All these activities are potentially useful for several respiratory conditions such as asthma, COPD, and recurrent respiratory tract infections.Usefulness of Pidotimod and its role as immunostimulant, has been discussed, we know, for several decades. Nevertheless, there is still much to know. Understanding its mechanisms and its potential usefulness in airway infections and its prevention, asthma both Th2 and non Th2 type, bronchiectasis, as adjuvant in vaccination and in allergen immunotherapy still remains to clearly unveil. The aim of this paper was to provide a useful updated review of the role of the main available immunostimulants, giving particular focus on Pidotimod use and its potentials utility in respiratory diseases. Pidotimod showed its usefulness in reducing need for antibiotics in airway infections, increasing the level of immunoglobulins  and T-lymphocyte subsets  endowed with immunomodulatory activity that affect both innate and adaptive immune responses. Higher expression of TLR2 and of HLA-DR molecules, induction of dendritic cell maturation and release of pro-inflammatory molecules, stimulation of T lymphocyte proliferation and differentiation toward a Th1 phenotype, as well as an increase of the phagocytosis have been demonstrated to be associated with Pidotimod in  Since the early 90’s more than a hundred papers have been published about Pidotimod, an immunostimulant drug. Due to its action mechanism and its potential, it has been used both in children and in adults, mostly for preventing respiratory tract infections, and asthma or chronic obstructive pulmonary disease exacerbations.Despite the use of antibiotics and vaccines, the frequency of respiratory tract infections is still high and these infections disturb a wide range of patients, from children to elderly, particularly these two extremes due to the deficiency of their immune system: immaturity in the first case and “immunosenescence” in the second one. For that reason, immunostimulant drugs steadily increased in the past few years, getting nowadays more importance and visibility in preventing the onset and reducing the duration of airway infections .Immunostimulants are a heterogeneous group of compounds that act non-specifically on the immune system by inducing its activation, either upregulating it or by favoring the activity of one of its components. By understanding deeply their biological function, they may be implemented in the clinical practice to shape the immune system favorably according to the different processes that want to be enhanced or hampered. According to these peculiar characteristics they have also been called “Biological Response Modifiers” (BRMs). Among this heterogeneous group of compounds which encompass both synthetic and naturally occurring substances, some of them deserve a brief discussion –31. ThisBacterial Lysates derive from a blend of bacterial cultures antigens throughout mechanical or chemical cell lysis. The selected bacteria include: Streptococcus pneumoniae, Streptococcus pyogenes, Hemophilous influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydia pneumonia, Streptococcus Aureus, Streptococcus viridans and Klebsiella pneumoniae. These bacterial antigens have shown to directly upregulate both innate and adaptive immunity, by stimulating Toll-like receptors, inducing dendritic cells (DC) maturation, increasing phagocytic activity, and by activating interleukin 2 receptor (IL2R) on different lymphocytes subsets, inducing therefore cytokines synthesis and enhancing the production of IgA antibodies [tibodies .A Cochrane review by Del-Rio-Navarro et al. concluded that immunostimulants reduce the incidence of acute respiratory tract infections (ARTIs) by 40% in susceptible children, but the included trials quality was generally poor with a high level of statistical heterogeneity ; howeverIn adults and elderly, polyvalent mechanical bacterial lysates (PMBL) have been shown to be superior respect to placebo and to polyvalent chemical bacterial lysates (PCML) in terms of reducing the number and the duration of infectious episodes, and reducing the need of antibiotic treatment in patients with recurrent respiratory tract infections (RRTI) . MoreoveLactoferrin is a natural iron-binding protein highly expressed by both epithelial cells in most exocrine secretions, and by neutrophils, which store the molecule in the secondary granules. It has been reported its functions as an alarmin-like molecule, promoting antigen presenting cells maturation and upregulation of proinflammatory cytokines synthesis [ynthesis , but alsynthesis ; other rynthesis , interfeynthesis , and an ynthesis .The clinical effect of lactoferrin supplementation as a preventive measure for infectious diseases has been mainly investigated in newborn and preterm infants. Notably, an Italian study conducted by Manzoni et al. showed a significant decrease in the incidence of sepsis in very low birth weight infants (< 1500 g) who received 100 mg/d of bovine lactoferrin from birth until the 30 or 45 day of life .Resveratrol is a natural polyphenolic compound contained in several plant species, such as peanuts, grapes and berries. It has been shown that resveratrol has an effect on a wide range of biological activities ranging from antimicrobial [icrobial , neuropricrobial  and antiicrobial . Resveraicrobial , to moduicrobial  and to dicrobial .in vivo with a septicaemia infection model system induced by nontypeable Haemophilus influenzae(NTHi), showing a reduction in NTHi viability and a concomitant reduction in airway inflammation [, resveratrol reduced mortality, lung injury and cytokine levels in the lung tissue of S. aureus pneumonia in murine models [Notably, it has been demonstrated that resveratrol has a remarkable anti-inflammatory effect in COPD patients and its antimicrobial and immunomodulatory effects have been studied ammation . Furthere models . Based oMonophosphoryl lipid A (MPLA) is a nontoxic, but still highly immunogenic derivative of lipid A, the biologically active part of lipopolysaccharide (LPS) endotoxin, and a Toll-like receptor 4 agonist. In vitro studies confirmed that MPLA is able to act, as its parent molecule LPS, as a potent adjuvant increasing the function of antigen presenting cells and an antibody response characterized by the production of IgG1 and IgG2 [and IgG2 . Becauseand IgG2 , in hepaand IgG2  and in pand IgG2 . Furtherand IgG2 , 30. Intand IgG2 , 32.in vitro studies [Pidotimod is a synthetic dipeptide molecule (3-l-pyroglutamyl-l-thiazolidine-4carboxilic acid) endowed with immunomodulatory activity that affects both innate and adaptive immune responses. Higher expression of Toll Like Receptors (TLR) 2 and of HLA-DR molecules, induction of DC maturation and release of pro-inflammatory molecules, stimulation of T lymphocyte proliferation and differentiation toward a Th1 phenotype, as well as an increase in the phagocytosis have been demonstrated to be associated with Pidotimod in  studies –35.in vitro and in vivo studies on human and animals showed that pidotimod is able to ameliorate both innate and adaptive immunity, and enhances the immune system capabilities to fight RRTI, especially in children with increasing resistance to viral infections [Several fections –40.Studies in different areas have demonstrated the benefit of Pidotimod, including its use in hepatitis C, HPV genital infection, Henoch-Schönlein Purpura, nephrotic syndrome, and immunodepressed individuals such as children and elderly . ConsideThe common end-point of these studies is that Pidotimod has an immunomodulatory activity which is able both to improve the clinical conditions of patients and to enhance and stimulate their immunity cells functions acting on both adaptive and innate immunity .The clinical efficacy of pidotimod in decreasing respiratory tract infections in paediatric age was assessed in multicenter randomized trials involving children with age ranged from 2 to 14 years old as regard fever duration, need to antibiotic treatment, hospitalization, school absence, recovery time and relapse rate. All these parameters were significantly reduced in the Pidotimod-treated groups, clarifying the supportive role of pidotimod in treatment of acute infections and its prophylactic role in prevention of RRTI , 43.It is well known that epithelial cells play a primary defensive role, not only providing a natural physical barrier but also being involved in innate and adaptive immune responses , by meanRecognition of PAMPs via TLRs results in the activation of NF-kB, which is a complex protein found in almost all animal cells and controls DNA transcription in response to variable stimuli, including bacterial and viral infections , 47. Actin vitro study to evaluate the immunomodulatory effects of pidotimod demonstrating that it is able to upregulate TLR2 expression, with significant increase in NF-kB protein expression and NF-kB nuclear translocation [Carta et al designed an location .Pidotimod is also able to induce phenotypic and functional maturation of mucosal DC, which plays an important role in the cross talk between innate and adaptive immunity, as pidotimod upregulates the expression of HLA-DR and co-stimulatory surface markers CD83, CD88.Pidotimod-induced DC maturation leads to release of pro-inflammatory mediators such as TNF-α leading to increase recruitment of inflammatory cells, activation of naïve T lymphocytes with proliferation and polarization toward Th1 phenotype. Besides its role on epithelial cells and DC, pidotimod also enhances cytotoxic activity of natural killer cells (NK) and phagocytic activity of neutrophils .Zhou and Dai proposed a study comparing the effect of Pidotimod to spleen aminopeptide  on Th1/Th2 cytokines balance in children with RRTI [Recent immunological data concluded that functional disorder of Th1/Th2 cells could be strictly related to development of RRTI in children. ith RRTI . Pidotimith RRTI .It is appropriate to mention that Pidotimod effects on decreasing Th2 cytokines against increasing Th1 cytokines would not only enhance the immune system capabilities to fight infections, but it has also a protective role against the development of atopy; pidotimod also was found to down regulate CD30 expression on cells which is linked to Th2 cells .A cohort randomized study held on children with Down syndrome (who are more prone to develop frequent respiratory tract infections), demonstrated that Pidotimod significantly upregulates genes involved in inflammation, chemotaxis and antimicrobial activity. Moreover, the ratio of flu-specific immunoglobulin G1/G3 (IgG1/IgG3) was skewed in Pidotimod treated individuals when it was given in association to seasonal virosomal adjuvanted influenza vaccine .Moreover, increased production of nasopharyngeal and salivary secretory IgA (sIgA) in children with respiratory tract infections treated with Pidotimod has been demonstrated, letting suppose a possible direct effect of pidotimod on B lymphocytes .Another study used nasal cytology to evaluate the ability of pidotimod to reduce nasal inflammation and improve children quality of life through a randomized controlled study involved 40 children with RRTI. Nasal cell motility, mucociliary clearance and the presence of supra nuclear-stria (SNS) were used as an indirect index of wellness of nasal ciliated cells: an improvement was seen in all these cytological features in the pidotimod treated group, with concomitant decreased frequency of respiratory tract infections and antibiotics consumption .The main mechanisms of action of pidotimod, at both innate and adaptive immunity levels, are summarized in Fig. Pidotimod follows a first order pharmacokinetic when administrated through oral route, while parenteral route follows second order pharmacokinetics, with a half-life of 4 h with and an oral bio-availability of 42–44%. It is eliminated through the kidneys without being metabolized. Plasma clearance is 5 l × hE-1 with apparent distribution volume 30 l. Severeal types of oral route of the drug are available  which are all bioequivalent .Pidotimod has a good safety profile without increased frequency of reported side effects or autoimmune disorders in the pidotimod treated patients. Moreover, a study conducted on mice showed that pidotimod has no mutagenic effects either . HoweverAcute respiratory infections (ARIs) are still pandemics despite the introduction of new antibiotics and vaccines which contribute to reduce the risk of mortality and morbidity and they remain widespread and affect both young and elder people . Furtherstreptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus), causing increase in disease severity, mortality and morbidity with progression to unfavoured sequela such as pneumonia [Most common causes of respiratory tract infections have viral origin especially human rhinoviruses (HRV), adenovirus, parainfluenza virus, respiratory syncytial virus (RSV), enterovirus, human metapneumovirus and coronavirus in addition to influenza viruses. , 59. Virneumonia .On the other hand, the host immune system tends to control viral infections via induction of apoptosis of infected cells using different mechanisms including: increasing TNF-alpha secretion, stimulation of natural killer (NK) cells to secrete perforin (making pores in the infected cells resulting in induction of cellular apoptosis) and stimulation of macrophages and neutrophils to produce reactive oxygen species causing oxidation of the infected host cell’s proteins, lipids and DNA leading to its death –67.Carta et al., held a study to assess the ability of pidotimod to induce cellular changes enhancing the host immune system to defend infections. This study has documented that pidotimod is able to upregulate TLR2, with no increase in ICAM or IL8 levels, thus playing a protective role in decreasing susceptibility to HRV infection and from neutrophil-mediated damage to the airway surface [ surface .in vitro study aiming to evaluate the effect of pidotimod on immune system demonstrated that it is able to down-regulate MCP-1, which is a master regulator in inflammatory response associated with severe recurrent viral bronchiolitis in the children [Another children . Moreovechildren .S. pneumoniae infection. Even if the incidence of LRTIs has decreased in the last decade in the pediatric population, LRTIs remain a leading, albeit preventable, cause of death among elderly patients [Lower respiratory tract infections (LRTIs), such as acute bronchitis and pneumonia, represent the most common cause of death from infectious diseases and the fourth overall most common cause of death worldwide. In 2016, it has been estimated that LRTIs have caused 2 million and 370 thousands deaths, among which more than 1 million where due to patients .in vivo immunomodulatory effect of pidotimod during acute community-acquired pneumonia (CAP) [Two different studies were planned to evaluate the ia (CAP) , 71. Oneia (CAP) , 71 Thesp < 0.00001) compared with the conventional treatment. Pidotimod could significantly decrease the duration of cough and fever. The number of patients using antibiotics was also remarkably decreased in the Pidotimod treatment group. Moreover, Pidotimod administration improved the levels of serum immunoglobulin  and T-lymphocyte subtypes . Besides, Pidotimod administration did not increase the risk of adverse events of any cause  [A recently published meta-analysis assessed a total of 29 RCTs consisting of 4,344 paediatric patients. Ten RCTs were published from Italy, Russia or Greece, and 19 RCTs were published by Chinese groups. Nonetheless, appropriate randomization methods were only used in 15 trials. Only one study had explicit allocation concealment. Since only eight RCTs were double-blind and placebo controlled, the evidence was not assessed as high quality. The meta-analysis indicates that treatment with Pidotimod resulted in a significant increase in the proportion of participants who had lower respiratory tract infections  and molecular risk factors. Molecular risk factors for COPD include a plethora of gene polymorphisms, dysregulations of the antioxidants pool, metalloproteinase abnormalities and uncontrolled role of elastase. COPD exacerbations are triggered most often by respiratory viral infections, mainly rhinoviruses, even if bacterial component is present –79. MoreIn terms of immunological response, aside from the inflammation, there is usually a neutrophilic recruitment; sputum eosinophilia can be also relevant, in particular during viral infections .Being the reduction of exacerbation frequency and intensity paramount for the disease control, it appears fundamental to act on the immune system in order to prevent infections and to reach the optimum of care.Due to its role of potentiating and regulating both the innate and acquired immune systems, Pidotimod appears to be promising also in COPD. In two Italian double-blind randomized trials Pidotimod showed significant reduction in infective COPD exacerbations in Pidotimod treated patients , 83. ThiPidotimod was found effective also in a particularly susceptible population: elderly COPD patients; a population patients with severe COPD with different degrees of immunological signs of “immunosenescence” achieved a reduced number of exacerbations by the concomitant use of Pidotimod with flu vaccination compared to those treated with vaccine only .In terms of bacterial exacerbations, another study showed that patients treated with Pidotimod plus amoxicillin/ clavulanic acid, had a faster remission of symptoms compared to those treated with antibiotics only . MoreoveAsthma phenotypes and endotypes based on inflammatory airway involvement have nowadays increasing medical interests thanks to the development of targeted therapies that aim to hamper directly the mechanism of the underlying pathologic processes . At leasPre-clinical evidence are already available: in a study investigating the ability of Pidotimod to affect in vitro the phenotype and cytokine profile of blood cells in relation to atopic asthma showed that it was able to down-regulate the expression of CD30 on mononuclear cells isolated from both atopic asthmatic children and healthy controls . BecauseT2-low asthma endotype patients tend to not react to targeted therapies for pathologic type 2 inflammation, such as glucocorticosteroids. Some, recent studies support the theory that NK cell cytotoxic function is disabled in part by steroids in severe asthma. These NK cells express specialized pro-resolving mediators (SPMs) that are pivotal signals for the resolution of tissue inflammation; nevertheless, SPM abundance or signalling receptors are disrupted in chronic inflammatory disease. SPM receptors and SPMs can in some instances counter the deleterious effects of corticosteroids on the effector function of NK cells. Together, these findings suggest that some severe asthma, patients already refractory to the beneficial actions of corticosteroids, may be harmed by the steroids, increasing susceptibility to viral infection and asthma exacerbations. Thus, leaving an unveiled indication for the use of Pidotimod in these patients once its acts in the prevention of viral infections and asthma exacerbations .In Europe, approximately 23% of the population is affected by allergic rhinitis (AR)  that is Allergen immunotherapy (AIT) is nowadays the only treatment specifically targeting the allergic inflammatory pathways and it represents a prototype of personalized medicine approach to respiratory disease patients . AIT is Adjuvants have the potential to modify the pharmacological and immunological effects of allergen vaccines. They may modulate allergen delivery, act as a depot, stimulate immune responses or limit antibody responses in order to reduce unwanted side effects.Adjuvants may be used in combination with potentially cumulative effects. Therefore, since Pidotimod action pathways include an effect against TSLP and TNF-α action Fig. , it coulA recent study, used Pidotimod in chickens vaccinated with Newcastle disease virus vaccine revealing that Pidotimod could significantly promote growth performance, lymphocyte proliferation, enhance serum antibody titer, CD4/CD8 cell ratios and improve serum IL-2 and IFN-γ concentrations, indicating that it significantly improves the immune efficacy of Newcastle disease vaccine, once again reinforcing the role of immunostimulant that already is well established in previous literature findings .In the last years several immunostimulants of natural or synthetic origins, and working with different mechanisms, have been created for the prevention of RRTIs . ClinicaFurther high-quality, large-scale randomized controlled studies and real-life experiences are essential to provide confirmatory evidence.


Stem cell (SC)-based tissue engineering and regenerative medicine (RM) approaches may provide alternative therapeutic strategies for the rising number of patients suffering from chronic kidney disease. Embryonic SCs and inducible pluripotent SCs are the most frequently used cell types, but autologous patient-derived renal SCs, such as human CD133+CD24+ renal progenitor cells (RPCs), represent a preferable option. RPCs are of interest also for the RM approaches based on the pharmacological encouragement of in situ regeneration by endogenous SCs. An understanding of the biochemical and biophysical factors that influence RPC behavior is essential for improving their applicability. We investigated how the mechanical properties of the substrate modulate RPC behavior in vitro. We employed collagen I-coated hydrogels with variable stiffness to modulate the mechanical environment of RPCs and found that their morphology, proliferation, migration, and differentiation toward the podocyte lineage were highly dependent on mechanical stiffness. Indeed, a stiff matrix induced cell spreading and focal adhesion assembly trough a Rho kinase (ROCK)-mediated mechanism. Similarly, the proliferative and migratory capacity of RPCs increased as stiffness increased and ROCK inhibition, by either Y27632 or antisense LNA-GapmeRs, abolished these effects. The acquisition of podocyte markers was also modulated, in a narrow range, by the elastic modulus and involved ROCK activity. Our findings may aid in 1) the optimization of RPC culture conditions to favor cell expansion or to induce efficient differentiation with important implication for RPC bioprocessing, and in 2) understanding how alterations of the physical properties of the renal tissue associated with diseases could influenced the regenerative response of RPCs. Kidney disease is a worldwide public health problem . The numEncouraging the intrinsic regenerative capacity of the kidney through pharmacological treatments that target populations of renal progenitor cells (RPCs) residing inside the kidney is an alternative RM approach . Indeed,The Pax2+ cells are the murine counterparts of a population of RPCs described and characterized in humans as a hierarchical population of CD133+CD24+ cells, containing parietal epithelial cells and scattered tubular epithelial cells ,17,18. HBoth RPC-based TE and RM approaches would greatly benefit from advances in our understanding of the factors that influence their behavior. Indeed, it is clear that inherent factors, present in the environment of the cell or in cell culture, are critical to maintaining their undifferentiated state, supporting specific RPC fate decisions or shift in phenotype. Recent developments emphasized that physical attributes of the SC niche and of the materials in the cell environment can influence SC behavior with a potency that rivals that of biochemical supplements ,22. In pIn this paper, we explore the impact of substrate stiffness on RPC viability, proliferation, and differentiation and try to identify the mechanisms that enable them to “feel” the stiffness and convert biomechanical stimuli into biochemical signals. We found that the morphology, proliferation and migration of RPCs were highly dependent on mechanical stiffness. Indeed, stiff matrix induced cell spreading and focal adhesion (FA) assembly trough a Rho kinase (ROCK)-mediated mechanism. Similarly, the proliferative and migratory capacity of the cells increased as stiffness increased and ROCK inhibition by antisense LNA-GapmeRs or Y27632 abolished these effects. The capacity of RPCs to differentiate into the podocyte lineage, in terms of acquisition of podocyte markers, was also modulated in a narrow range, by the elastic modulus of the substrate and involved ROCK activity.These results would help (1) to set-up new optimized RPC culture conditions to favor cell expansion or induce efficient differentiation with important implication for RPC bioprocessing in view of their application in TE; (2) to understand how the regenerative response of RPCs could be influenced by alterations of the physical properties of the renal tissue associated with diseases.Human RPCs were obtained and cultured as previously described  in agreeFor Rho-kinase inhibition, RPCs were seeded on the plate and, after 12 h, treated with vehicle (DMSO) or Y27632 (10 µM) .ROCK1 and ROCK2 antisense LNA-GapmeRs were designed by the Qiagen selection tool  and administered to RPCs using Xfect transfection reagent . In detail, 16,000 cells/well were seeded in a 24 well plate and transfected overnight with 1 µM antisense LNA-GapmeRs for ROCK1, ROCK2 or Negative control (scramble). The efficacy of mRNA knockdown was evaluated 48 h after transfection by qRT-PCR. For cell proliferation assessment, we transfected RPCs cultured on the Softwell G 96-Collagen coated plate and evaluated MTT after 48 h. Similarly, immunofluorescence staining for ROCK1 and ROCK2 was performed 48 h after transfection performed on RPCs cultured on Softslip.Cell viability assay was performed employing Calcein-AM  staining following manufacturer’s instructions.For analysis of RPC circularity, aspect ratio and spreading area, RPCs were cultured on Softwell of different stiffness for 7 days. At least 40 cells for each condition were analyzed using ImageJ. Cell spreading and circularity were measured manually tracing the outline of phase images and quantified using ImageJ. Major to minor axis ratio was analyzed using ImageJ. At least 70 cells were analyzed per condition.Podocyte differentiation was obtained as previously described , by cultQuantitative RT-PCR was performed as described  using coConfocal microscopy was performed on RPCs cultured on Softslip by using a Leica SP5 AOBS confocal microscope  equipped with a Chameleon Ultra-II two-photon laser . Cells were fixed for 10 min with paraformaldehyde. The following antibodies were used: Anti-paxillin , anti-nephrin , anti-ROCK1 , anti-ROCK2 . Staining with Alexa Fluor 546 phalloidin  was performed following manufacturer’s instructions. Double immunolabelling was performed as described [The intensity of phalloidin and nephrin stainings was quantified with ImageJ by measuring raw integrated densities of the signal. Background raw integrated densities were subtracted, and this net integrated density was then normalized to the total measured cell area. At least 10–15 cells were analyzed per condition in two independent experiments.RPCs were seeded on the Softslip and incubated 24 h to allow cells to fully spread. The Softslip was then transferred to the microscopy facility and time-lapse recordings were performed using Leica SP5 microscope equipped with a CO2 incubator at 37 °C. Microphotographs of a defined region were taken with a 10× objective at 5 min intervals for 9 h to generate a video. For random motility analysis, at least 20 cells for each conditions were manually tracked for at least 3 h using the ImageJ MtrackJ plugin. Data were used to calculate cell trajectories, average cell speed, and mean square displacement using the DiPer open source program .p-value < 0.05 was considered statistically significant.The results were expressed as mean ± SEM or as median (interquartile range). Comparison between groups was performed by the one-way ANOVA with Tukey or Bonferroni post-hoc test, as appropriate. Statistical analysis was performed using OriginPro statistical software. A The shape of individual cells is based on the balance between external biomechanical forces and internal cellular forces, and the level of internal forces is proportional to the elastic material properties of the surrounding extracellular matrix (ECM) . This suThe shape of cells is a fundamental signal of proliferation, a potent regulator of cell growth and physiology, and is adapted for specific functions ,35. We tCytoskeleton organization and FA formation are notoriously involved in converting mechanical cues into intracellular signals ,37,38, tThese results showed a strong correlation between the mechanical properties of the substrate and actin cytoskeleton reorganization and FA assembly in RPCs.To assess the effect of substrate stiffness on RPC motility, we monitored cells in real time using time-lapse microscopy and analyzed cell movement through the open-source computer program DiPer . FollowiThese results suggest that RPC migration rate is substrate-stiffness sensitive, with the 4 kPa elastic modulus being the cutoff for RPCs to shift from a low to a high migratory behavior.We already demonstrated that RPCs produce ECM components in response to transforming growth factor-β (TGF-β) . We thusDirect effects of matrix physical attributes such as matrix stiffness on SC lineage specification have been demonstrated . It is aWe then assessed the effect of substrate stiffness on the capacity of RPCs to differentiate into podocytes . To thisThese results demonstrate that the capacity of RPCs to differentiate into podocytes is modulated by the elastic modulus of the substrate and that stiffness variations over a limited range strongly influence the cellular response.The activation of ROCK is known to modulate the organization of the actin-based cytoskeletal systems, including the formation of stress fibers and FA. In order to understand the role of ROCK in RPC response to extracellular stiffness, we treated RPCs grown on stiff substrates (12 and 50 kPa) with the ROCK inhibitor Y27632, analyzed actin cytoskeleton organization, and paxillin distribution. Inhibition of ROCK resulted in the disassembly of stress fibers and FA, as evidenced by F-actin disorganization and reduction of paxillin clusters a–c.We next explored the involvement of ROCK activity on the stiffness-mediated increase in RPC proliferation and migration. In the MTT assay, we observed that treatment with Y27632 inhibited the proliferation of RPCs cultured on substrates with higher stiffness, while no effects were observed on RPCs cultured on soft substrates d, indicaSimilar results were obtained analyzing the effect of Y27632 treatment on RPC migratory capacity. Indeed, RPCs cultured on a 50 kPa substrate in presence of Y27632 showed a reduction in their overall migration efficiency, as demonstrated by the significant decrease observed in MSD e.Treatment of RPCs with Y27632 during the differentiation period inhibited the increase of nephrin expression induced by RA of around 80% f, while Y27632 is a non-isoform-selective pharmacological inhibitor of both ROCK1 and ROCK2 and also has non-selective effects. To more selectively target ROCK1 and ROCK2 isoforms and verify their roles on the stiffness mediated effects on RPC physiology, we treated RPCs with antisense-LNA-GapmeRs against ROCK1 or ROCK2. LNA-GapmeRs induced downreguation of ROCK1 and ROCK2 mRNA and protein expression a–c. MoreDiscovering the influence of matrix stiffness on SC behavior has important implication for SC biology and RM. The effects of matrix stiffness have been largely studied for ESCs, MSCs, neural SCs, corneal epithelial SCs, and muscle SCs. In this study, the use of collagen I-coated polyacrylamide hydrogels with variable stiffness indicated that, at least in vitro, the phenotype of human RPCs was highly dependent on the Young’s modulus of the substrate, with stiffer substrates promoting RPC proliferation and migration. Substrate stiffness modulated also the capacity of RPCs to differentiate toward podocytes, with a Young’s modulus of 12 kPa being optimal among those analyzed.Our results demonstrated that actin cytoskeleton reorganization with formation of actin stress fibers, FA assembly and ROCK activation represent important molecular elements underlying the mechanotransduction responses of human RPCs. There are many mechanisms at play at the cell/substrate interface, but the fundamental interaction that all cells must have is a link between the cytoskeleton and the substrate . FollowiTo our knowledge, this is the first paper focused on the effects of substrate mechanical properties on RPCs. Few studies have analyzed these effects on other renal cell types. Beamish et al. studied the in vitro effects of substrate mechanical properties on human renal proximal tubular cells (RPTEC) . In agreHu et al.  and, morWith this study, we demonstrated that substrate stiffness induced dramatic effects on RPC shape and activities. This knowledge could increase our ability to control RPC behavior at least in vitro using not only biochemical supplements, but also inherent material properties and thus help to circumvent cost barriers to efficient RPC expansion and lineage-specific differentiation. Moreover, the results described in this work could have important implications for the design of new renal assist devices, in which the surfaces could be coated or physically modified to encourage cell integration and promoting appropriate RPC responses. Finally, the comprehension of the physical forces that regulates RPC behavior are crucial for the development and optimization of glomerular-on-chip models. In this context, it is of interest that polyacrylamide hydrogels can be designed to simulate the in vivo environment and should be regarded as a promising scaffold material.RM strategies based on activation of endogenous regeneration operated by RPCs would also benefit from the enhanced understanding of their response to extracellular biophysical properties provided in this work. Indeed, in vivo mechanical cues are important in adult tissue homeostasis, where adult SCs require physical interactions with the ECM to maintain their potency and the alteration of tissue stiffness is associated with diverse array of pathologies . TissuesHIV/nl mice (HIV-associated nephropathy model), the glomeruli were significantly softer than normal [HIV/nl mice, glomeruli soften progressively despite an increased collagen deposition. This observation could be in agreement with our demonstration of increased mRNA levels of ECM components in RPCs cultured on softer substrates.Little is known about the biomechanical properties of healthy and diseased kidneys. Using atomic force microscopy, Wyss and colleagues found that the Young’s modulus of healthy rat and mouse glomeruli is ~2.5 kPa, and demonstrated that at an early stage of injury in Col4a3(-/-) mice (Alport model) and Tg26n normal . The oven normal . This wan normal  reportedOur results have potential relevance to understanding the mechanisms of glomerular regeneration operated by RPCs. We can hypothesize that, because of the softening of glomeruli in the early stage of glomerular injury, RPCs assume a more undifferentiated phenotype and start the deposition of matrix components. Increased matrix deposition induces increased stiffness of the tissue that may permit maintenance of structural integrity at the cost of function but allow for subsequent recovery, inducing RPC activation, and increasing RPC proliferation and migration. Indeed, the acquisition of a more migratory phenotype, allowed by the promotion of FA in stiff conditions, is an important feature in wound healing responses. Interestingly, most cells migrate preferentially to stiffer regions via unbalanced forces created by stiffness gradients, i.e., durotaxis. The adhesion of parietal epithelial cells to bare glomerular filtration membrane observed in the early stage of glomerular diseases following podocyte detachment could represent the mechanism by which RPCs localized on the parietal layer of the Bowman’s capsule “sense” the glomerular tuft stiffness. From our data, it is also possible to hypothesize that RPCs signal changes in biophysical cues to other cells within the glomerulus trough thin cytoplasmic bridges. Indeed, we observed in vitro the presence of long protrusions resembling nanotubes on RPCs grown on soft substrates similar to those observed in vivo in a mouse model of unilateral ureteral obstruction using multiphoton microscopy .Interestingly, enhanced expression of paxillin and FA kinase have been reported in glomeruli of rat models of glomerular immune injury and in experimentally induced nephrotic syndrome ,55, whilCreating an optimized microenvironment suitable to support growth and differentiation of regenerative SCs is becoming an area of intense research, in particular for biomaterial-mediated tissue regeneration. Indeed, with the advancement of material science, it is becoming possible to generate active biophysical signals to direct SC fate through specially designed material microstructures. These biophysical signals include material stiffness, tensile strength, micropatterned structure, material surface chemistry, ECM-coated material, and material-transmitted extracellular mechanical force. Future researches could unveil how these material properties can locally control the expansion and direct the differentiation of specific endogenous steSC populations, or serve as inductive regulators for de novo tissue formation, with important implications for in situ tissue engineering approaches.


Syndecans belong to a four-member family of cell surface heparan sulfate proteoglycans (HSPGs) abundantly present in various tissues. They are primarily recognized as extracellular matrix (ECM) receptors able to bind various ECM components and form gradients of morphogens and growth factors. Syndecans are composed of core protein with distinctive cytoplasmic, transmembrane, and extracellular domains to which several HS glycosaminoglycan (GAG) chains are covalently attached. In development of composite organs, such as teeth, expression patterns of syndecans display temporo-spatial shifts between epithelial and mesenchymal tissue compartments. Along with diverse functional properties of syndecans and generally large number of their interactors due to HS GAG chain content, this suggests possible involvement of syndecans in modulation of epithelial-to-mesenchymal crosstalk. Functional versatility of syndecans greatly depends upon the biochemical properties of attached HS GAG chains. These are specifically determined during the HS biosynthesis by the combinatorial action of glycosyl-transferases (Exts/EXTs) and bi-functional sulfotransferases (Ndsts/NDSTs), as well as by post-biosynthetic enzymatic cleavage of HS by the only active endoglucuronidase in mammals, heparanase 1 (Hpse1/HPSE1). Matching the essential requirement for HS during organogenesis, null-mutant animals for genes encoding these enzymes display severe developmental anomalies of mineralized tissues (including teeth) with embryonic or perinatal lethality. In this study, we analyzed expression of syndecan HSPGs , enzymes involved in HS biosynthesis  and HS cleavage (HPSE1) in human tooth germs during the early stages of odontogenesis. All of the investigated factors displayed temporo-spatial differences in expression patterns, and some of them showed distinctive asymmetries of expression domains. Our findings suggest that these factors might be differentially involved in cellular processes which take place during the early odontogenic sequence in humans. Syndecans belong to a small family of cell surface heparan sulfate proteoglycans (HSPGs) and are one of the most abundant cell surface receptors . BiochemIn developing tissues, syndecans are involved in regulation of cellular responses to challenges from microenvironment (ECM dynamics), which determine cell fate, cell shape, proliferative potential and the ability of cells to migrate. This functional versatility of syndecans is intricately associated with biochemical properties of their HS GAG side chains . BiosyntSeveral studies have already indicated that Sdc1 is involved in modulation of multiple cellular processes during the odontogenesis in experimental animals and humans , 2015. Tn = 3), 11/12 (n = 3), and 14 weeks of gestation (n = 4), containing tooth germs in the late bud, cap and early bell stages of development, respectively. Fetal tissue was obtained after spontaneous abortions and tubal pregnancies from the Department of Pathology, University Hospital Split, Split, Croatia. The samples were stored as histological sections at -24oC as a part of fetal human tissue archival collection of the Department of Anatomy, Histology and Embryology . Approval for tissue processing was given by the Ethical and Drug Committee of University Hospital Split  in accordance with Helsinki Declaration  and equalization of signal was firstly performed on positive control images by triple dark point level adjustment using custom made presets in Adobe Photoshop® CS6. After each step, pixel intensity values were measured  at multiple marker points placed in structures known to be negative for staining with primary antibodies (cell nuclei), or DAPI (inter-nuclear spaces). Depending on the tissue type  and structure , background reduction within 0–15 pixels was considered sufficient. Next, the fluorescence leakage reduction was performed by subtraction of counter-signals (red for green fluorescence and green for red fluorescence) with subsequent median signal intensity filtering in ImageJ.Photo-micrographs were shot by SPOT Insight digital camera , mounted on Olympus BX61 fluorescence microscope . Camera settings were set using image acquisition software CellA® CS6) . However, the additional sample unit threshold 8-bit images were made for each investigated factor within the same intensity range (15–95 pixels). The images were processed as described above, with exception that the subtractions of overlapping yellow areas were performed at five pixel increments . Thus, the total number of 204 sample unit threshold 8-bit images were made for each investigated factor  and were subsequently used for statistical analysis of expression domain area distribution.Preparation of merged image doublets used for co-localization analysis, and determination of region of interest (ROI) was done as described previously . AccordiIn order to create 3D surface plots and plot profiles, photo-micrographs were first calibrated to measurement scale in centimeters. Following that, the intensity in pixel values was converted to optical density (OD) units using Kodak No. 3 calibrated step tablet scanned with Epson Expression 1680 professional scanner. The tablet has 21 steps with a density range of 0.05–3.05 OD, however, total of 19 measurements is sufficient in order to obtain conversion curve with the list of corresponding intensity values in pixels and OD (ImageJ). Opposite from pixel value scale, increasing values in OD scale correspond to decreasing intensity. 3D surface plots were used to display the spatial distribution of signals represented by colored spikes corresponding to variations in intensity. Profile plots were used for the comparison of the overall intensity of expression, and to provide general information about the texture of staining patterns. Merged plot profiles with four intensity curves (corresponding to different stages of development and structures of interest) were made for each investigated factor.p < 0.05.Total surface covered by expression domain and the distribution of intensity (both expressed in percentage points) were calculated for individual factors, as well as for two groups of factors (syndecans/HSPGs and HS biosynthesis/degradation) in each ROI  (ImageJ). Thus, the comparative analyses were performed for three stages of development: late bud (7/8 weeks), cap (11/12 w), and early bell stage (14 w). Data distribution was analyzed by Kruskal–Wallis test, followed by Dunn’s Multiple Comparison Test . Statistical significance was set at Figure 1).Tooth germ undergoes several histologically distinctive stages of development during the early odontogenic sequence  in which morphogenesis of enamel organ takes place. In this study, we analyzed expression domains and patterns of syndecans  and enzymes involved in biosynthesis  and cleavage (HPSE1) of HS GAGs in human incisor tooth germs during the late bud , cap , and early bell stage  of development . Similar pattern was observed in the early bell stage, albeit with slightly reduced intensity of staining in dental papilla and dental follicle surrounding the cervical loops. Stellate reticulum, as well the newly differentiated stratum intermedium , were positive to Sdc1 . Inner enamel epithelium, outer enamel epithelium, and cervical loops were negative to Sdc1 staining during the cap and early bell stage. Expression domains of Sdc2 and Sdc4 displayed only partial overlapping with expression domain of Sdc1. However, unlike Sdc1, both factors were expressed on the epithelial side of epithelial-mesenchymal interface  with variable intensity throughout the entire investigated period . In contrast to Sdc2, Sdc4 was expressed in parts of dental follicle adjacent to cervical loops, and expression of both Sdc and Sdc4 was visible throughout the inner enamel epithelium and stratum intermedium in the early bell stage. Intensity correlation analysis, performed on single and double immunofluorescence images , disclosed non-nuclear expression pattern for Sdc1, Sdc2, and Sdc4  . Also, Sdc1 did not co-localize with Sdc2 throughout the investigated period, whereas the co-localization of Sdc1 and Sdc4 was observed only in the dental papilla during the cap stage (data not shown).In the late bud and cap stages, Sdc1 was transiently expressed in the mesenchymal parts of tooth germ . Expression of Sdc1 in the epithelial parts of human tooth germs was almost absent during the bud stage, but was visible in the differentiating stellate reticulum during the cap stage, especially in its distal/posterior portion . The expressions of EXT1 and NDST2 were of comparable intensity, albeit the expression of NDST2 was generally higher during the bud and cap stage. Furthermore, both factors were strongly expressed at the epithelial-mesenchymal interface, in the inner enamel epithelium (stronger EXT1) and cervical loops (stronger NDST2) . Surprisingly, expression domain of NDST1 was extremely limited throughout the investigated period . During the bud stage, the expression domain of NDST1 slightly overlapped with the expression domains of EXT1 and NDST2. It was located in a portion of the tooth bud and in the restricted area of condensing dental mesenchyme underlying the tip of the tooth bud . In fact, the strongest expression of NDST1 was observed in jaw mesenchyme remotely surrounding the tooth germ, as well as in Meckel’s cartilage (mc) during that stage of development (data not shown). In cap and early bell stages, NDST1 was mostly expressed in the inner epithelium and cervical loops. However, the intensity of expression was weaker than that of EXT1 and NDST2. Intensity correlation analysis disclosed non-nuclear expression pattern , except for a slight co-localization with DAPI in cervical loops (EXT1 and NDST2) and inner enamel epithelium   in the early bell stage. This was attributed to the extreme cell nuclei density of the inner enamel epithelium. Intensity correlation analysis performed at maximum magnification (×100) confirmed cytoplasmic expression pattern of all three factors (data not shown). As expected, color scatterplots for double immunofluorescence with Sdc1  disclosed no co-localization, and thus reconfirmed the cytoplasmic expression pattern of EXT1, NDST1, and NDST2, since HS biosynthesis enzymes normally reside within the ER and Golgi apparatus . The most intense staining was observed in the cervical loops during the cap stage, and in small sections of stellate reticulum adjacent to the cervical loops and outer enamel epithelium . In the early bell stage, expression of HPSE1 in the cervical loops was less intense and limited to their tips. Furthermore, the expression of HPSE1 in the area of future cusp tip was visible in the inner enamel epithelium and overlaying stratum intermedium. In this area, the expression of HPSE1 was clearly demarcated on one side, whereas on the other side it displayed gradual decrease in intensity toward the cervical loop . However, throughout the entire investigated period, epithelial aspect of the epithelial-mesenchymal interface between the enamel organ and underlying dental papilla was positive to HPSE1. Intensity correlation analysis disclosed non-nuclear expression pattern , except for a slight co-localization in cervical loops and the inner enamel epithelium in the early bell stage. No co-localization with DAPI was found when the intensity correlation analysis was performed on maximum magnification (×100). In contrast to EXT1, NDST1, and NDST2, color scatterplots for double immunofluorescence with Sdc1 disclosed co-localization during the cap stage, and in cervical loops during the early bell stage . Taken together, this means that HPSE1 displays both cytoplasmic and intercellular expression patterns in developing human tooth germ. Serial mergers of four HPSE1 photo-micrographs  with Sdc1 photomicrograph  disclosed the co-localization patterns at low intensity of HPSE1 expression. Co-localization pattern was particularly visible during the cap stage in a wide area of dental papilla arching toward the distal/posterior part of the enamel organ. Thus, co-localization of Sdc1 and HPSE1 expression domains displayed somewhat asymmetric distribution .Heparanase 1 was mostly expressed in the epithelial parts of human tooth germ, apart from its ubiquitous expression during the cap stage . These may be attributable to the involvement of investigated factors in highly dynamic cellular processes during the progression of morphogenesis of enamel organ. Among syndecans, the expression of Sdc1 continuously displayed the greatest range in both the intensity and spatial distribution. In contrast to Sdc2, the intensity of Sdc1 expression slightly decreased in intensity as the tooth germ development progressed and odontogenic tissues began to differentiate. Sdc4 displayed relatively balanced intensity profile. Intensity profiles of EXT1, NDST1, and NDST2 disclosed that the HS biosynthesis is differentially regulated in a stage-specific manner. While the expression of EXT1 and NDST2 displayed well-matched profiles, that of NDST1 indicated more fluctuations in intensity and limited spatial distribution. HS biosynthesis enzymes operate in physical complexes, whose composition greatly determines the structure of HS . By comparison of these data with relatively balanced intensity of Sdc4 profile displayed throughout the investigated period, the upregulation of Sdc4 in cervical loop detected by quantitative analysis seems to be mostly due to the increased size of Sdc4 expression domain, and much less due to variation in intensity range of its expression .Quantitative analysis of expression domain and intensity distribution disclosed statistically significant differences only for Sdc4 between late bud and early bell stage , as well as in the early bell stage between the cervical loop and inner enamel epithelium   knockout mice even though Hpse1/HPSE1 is the only known active endo-β-glucuronidase in mammals found to be ubiquitously expressed during development . Therefore, while these enzymes perform similar functions in mice and humans due to their homology, the redundant mechanisms covering for their loss-of-function might significantly differ between the two species.Differential expression of syndecans and enzymes involved in HS biosynthesis and degradation during human odontogenesis must be viewed within the context of a vast regulatory network whose operating and redundant mechanisms are far from being understood . Althougesponse) . In cont to date . No discelopment . The absout mice . Loss-ofout mice . It is iDue to ethical considerations and scarcity of specimens, research of early human development is by default methodologically limited and descriptive. Therefore, based on results presented in this study, it is not possible to fully assess the functional importance of investigated factors for normal development of human tooth germs. Most of what we know about molecular regulation of odontogenesis comes from studies of developing mouse molars. However, inter-species differences in dental formula, duration of odontogenic sequence and morphology of fully developed teeth, do suggest that more or less subtle differences in molecular regulation of odontogenesis in different species should be reflected on molecular level. For that matter, the present and future studies of involvement of syndecans and functionally related factors in cellular processes during human tooth development are not without the intrinsic value, because they might also provide some clues into how these differences are established.DK designed the study, devised and selected image analysis methods, performed selection of primary antibodies and control immunofluorescence staining, processed and interpreted data, reviewed the literature, and wrote the manuscript. TSB performed and supervised immunofluorescence staining, acquired and interpreted data , wrote parts of the manuscript , reviewed part of the literature, and proof-read the manuscript. LLA performed immunofluorescence staining, assisted in acquisition of data and initial photo-micrograph processing, reviewed part of the literature, and proof-read the manuscript. KV supervised immunofluorescence staining, reviewed part of the literature, interpreted data , and proof-read the manuscript. MS-B provided theoretical background for the study design and revised and co-edited the manuscript with DK.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.


Climate change represents one of the most significant threats to human and wildlife communities on the planet. Populations at range margins or transitions between biomes can be particularly instructive for observing changes in biological communities that may be driven by climate change. Avian communities in lowland boreal habitats in the Adirondack Park, located at the North American boreal-temperate ecotone, have been the focus of long-term monitoring efforts since 2007. By documenting long-term changes in community structure and composition, such datasets provide an opportunity to understand how boreal species are responding differently to climate change, and which habitat characteristics may be best able to retain boreal avian communities. We examined three specific questions in order to address how well current biological communities in Adirondack boreal wetland habitats are being maintained in a changing climate: (1) how do trends in occupancy vary across species, and what guilds or characteristics are associated with increasing or decreasing occupancy? (2) how is avian community composition changing differently across sites, and (3) what distinguishes sites which are retaining boreal birds to a higher degree than other sites? Our analysis revealed that (1) boreal species appear to exhibit the largest changes in occupancy among our study locations as compared to the larger avian community, (2) dynamics of community change are not uniform across sites and habitat structure may play an important role in driving observed changes, and (3) the particular characteristics of large open peatlands may allow them to serve as refugia for boreal species in the context of climate change. Climate change is among the most significant threats to ecological communities worldwide and can lead to profound changes to the structure of biological communities . DocumenPicoides dorsalis), boreal chickadee (Poecile hudsonicus), Canada jay (Perisoreus canadensis), Lincoln’s sparrow (Melospiza lincolnii), palm warbler , olive-sided flycatcher (Contopus cooperi), rusty blackbird (Euphagus carolinus), and yellow-bellied flycatcher (Empidonax flaviventris). The habitats of these boreal specialists are thought to be particularly vulnerable to climate change [Populations at range margins or transitions between biomes can be particularly instructive for observing changes in biological communities that may be driven by climate change ,10. The e change ,13 and de change ,15.Lowland boreal habitats in the Adirondack Park consist of open peatlands, conifer dominated forested peatlands, and neighboring conifer dominated upland communities. These habitats are patchily distributed and landscape characteristics such as size, connectedness, structure, and anthropogenic disturbance to wetland habitats appear to interact with climate to influence dynamics of birds in these locations. Because they tend to be cooler than surrounding landscapes , peatlanWe have found that boreal habitat specialists in these communities are in decline and appear sensitive to changing climate . Boreal Climate change does not operate uniformly across landscapes. There is high variability in temperature and precipitation characteristics across our study site locations in the Adirondack Park, NY and some sites may be better suited to harbor species over the long term than others. Climate refugia are defined as areas relatively buffered from contemporary climate change over time such that important physical, ecological, and social characteristics are able to persist . One pos2 located in the northern part of New York State in the US . The predominant habitat type in the park is Northern Hardwood and Conifer Forest, followed by Boreal Upland Forest and Northern Swamp [Picea mariana) and tamarack (Larix laricina), ericaceous shrubs such as leatherleaf  and Labrador tea (Ledum groenlandicum), herbaceous plants such as Sarracenia purpurea (pitcher plant) and sedges , and Sphagnum mosses.Our study occurred in the Adirondack Park, an area of 19,700 kmrn Swamp ,22. Thourn Swamp . This stAs described in , an initWe conducted unlimited distance point counts to assess presence/absence of passerines and woodpeckers along transects of 5 points spaced at least 250m apart within boreal wetland habitats . We emplt+1/ψt and obtained a rate of change in occupancy for the study period by calculating the geometric mean of individual growth rates (λt) between all years [To identify the characteristics of individual species whose occupancy is changing more or less rapidly in boreal habitats (Question 1), we first used the multi-season model implemented in program Presence  to calcull years . A totalIn order to identify potential mechanisms underlying individual species’ changes, we used analysis of variance and regression to examine a set of species characteristics for their association with trend parameters. We tested 9 categorical and 5 continuous species characteristics including (1) boreal, (2) southern, (3) feeding guild, (4) foraging guild, (5) nesting location, (6) primary habitat, (7) migratory strategy, (8) winter geography, (9) single- or multiple-brooded, (10) mean clutch size, (11) mean body weight, (12) mean latitude of New York State distribution, (13) estimated NY population, and (14) mean arrival date .Catharus ustulatus), blackpoll warbler (Setophaga striata)) but are more commonly associated with high elevation conifer communities and uncommon in the habitats sampled here.Both “boreal” and “southern” are categorical descriptions of species distributions. Boreal species are those which have been the specific targets of long-term monitoring in boreal habitats in the Adirondacks  and are Southern species were those defined as having a latitudinal center of their distribution that is south of the southern boundary of the Adirondack Park. We used the mean latitude and longitude of Breeding Bird Atlas blocks in which each species occurred within New York State during the most recent atlas  to identInformation related to feeding , foraging , nesting , primary habitat , migratory strategy , winter geography , number of broods , clutch size, and body weight was obtained from a variety of sources ,32,33,34The questions of concern in the individual species analysis were: (1) which species are exhibiting patterns of increasing or declining occupancy in boreal wetlands (2) are there commonalities among species exhibiting particular patterns of change, i.e., to what degree to guild types explain changes among individual species?The purpose of the second part of our analysis was to examine the process of avian community change individually at each study site and to determine whether observed patterns of change differ among these sites. In contrast to the first part of our analysis in which we examined individual species occupancy patterns and associated guilds/characteristics, here we model each site independently and examine changes in species richness, such that it is possible for processes of avian community structural change to vary across sites.MacKenzie et al.  describeWe took this approach to investigate characteristics of change among bird guilds. For each of our individual study locations, we ran two model sets to examine increases (colonization) and decreases (extinction) in relative species richness for the decadal period of 2007–2016, and used covariates to determine how changes in relative species richness were driven by particular species characteristics (as described previously under Question 1). Specifically, for each individual location, we accounted for detection and, using the same set of factors explored above , restricted each model to a single covariate placed on either colonization or extinction . With coThe purpose of the final part of our analysis was to determine what distinguishes those locations which are retaining boreal birds to a higher degree than others and therefore may be refugia for these species. The primary aim of our long-term monitoring has been to identify trends and drivers of change for a suite of boreal birds in Adirondack peatlands, a group for which declining patterns of occupancy have been documented in 6 of 8 tracked species . As suchOur initial aim was to examine individual species trends across sites and to identify the characteristics of individual species whose occupancy is changing more or less rapidly in boreal habitats. Across all species , we identified 35 species which had λ > 1 and 22 with λ < 1 . A growtPiranga olivacea), American goldfinch (Spinus tristis), and least flycatcher (Empidonax minimus). Among those with highest λ were pine warbler (Setophaga pinus), pileated woodpecker (Drycopus pileatus), Eastern bluebird , and Swainson’s thrush  and decreases (extinction) in relative species richness for 2007–2016, and used covariates to determine how changes in relative species richness were driven by species characteristics. Combining information from results of model selection at all sites led to a number of general patterns. We found that (1) the process of community change among these habitats is highly variable, with no one factor most critical in driving increases or decreases in species richness across all sites, and (2) for most groups, loss of species richness appears more likely than gain .Several characteristics appear to be important to driving patterns of increasing or decreasing species richness among our study locations . Across With respect to extinction, rates were highest for open land birds and lowest for conifer forest species, and, by nest location, extinction was highest for shrub nesting birds, followed by cavity, tree, and ground nesters. Combining information from both dynamic rates, it appears that conifer species and commensal/generalists are among the most likely to colonize boreal wetland habitats and among least likely to leave them, while birds of open lands are the least likely to colonize and most likely to abandon these habitats. Though birds with distributions centered to the south of the Adirondacks were important in changing community structure within our study sites, they remain less likely to colonize these habitats than they are to leave them . Among nNoncategorical species characteristics such as clutch size also influenced colonization and extinction rates, but results for most continuous variables were mixed across our sites. Among these variables, mean body weight occurred most often in top models for colonization or extinction (n = 22 sites), followed by NY population (n = 18), latitude (n = 10), mean clutch size (n = 8), and arrival date (n = 5). In all cases, the influence of these characteristics on changes in species richness was mixed among sites, exerting a positive influence on colonization or extinction in some sites and a negative influence in others . The mosWe utilized the third component of our analysis to attempt to discern factors associated with decreasing representation of the boreal guild, and to determine the characteristics of sites that may be serving as refugia for these species. The primary focus of long-term monitoring in these habitats  has beenWe found that several factors were important in distinguishing sites characterized by high (mean = 0.73), medium (mean = 0.39), and low (mean = 0.11) levels of extinction . We founThe purpose of our work was to explore temporal changes in avian community composition in lowland conifer habitats with the aim of understanding the characteristics of both species and individual sites that are associated with high and low degrees of change, and to determine how these characteristics may help to identify refugia for boreal species. We asked, specifically, (1) how do trends in occupancy vary across species, and what guilds or characteristics are associated with increasing or decreasing occupancy (2) how is avian community composition changing differently across sites, and (3) what distinguishes sites which are retaining boreal birds to a higher degree than other sites.Empidonax alnorum), which we found to have a positive trend, least flycatcher (Empidonax minimus), which had a negative trend, and great-crested flycatcher (Myiarchus crinitus), for which insufficient data precluded trend assessment. The decline of aerial insectivores has been highlighted in several regions [We found, on the whole, a greater proportion of species increasing in occupancy over time than those that exhibited patterns of decline. Increases and decreases among individual species mimic those that have been found in other locations, but in general the only strong pattern observed was a loss of boreal bird species that have been the focus of long-term monitoring. Work in other high latitude temperate and boreal systems has similarly detected declines among species associated with northern systems. Niemi et al. , working regions ,47, and  regions , neonico regions , and cli regions  have all regions  lands inWe explored changes in avian communities in boreal wetlands by modeling changes in relative species richness within each of our study locations independently. We found that the process of community change was not uniform across sites and that different factors appear to operate at different locations in structuring community change. Broadly, however, it appears that habitat affinity plays an important role in shaping which birds are colonizing and which are leaving these habitats.th century [Results suggest that conifer-associated species and commensal/generalist species are most likely to be increasing as a proportion of the bird community, while open land birds are more transient in low elevation boreal wetlands. These patterns may reflect broad-scale abundance changes documented in other studies. Both commensal species and forest birds are among the species with the most robust populations and least conservation concern based on North American population trends . Forest  century ,55. Incr century ,57, with century , climate century . The obs century , as well century . Biotic  century .Migration strategy, nesting location, and southern vs northern distribution were also important characteristics linked to long-term changes in relative species richness in boreal wetlands. Because they are present on territories year round, resident birds are often theorized to be less sensitive to and more capable of tracking changing conditions over time , and incIn concert, our findings with respect to the first two questions point toward potential long-term structural change. Though the majority of individual species were determined to have relatively stable occupancy trends (Question 1), with respect to changes in species richness in these communities, most guilds exhibited higher extinction rates than colonization rates (Question 2), suggesting potential overall declines in species richness over time. It is possible that, although most individual bird species are not exhibiting large changes in occupancy, some of the more specialized members of the community are declining  and these communities as a whole are shifting toward a smaller number of more common species.We found that extinction rates of boreal birds were lowest in sites with the largest amounts of Northern Peatland and lowest amounts of Boreal Upland Forest. Among our study sites, high amounts of Northern Peatland are associated with large wetland sites; some of the largest boreal wetlands in our system are those dominated by Northern Peatland habitat types such as Boreal Laurentian Bog and Boreal Laurentian-Acadian Acidic Basin Fen . They arWe also found that sites with low extinction rates for boreal birds were characterized by lower deviation in winter precipitation from normal conditions. Separate analyses of these data have found that boreal birds are sensitive to current temperature and precipitation patterns and that winter precipitation characteristics influenced the persistence of resident boreal bird species including black-backed woodpecker, boreal chickadee, and Canada jay . VariabiIt is important to understand the degree to which changing avian species composition overall may also be contributing to the loss of boreal birds. We found sites with high extinction rates for the boreal bird guild had significantly higher colonization rates of both southern and commensal species than did sites with low or medium extinction rates. Increases among both groups represent possible responses to climate change and additional pressures on boreal birds in the form of competition for resources. Climate change may explain increasing southern bird occurrence  and is sOur analysis revealed that (1) boreal species may be exhibiting the largest changes in occupancy among our study locations as compared to the larger avian community, (2) processes of community change are not uniform across sites but habitat structure may play an important role in driving observed changes, and (3) particular characteristics may allow some locations  to serve as refugia for boreal species in the context of climate change. The largest open peatlands in the Adirondacks are generally well protected, but the network of smaller, more isolated boreal habitats exists among a variety of ownerships and land use types. Because boreal habitats in the Adirondacks are islands within a landscape dominated by temperate forest types, and because boreal birds within them may behave as metapopulations , protectS1 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Methane-producing microbial communities are of ecological and biotechnological interest. Syntrophic interactions among sulfate reducers and aceto/hydrogenotrophic and obligate hydrogenotrophic methanogens form a key component of these communities, yet, the impact of these different syntrophic routes on methane production and their stability against sulfate availability are not well understood. Here, we construct model synthetic communities using a sulfate reducer and two types of methanogens representing different methanogenesis routes. We find that tri-cultures with both routes increase methane production by almost twofold compared to co-cultures and are stable in the absence of sulfate. With increasing sulfate, system stability and productivity decreases and does so faster in communities with aceto/hydrogenotrophic methanogens despite the continued presence of acetate. We show that this is due to a shift in the metabolism of these methanogens towards co-utilization of hydrogen with acetate. These findings indicate the important role of hydrogen dynamics in the stability and productivity of syntrophic communities. InteracTowards deciphering ecological and evolutionary drivers of function and functional stability in microbial communities, methanogenic anaerobic digestion (AD) offers an ideal model system, where the production of methane from complex organic substrates can be taken as a proxy for a community function. AD communities are found in many environments including ocean and lake sediments, soil and animal guts and are used in biotechnological re-valuation of organic waste . It is w2 and acetate, which can be used by aceto- and hydrogenotrophic methanogens prod. need then be combined with the observed residual levels of these compounds in the system, to estimate the levels that were available to Mb ([Acetate]Mb and [H2]Mb):The estimated [Acetate]4]calc) by Mb, through reactions 1, 2 and 6. The actual amounts of acetate used in reactions 2 and 6, as well as the actual amounts of H2 used in reactions 1 and 6, are unknown. If we assume a full conversion through the three reactions, we would have the following stoichiometric balances:xi and yi denote the amounts of acetate and H2 used in reaction i, respectively. These three equalities can then be re-arranged to yield the overall theoretical methane production.We can now use these values to calculate the estimated stoichiometric, theoretical methane production ([CH


The aim of this study was to review aspects of the pathobiology of the meniscus in health and disease and show how degeneration of the meniscus can contribute to deleterious changes in other knee joint components. The menisci, distinctive semilunar weight bearing fibrocartilages, provide knee joint stability, co-ordinating functional contributions from articular cartilage, ligaments/tendons, synovium, subchondral bone and infra-patellar fat pad during knee joint articulation. The meniscus contains metabolically active cell populations responsive to growth factors, chemokines and inflammatory cytokines such as interleukin-1 and tumour necrosis factor-alpha, resulting in the synthesis of matrix metalloproteases and A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS)-4 and 5 which can degrade structural glycoproteins and proteoglycans leading to function-limiting changes in meniscal and other knee joint tissues. Such degradative changes are hall-marks of osteoarthritis (OA). No drugs are currently approved that change the natural course of OA and translate to long-term, clinically relevant benefits. For any pharmaceutical therapeutic intervention in OA to be effective, disease modifying drugs will have to be developed which actively modulate the many different cell types present in the knee to provide a global therapeutic. Many individual and combinatorial approaches are being developed to treat or replace degenerate menisci using 3D printing, bioscaffolds and hydrogel delivery systems for therapeutic drugs, growth factors and replacement progenitor cell populations recognising the central role the menisci play in knee joint health. Osteoarthritis is responsible for a large and rapidly increasing global disease burden that is challenging health-care systems worldwide. The need for improved and rapid processes to develop new therapies, is obvious. Disease progression in osteoathritis, is marked by increasing pain and loss of joint function, however the development of osteoarthritis is usually slow, taking many years from its first mild symptoms to severe debilitating end-stage disease. Although progression to this end-state occurs in only a minority of patients with osteoarthritis and disease in many patients remains stable for many years, each year osteoarthritis led to ~160,000 total joint replacements in England and Wales and 492,000 in the USA in 2017 [The semilunar meniscal fibrocartilaginous menisci provide joint congruity to the curved weight bearing surfaces of the articular cartilages and collectively these structures provide weight bearing properties to the human knee joint  Figure . The menThe β-defensins are 2–6 kDa cationic peptides which bind to meniscal proteoglycans and have anti-bacterial, ant-fungal and anti-viral activities which have protective roles to play in human meniscal tissues. β-Defensins are significantly upregulated during the inflammatory conditions which are generated in the knee-joint during OA and act in concert with DAMP (damage associated molecular pattern) and PAMP (pathogen associated molecular pattern) receptors such as the Toll-like and NOD-like receptors of the innate immune system which identify lipopolysaccharide and peptidoglycan components on cell membranes of invading organisms ,20 and tMeniscal biology in health and disease ,3,4,5 anSeveral prospective biomarkers have been correlated with various criteria of meniscal pathology including synovial fluid HMGB (high mobility group box)-1 levels ,37, inflProteomics approaches have identified meniscal markers associated with disease pathology ,47,48,49Gene profiling has established that traumatic and degenerative meniscal tears have different expression profiles  and thatThe underlying molecular changes underlying progression of OA are incompletely understood however some transcriptomic studies are now providing some insightful information into this disease process. Gene profiling of articular cartilage from knees with meniscal tears are distinctly different from knees with OA cartilage lesions but relatively intact menisci , the preGene expression studies in cartilage of surgically induced OA in wild-type (WT) mice and Adamts5Δcat mice, in which ADAMTS-5 activity is ablated and thus aggrecan loss and cartilage erosion is inhibited has been undertaken to distinguish gene expression changes that are independent of ADAMTS-5 activity and cartilage breakdown. Gene expression in meniscal cartilage from the developing lesion in the destabilized medial meniscus and corresponding regions in sham-operated joints were compared using whole-genome microarrays . PreviouAQP1, COL1A1 and CLEC3B and an increased distribution of these proteins confirmed in OA cartilage using immunohistochemistry [Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes has been used to identify cartilage genes and pathways involved in OA . Examinahemistry . Integrahemistry . The grohemistry . Histolohemistry .AQP1), tetranectin (CLEC3B) and COL1A1 [As already noted, three genes have been identified strongly associated with OA cartilage, aquaporin-1  for tissue repair became evident in 2003  when aduMany reviews have emphasized the great potential of MSCs for meniscal repair applications ,43,45,71The meniscus is a tissue which has very significant physical demands placed on it within the knee joint to protect and stabilise other knee joint tissues which provide knee joint articulation and weight bearing. Artificial scaffolds developed for partial or total meniscal replacement still do not totally reproduce the material properties of native meniscal tissues and further developments will be required to perfect this technology ,113,114.® (2-octyl cyanoacrylate), fibrin glue for pulmonary leaks, and the recently developed TissuGlu® (an FDA approved urethane-based adhesive) for abdominoplasty surgery [Notaden benetii  is a skin exudate secreted by the female frog during mating and ensures extended sexual union by the male for efficient egg fertilization. This frog glue [Knowledge of biological glues and tissue adhesives for the attachment of meniscal flaps or tears have undergone significant improvements in recent years. Tissue adhesives currently used in surgical applications, reviewed in ,105 incl surgery ,117. Tis surgery  and barn surgery  to marinrog glue ,121 provrog glue ,123. Thirog glue ,127,128.Moderate physical exercise has been examined as a means of positively regulating beneficial chondroprotective and anti-inflammatory genes in human knee joint tissues as a means of therapeutic intervention during inflammatory osteoarthritic conditions in the knee-joint . Such anThe menisci are major cartilaginous structures in the human knee with essential roles in the stabilization and weight bearing of this structure. A healthy meniscus is therefore a high priority to ensure the health of other knee joint tissues. A realization of the inter-dependance of all knee joint tissues to provide an efficient articulatory and weight bearing structure indicates that for any therapeutic measure to meet with success in the treatment of OA or RA it must be beneficial to all of the varied cell populations in the human knee. The large number of therapeutic measures being developed to specifically halt further degeneration, stimulate regeneration or replace meniscal tissues points to the central importance of the meniscus to knee joint function. Without a therapeutic measure effectively addressing all these parameters in OA there is little likelihood of a successful outcome in this most debilitating of conditions. Moderate exercise has been shown to be beneficial to knee joint tissues and dietary adjustment to include virgin olive oil has further beneficial effects in terms of a lowering of knee joint inflammation. With knee OA predicted to become a leading musculoskeletal condition within the next two decades there is an extreme need for effective measures to be developed and all avenues of therapy need careful evaluation.


Recent and rapidly developing movements relating to the increasing awareness and reports of gender bias, discrimination, and abuse have reached the academic environments. The consideration that negative attitudes toward women and abuse of power creates a hostile environment for female scientists, facilitating sexual harassment and driving women out of science, can be easily related to. Rationally inaccessible gender biases are not only evident at the level of the researchers, but are also paralleled by a corresponding imbalance at the level of the research subjects. Here, we focus on the maternal immune activation (MIA) animal model to illustrate exemplarily the current state of ex-/inclusion of female research subjects and the consideration of sex as biological variable in the basic neurosciences. We demonstrate a strong sex disparity with a major emphasis on male animals in studies examining behavioral and neurochemical alterations in MIA offspring. We put forward the hypothesis that this neglect of female subjects in basic research may stem from a hard-wired sex/gender bias, which may also be reflected in a similar attitude toward female scientists. We suggest exploring the possibility that by dismantling sex bias and male dominance in basic research one would get an additional handle on favorably modifying the perception and appreciation for women in science. The publishing situation itself may be influenced by gender biases during the review process3. In addition, subtle and unconscious acts of discrimination during the recruitment process5 are also in play, preventing women from becoming laboratory heads and reaching positions, which would naturally allow them to feature as the prominent “last” or “senior” author on scientific publications.Despite decade-long efforts, including officially declared intentions and a variety of initiatives for enhancing gender equality, gender biases in scientific environments still exist today and powerfully impact diverse facets of research and academic life and career. The dramatic underrepresentation of female scientists in positions of power and status, in academic and private institutions, is mirroring the gender mismatch in authorships on scientific papers9. It has been concluded that in academia cases of sexual harassment are more frequent than in most other areas of public life10. Consequently, several scientific societies and funding agencies have published declarations concerning their policies toward sexual harassment and issued statements regarding ensuing consequences within the framework of the respective infrastructures. The consideration that negative attitudes toward women and abuse of authority and power creates a hostile environment for female scientists, on the one hand facilitating sexual harassment and abuse, on the other hand driving women out of science can be easily related to.Over the past months the rapidly developing #Metoo movement has reached the academic environments and several prominent cases have unfolded since11 still so vulnerable to making biased decisions when it comes to the evaluation of female scientists and scientific results produced and/or presented by a woman? The answer can be found at the very core of the issue itself: Gender bias, like other forms of prejudices and unintentional discrimination, is not subject to conscious reflection and decision making but rather determined by automated emotional processes, subtly bypassing rational and critical internal reviewing structures14.Facing this situation in 2019 begs the question why even in science, where the ability to most objectively and rationally evaluate data is a core principle and prerequisite for true achievements and progress, such dramatic biases, nurtured by emotionality and subjective believes, persist so tenaciously. Why are some of the people who describe themselves as being fair and objectivehttps://grants.nih.gov/grants/guide/notice-files/NOT-OD-15-102.html). Basic research, however, is lagging behind, as it is still heavily concentrated on male model animals, largely ignoring and/ or not addressing sex as possible variable in scientific data sets. It is more than obvious that preclinical work, which may eventually inform translational research and clinical research questions and therapeutic developments, needs to strive for the inclusion of aspects of sex and sex-dependent effects and functions if both women and men are considered as final beneficiaries of biomedical research.These deeply rooted and rationally inaccessible biases are not only evident at the level of the researchers but penetrate into and are paralleled by a corresponding imbalance at the level of the research subjects. Sex and gender differences in drug metabolism, reactions, and side effects are well-described, generally accepted and have led to official requirements to integrate men and women in all study populations comprising human subjects  population remaining to be further determined.The misrepresentation of female subjects in basic research can be “historically” explained, in part founded on incorrect perceptions, such as that experiments conducted in female animals are inherently more variable than those relying on males18. In the current article, we are focusing on the experimental animal maternal immune activation (MIA) model to exemplarily illustrate the current state of sex-/inclusion of female research subjects and the consideration of sex as biological variable in basic (neuroscience) research. We summarize, integrate, and discuss MIA literature for the time period of 2000–2018. Focusing on three levels of phenotypic characterization of MIA offspring , we demonstrate a strong sex disparity with a major emphasis on male animals in the available studies and reflect on consequences and future perspectives.To date, most preclinical biomedical research has been conducted with inadequate consideration of the sex of the experimental subjects studied26 diagnoses. Recently, the range of psychiatric disturbances associated with gestational infections has been extended to include an increased risk for offspring bipolar disorder, major depression, epilepsy, and cerebral palsy28. It is considered that rather than damage being induced by the infectious agent itself, maternal infection disrupts the delicate immune balance between the maternal and fetal environments. Hereby, the milieu for the developing fetal nervous system is altered paving the way for the occurrence of aberrant brain structures and functions, which form the basis for an augmented risk for offspring mental disorders23. Several animal models of MIA based upon the administration of immunogenic substances to the pregnant female have been developed in search for the pathophysiological mechanisms underlying the detrimental effects of gestational infection.The MIA model of neuropsychiatric disorders was founded based upon a series of epidemiological studies, which had demonstrated an association between maternal infection during pregnancy and offspring autism spectrum disorder or schizophrenia29 and used live human influenza virus to induce MIA in mice30. Although directly employing a pathogenic virus has the advantage that a full spectrum of naturally occurring immune response can be elicited, the field has since moved toward mainly using Poly (I:C) (polyinosinic-polycytidylic acid) and LPS (lipopolysaccharide) to induce MIA in laboratory animals. These substances, which mimic viral and bacterial infection, respectively27 induce a limited, but well-defined immune response allowing for a precise control over the intensity and timing of MIA as critical determinants for offspring phenotype33. In addition, variations in other experimental parameters including the experimental animal’s strain and age, housing conditions, and other environmental variables contribute to the heterogeneity of MIA paradigms and the effects observed36. For the sake of enhancing transparency and reproducibility and to minimize bias in the MIA mouse model, the request to standardize and meticulously report on the applied MIA model has been recently been put forward, including the integration of major experimental variables at the side of the offspring37. Here, the age at which the animals are being examined, the brain region analyzed and, particularly, offspring sex is considered as significant modulatory impact. Sex-dependent time points of vulnerability and resilience and sex-specific responses to prenatal stress are known to shape an individual’s response to stress over the lifetime and determine the risk for the development of mental illness22. These examples illustrate a general principle, which demands the inclusion of sex as a biological variable (SABV) in biomedical research as prerequisite for improving our understanding of disease mechanisms and the development of preventive and therapeutic approaches.Pioneering studies were designed based upon the reported association between prenatal influenza infection and a diagnosis with schizophrenia in the adult lifen = 733) and ScienceDirect (n = 676) limited to the time period between 01 January 2000 to 01 September 2018. After removal of duplicates, the total number of records was n = 1003. Eight hundred and twenty-eight manuscripts ) were screened and included in the subsequent analysis. Of these articles, only manuscripts using mammals as experimental animals and MIA induction based upon LPS and/or Poly (I:C) injection, IL-6, Escherichia coli, stress-induced stimulation or influenza virus infection, n = 432 were considered. Studies focused only on the pregnant mothers, placental studies only, preterm labor, effect of infection on abortion rates/fetus absorption, mother treated with ethanol and/or high/low-fat diet, trial studies on human (women and/or child) were not included (n = 396). The PRISMA flow diagram was used in order to summarize the search and study selection processes  using PubMed . Effective 25 January 2016, the NIH implemented a policy aimed at integrating sex and gender science as an integral component of methodological rigor and reporting in health research (NOT-OD-15-005 https://grants.nih.gov/grants/guide/notice-files/not-od-15-005.html), which expects scientists to account for the possible role of SABV in vertebrate animal and human studies. Moreover, on 9 June 2018, the Human Subjects System replaced the Inclusion Management System for reporting participant sex/gender, race, and ethnicity information (NOT-OD-18-179 https://grants.nih.gov/grants/guide/notice-files/NOT-OD-18-179.html). Nonetheless, single sex studies of males still largely predominate in the MIA literature during the 2016–2018 period increasing to ~ 43%  analysis of male and female data separately (M/F); (ii) inclusion of male and female data with an analysis by sex (M + F); (iii) merging of male and female data indicating that no sex effects had been found without showing the respective analysis (M + F comb) and (iv) studies using both male and female subjects without indicating whether an analysis by sex had been conducted (M + F NR). We found that the MF group is composed of 37.1% of M/F, ~ 17% of M + F and a 14% studies with both M/F and M + F analysis, followed by a 10% of M + F comb and 21.7% of M + F NR Fig. . Of note43% Fig. , whereas38. As depicted in Supplemental Fig. n = 12). However, given the overall small number of manuscripts included herein it is important to unmistakably emphasize that this first glance on the potential relevance of author gender for the choice of sex of the experimental subjects used, is not meant to and cannot provide any definitive answer on this matter. Larger-scale, specifically designed studies with different and dedicated analytical instruments will be needed in order to properly address this question.In order to examine whether there could be a tentative association between the gender of the senior (lead) author and the sex of the experimental animal in the MIA studies, authors' gender on all studies included in the present analysis was inferred based upon first name, published photos and other text references as previously described46. The tradition of performing behavioral experiments mainly in male rodents can be related to the early discovery of estrous-linked changes in locomotor activity in rats47, which alerted experimenters to hormonally programmed changes in females as possible confounds of non-reproductive traits. However, few systematic studies have directly compared the variability in behavioral studies conducted in male versus female rodents. A meta-analysis of 40 inbred strains of mice concluded that the assumption that female mice exhibit more variable response than males is not substantiated by experimental evidence15. A recent review supported this notion stating that “the belief that non-human female mammals are intrinsically more variable than males and too troublesome for routine inclusion in research protocols is without foundation” and further reported a particular sex bias in neuroscience research where studies relying exclusively on male animals surpassed those using females 5.5–149.Offspring of mothers experiencing gestational immune activation have been repeatedly demonstrated to feature behavioral impairments related to several neuropsychiatric disorders. The behavioral phenotypes revealed include abnormal social behaviors, repetitive behaviors, depression-like, and anxiety-like behaviors as well as memory deficits51 albeit apparent gender-dependent distribution patterns in the prevalence of several psychiatric disorders, including those whose symptoms are being modeled in MIA offspring , whereas only 3.4% had conducted studies solely in female MIA offspring. Of the ~ 38.1% of MF studies, the M/F group comprised 35.6%, the M + F group ~ 20% and the combination of both ~ 17%, mirroring the overall trend  injected mothers but not their female littermates, exhibited abnormally persistent latent inhibition and slower reversal compared with controls. Reversely, only female offspring exhibited increased immobility and decreased saccharine preference in the forced swimming test and saccharine preference test. Interestingly, employing a different MIA protocol anxiety- and depression-related behavior were also exclusively observed in adult male offspring56. On the other hand, no effect of gestational Poly(I:C) challenge on prepulse inhibition (PPI) was found in male rat offspring exclusively in one study57, whereas others reported no effect in either sex58. Interestingly, in the LPS MIA model a PPI deficit was observed in male offspring only31.Indeed, the influence of sex on brain function health and disease is underexplored in neuroscience researchend Fig. . However59. Serotonin and dopaminergic signaling is altered in offspring across different MIA models29. In addition, specific changes in inhibitory neurotransmission have been linked to both schizophrenia and autism spectrum disorders60 and similar reductions in several components of the GABAergic system have been repeatedly identified in the brain of MIA offspring62.MIA offspring exhibit neurochemical changes that are characteristic of several neuropsychiatric disorders63) > 43% of MIA studies examining neurotransmitter changes focused on male offspring only and 30% were MF studies. Of these, 37% were M/F and 10% only M + F studies, including a direct statistical analysis of SABV .Here we found that despite well-established sex differences in sex hormones and their interactions with neurotransmitter systems , the increase in workload and costs that comes with doubling animal number by including both sexes etc.However, what if this sex bias in basic research stems from a more deeply rooted, hard-wired, consciously inaccessible, and logically uncontrollable neglect of the relevance of female research subjects? What if this sex bias was also reflected in a similar attitude toward female scientists and women in society in general? This hypothesis can neither be experimentally falsified nor verified to date. However, do the lean numbers of women in leadership positions (in science) and their modest increases despite great efforts, together with the tip of the iceberg of sexual misconduct and assault revealed by the #Metoo movement, not suffice to explore the possibility that by dismantling sex bias and male dominance in basic research we would get an additional handle on favorably modifying the perception and appreciation for women in society? Would equity not provide a short-cut to excellence, if the full potential of ~ 50% of people could add an additional/ different source of intellectual insight and execution of leadership tasks?How long would it take to observe measurable changes?Evolutionary biology holds an answer to this question: the rate of change is largely dependent on the environmental pressure.Supplemental Figure 1Supplemental Figure 2Supplemental Figure LegendsSupplemental Table 1Supplemental Table 2Supplemental Table 3