in situ hybridization, and by taking advantage of the generated transgenic lines we hope to understand the role of thermospermine in the vascular tissues formation in this woody species.Overall, our results suggest that thermospermine has a regulatory role in xylem differentiation/maturation in poplar. Although a feedback control of thermospermine synthesis seems to be present in


During the recent years, numerous studies contributed to clarify the role of somatostatin systems, especially long-range somatostatinergic interneurons, in several functions they have been previously involved in. New advances have also been made on the alterations of somatostatinergic systems in several brain diseases and on the potential therapeutic target they represent in these pathologies.Somatostatin is highly expressed in mammalian brain and is involved in many brain functions such as motor activity, sleep, sensory, and cognitive processes. Five somatostatin receptors have been described: sst CST peptides are predicted to occur as 14-AA or 17-AA short forms in rodents and humans, respectively, and a 29-AA extended form in both species. CST is mainly restricted to the cerebral cortex and the hippocampus in the central nervous system (CNS). CST has been implicated in several brain functions such as learning and memory, regulation of sleep/wakefulness rhythms and it is suspected to have an anticonvulsant activity  has been cloned in 1996  and sharin situ remains an intense field of study. The last decade showed increasing progress in understanding the role of SRIF in brain functions using molecular, pharmacological, and behavioral approaches. The development of innovative molecular, genetic, and imaging tools now allows to go a step further and to assess the cellular contribution of SRIF-expressing cells in neuronal networks ex vivo and soon in vivo. In this review we will give an overview of the latest findings concerning SRIF systems in brain, report some recent data concerning their synaptic actions and their physiological roles within the brain, in normal or pathological conditions.Somatostatin induces many transduction mechanisms in transfected systems . SRIF-immunoreactivity is found at high level in the mediobasal hypothalamus and median eminence, amygdala, preoptic area, hippocampus, striatum, cerebral cortex, sensory regions, and the brainstem  under the control of the GAD67 promoter to visualize GABAergic populations more easily. In the GIN (GFP-expressing inhibitory neurons) strain , a major inhibitory neurotransmitter and labels mostly non-glutamatergic cells in the brain. In order to target SRIF interneurons ) strain  nearly a) strain . The rec) strain  opens prPrevious immunohistochemical and tracing studies have identified two main categories of SRIF neurons: those acting locally in a given structure within microcircuits (interneurons) and those projecting to a distant structure (long-projecting neurons). Nevertheless, recent data using GFP transgenic mice revisit previous anatomical records by demonstrating that some formerly called interneurons also belonged to the projecting neurons category. The different kinds of GABAergic interneurons are classified according to their molecular, physiological, and morphological properties. Immunohistochemical characterization of neuronal populations in rat cortex initially stated, based on calcium-binding proteins and peptides expressions that parvalbumin (PV), SRIF, calretinin, and cholecystokinin labeled four main non-overlapping chemical classes of interneurons . HoweverRecent morphological and electrophysiological studies using GIN mice focused on SRIF-expressing populations in cortical circuits. In mouse cortex, parvalbumin- and SRIF-expressing neurons respectively constitute 40 and 30% of the total GABAergic neurons, calretinin being expressed in 50% of the somatostatinergic population . The remThe major class of SRIF interneurons, the Martinotti cells, have ascending axons that arborize and spread horizontally in layer 1, targeting the distal dendritic parts of excitatory pyramidal neurons  belonging to the G-protein-coupled receptors (GPCRs) family were cloned and characterized from various species. Sequence homology is 39–57% among the five subtypes, each being highly conserved across species. They activate multiple intracellular targets  and SRIF-2 (sst1 and sst4 receptors). Figure 2B represents the wide expression of SRIF receptors in the CNS. In contrast to most GPCRs, sst1-5 are unique because their gene coding sequence is devoid of introns. However, this does not preclude the generation of spliced variants such as a shorter isoform of mouse sst2, named sst2B, originating by the excision of a cryptic intronic sequence /G(o) protein. Somatostatin can also inhibit N-type Ca2+ channels in the dentate gyrus  and activates large conductance g(K)Ca2+ (GK channels). These results highlight the fact that SRIF is a regulator of cellular function in the striatum. The numerous effects of SRIF on Ca2+ and K+ channel conductance in different structures are reviewed by In medium spiny neostriatal neurons, SRIF produces a qualitative change in the firing pattern from a tonic regular to an interrupted “stuttering”-like pattern . These a2/sst3, sst2/sst4, sst3/sst4  and motor functions as well as cognitive functions such as learning and memory and emotion  mice  mice . Therefo2 receptor since both intra-septal and intra-amygdala SRIF microinfusions induced anxiolytic effects that were completely reversed by selective sst2 receptor antagonist injection in these brain areas  pathology for a number of years. Indeed, since the early 1980s, it is known that SRIF levels in cortex and hippocampus are decreased in AD patients . Later, 1, sst3, and sst4 receptors whereas sst2 and sst5 receptors were unchanged  is altered in frontal and temporal cortices, other putative cortical SRIF receptor classes  are not as broadly affected  suggests an impaired excitation/inhibition balance that is potentially mediated by decreased GABA content . More sp effects , a close2, but not sst1, mRNA is preferentially lower in layers 5–6, and in larger, putative pyramidal neurons in those layers. These authors suggested converging pre- and postsynaptic mechanisms to reduce inhibitory neurotransmission in pyramidal neurons in the PFC, which could alter the synchronization of low frequency oscillations and disturb working memory performance in subjects with schizophrenia.One of the most consistent findings in schizophrenia neuropathology is deficits in cortical inhibitory interneurons across multiple cortical regions . It has 2 receptor is likely to mediate the anticonvulsant effects of SRIF in rat hippocampus (4 (1 receptors (2 is also activated while the infarct size is significantly reduced in sst2 KO mice (1 receptors do not appear to mediate the in vivo anticonvulsive effect of SRIF (3 and sst4 mediate this effect through a functional interaction with sst2 receptor (Somatostatin is highly expressed in brain regions associated with seizures and has been implicated as playing a prominent role in epilepsy  based onpocampus , observaampus (4  and/or seceptors , 2006 in KO mice . However of SRIF , whereasreceptor .Somatostatin systems are widely expressed in the different brain regions and are involved in numerous processes from sensory to cognitive functions, suggesting that they play major roles in brain functioning. These key roles are illustrated by the decrease of SRIF concentrations observed in neurodegenerative diseases such as AD and Parkinson’s disease but also in psychiatric diseases such as schizophrenia and MDD. From this perspective, SRIF systems represent a potential and challenging therapeutic target. Further studies need to be carried on to unravel the role of SRIF systems in all functions they have been implicated in.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 developing central nervous system (CNS) is vascularized via ingression of blood vessels from the outside as the neural tissue expands. This angiogenic process occurs without perturbing CNS architecture due to exquisite cross-talk between the neural compartment and invading blood vessels. Subsequently, this intimate relationship also promotes the formation of the neurovascular unit that underlies the blood–brain barrier and regulates blood flow to match brain activity. This review provides a historical perspective on research into CNS blood vessel growth and patterning, discusses current models used to study CNS angiogenesis, and provides an overview of the cellular and molecular mechanisms that promote blood vessel growth and maturation. Finally, we highlight the significance of these mechanisms for two different types of neurovascular CNS disease. The vertebrate central nervous system (CNS), comprised of brain, spinal cord, and retina, is vascularized during its development to provide oxygen and nutrients to newly born neurons, long before they extend axons and dendrites. The neural tube acquires its own vascular network prior to birth via angiogenic sprouting from vessel networks that form immediately outside the CNS; the intraneural blood vessel network then expands as the neural tissue grows. In contrast, the multilayered retina is initially supplied by a combination of two extra-retinal vascular systems, the choroidal vasculature that supplies the outer retina, and the hyaloid arteries that supply the inner retina and lens; the choroidal vasculature persists, but late in mammalian development, the hyaloid arteries are replaced with a dedicated intraretinal vascular system. In this review, we will provide a historical perspective on research into neural tube and retinal angiogenesis, discuss current models available to study CNS angiogenesis, and summarize recent progress in uncovering the cellular and molecular mechanisms of blood vessel growth and maturation in the CNS.The process of CNS vascularization was first studied in the fetal chick brain and both rat and rabbit cerebral cortex with a combination of India ink perfusion and electron microscopy to reveal the structure of patent blood vessels –3. In coFollowing on from the pioneering studies, subsequent research into the mechanisms of CNS vascularization used antibody-based and embryological techniques to trace the behavior of developing blood vessels in the neural tube. Initially, the QH1 antibody, which recognizes quail angioblasts and endothelial cells , was useSince the discovery of QH1, additional vascular markers have become available that facilitate the study of brain and retinal angiogenesis in other vertebrates, including mouse. Accordingly, immunohistochemical, immunofluorescent, and immunoblotting techniques are now commonly combined with traditional methods to study the molecular and cellular mechanisms of vessel growth in the CNS. In particular, immunological techniques have been used to compare CNS angiogenesis in normal development and after genetic modification of candidate vascular growth and patterning factors.kdrl (flk1) or fli1 are used extensively, and can be used to study CNS vascularization in normal and genetically altered fish embryos.Not long after the discovery of brain vascularization through angiogenic vessel ingression, it was shown that a comparable mechanism also operates in the mammalian retina, with angiogenic vessel ingression from the optic nerve vasculature , 13. CurThe vascularization of the mouse embryo hindbrain is initiated around embryonic day (E) 9.5, when a few vascular sprouts emerge from the PNVP and invade the hindbrain parenchyma .Endothelial tip cells respond to signals by initiating migration, while endothelial stalk cells follow behind the tip cell and respond to signals with proliferation and lumen formation to form the main body of new vascular sprouts. Initial experiments linked tip cell and stalk cell behaviors to signals provided by the vascular endothelial growth factor VEGF-A (referred to as VEGF in the remainder of this review) , 34. SubConsistent with a key role for VEGF in tip cell induction in the retina and hindbrain in vivo, a high level of VEGFR2 and low level of VEGFR1 relative to neighboring endothelial cells promotes tip cell behavior in chimeric embryoid bodies . Recent GLUT1 gene that lead to GLUT1 deficiency cause a rare autosomal dominant disorder characterized by a low cerebrospinal fluid glucose concentration due to reduced transport across the BBB ).Flk1 knockout mice are not suitable to study the specific roles of FLK1 signaling in CNS vascular development. However, use of a function-blocking antibody revealed that FLK1 is essential for tip cell formation and vascular outgrowth in the retina [Flk1 is deleted in endothelial cells, retinal angiogenesis depends on the alternative VEGF family receptor tyrosine kinase FLT4 (VEGFR3), which is best known for its role as a VEGF-C receptor in lymphangiogenesis [The tyrosine kinase FLK1  is the main signal transducing VEGF receptor in endothelial cells in vitro and essential for endothelial cell survival and blood vessel formation, with its loss leading to embryonic lethality at E9.5 in the mouse (reviewed in ). Due toe retina . FLK1 waogenesis .Neuropilin 1 (NRP1) is a non-catalytic transmembrane protein whose genetic loss, either globally or specifically in endothelial cells, severely inhibits CNS vascularization, as shown for mouse in the spinal cord , hindbraBoth VEGF165 and SEMA3A have been implicated as modulators of endothelial cell behavior. However, studies of mouse knockouts lacking SEMA3A have shown that this NRP1 ligand is dispensable for normal brain vascularization and blood vessel formation elsewhere in the developing mouse , 73. In The above studies did not identify roles for SEMA3A in developmental angiogenesis, but tumor studies implicated SEMA3A as a modulator of pathological angiogenesis. Thus, SEMA3A reduces the overall vascularity of tumors and “normalizes” tumor vessels, in part by recruiting myeloid cells that stimulate vessel maturation . MoreoveVegfa120/120 mice lacking heparin/neuropilin binding VEGF isoforms have milder CNS vascular defects than mice lacking NRP1 [Semaphorin signaling through NRP1 does not impair brain vascularization, but loss of NRP1 from endothelial cells causes vascular brain defects similar to those caused by loss of NRP1 in all cell; accordingly, it was proposed that VEGF-A rather than SEMA3A signaling through NRP1 is essential for vascular development . Yet, diing NRP1 , 67. Thiing NRP1 –81. WhicSEMA3E is the only class 3 semaphorin that does not bind to a neuropilin receptor, but instead binds directly to the plexin PLXND1 . In the Several additional signaling pathways have recently been identified as important for CNS angiogenesis, with roles in blood vessel ingression and/or survival. The genetic loss of both WNT7a and WNT7b reduces neural tube angiogenesis, and utilizes the canonical Wnt/β-catenin pathway, as vascular-specific loss of β-catenin resulted in reduced neural tube vessels, and those that ingressed were dilated and hemorrhagic , 86. EffSeveral studies revealed a requirement for the orphan G protein coupled adhesion receptor GPCR124 in endothelial cells for proper neural tube vascularization –90. SpecThe many facets of neurovascular disease have recently been reviewed extensively . Here, wCavernous hemangiomas in the brain consist of thin-walled, fragile blood vessels with poor blood flow that are known as CCMs. They are caused by mutations in proteins that are important for cerebral vascular integrity . ThAlthough cancerous brain cells emerge independently of the brain vasculature, the close functional relationships between CNS vessels and neural cells can lead to brain tumors that are highly vascularized and difficult to treat, primarily in the case of glioblastoma . ReThe cellular interactions and molecular signals critical for CNS vascularization and the formation of the neurovascular unit are being elucidated at an accelerating speed, and we are beginning to appreciate the significance of defective vascular development for the emergence of CNS pathologies. Yet, it is likely that additional signaling mechanisms and interactions remain to be identified before we will fully understand how the cross-talk of neural and vascular cells regulates blood vessel ingression into the CNS and the formation of a fully functional BBB. Ultimately, understanding these signaling pathways will reveal how a defective neurovascular unit impacts on CNS function during aging and in neurological disease. It will also benefit the development of new therapeutic strategies aimed at restoring or improving vascular supply to ischemic retina and brain in diseases such as diabetic retinopathy, age-related neurodegeneration and stroke. In the mature brain, neuronal activity stimulates changes in blood flow that can be measured by fMRI, but how this flow information is sensed and leads to structural changes in blood vessels is largely unknown and therefore requires further research. Finally, the specific features of the tumor microenvironment that promote the differentiation of tumor cells into endothelium, and the functional consequence of this transdifferentiation for glioblastoma remain to be determined.


Despite the numerous studies proposing early human population expansions from Africa into Arabia during the Late Pleistocene, no archaeological sites have yet been discovered in Arabia that resemble a specific African industry, which would indicate demographic exchange across the Red Sea. Here we report the discovery of a buried site and more than 100 new surface scatters in the Dhofar region of Oman belonging to a regionally-specific African lithic industry - the late Nubian Complex - known previously only from the northeast and Horn of Africa during Marine Isotope Stage 5, ∼128,000 to 74,000 years ago. Two optically stimulated luminescence age estimates from the open-air site of Aybut Al Auwal in Oman place the Arabian Nubian Complex at ∼106,000 years ago, providing archaeological evidence for the presence of a distinct northeast African Middle Stone Age technocomplex in southern Arabia sometime in the first half of Marine Isotope Stage 5. The Nubian Complex is a regionally distinct Middle Stone Age (MSA) technocomplex first reported from the northern Sudan in the late 1960 s Nubian Complex industries are distinguished by a characteristic and highly standardized method of preferential Levallois reduction, “mass-produced from an elaborate archetype” The early Nubian Complex is distinguished by a higher frequency of Nubian Type 2 cores in conjunction with bifacial foliates and handaxes Taking into account its distinct, regionally-specific characteristics, Marks There are claims for the presence of Nubian technology in eastern Arabia. One “possible” Nubian Type 1 core was reported at Jebel Barakah, UAE, illustrated in Wahida et al. Despite published reports of Nubian At present, the northernmost extent of Nubian Type 1 cores is demarcated by assemblages found in the vicinity of Jebel Urayf and Naquah, in central-east Sinai In light of these tantalizing, yet inconclusive findings, the Dhofar Archaeological Project (DAP) was initiated in 2010 to explore the Late Pleistocene archaeological record of the Dhofar region in southwestern Oman. During the 2010 fieldwork campaign, a surface scatter with Nubian Type 1 and Type 2 Levallois cores was discovered in Wadi Aybut, central Dhofar. Subsequent research by DAP has focused on geoarchaeological investigation of the Aybut drainage system and surrounding landscapes, optically stimulated luminescence (OSL) dating of cemented fluvial sediments at Aybut Al Auwal that contained a handful of diagnostic Nubian Levallois artifacts, systematic survey to articulate the distribution of Nubian Levallois core technology throughout Dhofar, and techno-typological analysis of Nubian Levallois reduction strategies in Dhofar to assess the relationship of these assemblages with the African Nubian Complex.2. The landscape of this region encompasses a variety of geomorphic settings, partitioned into four ecological zones  SalaThe coastal plain, stretching some 90 km in length, is a crescent-shaped landmass up to 15 km wide, bounded by the Arabian Sea and the Jebel Qara escarpment. The plain is composed of alluvial fans up to 100 m thick, which are cut by short wadis draining southward from the escarpment toward the sea. Several springs emerge along the foot of the escarpment, around which grow date and coconut palms, bananas and other tropical fruits, and grasses North of the plain, the Jebel Qara-Jebel Samhan mountain chain rises abruptly to a maximum height of 850 m above sea level. The high escarpment forms an orographic barrier that traps moisture from the Indian Ocean Monsoon (IOM), generating up to 200 mm of annual rainfall across the mountains. Due to moisture brought by the IOM, the Dhofar highlands are covered in a mantle of dark brown clay soil that supports a subtropical cloud forest belonging to the Somalia-Masai center of endemism Northwards, past the current watershed divide, the escarpment flattens out onto a deeply incised limestone plateau called the Nejd, which extends approximately 250 km east-west and 150 km north-south. Around its southern extent, the Nejd is a barren scabland marked by an intricate series of minor wadis dissecting the plateau. These smaller drainage systems converge into larger and more deeply incised canyons that extend up to 100 km northward across the plateau, running parallel to one another. As they reach the northern extent of the Nejd, the wadis empty onto a gently undulating gravel plain flanking the Rub' Al Khali desert.Mudayy member, which is the highest quality on the plateau, outcropping in the southern and central regions. Chert-bearing units within the overlying Rus formation are concentrated in the southern Nejd, including the lower chalky Aybut member and upper marly-carbonate Gahit member. The quality and size of this raw material is quite variable; due to post-depositional displacement, most of the Aybut member chert is highly fractured. Thin, high-quality grey chert plaquettes are found within the Gahit member Three high-quality Eocene chert beds outcrop widely throughout the Nejd plateau, making the landscape appealing for prehistoric toolmakers. Fine-grained, large, banded chert slabs are found within the The drainage channels incising the Nejd plateau formed during wet climatic regimes throughout the Quaternary These data indicate that the monsoon increased in intensity during three intervals within MIS 5. Among these humid episodes, the last interglacial  appears to represent the most significant wet phase within the entire Late Pleistocene, with rainfall surpassing all subsequent pluvials Climate records indicate that MIS 4 (74–60 ka) was a period of rapid global cooling, at which time much of Arabia was beset by prolonged aridity caused by the southward displacement of the IOM. Records from the Arabian Sea attest to a period characterized by cooler sea surface temperatures, low productivity, and increased terrigenous (aeolian) input Ziziphus ziziphus (Jujube), Adansonia digitata (Baobob), Ficus sp. , Calotropis procera (Sodom's Apple), and Adenium obesum (Desert Rose) a fortiori, that the archaeological record will demonstrate cultural affinities at such times.Activation of Arabian fluvial systems during humid phases eg., DAP fieldwork was conducted over the course of two seasons in the winter of 2010 and 2011; required permits to carry out survey and excavation were granted by the Ministry of Heritage and Culture in Oman. To date, DAP has mapped 110 occurrences with Nubian Levallois technology across the Nejd plateau, ranging from occasional isolated cores to high-density scatters . Lithic Surveys were conducted along 40 transects throughout the Nejd plateau, Jebel Qara escarpment, and Salalah coastal plain . TransecFrom the distribution of findspots in Dhofar exhibiting Nubian Levallois technology, it appears that occurrences are confined exclusively to the Nejd plateau, where they are most often found near stream channels and raw material outcrops. Survey transects did not produce evidence for any kind of MSA/MP occupation along the coastal plain or the fringes of the Jiddat Al Harassis gravel plain bordering the eastern Nejd. The westernmost occurrence (TH.102) was an isolated Nubian Type 1 core in Wadi Tanfarut along the Yemeni border, while the easternmost site (SJ.56) was a low density Nubian Levallois scatter in Wadi Qaharir, 250 km to the east. In the north, a small number of Nubian Type 1 and Type 2 cores were discovered around Shisur Farms (TH.38), on an ancient fluvial terrace 7 km east of Wadi Ghadun. Given the logistical difficulties of survey within the Rub' al Khali desert, we were not able to investigate this zone and cannot yet address the northern distribution of such sites in Dhofar. The southernmost occurrence (TH.78) was an isolated Nubian Type 1 core on a low terrace above Wadi Nirin, less than 2 km from the northern slopes of Jebel Qara. In every assemblage encountered, Nubian Type 1 cores were by far the most prevalent, and Nubian Levallois technology was never found in conjunction with a bifacial component.Mudayy member chert beds as the channels developed. The Aybut-Banut-Amut-Ghadun drainage system is unlikely, however, to be the only center of MSA occupation on the Nejd. We have systematically surveyed less than 1% of the 33,000 km2 plateau, so it is likely that there are other catchments with similarly high concentrations of MSA artifacts.Of the MSA sites with Nubian Levallois technology mapped by DAP, 39 findspots (<1 artifact per sq m), 55 low density scatters (1–10 artifacts per sq m), and 16 high density scatters (>10 artifacts per sq m) were recorded. While isolated findspots and low density scatters are found across the entire plateau, evidence for intensive/recurrent settlement is concentrated in the west-central Nejd, around a large catchment system made up of Wadis Aybut, Banut, Amut, and Ghadun. This may be linked to the presence of ancient and modern groundwater-fed springs that emerge around the village of Mudayy, at the confluence of Aybut and Banut. Not only would this zone have provided a considerable amount of water in both its rivers and springs, but also fluvial downcutting would have continually excavated fresh Mudayy chert and chipping debris, and is incised by a series of small stream channels  is an open-air site that contains artifacts on the surface and buried within fluvial sediments in Wadi Aybut, west-central Nejd. The site was found on the second terrace, ∼20 m above a relict tributary channel feeding the main wadi system. The terrace is blanketed in a pavement of naturally occurring channels . Lithic channels , while tchannels . AlthougMudayy member) and Tertiary limestone (Umm Ar Radhuma formation) The Aybut Al Auwal terrace is formed by unconformities within horizontal strata of the underlying bedded chert  limestone and chert clasts within their course.One such lateral channel-fill deposit was excavated to a depth of 92 cm and is comprised of four distinct stratigraphic units, which overlie the limestone channel bed . The uppThe depositional age of the artifact-bearing sediments in Unit 3 was estimated by OSL dating of buried quartz grains 2. An additional 10 pieces were excavated from ∼1 m3 of highly-cemented sediment comprising stratigraphic Unit 3, and 11 desilicified artifacts were collected nearby eroding from the side of the channel  is a Nubian Complex surface scatter situated on a gravel plain some 5 km northeast of Aybut Al Auwal. The site is positioned at the headwaters of two large tributary systems, with prominent views of wadi channels to the east and west . Althoug2. A 10×10 m area was systematically collected in 1 m2 units, and all cores, tools, and a 25% sample of debitage were analyzed. Cores and larger pieces of debitage are only moderately weathered, however, the smaller material is in exceptionally poor condition, due to a combination of taphonomic processes including deflation, winnowing, surface runoff, chemical alteration, and thermal fracturing. While striking platforms and scar patterns are clear and permit technological analysis, the resulting edge damage caused by these destructive processes has obscured possible retouch, hindering typological identification. Given this problem, the Aybut Ath Thani tool type list should be approached with caution.The small lithic scatter observed at Aybut Ath Thani is constrained to no more than 400 mOf the 1,734 artifacts comprising the Aybut Ath Thani assemblage, 157 (9%) are cores . Nubian Although the site is positioned slightly away from a source of raw material, there is a relatively low ratio of non-cortical pieces to cortical pieces . There aSome blanks were identified with sufficiently consistent retouch to be classified as tools, despite the heavy edge damage on many of the pieces in this assemblage. These types, presented in Mudayy member chert beds, as the soft limestone hills were broken down by wind and surface runoff.Mudayy As Sodh (“Mudayy's Rooftop”) is located on a high plateau above the village of Mudayy. The site consists of multiple surface scatters just over 1 km east of Aybut Al Auwal, around a series of shallow basins that debouch into the main Aybut tributary. Small gullies (<50 cm deep) incise the silicate gravel covering the plateau, where a variety of assemblages were observed in discrete patches across the landscape. Nubian Complex scatters were identified closer to the edge of the plateau overlooking the drainage systems below, while less weathered Nejd Leptolithic 2 was systematically collected from one Nubian concentration at Mudayy As Sodh, chosen for its high density of cores and debitage. 965 artifacts were recovered in total, including 92 cores, 69 tools, and 804 pieces of debitage  is situated 6 km southeast of Aybut Al Auwal. The site consists of several concentrated lithic scatters on the first erosional terrace (about 5–15 m wide), perched ∼10 m above a steeply-incised wadi channel . Excepti2. As there are no obvious differences between the two areas, we have combined them into a single assemblage for the purposes of this analysis. The composite assemblage is comprised of 449 artifacts; Nubian core conjoins indicate there has been minimal post-depositional disturbance  and/or steep bilateral (Type 2) removals. Specimens from Dhofar exhibiting this characteristic distal median ridge are shown in cross section in Nubian Complex assemblages in northeast Africa exhibit multiple core types, including Nubian Type 1, Nubian Type 2, preferential centripetal Levallois, bidirectional, and single platform . In DhofThe most common tool types found within Nubian Complex assemblages in Dhofar are Levallois points, flakes, and blades, which show a propensity toward elongation and converging lateral edges. The relatively few retouched tools include sidescrapers, endscrapers, denticulates, notches, and miscellaneous retouched pieces, with a trace number of burins and perforators . This saGiven these technological and typological similarities, we classify the Dhofar assemblages as late Nubian Complex. It is more likely that the high degree of overlap observed in southern Arabian and northeast African Nubian Complex assemblage – a continuous phytogeographic zone divided only by the Red Sea – is the result of cultural exchange, rather than the synchronistic result of concurrent technological evolution. For the time being, the apparent distribution of Nubian Levallois technology in Arabia is limited to the Nejd plateau and, perhaps, Hadramaut valley . ArchaeoWhile any explanation must be speculative, we suggest that the significantly higher frequency of Nubian cores in Dhofar, as opposed to the Nile Valley, may be the result of variations in hunting behavior across the two landscapes. These differences, in turn, are the function of hydrology and the area of exploitable land in Dhofar versus the Nile Valley. In southern Arabia during MIS 5c, there were extensive grasslands cut by drainages, but none so big as to limit faunal distributions or impede hunter-gatherer mobility. In the Nile Valley, on the other hand, exploitable land was limited to the valley itself and to a narrow strip of land along its sides. Both to the east and west of the Nile Valley, the flat gravel plains would not have been appropriate hunting terrain, as confirmed by the lack of sites even a few kilometers from the valley Homo sapiens found in the Omo river valley, Ethiopia  from Africa is the subject of considerable debate If MSA inhabitants of northeast Africa were AMHs, then the presence of a regionally-specific African MSA industry in Dhofar is relevant to the question of modern human expansion. The route and timing of Movement through the northern dispersal route is based on AMH remains discovered at Skhul and Qafzeh in Israel dating to early MIS 5 Genetic studies of human mtDNA favor the southern dispersal route as the primary conduit for early modern human expansion(s) out of Africa To some degree, the discovery of late Nubian Complex assemblages in Dhofar upholds this model. The distribution of this technocomplex in the middle and lower Nile Valley, the Horn of Africa, Yemen, and now Dhofar provides a trail of diagnostic artifacts - stone breadcrumbs - spread across the southern dispersal route out of Africa. The close similarity between African and Arabian late Nubian Complex assemblages suggests that these sites are more or less contemporaneous; they were separated for an insufficient amount of time for independently derived technological traits to develop between regions. As the late Nubian Complex at Aybut Al Auwal is dated to MIS 5c, slightly earlier than the late Nubian Complex in Africa Coalescence ages for non-African mtDNA lineages range from 70 to 45 ka, depending on the use of different mutation rates, calibration methods, and statistical models A second possibility is that the mtDNA coalescence age of L3 would appear younger than the time of initial expansion if pioneering groups moving into Arabia had been sex-biased toward a low number of females The archaeological evidence does not yet permit us to evaluate what became of the late Nubian Complex in Arabia. Our study only documents the presence of this industry in Dhofar during MIS 5c; we do not yet know when Nubian Complex toolmakers arrived on the subcontinent or what became of them over the course of the Late Pleistocene. The eastern distribution of the Nubian Complex appears to terminate at the edge of Nejd plateau. Surveys throughout the rest of Oman and eastern Arabia have not produced any evidence of Nubian Complex technology. Assemblage C from the last interglacial site of Jebel Faya is classified as a generalized East African MSA technological complex  and is ascribed to AMH toolmakers. Its small assemblage size and limited workshop characteristics, however, preclude attribution to any specific, contemporaneous East African industry The presence of seemingly Nubian-derived assemblages around the Wadi Aybut-Banut-Amut-Ghadun drainage systems, discovered during the DAP 2011 fieldwork campaign, hints at the survival of some aspects of the Nubian Complex technological tradition within Dhofar. These ‘Developed Nubian’ assemblages exhibit a suite of core reduction strategies including Nubian Levallois, ‘microlithic’ Nubian, and flat cores with bidirectional blades struck from faceted platforms. Such assemblages, however, must still be adequately defined and placed within a chronological framework.Although southern Arabia experienced successive periods of extreme aridity after MIS 5, terrestrial archives document another increase in precipitation across the interior of Arabia during early MIS 3 Figure S1Example OSL decay and dose-response curves from AYB1-OSL1. Decay curve (a) and dose-response curve (b) for a single aliquot of quartz (∼50 grains). The De of ∼70 Gy is obtained by interpolation of the sensitivity-corrected natural OSL signal, shown in red on the y-axis of the inset plot. The data in (a) and (b) were collected after preheating the natural and regenerative doses at 260°C for 10 s. Panel (c) shows the De values obtained from aliquots preheated at a range of temperatures , along with the extent of recuperation ; these data indicate that the measured De value is not sensitive to the chosen preheat temperature. The De values obtained from 42 separate aliquots of AYB1-OSL1 are displayed in (d); each aliquot was preheated at 260°C for 10 s. The filled circles and open triangles denote the values obtained using the ‘late light’ and ‘early background’ subtraction approaches, respectively, and the shaded band is centred on the weighted mean De value (∼58 Gy) used to calculate the OSL age of this sample. Plot (e) shows the De values obtained from 22 single aliquots of AYB1-OSL2: the symbols are the same as in (d) and the shaded band is centred on the weighted mean De value (∼61 Gy) used to estimate the sample age.(TIF)Click here for additional data file.Table S1e) values, environmental dose rates, and OSL ages of the sediment samples from Aybut Al Auwal.Equivalent dose  uncertainty, calculated as the quadratic sum of the random and systematic uncertainties. The De uncertainty includes a relative error of 2% to allow for possible bias in the calibration of the laboratory beta source.(DOC)Click here for additional data file.Appendix S1Optically stimulated luminescence (OSL) dating.(DOC)Click here for additional data file.


Peromyscus maniculatus) population density and season on transmission dynamics of Sin Nombre hantavirus. In early summer, mid-summer, late summer, and fall 2007–2008, predetermined numbers of infected and uninfected adult wild deermice were released into enclosures and trapped weekly or bi-weekly. We documented 18 transmission events and observed significant seasonal effects on transmission, wounding frequency, and host breeding condition. Apparent differences in transmission incidence or wounding frequency between high- and low-density treatments were not statistically significant. However, high host density was associated with a lower proportion of males with scrotal testes. Seasonality may have a stronger influence on disease transmission dynamics than host population density, and density effects cannot be considered independent of seasonality.Surveys of wildlife host-pathogen systems often document clear seasonal variation in transmission; conclusions concerning the relationship between host population density and transmission vary. In the field, effects of seasonality and population density on natural disease cycles are challenging to measure independently, but laboratory experiments may poorly reflect what happens in nature. Outdoor manipulative experiments are an alternative that controls for some variables in a relatively natural environment. Using outdoor enclosures, we tested effects of North American deermouse ( Peromyscus maniculatus; hereafter deermouse) populations in Montana show maximum Sin Nombre hantavirus (SNV) infection  in the spring, and this peak is often positively associated with the size of the deermouse population the preceding fall In the past 30 years, numerous theoretical models have been proposed to explain how pathogens become established and spread in host populations. Early models assumed that the driving force behind directly transmitted parasites was population density (density-dependent transmission) and, because these models were useful to understanding many human diseases, they were applied to wildlife populations The effects of seasonality on disease dynamics in wildlife are another focus of disease ecologists. Seasonal variation in precipitation, temperature, and resource availability can influence host population dynamics, host physiology, and disease dynamics in wildlife host populations Hantaviruses are directly transmitted, specialist microparasites endemic in natural rodent and insectivore populations; some, including SNV, are pathogenic for humans. Hantaviruses generally establish a persistent infection with long-term shedding in a single natural host species The individual effects of seasonality and density on natural disease cycles are often hard to tease apart from each other and from other confounding factors driving host parasite systems. One way to explore and quantify these effects is through manipulative field experiments using a well studied host-pathogen system. The deermouse-SNV host-pathogen system has been a subject of intensive longitudinal studies that have improved understanding of the relationships between SNV transmission dynamics with seasonal factors and with host population density A partial solution to these problems is the use of outdoor, semi-natural enclosures that approximate natural field conditions more closely than does a laboratory. Such studies also allow working with a closed population of a limited number of individuals of known sex, age, physical condition, and infection status, and the events observed during the experiment are largely a consequence of those well known experimental conditions. We used the deermouse-SNV host-pathogen system in Montana to explore the effects of density and seasonality on pathogen transmission. Longitudinal field studies in Montana have demonstrated that the greatest number of seroconversions and the greatest proportion of deermice with detectable SNV RNA are found in the mid-to-late breeding season . The study was also reviewed and approved under Emory University Biosafety protocol #100-2008. No trapping permit is required for trapping rodents in Montana.This study was conducted in shrub-steppe grassland near Butte, Montana, USA, May–October 2007 and August–September 2008. We conducted 4 experiments–1 preliminary transmission experiment (experiment A) and 3 density experiments  and a predetermined number of uninfected (susceptible) mice were released into each enclosure according to the study design . As we wAll mice released into the enclosures were ear-tagged with sequentially numbered, metal, fish fingerling tags . Mice were provided grain, apple chunks, and water weekly or as needed. Food was scattered widely throughout the enclosure to avoid unnatural aggregations at feeding stations; water was provided in water-bottles, as required by our IACUC protocol, in the burrows. Rodents in enclosures were trapped weekly (2008) or biweekly (2007) to collect blood samples using standardized protocols for SNV surveillance Mice trapped within 5 km of the study area were assigned to 1 of 3 age classes according to body weight: mice <14 g were juveniles; mice 14–17 g were subadults; mice >17 g were adults Rodent hosts of other hantaviruses are most infectious 2–5 weeks post-infection, but are known to shed infectious virus for much longer After the start of each experiment, if a susceptible mouse in an enclosure was found positive for either SNV RNA or SNV antibody, he was designated as a transmission-event (TE) mouse. Every TE mouse was found positive for both SNV RNA and antibody, except for 3 mice that did not develop detectable IgG antibody before either dying or the end of experiment. For these 3 mice, we confirmed infection with SNV by detecting SNV RNA in 2 or more blood samples collected on different dates, or by sequencing the samples (2008 mice).Because the mice in 2007 were not quarantined, it is possible that some were infected prior to release into the enclosures. The majority of TE mice had negative SNV antibody and RNA results for at least 2 weeks post-release and seroconverted or had detectable SNV RNA in their blood 1 month post-release. Our 2008 quarantine results indicated that mice that were previously exposed seroconverted within the first 2 weeks. Three of the TE mice seroconverted within two weeks after introduction into the enclosures. Two of these three mice had very low antibody titers , Mouse 2: titer of 200, positive for SNV RNA), which is consistent with the blood profile of a very recently infected mouse (Bagamian 2012). Also, both mice were from the same enclosure, suggesting close temporal exposure to the same donor. Fifteen of the 18 transmission events (excluding these two mice: 13/16) involved multiple mice in the same enclosure (Bagamian 2012). Thus we feel that infection prior to release into the enclosures is unlikely for these two mice.Nevertheless, we analyzed our data both including and excluding these mice and report both sets of results. A third mouse was SNV RNA-positive and had a high antibody titer (1600) two weeks post-release into enclosures. Because infection prior to release into the enclosure seemed possible, this mouse was excluded from our analyses of transmission incidence. Although we feel that these criteria for excluding potential transmission events prior to the experiment are reasonable, we emphasize that we have no way of excluding such events with 100% certainty. We report transmission incidence as the number of new infections (transmission events/sum of the number of mouse-weeks of observations) see .4. For mWe conducted statistical analyses  using Microsoft Excel 2007 and R . Details of the analyses and variable derivation are provided in For all analyses, we excluded data from mice that were released into the enclosures and never recaptured . We also excluded data from 1 low-density enclosure (Enclosure 3) in Experiment 1 in analyses of transmission incidence, because it was unclear whether the donor mouse was truly infected. His blood was positive for SNV RNA in 1 of 2 samples, but he was not recaptured again to reconfirm infection status. There were no TE mice in Enclosure 3 in Experiment 1. In analyses of wounds, scrotal condition, and weight gain for the 2008 experiment, we also excluded information from 2 mice that were in the experiment for less than 2 weeks, because of insufficient data.We analyzed the total number of new wounds on each mouse per experiment, season, or density treatment. The total number of new wounds was counted on an individual animal over the course of the experiment, and each animal was represented only once in any analysis. This conservative measure only includes wounds detected on a new location on the mouse (tail vs. ear) and increases in the number of wounds from the previous sampling session. This ensured that the same wound was not counted twice for any animal, but also allowed for the possibility that some new wounds in the same area as a previous wound may not have been counted. We ran a linear regression model with the season as a categorical predictor variable, and the outcome variable was the number of new wounds per experiment.We documented 18 transmission events over 4 experiments . BecauseWe found statistically significant differences in the incidence of transmission between each summer period  and the fall demonstrated by both rate ratio confidence levels and by the test of differences of proportion including all 18 transmission events, and when the 2 potentially questionable events were removed from the analyses . Transmi103 = −1.998, p  = 0.048, βfall  = −1.1522, SE = 0.5767), and suggestively higher in comparison to late summer , but not in comparison to mid-summer , as determined by a linear regression comparing each season to early summer  or in each experiment .We found no significant differences in the average number of new wounds on individual mice between high- and low-density treatments overall . The proportion of scrotal males in the low-density group was generally higher than that of the high density population, and lower than the outside mouse populations, but none of these differences were significant  was significantly higher than the proportion of abdominal males with wounds . Wound frequency did not differ significantly between adult males in the enclosures compared to those captured outside the enclosures (FET: p = 1.00).35 = 0.1675, p>0.05).When measured at the beginning of each experiment and at the end of each experiment, no statistically significant differences were found in the mean animal weight between treatments (high- vs. low-density) or between locations (inside vs. outside enclosures) in any of the experiments . During Experiment 2, there were no significant differences between treatments in the mean rate (g/week) of weight gain . Transmission of SNV in low-density enclosures occurred in only 1 of the 3 density experiments, and the overall transmission incidence in the high-density treatment was 2-fold higher than in the low-density treatment. Nevertheless, this difference between the density treatments was not statistically significant. Sample sizes were low and any effect of density on transmission frequency might have been obscured by the number of observed zeros. Alternatively, SNV transmission may be frequency dependent. Six years of cowpox virus dynamics in bank voles showed that transmission appeared to be density dependent during the winter, but frequency dependent in the summer–emphasizing the importance of seasonal variation in host behavior and susceptibility on disease processes Our objectives were to observe natural transmission of SNV in et al.The incidence of SNV transmission decreased significantly as experiments were conducted later in the breeding season. This observation is consistent with previous mark-recapture studies, which have indicated strong seasonal trends in seroconversion and increased prevalence of infection during the breeding season P. maniculatus populations are highly variable and depend on photoperiodic cues, temperature, and food availability et al.Initiation and cessation of the breeding season for Microtus montanus and Microtus pennsylvanicus), and P. maniculatus bairdii have shown a strong and significant effect of high population densities in suppressing reproduction in both males and females Studies of caged albino and wild-type house mice, free-roaming vole populations  after release in comparison to before they were released into the enclosures . The proportion of scrotal adult males in the enclosures at T1 was also significantly lower than the proportion of scrotal adult males captured outside the enclosures . Additionally, in the second-longest running experiment , while approximately 30% of males in the high-density group and 10% of males in the low-density group had scrotal testes at T0 (data not shown), no males with scrotal testes were captured at T1 and at the next 2 trapping sessions, although breeding males were captured outside the enclosures at T1 see . One impAlthough population density clearly affected the ability to maintain breeding condition, it had no statistically significant effect on the rate of weight gain. The supplementary food and water in the enclosures may have contributed to weight maintenance.The average number of new wounds per mouse was significantly higher in the early summer than late summer and fall. As the breeding season begins, males often respond to seasonal cues and establish and defend territories The fact that incidence of transmission and average number of new wounds per mouse peaked at the beginning of the breeding season and decreased over time provides some support to the current view that direct contact may be the primary mode of transmission in wild deermouse populations, because the most transmission occurred during times where the mice were most aggressive. We cannot rule out the possibility that SNV may have been transmitted both directly and indirectly in the enclosures. Future studies could implement cameras, pit tag recorders, and fluorescent marking powder A major limitation of our experiments was small sample size. We were able to maintain a limited number of mice per enclosure, and we observed 18 transmission events total in all 4 experiments. However, as natural transmission events are rare by nature, recording 18 events in a semi-controlled setting could be considered very successful. A previous laboratory study reported only 1 SNV transmission event out of 54 attempts Our results, especially in the light of previous mark-recapture studies of effects of season and density on infection dynamics in wildlife populations, emphasize the importance of considering the strong effects of season as a confounder when making comparisons of density effects in natural populations. Seasonality, even when only evaluated within the timeframe of the breeding period (spring to autumn), may be more influential in disease dynamics than population density. Season influences host behavior, susceptibility, host reproduction, and other physiological processes, all of which are critical in maintaining disease transmission cycles in nature. Although there is a likely effect of host population density on disease transmission, density processes cannot be considered independently of seasonal factors when exploring natural host-pathogen systems.We successfully conducted large-scale manipulative experiments that followed SNV transmission in deermice under controlled conditions. Our experiments provided further insight into the effect of seasonality and density on hantavirus transmission, reservoir host aggression, and host reproductive processes. Our successful methodologies might be used to address other questions in the field of wildlife disease ecology or in similar zoonotic host-pathogen systems.Figure S1Sheet metal enclosure array used for containing deermice in Montana.(JPG)Click here for additional data file.Figure S2Interior corner of one enclosure showing Sherman trap.(JPG)Click here for additional data file.Appendix S1Supplementary methods for Population Density and Seasonality Effects on Sin Nombre Virus Transmission in North American Deermice (Peromyscus maniculatus) in outdoor enclosures.(DOCX)Click here for additional data file.