Thirty years ago Cairns and Potten first noted that intestinal epithelial cells fail to produce carcinomas at a rate proportionate to the number of divisions they undergo throughout their lifetimes ,2. This 3]-thymidine incorporation into the DNA of developing mice showed that label-retaining cells persist within intestinal crypts and hair follicles over an extended period of time [An extension of this hypothesis holds that somatic stem cells retain thymidine analog labels due to slow division rates or asymmetric segregation of DNA . Prelimi of time . Followi of time –20.Nevertheless, the immortal DNA strand hypothesis remains a subject of contention, proving difficult to confirm or refute. This controversy is due in part to ill-defined or heterogeneous stem cell populations and the complexity associated with the use of thymidine analog labeling to track DNA segregation in metazoans. More importantly, few studies to date have established the fate of both parent and daughter cells following asymmetric division.Thymidine analog labeling relies on the assumption that immortal DNA templates are labeled during symmetric stem cell divisions and that subsequent divisions, in which the labeled template is retained, will be asymmetric. However, stem cells may undergo symmetric, asymmetric, or a combination of symmetric and asymmetric divisions during development and adulthood. Therefore, the time frame of label delivery is critical for proper experimental design, and thymidine analog labeling requires precise knowledge of the time during development or regeneration when stem cells switch from symmetric to asymmetric divisions. Untimely administration or withdrawal of a label will result in its failed incorporation into template DNA strands or premature dilution, confounding experimental analysis.To date, both growth and injury models have been employed in an attempt to overcome these obstacles. Growth models rely on the administration of a label at some time point during development, when the majority of stem cells are thought to be undergoing symmetric divisions. In contrast, injury models utilize physical or chemical means to induce a proliferative state in which symmetric stem cell divisions can occur. Recently, it has been suggested that the toxicity associated with 5-bromo-2′-deoxyuridine (BrdU) and other thymidine analogs is sufficient to induce the degree of injury required to stimulate symmetric stem cell divisions and label incorporation . HoweverProliferative markers like Ki67, phospho-histone H3, and proliferating cell nuclear antigen (PCNA) have been used in conjunction with stem cell markers to ensure continuous cell division, although one caveat to this strategy is the lack of appropriate markers for many stem cell populations . With thin vitro using live video microscopy, they will remain problematic for in vivo studies where division times may remain rough estimates. A low number of chromosomes in the model organism compounds this problem [Recent evidence also indicates that chromosome number, as well as the number of divisions prior to analysis, can influence experimental outcomes . Success problem ,21. Althin situ hybridization (CO-FISH) has made this possible [Some chromosomes may be distributed randomly, necessitating chromosomal resolution of DNA to verify asymmetric segregation . Recentlpossible ,24. Origpossible  used CO-possible  provided− HSCs outperformed CD34+ cells in serial transplantation and competitive reconstitution assays.In addition to the limitations of thymidine analog labeling, ill-defined stem cell populations may include a mix of stem and progenitor cells, resulting in a failure to identify the appropriate subset of cells undergoing non-random chromosome distribution. Even in the hematopoietic compartment, where stem cells are defined by numerous surface antigens, additional characterization has led to new evidence in favor of asymmetric chromatid segregation ,8. AlthoStudies have often failed to provide direct evidence that parent and daughter cells adopt distinct fates following asymmetric DNA segregation. Drosophila germ line stem cells (GSCs) and their progeny can be identified by established markers and their unique anatomical position. As GSCs undergo mitosis, one daughter remains attached to a fragment of somatic tissue called the hub, maintaining its identity as a stem cell, while its sister differentiates and migrates outward . ExploitWhether stem cells distribute their DNA asymmetrically will most likely depend on the cell population, tissue type, and the period of development under examination. It seems unlikely that chromatid segregation will follow a universal pattern. However, recent developments have exposed the limitations of current techniques and highlighted the need for appropriate experimental design. It may be premature to draw conclusions about the asymmetric segregation of DNA for even the most well characterized stem cell systems without identification of an appropriate subpopulation through the use of suitable markers and functional tests.Based on the limitations of thymidine analog labeling outlined above, and data generated from previous studies, we suggest that future analysis of somatic stem cell chromosome segregation should strive to meet the following criteria:The label must be shown to be present throughout the course of the chase.The label must be incorporated into the “immortal” template DNA strand.Characterization of label retaining stem cells must be rigorously established through the use of appropriate markers or functional tests.The fate of both parent and daughter cells must be determined after asymmetric division.in vivo or in vitro and growth should not exceed two or three divisions.The cell cycle time must be determined Labeled cells should be shown to continually divide during the chase period.The toxicity of the label should not affect the cell cycle.Both growth and injury models should be utilized when possible.The evidence in support or opposition of asymmetric DNA segregation is mixed. However, the biological advantage this distribution may impart to the parent cell remains uncertain. Retention of DNA could prevent the accumulation of mutations as initially suggested, but it may also play a critical role in maintaining stem cell quiescence and identity through epigenetic memory . Future


Despite significant progress in recent years in characterizing the response properties of neurons in the auditory cortex, the exact role that auditory cortical activity plays in sound processing remains highly debated. One hypothesized function is to improve the detection, discrimination or categorization of sounds that are behaviorally salient. We explore this possibility in a mouse model using species-specific ultrasonic calls. Single unit cortical recordings were taken in awake, head-restrained virgin and mother mice during playback of a variety of conspecific pup and adult calls. Previously , we had We used the Van Rossum and Victora-Purpura distance metrics to quantify differences between the “collapsed” spike trains  evoked by different calls. We noticed that the magnitude of these distances were often dominated by overall evoked spike rate, which complicated comparisons across units with different degrees of call responsiveness. Hence, to obtain a clearer picture of changes between animal groups regardless of overall responsiveness (which was variable), we created for the distance between any pair of calls, a normalized spike distance between their “collapsed” spike trains. Specifically, the usual distance was divided by the average of the distance between spontaneous spike trains and those evoked by each call, thus quantifying average responsiveness to calls in terms of evoked spike train distances from silence.Our results continue to support the idea that the contrast in neural population encoding for these calls across core auditory cortical fields has improved. As in the case of evoked inhibition, the most pronounced changes arise in the lateral-band areas not normally tuned for the ultrasonic frequencies of the calls studied See Fig. . Taken t


Learning to choose adaptively between different behavioral options in order to reach goals is a pervasive task in life for people of all ages. Individuals are often confronted with complex, uncertain situations that nonetheless require decisive actions that would facilitate the pursuit of short-term or long-term goals. Adaptive decision making as such entails interactions between processes that monitor the choice-outcome relations as well as processes that evaluate these relations with respect to goal relevance. These dynamics implicate close interplays between attention, learning, memory, motivation, and emotion, which are subserved by cortical-subcortical networks and are neurochemically regulated by transmitters, such as norepinephrine, dopamine, and serotonin. Across the life span, these functional brain circuits as well as neurotransmitter systems undergo basic biological maturation and senescence as well as plasticity due to the accumulation of experience or changes in motivational goals (Braver and Barch, Multidisciplinary research on decision making and aging is growing at a rapid pace (Brown and Ridderinkhof, Here we feature empirical work ranging from behavioral (Mather and Schoeke, Despite stereotypes of old age as a life period characterized by global cognitive declines, there are many decision-related processes that remain intact or might even be enhanced in older adults. In this Research Topic, Mather and Schoeke  and CavaThe perspective piece by Mata et al.  in this Given the evidence for the adaptability of choice in old age, these findings might suggest that it would be beneficial to provide decision strategies to older adults in scenarios where they would be most vulnerable to making mistakes (Samanez-Larkin et al., Two of the papers in this series examine age differences in discounting of temporal delays (Samanez-Larkin et al., Older adults are not always more willing to wait. Shivapour et al.  in this Overall the series of papers identifies areas of potential improvement, preservation, and decline in decision-related processes across adulthood. A potentially interesting area that we did not cover in this series but one that is gaining recent attention is the examination of differential effects of genetic variability on decision making across the life span (Hämmerer et al.,


In this review, some of the challenges and opportunities encountered during protein comparability assessments are summarized with an emphasis on developing new analytical approaches to better monitor higher-order protein structures. Several case studies are presented using high throughput biophysical methods to collect protein physical stability data as function of temperature, agitation, ionic strength and/or solution pH. These large data sets were then used to construct empirical phase diagrams (EPDs), radar charts, and comparative signature diagrams (CSDs) for data visualization and structural comparisons between the different proteins. Protein samples with different sizes, post-translational modifications, and inherent stability are presented: acidic fibroblast growth factor (FGF-1) mutants, different glycoforms of an IgG1 mAb prepared by deglycosylation, as well as comparisons of different formulations of an IgG1 mAb and granulocyte colony stimulating factor (GCSF). Using this approach, differences in structural integrity and conformational stability profiles were detected under stress conditions that could not be resolved by using the same techniques under ambient conditions . Thus, an evaluation of conformational stability differences may serve as an effective surrogate to monitor differences in higher-order structure between protein samples. These case studies are discussed in the context of potential utility in protein comparability studies. Protein based drugs are structurally complex, highly specific macromolecules used therapeutically to compensate for body deficiencies , as medical treatments  as well as to prevent diseases   or in the finished drug product   and the final drug product  is typically the first step of a comparability evaluation. Peptide mapping is typically the initial step, usually used to assess the integrity of the amino acid residues and sequence within a specific protein molecule. This technique involves the chemical or enzymatic treatment of the protein to generate a specific set of peptide fragments, which in turn are separated using reversed phase high performance liquid chromatography (RP-HPLC) equipped with diode-array ultraviolet (UV) for detection. Isolated peptide fragments are also mass characterized using online mass spectrometry. Peptide mapping has proven to be an especially valuable technique in comparability studies, since each protein upon digestion with specific proteolytic enzymes will give specific peptide fragments that are used as a high resolution fingerprint for the protein under evaluation  such as N-glycosylation, O-glycosylation and/or C-terminal lysine clipping, are frequently observed upon changing the cell-culture manufacturing processes. This is partially responsible for the heterogeneous nature of pharmaceutical proteins, since they are usually produced by cells in culture  in a protein produced from both pre- and post-change manufacturing processes is essential since the overall three dimensional configuration not only defines a protein's stability and functionality, but also its efficacy and safety when being used as a therapeutic. Differences in higher order structures could stem from physical changes in structural integrity  as well as from modifications in either the side chains  and/or PTMs such as glycosylation. These differences in higher order structure (HOS) are sometimes only detected during long-term storage or accelerated storage which may involve changes to the surrounding environment . In addition to the properties of the protein molecule itself, pharmaceutical additives , solution conditions (such as pH and ionic strength) as well as the nature of the final dosage form (liquid vs. lyophilized) can also impact the HOS of a protein.The assessment of higher order structural integrity and conformational stability of proteins in comparability studies is best performed using multiple biophysical and calorimetric techniques. Far-ultraviolet circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) have been widely used to analyze the overall secondary structure content of proteins. These methods produce distinctive signals that correspond to the different secondary structural folds . Detailed studies regarding the use of CD spectra for structural comparisons in comparability studies have been published recently  and surface plasmon resonance (SPR). Another group of bioassays, that directly measures biological functionality, are often also used with protein drugs including in vitro cell proliferation and in vivo animal models. Using functionality bioassays (if feasible) in comparability studies is typically preferred over simple antigen binding assays because some degraded, modified protein molecules could still bind non-specifically to the target molecule in a simple binding assay. Protein molecules containing multiple regions with different functionalities should have multiple functionality and/or binding bioassays reflecting each and every structural region. For example, monoclonal antibodies are composed of two antigen binding sites (Fab), responsible for antigen binding, and one crystallizable region (Fc), responsible for immune effector functions. Each of these regions may need to be tested for its ability to bind to its specified target to perform its desired function.Techniques used to characterize the biological activity of a protein serve as a critical complement to physiochemical assays in confirming the correct higher order structure of a protein. Determining bioactivity for monoclonal antibody based protein drugs is usually done using a variety of Case studies examining protein functional activity changes due to changes in manufacturing process have been reported. An oxidized form of the protein filgratism (a human granulocyte-colony stimulating factor used primarily for patients with severe neutropenia) was found to lose 75% of its potency, whereas, filgratism dimer had 67% potency compared to the intact molecule  of protein degradation. Accelerated stability studies measure the rate of given degradation process over time at various temperatures in specific formulations and primary containers. Evaluating the effect of storage period, excipients and environmental stress on the accelerated (and long term) stability of protein drug products is an essential part of formulation development and comparability evaluations. Protein drugs encounter different environmental stresses during production, storage, shipment and patient administration. Thus, accelerated stability studies and forced degradation studies are needed to determine the stability profile of a protein drug by subjecting the protein to various stresses including elevated or changing temperatures, freezing, thawing, agitation, oxidative environment, light, and the presence of different interfaces and pH changes.  studies may need to be evaluated, in the form of either non-clinical or clinical studies, in which the pharmacokinetic profile of the protein drug is evaluated using animal models (rodents or primates) or in humans, respectively. In cases involving an observed non-similarity between the products under comparison, it may be necessary to perform pharmacokinetic studies in human subjects to demonstrate comparability  was observed after a 20% reduction in sialic acid content of the glycan attached to the protein  have been demonstrated to have no effect on the pharmacokinetic profile  in addition to the dose and route of administration are all considered extrinsic factors that may be related to the immunogenicity of a protein based drug  , the magnitude and type of manufacturing changes, and the analytical tools available to monitor the structural integrity of a protein. An in-depth understanding of the protein including its inherent susceptibility to the different chemical and physical degradation mechanisms, combined with accumulated clinical and manufacturing experience with a product, is essential for a successful comparability study. Accurate structural determination for protein-based drugs is a difficult task for the pharmaceutical and biotechnological industry, in contrast to small molecule drugs, because of their complex three-dimensional structures, sensitivity to their environment and their inherent microheterogeneity. Major advances have been made in developing analytical tools for primary structure analysis, ranging from chromatographic  and electrophoretic (capillary isoelectric focusing and capillary sodium dodecyl sulfate) separation methods that are typically linked with mass spectrometry detection  to accurately characterize a protein's primary structure including any post-translational modifications.H2 domain of the mAb was seen upon using thiocyanate (as an anion) and arginine (as an excipient) that was correlated with decreased thermal stability of the CH2 domain and an increase in aggregation propensity. These results demonstrate the intricate interrelationships between different excipients and their effect on protein dynamics and physiochemical stability of a monoclonal antibody. Recent advances in both H/DX-MS , the use of higher resolution analytical techniques such as NMR, X-ray crystallography, cryo EM and H/D exchange mass spectrometry that provide structural details about a protein's folded structure and its dynamic behavior remains limited as part of analytical comparability studies. Although these techniques are very powerful in terms of obtaining detailed information about a folded protein's structure, their use has been restricted due to some drawbacks including protein size and formulation composition (presence of excipients) in addition to being technically challenging, expensive and time consuming techniques. Research is currently being performed to overcome these drawbacks. For example, the effect of different anions and excipients on an IgG1 mAb local flexibility was evaluated using H/DX-MS . Additionally, the evaluation of conformational stability differences may not only be an effective surrogate to monitor differences in higher-order structure between protein samples, but also a useful complement to traditional accelerated stability and forced degradation studies often used in analytical comparability studies as described above.Although lower resolution biophysical techniques such as CD, intrinsic/extrinsic fluorescence spectroscopy, DSC and static light scattering measurements are commonly used to monitor the structural stability of protein-based drugs, no single technique provides sufficient information to establish the higher order structural integrity of complex macromolecules such as proteins. Therefore, the use of more than one technique is generally needed for better characterization. The multi-dimensional nature of such analysis makes collection, analysis and visualization of larger data sets desirable. Historically, data analysis of protein conformational stability data was performed by either visual inspection or data fitting of thermal unfolding curves to sigmoidal functions of results from individual instruments. These types of approaches can result in data interpretation that is not only subjective, but limited in terms of their scope and utility.Tm/Tonset values to determine differences between the different formulations under comparison. This makes data interpretation across multiple samples difficult and potentially highly subjective. Using bovine granulocyte colony stimulating factor (bGSCF) as a model protein combined with second derivative absorbance spectroscopy as a model biophysical method, the physical stability of the protein was characterized across multiple pH and temperature conditions, and the first EPD was constructed  and n represents the experimental conditions . After data normalization, singular value decomposition (SVD) of the m × n experimental data matrix is calculated to extract three orthogonal basis vectors associated with the three largest singular values. These results are then mapped to red-green-blue (RGB) color scheme that can be visualized as a function of the stress conditions . After an EPD is generated, what remains is the interpretation of the change of colors of the different areas of the EPD. The colors of the different areas within an EPD have no physical meaning. Instead, the change in color signifies a structural change in the protein as detected by the data from the individual experimental techniques. A scientist must refer to the original experimental biophysical data and consider the physical process that generated regions with different colors for further interpretation of the molecular origin of the color change.An EPD analysis involves the collection of protein physical stability data under different experimental conditions using multiple low-resolution biophysical techniques. The stability data from the experimental conditions and techniques are then entered into The EPD method employs SVD and linear algebra to extract distinctive patterns from a large collection of data. The EPD method requires a number of steps as described in Figure EPDs have been extensively used in formulation development and stabilization studies with a wide variety of protein based drugs and vaccines as reviewed in detail elsewhere . This three-index EPD method can be extended to visualize data from any three biophysical techniques, if a proper color legend is provided.Second, Radar charts and Chernoff face diagrams have been utilized as color-free data visualization tools. They use certain icons such as polygon axes and facial expressions, respectively, to display large data sets under given conditions .Finally, comparative signature diagrams (CSDs) , an IgG1 monoclonal antibody, and granulocyte colony stimulating factor (GCSF) were characterized using various biophysical methods as a function of temperature and solution pH. The large physical stability data sets obtained were combined into various types of diagrams for data visualization  was monitored as a function of temperature to reflect the fluorescence signals from both Trp and Try residues. The intensity at 305 nm was primarily due to Try residues whereas 330 nm corresponds to Trp residues. Because a single Trp residue is quenched in the native conformation of FGF-1, higher values of I305/I330 indicate native like structure whereas low values reflect an altered conformation. Extrinsic fluorescence measurements using 1-Anilino-8-naphthalnesulfonate (ANS) were also performed to monitor the exposure of apolar sites as an indicator of conformational alteration in the protein. ANS fluorescence intensity was measured at 480 nm as a function of temperature. Aggregation behavior of the protein at different pH values was evaluated by measuring static light scattering at 280 nm as a function of temperature.Both WT and mutant FGF-1 proteins were characterized as a function of solution pH (3–8) and temperature (10–90°C) using various biophysical techniques. Far-ultraviolet circular dichroism (far-UV CD) was used to characterize secondary (and tertiary) structural changes in the WT and mutant FGF-1 at each pH value. The CD signal at 228 nm, presumably reflecting a signal from a combination of β-turns, loops and aromatic side chains, was monitored as a function of temperature from 10 to 90°C. Intrinsic fluorescence measurements were carried out to monitor tertiary structural changes. FGF-1 contains eight Tyr and one Trp and the latter is quenched in the native state. Samples were excited at 280 nm and the ratio of fluorescence intensity at 305 and 330 nm  was used to measure soluble aggregates, resonance mass measurements (RMM) to measure sub-micron particles (~0.3–2 μm) and MFI to measure sub-visible particles (2–70 μm). These “multiple-instrument radar charts” were prepared for the heat and stirring induced aggregation of the mAb under varying salt concentrations and the mechanistic basis and kinetics of aggregates/particles formation was evaluated. Such types of data presentation further diversify the applicability of the radar chart based data visualization technique in comprehensive characterization of different sizes of aggregates/particles in various formulations under a variety of stress conditions.This case study illustrates the capability of using radar charts to summarize and compare large data sets from MFI analysis of particle size distribution, concentration, and morphology across different protein formulations in a single figure. This work also demonstrates the potential utility of radar chart analysis to compare the profiles of aggregate and particle formation of varying size ranges and morphologies as measured by a variety of analytical methods, across different protein samples in analytical comparability assessments.The analysis of protein higher-order structure is an essential part of analytical comparability assessments of biopharmaceutical drug candidates. Although there are a number of higher resolution analytical tools potentially available to examine and compare the higher-order structure of protein molecules, many of these techniques have practical limitations in their routine use including technical complexities, low-throughput, high costs and difficult interpretability. Additionally, the size and concentration of a protein as well as the presence of various additives in the formulation (which can interfere with some of these assays) further restrict their use. While lower resolution biophysical techniques are more readily available to analyze various structural aspects of protein molecules in pharmaceutical formulations, such methods may be unable to detect minor structural alterations in protein molecules even if they have significant implications for their functionality. In this review, we have illustrated that the biophysical characterization of various protein molecules , by using a combination of lower resolution, high-throughput techniques combined with various data visualization tools, can detect physical stability differences in proteins with minor structural/conformational changes. The case studies of FGF-1 mutants and differentially glycosylated IgG-1 mAbs demonstrated the application of this approach to compare the effect of relatively minor modifications in protein structure on their conformational stability. The GCSF case study illustrated a data visualization technique to statistically compare the structural/conformational stability differences measured with the same protein in different formulations. The final case study presented a radar chart data visualization technique to more comprehensively characterize protein aggregate and particle formation as generated by two different agitation stresses applied to IgG1 mAb solutions containing varying levels of sodium chloride. In conclusion, characterization of the physical stability of proteins using various lower resolution, higher throughput biophysical techniques, combined with different environmental stresses and data visualization tools, can potentially as serve as a useful tool to evaluate the higher-order structural integrity of proteins for comparability purposes.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.


Intravenous injection of therapeutics is required to effectively treat or cure metastatic cancer, certain cardiovascular diseases, and other acquired or inherited diseases. Using this route of delivery allows potential uptake in all disease targets that are accessed by the bloodstream. However, normal tissues and organs also have the potential for uptake of therapeutic agents. Therefore, investigators have used targeted delivery to attempt delivery solely to the target cells; however, use of ligands on the surface of delivery vehicles to target specific cell surface receptors is not sufficient to avoid nonspecific uptake. PEGylation has been used for decades to try to avoid nonspecific uptake but suffers from many problems known as “The PEGylation Dilemma.” We have solved this dilemma by replacing PEGylation with reversible masking using low-molecular-weight neutral lipids in order to achieve optimal-targeted delivery solely to target cells. Our paper will focus on this topic. We use bilamellar-invaginated vesicles (BIVs) that are unique liposomal nanoparticles (NPs) providing highly efficient delivery for intravenous (iv) injection of encapsulated therapeutics including plasmid DNA –5. In adMuch research has been directed toward the synthesis of new cationic lipids. Some new formulations enable more efficient transfection of cells in culture. However, their efficiency measured in vitro did not correlate with their ability to deliver DNA after administration in animals. Functional properties defined in vitro do not assess the stability of the complexes in plasma or their pharmacokinetics and biodistribution, all of which are essential for optimal activity in vivo. Colloidal properties of the complexes, in addition to the physicochemical properties of their component lipids, also determine these parameters. In particular, in addition to efficient transfection of target cells, nucleic acid-liposome complexes must be able to traverse tight barriers in vivo and penetrate throughout the target tissue to produce efficacy for the treatment of disease, that is, countercurrent to increased intratumoral pressure gradients for the treatment of cancer. These are not issues for achieving efficient transfection of cells in culture with the exception of polarized tissue culture cells. Therefore, we are not surprised that optimized liposomal delivery vehicles for use in vivo may be different than those used for efficient delivery to some cells in culture. In summary, in vivo nucleic acid-liposome complexes that produce efficacy in animal models of disease have extended half-life in the circulation, are stable in serum, have broad biodistribution that can be focused, efficiently encapsulate various sizes of nucleic acids, are targetable to specific organs and cell types, penetrate across tight barriers in several organs, penetrate evenly throughout the target tissue, are optimized for nucleic acid : lipid ratio and colloidal suspension in vivo, can be size fractionated to produce a homogenous population of complexes prior to injection, and can be repeatedly administered. Recently, we demonstrated efficacy of a robust liposomal delivery system in small and large animal models for lung , breast Efficient in vivo nucleic acid-liposome complexes have unique features including their morphology, mechanisms for crossing the cell membrane and entry into the nucleus, ability to be targeted for delivery to specific cell surface receptors, and ability to penetrate across tight barriers and throughout target tissues. Liposomes have different morphologies based upon their composition and the formulation method. Furthermore, the morphology of complexes can contribute to their ability to deliver nucleic acids in vivo. Formulations frequently used for the delivery of nucleic acids are lamellar structures including small unilamellar vesicles (SUVs), multilamellar vesicles (MLVs), or the bilamellar invaginated vesicles (BIVs) recently developed in our laboratory Some investigators have loaded nucleic acids into SUVs using a variety of methods; however, the bulk of the DNA does not load or stay within the liposomes. Furthermore, most of the processes used for loading nucleic acids within liposomes are extremely time-consuming and not cost-effective. Therefore, SUVs are not the ideal liposomes for creating nonviral vehicles for targeted delivery.Complexes made using MLVs appear as “Swiss rolls” when viewing cross-sections by cryo-electron microscopy . These cUsing a formulation developed in our laboratory, nucleic acids are efficiently encapsulated between two bilamellar invaginated vesicles, BIVs . We creaBIVs condense unusually large amounts of nucleic acids of any size α dioleoyl phosphatidylethanolamine (DOPE) as a neutral lipid creates liposomes that cannot wrap or encapsulate nucleic acids. Several investigators have reported efficient transfection of cells in culture using DOPE in liposomal formulations. However, our data showed that formulations consisting of DOPE were not efficient for producing gene expression in vivo [Choosing the best cationic lipids and neutral lipids are also essential for producing the optimal in vivo formulation. For example, using our novel manual extrusion procedure does not produce BIVs using the cationic lipid dimethyldioctadecylammonium bromide (DDAB). Furthermore, DOTAP is biodegradable, whereas DDAB is not biodegradable. Use of biodegradable lipids is preferred for use in humans. Furthermore, only DOTAP and not DDAB containing liposomes produced highly efficient gene expression in vivo . DDAB di in vivo .Investigators must also consider the source and lot variability of certain lipids purchased from companies. For example, different lots of natural cholesterol from the same vendor can vary dramatically and will affect the formulation of liposomes. We use synthetic cholesterol instead of natural cholesterol that is purified from the wool of sheep. Synthetic cholesterol is required by the Food and Drug Administration for use in producing therapeutics for injection into humans.Our BIV formulations are also stable for a few years as liquid suspensions. Freeze-dried formulations can also be made that are stable indefinitely even at room temperature. Stability of liposomes and liposomal complexes is also essential particularly for the commercial development of human therapeutics. A common belief is that artificial vehicles must be 100 nm or smaller to be effective for systemic delivery. However, this belief is most likely true only for large, inflexible delivery vehicles. Blood cells are several microns (up to 7000 nm) in size and yet have no difficulty circulating in the blood including through the smallest capillaries. However, sickle cell blood cells, that are rigid, do have problems in the circulation. Therefore, we believe that flexibility is a more important issue than small size. In fact, BIV DNA-liposome complexes in the size range of 200 to 450 nm produced the highest levels of gene expression in all tissues after iv injection [We believe that colloidal properties of nucleic acid-liposome complexes also determine the levels of gene expression produced after in vivo delivery , 34. TheOur in vivo transfection data showed that an adequate amount of colloids in suspension was required to produce efficient gene expression in all tissues examined . The colA primary goal for efficient in vivo delivery is to achieve extravasation into and penetration throughout the target organ/tissue ideally by minimally invasive systemic administration. Without these events, therapeutic efficacy is highly compromised for any treatment including gene and drug therapies. Achieving this goal is difficult due to the many tight barriers that exist in animals and people. Furthermore, many of these barriers become tighter in the transition from neonates to becoming adults. Penetration throughout an entire tumor is further hindered due to the increased interstitial pressure within most tumors –37. We bOur delivery system is efficient because we have optimized the overall charge of complexes to produce the highest delivery into cells, that is approximately 45.5 mV measured by a zeta potential analyzer . Our comHowever, the positive charge on the surface of delivery vehicles also results in uptake in nontarget cells as well. Therefore, the charge must be shielded briefly until the complexes arrive at the target cell. As stated above, we believe that maintenance of adequate positive charge on the surface of complexes is essential to drive cell entry by direct fusion. Therefore, we created a methodology to achieve targeted delivery of our complexes in vivo without the use of PEG. These ligand-coated BIV complexes reexpose the overall positive charge of the complexes as they approach the target cells. In addition, through covalent attachments, we have added small molecules to the surface of our preformed complexes that mimic protein-protein interactions . These sMuch effort has been made to specifically deliver nucleic acid-liposome complexes to target organs, tissues, and/or cells. Ligands that bind to cell surface receptors are usually attached to PEG and then attached to the cationic or anionic delivery vehicle. Due to shielding of the positive charge of cationic complexes by constitutively incorporated PEG, delivery to the specific cell surface receptor can be accomplished by only a small fraction of complexes injected systemically. Furthermore, delivery of PEGylated complexes into the cell occurs predominantly through the endocytic pathway, and subsequent degradation of the bulk of the nucleic acid occurs in the lysosomes. Thus, gene expression is generally lower in the target cell than when using the nonspecific delivery of highly efficient cationic complexes. Recent efforts to use cleavable PEG are unimpressive and have not solved these problems  10, 12–, 32.As discussed above, the vast majority of the injected PEGylated complexes bypass the target cell, including those using cleavable PEG. Apparently, the PEGylated complexes cannot utilize critical charge interactions for optimal transfection into cells by direct fusion due to the overall low or neutral charge. The inability to expose positive charge on the surface of optimized delivery vehicles results in the transfection of fewer cells. PEGylation was first used to increase the half-life of complexes in the circulation and to avoid uptake in the lung. However, this technology also destroys the ability to efficiently transfect cells. We were able to increase the half-life in circulation of BIVs to five hours without the use of PEG. Because the extended half-life of BIVs is not too long, this delivery system does not result in the accumulation of complexes in nontarget tissues that occurs with circulation half-lives of one to three days as seen with PEGylated liposomal delivery systems. Some investigators have now reported targeted delivery that produces increased gene expression in the target cell over their nontargeted complexes. However, these nontargeted and targeted delivery systems are inefficient  comparedn-dodecyl-β-D-maltopyranoside, approximately 511 MW, just prior to iv injections [In using the extruded BIV DOTAP : Chol nucleic acid : liposome complexes, we produced an optimal half-life in the circulation without the use of PEG . Extendejections (Templetoin vitro by coculturing primary human endothelial cells with human lung or pancreatic cancer cells. The model was confirmed by increased expression of tumor endothelial phenotypes including CD31 and VEGF-A and prolonged survival of endothelial capillary-like structures. The cocultures were used for high-throughput screening of a specialized small-molecule peptidomimetic library to identify ligands specific for human tumor-associated endothelial cells in vitro. We identified small molecules that enhanced the transfection efficiency of tumor-associated endothelial cells, but not normal human endothelial cells or cancer cells. IV administration of our targeted, reversibly masked complexes into mice, bearing human pancreatic tumor and endothelial cells, specifically increased transfection to this tumor microenvironment about 200-fold. Efficacy studies using our optimized targeted delivery of a plasmid encoding thrombospondin-1 eliminated tumors completely after five intravenous injections administered once weekly. We plan to use our targeted, reversibly masked delivery system in the clinic to treat metastatic cancer.We have developed a multidisciplinary approach combining molecular biology, delivery technology, combinatorial chemistry, and reversible masking to create improved systemic, targeted delivery of plasmid DNA while avoiding nonspecific uptake in vivo. We applied this technology to efficiently target delivery to a human tumor-microenvironment model. We achieved efficient, targeted delivery by attachment of specific targeting ligands to the surface of our BIV complexes in conjunction with reversible masking to bypass nonspecific tissues and organs. We identified ligands that target a human tumor-microenvironment created


Podarcis muralis) to test whether anthropogenic disturbance causes an increase in FA. Compared to rural populations, urban ones showed higher levels of FA in all analyzed traits, thus providing evidence that FA can respond to anthropogenic disturbance. However, we also found significant differences in FA among traits, where femoral pores and subdigital lamellae, traits with a functional relevance, were more stable developmentally compared to supracilliar granules which have no evident function. Unsigned FA [abs(right-left)] exhibited significant, but weak, positive correlations among traits, indicating that developmental noise does not have a uniform effect across characters and thus questioning the view of developmental stability as an organism-wide property. The degree of signed FA (right-left) was more similar between structurally associated traits, possibly as an outcome of morphological integration. In conclusion, our results demonstrate that FA can be a reliable indicator of disturbance provided that it is analyzed on multiple traits simultaneously and examined at the population level.The increase in human activities that leads to wildlife decline and species extinction poses an urgent need for simple indicators of environmental stress in animal populations. Several studies have suggested that fluctuating asymmetry (FA) can be an easy, direct measure of developmental instability because it is associated to environmental stress and, as such, it can be a useful indicator of population disturbance. We examined three different morphological traits in urban and rural populations of the common wall lizard ( Environmental stress  caused by human activities can have significant detrimental effects on animal populations Fluctuating asymmetry (FA), defined as small, random deviations from perfect bilateral symmetry, has been widely accepted as a measure of developmental instability Elevated levels of FA have been found both in laboratory experiments and in natural populations of plants and animals exposed to different types of stressors Psammodromus algirusIberolacerta cyreniIberolacerta cyreni, where females were reported to prefer the scent of more symmetrical males Podarcis muralisAmphibolurus muricatusAnolis carolinesisIn lizards, high levels of FA have been associated with small island size and inbreeding Such contradictory results have led to question the validity of FA as a bio-indicator Podarcis muralis . This species has a great potential as a model organism for studying environmental disturbance in a broader context, as its populations are abundant and widespread across Europe  in urban and rural populations of the common wall lizard, s Europe . We specPodarcis muralis were collected by noosing P. muralis. In contrast to the urban populations, however, these were localities with a very low level of air contamination and where human activities potentially creating pollution are few. The distribution of P. muralis can be considered continuous in the whole region with no obvious barriers neither between or within urban and rural populations (see below).Adult individuals of th toe of the hindlimb and the supracilliar granules (SCGN), which have no evident function, were taken on both sides of the body using a digital camera . These traits were selected because they are easy and fast to quantify, and they present extensive variability in Podarcis populations and species After capture, animals were transported to the laboratory of the Faculty of Sciences and Mathematics of the University of Niš, where they were sexed, weighed and measured. Snout-vent length was measured to the closest 0.01 mm using dial calipers. We considered as adults animals larger than 50 mm SVL An asymmetry index (AI) was calculated for all examined traits as the value of the trait on the right side of the body, minus that on the left side (AI = R−L). AI values did not significantly deviate from normality within each population for any of the traits examined . Trait size dependence was examined by linear regression of unsigned AI values on SVL  and on (R+L)/2 (to test for dependence on trait size). To test for the presence of directional and/or fluctuating asymmetry, while taking measurement error into account, we used a two-way ANOVA design on log-transformed trait values of each of the three traits separately, with side as a fixed factor, individual as a random factor and their interaction as an additional term. In this ANOVA design, a significant effect of “side” alone would indicate the presence of directional asymmetry (DA); a significant interaction between side and individual, would point to the existence of fluctuating asymmetry. ANOVA analyses were conducted separately for each population.As our results indicated the existence of fluctuating asymmetry (FA) in all populations for all traits (see Results), we calculated an individual asymmetry index for each trait as the unsigned R−L difference between the log-transformed average of trait values across the two replicate counts of each individual, to account for measurement error (|ln(Raverage)−ln(Laverage)|) We examined the correlation between traits in unsigned FA [abs(right-left)] to test whether asymmetry is organism-wide, in which case FA in a single trait can be used as an indicator of individual quality. We also examined the correlation observed in signed FA (right-left) to test whether traits developing in the same body part  show similar asymmetry patterns. This would be the case if perturbations are transmitted between associated traits during development All statistical analyses were conducted in STATISTICA version 8.0 Lizards were collected and handled with permits of the Ministry of Environment and Spatial Planning of Republic of Serbia No.: 353-01-505/2012-03. All individuals were released in the capture sites upon completion of the procedures.Linear regression of |R−L| on (R+L)/2 and SVL revealed no trait size or body size dependence for any of the analyzed traits . Two-wayThree-way ANOVAs on the log-transformed average of trait values across the two replicate counts of each individual revealed differences both between populations and between population types , where uSignificant, though weak, correlations were found between all traits in unsigned FA, and between FPN and SDLN in signed FA .The results obtained throughout this study provide important insights for the study of asymmetries, both from a biological and a methodological perspective. As hypothesized, increased levels of FA were observed in urban populations of the common wall lizard compared to rural ones, supporting the idea that population-level FA can be used as an early indicator of environmental stress. However, our results also indicate that such inference requires the examination of FA in multiple traits at the population, not the individual, level, in order to obtain an accurate evaluation of developmental instability. This has important implications in the moment of using FA of morphological traits as a robust indicator of environmental disturbance for conservation purposes.Sceloporus occidentalis inhabiting areas with high use of motor vehicles Podarcis bocagei inhabiting agroenvironments Both environmental and genetic factors can cause an increase in FA 3 of oil and oil products from oil storage units (located less than four kilometers from the city center) and from fires in tobacco processing factories (less than two kilometers) in 1999, when high quantities of pollutants were released and spread across the city. Additionally, the increased use of motor vehicles and low quality of petrol also increase the emission of air pollutants  in cities, allowing more asymmetrical individuals to survive to adult age or to get successful mattings Although general FA patterns were concordant in all the traits examined, providing strong evidence for the existence of developmental instability in urban populations, the degree of FA visibly varied across traits. Specifically, SCGN exhibited higher FA values compared to FPN and SDLN across all populations. Consistent differences in FA between traits and across populations indicate differences in their developmental stability Drosophila flies suggesting strong developmental connection Further, the developmental integration among traits might also explain variation in FA across different traits. Correlation analyses reinforce this view. Significant but weak correlations were found between all traits in unsigned FA, but only between FPN and SDLN when signed FA was examined. The significant correlation between FPN and SDLN in signed asymmetries suggests that there are interactions between these traits during development. This result comes as no surprise, as both traits are located in the same body part (hindlimbs) and perturbations during development can be transmitted between them Further, correlations between trait asymmetries have been used to evaluate organism-wide developmental instability. If an individual asymmetry parameter (IAP) exists P. muralis. As such, it can serve as a sensitive bioindicator in this species. This conclusion is reinforced by the observation that general trends of FA, even if variable across populations, are consistently higher in urban localities as compared to rural ones, for several traits. However, the thorough cross-examination of different traits provides an important cautionary tale for conservation practice: methodological, developmental and/or evolutionary factors may cause a dissociation of FA levels across different traits and result in high levels of individual variation. As such, the examination of multiple traits at the population level should be a mandatory requisite, at least as a preliminary procedure, when using asymmetry patterns to establish environmental disturbance and implement conservation measures.Put together, our results provide evidence that FA responds to anthropogenic stress in populations of


Learning is often considered to be governed by reinforcement, causing animals to increase the probability of performing rewarded behaviours, and decrease the probability of behaviours leading to harmful outcomes. However, not all learning is governed by reward. Mere exposure to a novel environment or set of sensory stimuli, unpaired with any behaviour or reinforcement, leads to perceptual learning that allows the animal to more readily form behavioural associations with these stimuli, should the need later arise.In statistics and machine learning, the problem of forming representations of a data set without any explicit training is called "unsupervised learning". Artificial neural networks that perform unsupervised learning have been described, primarily for the case of firing rate neurons. Many of these learning rules allow the networks to perform analyses equivalent to standard statistical techniques. For example, a variant of the Bienenstock, Cooper and Monro (BCM) theory of plasticity allows neurons to implement projection pursuit, a statistical technique for searching for non-Gaussian projections of input data. Here we describe a synaptic rule for unsupervised learning in spiking neurons. The rule is derived as gradient optimization of the time integral of a nonlinear function of membrane potential. This function is shaped like a valley, leading to a preference for membrane potentials near rest or spike threshold, but avoiding intermediate-level potentials . To avoid saturation, the rule is stabilized by a homeostatic mechanism that enforces a constant firing rate using a PI controller that scales synaptic weights. This combination causes neurons to develop a skewed, non-Gaussian distribution of membrane potentials analogously to the projection pursuit algorithm.Using the TIDIGITS database of spoken digit utterances, we show the rule allows a recurrent network of spiking excitatory and inhibitory neurons to develop selective representations of these digits. Applying a linear classifier downstream of the recurrent network shows the Convallis rule allows substantially better readout than a purely random network ("liquid-state machine"), the homeostatic rule alone, or various other plasticity rules for spiking neurons. Applying the rule to simulations of in vitro plasticity paradigms, we find that it reproduces several published results including STDP, although STDP alone cannot produce similar performance in speech recognition. We suggest that ability to perform real-world information processing tasks provides a useful way to constrain theories of synaptic plasticity.


The challenges associated with demonstrating a durable response usingmolecular-targeted therapies in cancer has sparked a renewed interest in viewingcancer from an evolutionary perspective. Evolutionary processes have threecommon traits: heterogeneity, dynamics, and a selective fitness landscape.Mutagens randomly alter the genome of host cells creating a population of cellsthat contain different somatic mutations. This genomic rearrangement perturbscellular homeostasis through changing how cells interact with their tissuemicroenvironment. To counterbalance the ability of mutated cells to outcompetefor limited resources, control structures are encoded within the cell and withinthe organ system, such as innate and adaptive immunity, to restore cellularhomeostasis. These control structures shape the selective fitness landscape anddetermine whether a cell that harbors particular somatic mutations is retainedor eliminated from a cell population. While next-generation sequencing hasrevealed the complexity and heterogeneity of oncogenic transformation,understanding the dynamics of oncogenesis and how cancer cells alter theselective fitness landscape remain unclear. In this technology review, we willsummarize how recent advances in technology have impacted our understanding ofthese three attributes of cancer as an evolutionary process. In particular, wewill focus on how advances in genome sequencing have enabled quantifyingcellular heterogeneity, advances in computational power have enabled explicittesting of postulated intra- and intercellular control structures against theavailable data using simulation, and advances in proteomics have enabledidentifying novel mechanisms of cellular cross-talk that cancer cells use toalter the fitness landscape. The transformation of a normal cell into a cancerous cell involves the acquisition ofa series of genetic and epigenetic changes that daughter clones inherit . Next geKRAS mutations are also a commonoccurrence in colorectal cancer. In a recent clinical study with panitumumab, 38% ofpatients that were initially negative for KRAS mutations developedcirculating tumor cells that harbor detectable mutations in KRASwithin 5–6 months  is observed in three-fourths of primary colorectal tumors and prov6 months . A mathe6 months . While t6 months .Inherent in the view of cancer as an evolutionary process is that: (1) tumors consistof a heterogenous population of cells with different fitness for survival, (2) thecompetition among cells of a population is a dynamic process, and (3) there is acompetitive landscape in the tumor microenvironment that select for variants withimproved fitness. The fitness landscape includes competing for limited resources andintra- and extracellular mechanisms that are designed to maintain cellularhomeostasis. While genetic sequencing technology has revealed the complexity andheterogeneity of oncogenic transformation, understanding the dynamics of oncogenesisand how cancer cells alter the selective fitness landscape remain unclear. In part,this uncertainty has been due to a scientific focus on how somatic mutations alterthe inherent fitness of a cell to compete for limited resources and evadeintracellular control structures . Given tCellular heterogeneity within tumors has been recognized for several decades . While eGenetic sources of heterogeneity among malignant cells arise from the action ofmutagens, such as compounds found in tobacco and UV radiation. While differentmutagens have different signatures of DNA damage , the ranWhile these sequencing efforts have focused on clinically diagnosed tumors, autopsystudies suggest that alterations in the somatic genome may be much more prevalentwithin an organism than has been thought previously, a stage termed “occultcancer.” Nearly forty percent (39%) of women in their forties havehistologic breast cancer and a similar percentage of men in their forties havehistologic prostate cancer . In suppThe second attribute of evolutionary processes is that the different cell typescontained within the tumor microenvironment – stromal cells, malignantclones, and cells of the immune system – and their collective interactionscreate a dynamic system. This dynamic system interacts with a control structureassociated with tissue homeostasis. Homeostasis is a central theme in physiology,where causal mechanisms are used to maintain the physiological state associated withlife in the presence of external perturbations. These causal control mechanisms spanmultiple levels of organization  – from tincerebello model-based inference. There are five challenges with theconventional approach to identifying the control structure associated with tissuehomeostasis and oncogenesis: (1) the interactions among cells occur locally in thetumor microenvironment, (2) robust control typically involves redundant mechanisms,(3) the control structures can be non-linear, (4) the roles that specific mechanismsplay in regulating system response can change with time, and (5) many controlstructures are still unknown . To address thesechallenges, we will first examine the weaknesses associated with the conventionalin cerebello model-based inference and propose an alternativeapproach for inference that leverages contemporary advances in computationalpower.To identify these control structures, one frequently creates a mental model of howone thinks a system behaves based upon prior knowledge of the system ; designs a controlled experiment; and acquires data to infer usingstatistics whether the mental model is a valid representation of the causalmechanisms that regulate system behavior. Conventionally, the mental models are“tested” against the observed data using tools of inferentialstatistics that were originally developed in the early 1900s . CollectOne particular challenge in how classical tools of inferential statistics are used inpractice is that one formulates the inference test in terms of two alternativehypotheses: the null hypothesis – the experimental perturbation introducesno change in the system – and an alternative hypothesis – theobserved response is consistent with the proposed mechanistic hypothesis. If thedata observed under control and perturbed conditions are sufficient different, thenull hypothesis is rejected. Conventionally, the alternative hypothesis is thenaccepted. This conclusion depends on assuming that there are no other lurkingmechanisms at work in the system. To highlight the problematic nature of thisassumption, we consider recent controversial findings related to anti-tumorimmunity. Two recent papers suggest that the adaptive immune system does notinfluence tumorigenesis and metastasis formation nor chemotherapy response in aspontaneous HER2-driven genetically engineered mouse model for breast cancer , 2012. T1. So while the MMTV promotes the expression of theoncogene, the available data suggests that the MMTV-NeuT GEMM of breast cancer doesnot reproduce the degree of mutational heterogeneity observed in human breastcancers. Moreover, HER2/Neu overexpression has been suggested to downregulate majorhistocompatibility complex (MHC) class I expression based upon clinical data  can be implemented in the model in the form of a reduced value fordAk. Then as the value of kdA goes tozero, the presence or absence of adaptive immune cells does not alter the tumorgrowth trajectory. As these papers provide no information regarding the killingefficacy of cytotoxic T cell–tumor cell interaction, the data presented areinsufficient to support the stated conclusions. As alluded to in this example, thereare new methods for model-based inference that involve the use of mathematicalmodels and simulation to test hypotheses.where kin cerebello model-based inference, insilico model-based inference is the statistical reasoning about ourunderstanding of cause and effect in natural systems from experimental observationusing computer simulation. Similar to a microscope that assists our natural abilityto see small objects, mathematical models assist our natural intuition as theyrequire an explicit statement of underlying assumptions and establish formalrelationships between cause and effect. While mathematical modeling, perse, is not new to biology, there are recent advances in how our currentunderstanding of a reactive system can be tested against the observed data.Conventionally called scientific hypothesis testing, this process aims to protectagainst the possibility that a discovery is based upon natural chance alone and notupon a new mechanism. The methods used for scientific hypothesis testing weredeveloped in the early 1900s. These methods were well suited to the questions of theday, as we had very limited knowledge of biological systems and we were limited topencil-and-paper calculations. Today, the intellectual landscape is different. Highperformance computing and high-throughput assays have fundamentally changed the waywe study biology and motivate a contemporary approach. This contemporary approach iscalled in silico model-based inference and draws on ideas from highperformance computing, statistics, and chemical kinetics. The combination of highperformance computing with statistics is an active field of research that focusesprimarily on data regression problems using correlative  models -alpha feedback mechanism illustrates this result.Second, model predictions that are unable to match the observed data suggest thatthe topology is missing important connections, such as paracrine feedback mechanismsthat may be important in vivo but have no effect under conventionalin vitro conditions . Third, the model predictions are consistent with theobserved data but are unable to discriminate among competing topological hypotheses.The discovery of differential STAT1/STAT4 activation by interleukin (IL)-12illustrates the third type of result. According to the editor of Science Signaling,this work “serves as an example of how mathematical modeling can refine ourunderstanding of signaling pathways.” Ultimately, determining whether thetopology of a reaction network can be uniquely identified from the available data isessential for identifying the right control structures at work in biologicalsystems.As illustrated in The third attribute of evolutionary processes is that local cellular environmentprovides a selective fitness landscape for the retention or removal of malignantvariants from a population. This local fitness landscape includes competing forlimited resources – such as limited oxygen or glucose or stromal support– and active intra- and extracellular control mechanisms that aim to restorecellular homeostasis. Intracellular control mechanisms include p53, a protein thathelps control genomic integrity and is mutated in more than half of all cancers, and theFigure 2, mRNA expression results from 224 colorectal tumorand normal pairs reported as part of the Cancer Genome Atlas (TCGA) provide anoverview of the immunological bias present in colorectal cancer  and the corresponding phenotype ofimmune cells within the tumor microenvironment  and output  measurements . In contin vivo. Moreover, samples from the peripheral blood may not berepresentative of the local biological cues responsible for cell cross-talk.Conventionally, immunohistochemical methods have been used to identify localbiological cues present in the tissue microenvironment, a discovery processassociated with experimental bias. The experimental bias stems from the fact thatthe method for detecting a local biological cue must be selected apriori and that methods for detecting this biological cue must exist. Similar to the development of rule-based modelingmethods as a way to minimize bias, proteomics provide less biased methods foridentifying local signaling mechanisms that contribute to homeostatic control.There are two key differences in applying systems identification methods to helpidentify the control mechanisms that regulate anti-tumor immunity compared with thecontrol mechanisms that regulate substrate storage and metabolism. First, thebiological scales are different: coordinated substrate storage and metabolism is anorgan-level phenomenon while anti-tumor immunity is a cell population-levelphenomenon. Second, the cross-talk among components occurs locally through secretedproteins or cell-to-cell contact. While the dynamics of cell-to-cell interactionswithin the tumor microenvironment can be observed using intravital live imaging, thebioin vitro response of 45 different cancer cell to 35 anti-cancerdrugs while co-cultured with one of 23 different stromal cell lines  that malignant cells withinthe tumor microenvironment are heterogeneous, (2) that interactions among cellswithin the tumor microenvironment comprise a dynamic system, and (3) that intra- andextracellular control mechanisms constitute a selective fitness landscape thatdetermines the survival of cells within the tumor microenvironment. Innate andadaptive immunity function as important extracellular control mechanisms. Observedin a subset of melanoma patients, durable response to a new immunotherapy provideshope that restoring these extracellular control mechanisms can be used as aneffective weapon in the battle against cancer . HoweverThe author declares that the research was conducted in the absence of any commercialor financial relationships that could be construed as a potential conflict ofinterest.


Aromatase inhibitors effectively delay epiphysial maturation in boys and improve testosterone levels in adult men Therefore, aromatase inhibitors may be used to increase adult height in boys with gonadotropin-independent precocious puberty, idiopathic short stature and constitutional delay of puberty. Long-term efficacy and safety of the use of aromatase inhibitors has not yet been established in males, however, and their routine use is therefore not yet recommended. Over the past 15 years it has become evident that in men estradiol is responsible for a number of effects originally attributed to testosterone. Estradiol has an important role in gaining and maintaining bone mass, closing of the epiphyses and the feedback on gonadotropin secretion. This fact became particularly evident in men with aromatase deficiency. Aromatase is the enzyme responsible for conversion of androgens to estrogens. Men with estrogen deficiency caused by a mutation in the CYP19 gene suffer from low bone mineral density (BMD) and unfused epiphyses, and have high gonadotropin and testosterone levels ,3. This CYP19A1 gene. This gene is a member of the CYP gene family, encoding a class of enzymes active in the hydroxylation of endogenous and exogenous substances. The CYP19A1 gene is localized on chromosome 15 and comprises nine exons; the start codon for translation is located on exon 2. Transcription of the aromatase gene is regulated by several tissue-specific promoters. These promoters are under the influence of different hormones and growth factors such as gonadotropins  and interleukin-6, interleukin-11 and tumor necrosis factor-a  per day, of which approximately 20% is directly produced by the testes . Roughly5 μmol peTo date, eight males with aromatase deficiency have been described: seven adults -23 and oOn the other hand, several, mostly familial cases of aromatase excess have been reported. The clinical picture consists of gynecomastia, accelerated growth and premature bone maturation due to excessive peripheral estrogen synthesis. Stratakis et al.  describeThese case reports illustrate the important contribution of estrogens to male health and identify the possible indications and risks of aromatase-inhibitor treatment in men. Aromatase inhibitors may be used to treat or prevent gynecomastia. They may be used to increase gonadotropin secretion and thereby stimulate Leydig and Sertoli cell function. Aromatase inhibitors may be used to prevent or delay epiphysial closure and thereby increase adult height. A major concern of aromatase inhibition is the possible detrimental effect on bone mineralization.Aromatase inhibitors are classified as either steroidal or nonsteroidal, or as first, second or third generation. Steroidal inhibitors such as formestane and exemestane inhibit aromatase activity by mimicking the substrate androstenedione. Nonsteroidal enzyme inhibitors such as anastrozole and letrozole inhibit enzyme activity by binding with the heme iron of the enzyme. First-generation aromatase inhibitors such as aminoglutethimide are relatively weak and nonspecific; they can also block other steroidogenic enzymes necessitating adrenal steroid supplementation. Third-generation inhibitors such as letrozole and anastrozole are potent and do not inhibit related enzymes. They are well tolerated and apart from their effects on estrogen metabolism their use does not appear to be associated with important side effects in postmenopausal women . AlthougIt is well known from experimental evidence and from clinical observations that estradiol has powerful effects on gonadotropin release in men. Modulation of plasma estradiol levels within the male physiological range is associated with strong effects on plasma levels of LH through an effect at the level of the pituitary gland . LowerinAromatase inhibitors may be an attractive alternative for traditional testosterone substitution in elderly men because these compounds can be administered orally once daily and may result in physiological 24 h testosterone profiles. Additionally, misuse of aromatase inhibitors is unlikely since testosterone levels will not be stimulated to vastly supraphysiological levels. A small, controlled study demonstrated that anastrozole in a dose of 1 mg daily during 12 weeks will result in doubling of the mean bioavailable testosterone level in older men . A more Peripheral androgen aromatization is enhanced in subjects with increased body mass index . MassiveAlthough FSH release is primarily under the control of inhibin, circulating estradiol has a substantial effect on FSH levels in men . AromataEstrogens are essential for epiphysial maturation in boys. Aromatase inhibitors, therefore, may be used to lower estradiol levels and thereby slow down epiphysial maturation. This approach proved successful in rare conditions such as the aromatase-excess syndrome  and highAromatase inhibition has also been studied in boys with idiopathic short stature. Boys with a mean age of 11 years at the start of the study were treated with letrozole 2.5 mg once daily or placebo for 2 years . LetrozoBoys with constitutional delay of puberty are typically small for their age and final adult height is often in the low-normal range. These boys may be treated with androgens to induce puberty. Although testosterone induces growth velocity, the estrogens aromatized from testosterone will accelerate epiphysial maturation and for that reason reduce adult height further. The combination of testosterone and letrozole, therefore, was tested in boys with constitutional delay of puberty. This combination treatment effectively increased growth velocity but epiphysial maturation was slower in the letrozole-treated group, leading to a significant increase in predicted adult height ,65.Aromatase inhibitors are widely prescribed for hormone-responsive breast carcinoma in postmenopausal women. It is well known that aromatase inhibition results in a dramatic reduction of tumor estrogen concentrations . As gyneData on treatment of male mammary tumors with aromatase inhibitors are scarce and indicate that this treatment modality is unlikely to be successful because of the unwanted effect of increased levels of testosterone, making it impossible to reach the low estradiol levels obtained in postmenopausal women after this treatment . The comExtensive experience with third-generation aromatase inhibitors in postmenopausal women did not reveal major side effects related to their use. Long-term use in postmenopausal women is associated with a moderate increase in bone resorption and a modest decrease in BMD compared with placebo ,3. As ouOver the years compelling evidence has accumulated that in men estradiol has an important role in gaining and maintaining bone mass, closing of the epiphyses and feedback on gonadotrophin release. Aromatase inhibitors, mostly combined with agonists of gonadotrophin-releasing hormone proved effective for the prevention of premature epiphysial closure in boys with pubertas praecox of various etiologies. There is also evidence that aromatase inhibitors can be used in boys with idiopathic short stature and boys with constitutional delay of puberty to increase adult height. Aromatase inhibitors are not effective for the treatment of gynecomastia in pubertal boys and have limited efficacy for the prevention of gynecomastia in bicalutamide-treated men with prostate cancer. Although aromatase inhibitors increase FSH levels, there is no consistent evidence for a beneficial effect on spermatogenesis. In older men with so-called late-onset hypogonadism, aromatase inhibitors may emerge as an attractive alternative for traditional testosterone supplementation to improve testosterone levels. The long-term benefits of higher testosterone levels in older men remain controversial, however. Moreover, it is questionable whether aromatase inhibitors are able to stimulate testosterone production sufficiently in men with truly low testosterone levels for whom testosterone treatment is currently recommended. Although most of the recent studies with aromatase inhibitors in boys and adult men do not show major detrimental effects on bone long-term skeletal safety remains an issue of concern.The authors declare that they have no competing interests.WDR wrote the original version of this manuscript. FHDJ extended the text with more recent observations and conclusions. Both authors read and approved the final manuscript.


EEG rhythms reflect the synchronized activity of underlying biological neuronal network oscillations, and certain predominant frequencies are typically linked to certain behavioral states. For instance, slow wave activity characterized by sleep slow oscillation (SO) emerges normally during slow-wave sleep (SWS). In this mini-review we will first give a background leading up to the present day association between specific oscillations and their functional relevance for learning and memory consolidation. Following, some principles on oscillatory activity are summarized and finally results of studies employing slowly oscillating transcranial electric stimulation are given. We underscore that oscillatory transcranial electric stimulation presents a tool to study principles of cortical network function. The concept that oscillatory brain electric activity—as measured in the EEG or as local field potentials—is more than just an epiphenomenon and can directly impact biological neuronal network activity has existed for some time , which coordinates not only thalamo-cortical sleep spindle activity, but also hippocampal SWRs (Timofeev and Steriade, Outstanding experimental support at the level of cell-pairs for the relevance of SWRs for hippocampo-to-neocortical information transfer was given by Wierzynski et al. . During In the following we point out some essential principles of brain rhythms which indicate how studies employing transcranial weak oscillatory currents can contribute to understanding cortical network function.Single neurons involved in oscillatory activity do not necessarily fire once per cycle, nor even with the frequency of the network oscillation, but properties of neurons matter with regard to determining collective network synchrony (Jacobs et al., At the network level, the application of weak oscillatory currents is most effective at the resonance frequency of the network, characterized by the presence of an Arnold’s tongue (i.e., preferred enhancement occurs at this resonance frequency at weak amplitude of the applied current; Ali et al., The mechanisms and prerequisites responsible for resonant EEG activity induced by transcranial weak oscillatory stimulation are still in need of further research. While it is in line with theoretical concepts described above and in Figure The variability in results we and others have observed employing SO-tDCS (e.g., Eggert et al., Finally, up until now only rather short term effects have been considered, yet memory can improve across days with repeated learning. Constant tDCS has been shown to modify plasticity related products (Fritsch et al., Findings that the most consistent effect of SO-tDCS during SWS is on the endogenous sleep slow oscillatory rhythm implies that this oscillation of neocortical origin was primarily impacted by SO-tDCS, and causally affected memory consolidation and learning. But, selective activation and deactivation of other brain structures within the circuit, in combination with other methods, e.g., optogenetics, is furthermore required to highlight the specific function of the neocortical network for memory consolidation. Furthermore, the differential results of transcranial weak oscillatory stimulation due to brain state point out the necessity, as technical capabilities develop, to consider this state-dependency in research approaches investigating local networks and neuronal properties, e.g., by mimicking different brain states in slice preparations. Finally, development and extension of computational network models can help guide systematic studies on transcranial weak oscillatory stimulation investigating coupled rhythms (e.g., Reato et al., 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.


Neuroscientists are now discovering how hormones and brain chemicals shape social behavior, opening potential avenues for pharmacological manipulation of ethical values. Here, we review recent studies showing how altering brain chemistry can alter moral judgment and behavior, focusing in particular on the neuromodulator serotonin and its role in shaping values related to harm and fairness. We synthesize previous findings and consider the potential mechanisms through which serotonin could increase the aversion to harming others. We present a process model whereby serotonin influences social behavior by shifting social preferences in the positive direction, enhancing the value people place on others’ outcomes. This model may explain previous findings relating serotonin function to prosocial behavior, and makes new predictions regarding how serotonin may influence the neural computation of value in social contexts. How does the brain produce moral behavior? What biological mechanisms determine whether a given individual harms or helps others? And can our neurobiology be manipulated for good? Of late, questions like these have arisen at the interface of neuroscience, ethics, and the law. Here, we examine the contributions of a single brain chemical—serotonin—to moral judgment and behavior.Serotonin is a monoamine neurotransmitter that is evolutionarily ancient and well preserved across mammals. It is one of the most widely distributed neurochemicals in the mammalian nervous system, making its precise functions difficult to pinpoint; however, serotonin is more concentrated in certain structures than others. Anatomical studies illustrate the highest densities of serotonin concentrations in various limbic structures, such as the cingulate, entorhinal, insular, and temporopolar regions, along with the ventral and pallidal regions of the striatumNot surprisingly, then, serotonin has long been implicated in social behavior across species.22Despite abundant evidence linking serotonin to morally relevant social behaviors, the neurobiological and psychological mechanisms mediating these relationships remain unclear. A recent meta-analysis encompassing 175 independent samples and over 6,500 total participants found a reliable inverse relationship between serotonin and aggression, but failed to identify the specific factors explaining the heterogeneity in study outcomes.Moral codes dictate how people should treat one another, and most of these focus on two facets of social relationships. The first prescribes caring for others and prohibits harm; the second relates to the fair distribution of resources and reciprocity in social interactions.29et al.;30In the following, we present evidence that serotonin modulates human concerns for harm and fairness, and we examine the potential mechanisms. We first consider how serotonin influences harm aversion in moral judgment and aversive processing more generally, and then examine how serotonin shapes behavioral responses to fairness and reciprocity. Finally, we synthesize these findings into a theoretical process model whereby serotonin influences social behavior by shifting social preferences in the positive direction, enhancing the value people place on others’ outcomes. This is not a comprehensive review; instead, we focus specifically on studies conducted in humans employing controlled, experimental manipulations of the serotonin system in the laboratory. For a recent comprehensive review of serotonin and social behavior, we refer readers to Kiser harm aversion infuses moral judgments;36Violence toward others is restrained by a seemingly deep-rooted aversion to harmful actions.Why do people react so differently to the two scenarios? One influential hypothesis posits that personal cases like push elicit stronger emotional responses than do impersonal cases like switch, and these emotional reactions drive harm-averse judgments in the former to a larger extent than in the latter.32et al.et al.Note that most studies of moral judgment ask participants whether it is morally permissible to actively cause harm, for example, “Is it morally permissible to push the man?” In these studies, negative cues associated with the harmful action in question could trigger a withdrawal reflex, making subjects less likely to endorse active responses. We might expect this Pavlovian aversive withdrawal process to be particularly strong in personal scenarios that often involve lurid descriptions of the violent actions. There is some evidence supporting this hypothesis. Increasing the vividness of the descriptions of harm in moral dilemmas reduces endorsement of harmful actions.It turns out that serotonin has been implicated in precisely this aspect of aversive processing—namely behavioral withdrawal in the presence of aversive cues. Early studies in rats demonstrated that global brain serotonin depletion made them insensitive to punishment.54One current hypothesis is that serotonin plays a key role at the intersection of aversion and inhibition. Under normal conditions, the presence of aversive outcomes leads to behavioral inhibition, which can manifest in a reduced probability of action or in slowed response times. Modest depletion of brain serotonin in humans abolishes this aversively motivated behavioral inhibition,56Serotonin's involvement in these rather basic aspects of aversive processing suggests that its influence could translate upward into effects on moral judgment. There is some evidence pointing in this direction; neuroimaging studies of moral judgment have shown that imagining harmful acts against others engages brain regions with dense serotonergic projections, including the anterior cingulate cortex, ventromedial prefrontal cortex (vmPFC), amygdala, and striatum.et al.Crockett et al. further examined whether individual differences in empathy moderated the effects of the drug. On the basis of their scores on the Interpersonal Reactivity Index,Crockett et al.Notably, the effects of citalopram on moral judgment were specific to personal dilemmas involving emotionally salient harms. Recall the data suggesting that serotonin is critical for translating aversive Pavlovian cues into behavioral inhibition.BlairHumans are often selfish, but also care about the interests of others—for example, people are sometimes willing to incur costs to achieve fair outcomes, punish unkind behavior, and reward kind behavior.Cooperative behaviors in social dilemmas have been linked to serotonin function. One study found that after 2 weeks’ treatment with citalopram, participants were significantly less likely to behave in a self-interested manner in a modified version of the prisoner's dilemma that allowed participants to act selfishly, cooperatively, or charitably.Preferences for negative reciprocity and fairness have been extensively studied using the ultimatum game (UG). The UG consists of two players, a proposer and a responder, who must agree on a way to share a sum of money, or neither will receive anything. The proposer must offer a division of the sum to the responder, who must make a decision to either accept or reject this proposal. If the responder accepts the offer, both players are paid; if he rejects, neither is paid. Perfectly selfish responders will accept any nonzero offer, but responders with preferences for fairness or reciprocity will reject offers perceived to be unfair—typically less than about 30% of the stake.et al.Several studies have investigated the relationship between serotonin function and responder behavior in the UG. Emmanuele et al.Crockett et al.et al.Rejection of unfair offers in the UG can be explained by either preferences for fairness or preferences for reciprocity. Crockett et al.Meanwhile, negative reciprocity has been associated with activation in the dorsal striatum. Neuroimaging studies found activation in the dorsal striatum during retaliatory actions following both reception and observations of unfair behaviors.The association between serotonin and social reward processing dovetails with previous reports linking serotonin function to the processing of non-social rewardsOur synthesis of experimental findings advocates a causal role for serotonin in both harm aversion and social preferences for fairness and reciprocity. Could these two ostensibly distinct facets of morality instead reflect a single underlying dimension? More specifically, can harm aversion be thought of as a type of positive social preference?Often the social context determines whether social preferences are positive or negative: one notable example is inequality aversion.68et al.91We suggest that serotonin shifts the slope of the indifference curve in the direction of positive social preferences. Crockett Our model is primarily descriptive at the behavioral level, but makes predictions that can be tested at the neural level. For example, value-processing regions such as the ventral striatum and the medial PFC show a pattern of activation consistent with inequality aversion;Finally, it is worth mentioning that social cognitive and emotional processes concerning the representation of others’ mental states and emotions  clearly play a role in shaping concerns for harm and fairness.72Research into the neural basis of moral judgment and behavior has exploded over the past decade. The vast majority of this work has involved neuroimaging; these studies have provided valuable insights into the neural correlates of moral decisions, but are correlational in nature. More recent studies employing intervention methods, such as pharmacological manipulations and brain stimulation, have provided additional information about the brain systems that are causally involved in moral decisions. Future work employing these techniques will benefit from specified theoretical frameworks that generate novel predictions about how targeted interventions will modify moral judgment and behavior.


Maintenance of skeletal muscle is essential for health and survival. There are marked losses of skeletal muscle mass as well as strength and physiological function under conditions of low mechanical load, such as space flight, as well as ground based models such as bed rest, immobilization, disuse, and various animal models. Disuse atrophy is caused by mechanical unloading of muscle and this leads to reduced muscle mass without fiber attrition. Skeletal muscle stem cells (satellite cells) and myonuclei are integrally involved in skeletal muscle responses to environmental changes that induce atrophy. Myonuclear domain size is influenced differently in fast and slow twitch muscle, but also by different models of muscle wasting, a factor that is not yet understood. Although the myonuclear domain is 3-dimensional this is rarely considered. Apoptosis as a mechanism for myonuclear loss with atrophy is controversial, whereas cell death of satellite cells has not been considered. Molecular signals such as myostatin/SMAD pathway, MAFbx, and MuRF1 E3 ligases of the ubiquitin proteasome pathway and IGF1-AKT-mTOR pathway are 3 distinctly different contributors to skeletal muscle protein adaptation to disuse. Molecular signaling pathways activated in muscle fibers by disuse are rarely considered within satellite cells themselves despite similar exposure to unloading or low mechanical load. These molecular pathways interact with each other during atrophy and also when various interventions are applied that could alleviate atrophy. Re-applying mechanical load is an obvious method to restore muscle mass, however how nutrient supplementation  may further enhance recovery (or reduce atrophy despite unloading or ageing) is currently of great interest. Satellite cells are particularly responsive to myostatin and to growth factors. Recently, the hibernating squirrel has been identified as an innovative model to study resistance to atrophy. Skeletal muscle plays a significant role in quality of life and is essential for health and survival. It is highly organized at the micro- and macroscopic level and plays a major role in mobility of the human body. Skeletal muscle accounts for ~40% of body mass, permits precise movements and is highly adaptive. The characteristic of plasticity allows skeletal muscle to change and adapt depending on the stimuli placed upon it. Increases in mechanical load and increasing workload will stimulate muscle hypertrophy, while removal of mechanical load will lead to muscle atrophy as an appropriate adaptation to hypogravity  is seen in disease states such as cancer, AIDS, renal failure, congestive heart failure, chronic obstructive pulmonary disease (COPD) and burns. In addition to disuse atrophy, cachexia involves an intricate cytokine and inflammatory response inducing signaling cascades and gene transcription. This review will focus on disuse atrophy and selected growth factor-related signaling. Human models of disuse, such as immobilization and sedentary lifestyle (inactivity) include decreased mechanical loading. Immobilization can occur as a result of various injuries, mostly not of skeletal muscle so the influence on skeletal muscle is considered a side effect. Immobilization can also be used as a model to investigate disuse and load reduction on skeletal muscle as a research intervention. Unilateral lower limb suspension involves suspension of one limb while the other is used for movement assisted by crutches. This model resulted in 5–10% reduction in CSA of Quadriceps muscle after 4 weeks , the response of the myonuclear number and myonuclear domain size with atrophy is less conclusive. Despite less being known about the influence of disuse on myonuclei, the alterations in muscle morphology, biochemistry and physiology suggest that the myonuclei are influencing features of change within the muscle as well as being influenced by these alterations. Furthermore, atrophy from disuse occurs rapidly and its development is not simply the reverse of skeletal muscle hypertrophy. Myonuclear loss may follow a different timescale, occurring at a slower pace than the loss in muscle fiber size  in the maintenance of muscle mass, and the response to muscle loss.Myonuclei in skeletal muscle are post-mitotic and cannot replicate. Therefore, any increase in myonuclear number such as required for growth and repair is a result of SC fusion, although also to a very minor extent other stem- or stem cell-like cells  of remaining SCs are is altered during atrophy and if so, whether their functionality is can be restored with reloading. In studies investigating alterations in SC content and proliferative ability, the age and growth stage of the animals is important. To investigate the role of SCs and the effect which atrophy has on their function, using young animals whose growth has not stabilized can shed light on factors not investigated in other models. For example, the SC may be more responsive during growth and this may alter their response to the atrophy-inducing intervention  after 14 days of HS in mice , it should be highlighted that alterations in MRFs have been documented. During 14 days of immobilization, Snijders et al.  report mInvestigations into treatments to reduce atrophic response during HS have found that application of low-frequency electrical stimulation (LFES) partially rescues the loss of SCs and lessens the reduction in muscle cross-sectional area  and some evidence exists that SC are also influenced significantly by these pathways.The molecular mechanisms underpinning muscle atrophy with disuse remain to be fully elucidated. The next section aims to describe 3 of the key molecular pathways which are linked to skeletal muscle atrophy: Atrogin-1/MAFbx and MuRF1; the IGF-1-AKT-mTOR pathway; and the Myostatin Pathway. Existing authoritative review articles will be highlighted. Further, we aim to discuss how these pathways are related to SCs and myonuclear responses to atrophy.The ATP-dependent ubiquitin proteasome pathway is the primary degradation pathway of skeletal muscle in response to inactivity and disuse. The ubiquitin-proteasome pathway is involved in breakdown of short-lived proteins or long-lived myofibrillar proteins in skeletal muscle. Three distinct components are required for muscle breakdown using the ubiquitin proteasome pathway. E1 ligases which activate ubiquitin, E2 ligases that are responsible for transferring the activated ubiquitin to the protein molecule that is then targeted for degradation and the E3 ligases which regulate the actual transfer of ubiquitin to the protein. Two important skeletal muscle specific ubiquitin E3 ligases are Muscle-specific RING Finger protein1 (MuRF1) and Muscle Atrophy F-box (MAFbx/atrogin-1).MAFbx and MuRF1 are both primarily expressed in skeletal muscle and are upregulated in several models of disuse  is a member of the transforming growth factor (TGF)β superfamily and a negative regulator of muscle mass. Myostatin appears to be primarily found in muscle tissue , upregulating the ubiquitin proteasomal pathway via FOXO (more protein breakdown) . The increase in adipose may be due to the reduction of regenerative capacity of SCs  on the potential of both SCs and pluripotent stem cells to differentiate into different lineages, is of extreme significance for skeletal muscle health as well as for prospects of clinical medicine and therapies aimed at maintaining and positively influencing skeletal muscle.Reapplying mechanical load appears to be the most effective method to restore muscle mass, and increase myonuclear number, SC number and regenerative capacity.Resistance exercise increases muscle mass and by increasing the load placed on the muscle which activates the PI3-AKT-mTOR pathway and increases protein synthesis. As little as one bout of resistance exercise in healthy individuals has been reported to increase IGF1 gene expression , the SCs are not quiescent when their environment is changing but rather they are responding to the alterations in the niche area. This review brings together the current knowledge of myonuclear and SC response to disuse atrophy and highlights the complexity of the response in animals and humans.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.