Calcium (Ca2+) is a ubiquitous intracellular mediator involved in various cellular functions such as releasing neurotransmitters from neurons, contraction of muscle cells, fertilization, and cell differentiation and morphogenesis. The multiple roles played by this ion in mediating signals can be primarily explained by its spatiotemporal dynamics that are kept tightly checked by mechanisms that control its entry through plasma membrane and its storage on intracellular stores. Given the large electrochemical gradient of the ion across the plasma membrane and intracellular stores, signals that can modulate Ca2+ entry channels or Ca2+ receptors in the stores will cause Ca2+ to be elevated in the cytosol and consequently activating downstream Ca2+-responsive proteins resulting in specific cellular responses. This review aims to provide an overview of the reported neurotransmitters and neuropeptides that participate in early stages of development and their association with Ca2+ signaling.A primary reason behind the high level of complexity we embody as multicellular organisms is a highly complex intracellular and intercellular communication system. As a result, the activities of multiple cell types and tissues can be modulated resulting in a specific physiological function. One of the key players in this communication process is extracellular signaling molecules that can act in autocrine, paracrine, and endocrine fashion to regulate distinct physiological responses. Neurotransmitters and neuropeptides are signaling molecules that renders long-range communication possible. In normal conditions, neurotransmitters are involved in normal responses such as development and normal physiological aspects; however, the dysregulation of neurotransmitters mediated signaling has been associated with several pathologies such as neurodegenerative, neurological, psychiatric disorders, and other pathologies. One of the interesting topics that is not yet fully explored is the connection between neuronal signaling and physiological changes during oocyte maturation and fertilization. Knowing the importance of Ca Based on this, we focus this literature review on the association of neuronal signals with Ca2+ in oocyte mutation and early development.Oocyte maturation and embryogenesis has always been an area of interest for many researchers. Despite the abundant research on the topic, many aspects of the development process are yet to be studied. One specific area of interest is investigating the interplay between neurotransmitters and neuropeptides and oocyte maturation and early development . SimilarOocyte maturation is a process by which an immature oocyte arrested at prophase of first meiotic division resumes meiosis to reach competence for normal fertilization after ovulation . In almo2+ signaling in the oocyte physiology is described in many animal species .2+ signaling during oocyte maturation and fertilization . On the other hand, neuropeptides are a specific type of large-molecule neurotransmitters identified as dense-core vesicles for their appearance under electron microscopy .There are two different classes of neurotransmitter receptors, ionotropic and metabotropic. Ionotropic receptors are neurotransmitter-gated ion channels. This class of channels will open after neurotransmitter binding, and messages between neurons using ionotropic receptors will be delivered in less than a millisecond . On the 2+ signaling during these important processes.Different signaling pathways have been recognized as vital regulators of early organism development, such as fibroblast growth factor (FGF), Hedgehog, Wnt, transforming growth factor-β (TGF), and Notch . These sIn mammals, the gonads of both males and females and other reproductive organs are innervated by the parasympathetic and sympathetic efferents . A growiInnervation of the ovaries is mainly through the nerves from the ovarian plexus. Most of these nerves are noradrenergic nerves . Some stApostichopus japonicus, injected two neuropeptides, NGIWYamide and NGLWYamide into the abdominal cavities of sexually mature males and females, which resulted in induction of gamete spawning. Despite these valuable findings, the exact sites of action and synthesis of NGIWYamide are still unknown. The study was only able to illustrate the importance of the ovarian wall for the function of the peptide  and octopamine (OA)  .Different hormonal signals have been recognized for their vital role in the various reproductive processes, such as ovulation. Oocyte maturation in vertebrates is triggered by luteinizing hormone (LH) surge secreted by the anterior pituitary in response to the pulsatile release of gonadotropin releasing hormone (GnRH) by the hypothalamus . Howeverαs and Gαolf  is another important neurotransmitter that plays a significant role in regulating the immune system as it controls inflammation by preventing specific inflammatory cells’ effects in non-neuronal cells . Moreoveq or Gi . Ach is A, or the metabotropic receptor, GABAB. GABAA receptor is a chloride ion channel that when activated, results in cell membrane hyperpolarization. In contrast, GABAB is a G-protein coupled receptor that decreases presynaptic Ca2+ conductance and increases postsynaptic K+ conductance , was reported to play a role in promoting synapse formation, cell survival, and plasticity, specifically in the olfactory bulb. The study by Garcia et al. revealed that manipulating the CRH signaling levels can affect the formation of synaptic proteins and the maintenance of active synapses. .2+ concentrations. Eshra et al. studied the effects of different Ca2+ concentration levels on vesicle priming, fusion, and replenishment at mossy fiber synapses in the rodent cerebellum. This study showed Ca2+ dependent vesicle fusion, as demonstrated in previous studies. In terms of vesicle priming, the results indicated the presence of a high-affinity Ca2+ sensor at the studied synapse that initiated the process of priming at intracellular Ca2+ concentrations in the range of 30 and 180 nM. Finally, there is a low association between intracellular Ca2+ concentration and vesicle recycling. Overall, the study illustrated the high dependence of the neuronal signaling process, specifically the phases of priming and fusion, on the intracellular Ca2+ levels  mainly regulated intracellular Ca2+ release, which led to the activation of IP3 receptors and, subsequently, the ryanodine receptors attached to the endoplasmic reticulum, increasing Ca2+ concentrations in the cytosol. Moreover, to exclude the presence of other pathways triggered by NPSR1 activation, the researchers incorporated into the culture receptor-specific and PLC blockers and noted that the Ca2+ concentration levels did not increase, thereby concluding that NPSR1 mediates intracellular Ca2+ release only through Gq signaling (IP3-DAG Pathway). Overall, several studies link the NPSR1 gene to the development of panic and anxiety disorders. Thus, the authors indicated that their study design and findings will benefit future research in developing a better understanding of the clinical implications of the gene in anxiety and panic disorders (The role of the neuropeptide S (NPS) and NPS receptor 1 (NPSR1) complex in activating the intracellular pathways of Caisorders .2+ signaling in serotonin (5-HT) mediated neurite outgrowth  neurons  promoted a synchronic, transient increase in Ca2+ in the nucleus of hippocampal neurons. This increase translated into elevated levels of phosphorylated CREB, Nps4, and RyR2 mRNA. This study proved the imperative role of RyR2 and Ca2+ induced- Ca2+ release in the generation of nuclear Ca2+ transients following neuronal stimulation either through GBZ or glutamate uncaging  or Norepinephrine (NE). In Caenorhabditis, the pivotal role of OA in maintaining oocytes’ quiescence was proven through mutant nematodes lacking endogenous OA. The study identified SER-3, a G-protein-coupled receptor (Gq), as the receptor targeted by OA and allowing the transduction of downstream inhibitory signals. In Drosophila melanogaster, the balance between this noradrenergic and the nutrient signals determines the onset of oogenesis. Mutant forms of Drosophila melanogaster revealed the importance of OA in inhibiting the progression of egg chambers during nutrient insufficiency. This action, in turn, is mediated via either alpha or beta-adrenergic receptors. Like Drosophila melanogaster, the maintenance of oocyte quiescence in Danio rerio females is governed by the balance between the nutrient and noradrenergic signals. The only difference is that the ovaries in this species are innervated by NE-producing cells  release   acts on the pituitary gonadotrophs to induce biphasic Ca release . The Ca2channels . Other schannels , and indchannels . In addi C (PKC) .2+ dynamic in oocytes expressing rat TRH receptors  generation, they induce Ca2+ mobilization from distinct cellular stores in the Xenopus oocyte, suggesting that these different compartments are associated with different receptors  is another peptide that can modulate pituitary secretion and was shown to trigger a biphasic Caeceptors . TRH receceptors . TRH-medectively . Shapiraeceptors . Unfortu2+ signaling and the neurotransmitter serotonin (5-HT), in triggering oocyte maturation, spawning, and early development in bivalve mollusks , that can suppress 5-HT-induced oocyte maturation. The factor was detected with serotonin-containing neurons in the cerebral and pedal ganglia in the CNS of these bivalve mollusks  concentrations  and Neuronal Per Arnt Sim domain protein 4 (Npas4)  . On the ogenesis . Sato ettal rats . Additioelopment .2+ changes in modulating processes during oocyte maturation and early development. Further studies are needed to understand the molecular machinery modulated by Ca2+-dependent neuropeptides signaling. Equally important is to identify new gonadal neuropeptides and their receptors. Understanding the role of neuropeptide signaling in abnormalities associated with fertility and early development could pave the way for targeting these pathways during therapeutic interventions.Several neurotransmitters including neuropeptides are known to play a role in oocyte maturation and early development in vertebrates and invertebrates. Examples are noradrenergic molecules, serotonin, neuropeptides coupled to G-protein coupled receptor, GABA, and dopamine. However, few of these neurotransmitters were demonstrated to be dependent on Ca


The scale with which pharmaceutically relevant African genetic variants can be stratified, the expediency with which these platforms can be established, and their subsequent sustainability suggest that they will have an important role to play in the democratization of stratified healthcare in Africa. Here we discuss the requirement for African hepatic models, and their implications for the future of pharmacovigilance on the African continent.Pharmaceuticals are indispensable to healthcare as the burgeoning global population is challenged by diseases. The African continent harbors unparalleled genetic diversity, yet remains largely underrepresented in pharmaceutical research and development, which has serious implications for pharmaceuticals approved for use within the African population. Adverse drug reactions (ADRs) are often underpinned by unique variations in genes encoding the enzymes responsible for their uptake, metabolism, and clearance. As an example, individuals of African descent (14–34%) harbor an exclusive genetic variant in the gene encoding a liver metabolizing enzyme (CYP2D6) which reduces the efficacy of the breast cancer chemotherapeutic Tamoxifen. However, CYP2D6 genotyping is not required prior to dispensing Tamoxifen in sub-Saharan Africa. Pharmacogenomics is fundamental to precision medicine and the absence of its implementation suggests that Africa has, to date, been largely excluded from the global narrative around stratified healthcare. Models which could address this need, include primary human hepatocytes, immortalized hepatic cell lines, and induced pluripotent stem cell (iPSC) derived hepatocyte-like cells. Of these, iPSCs, are promising as a functional  The global population is becoming increasingly reliant on pharmaceuticals as healthcare systems are burdened by lifestyle diseases, drug resistance, mental health issues, and innumerable orphan diseases. While substantial progress has been made to ensure patients in Africa have access to treatments, the majority of pharmaceuticals are trialed on narrow genetic populations using both preclinical models and population cohorts that are not inclusive of global, especially African, diversity. This is well exemplified by the fact that the African continent currently contributes only ∼3.3% of the estimated 393 000 active clinical trials globally . Yet, deIt has long been understood that genetic variants in drug metabolizing genes are a major factor resulting in altered drug responses . ConsequIn the context of ADRs, precision medicine aims to maximize the probability of clinical success, by matching patients with predicted best treatment options, either for individuals  or well-defined subsets of the population (stratified medicine). The mitigation of ADRs through regulatory interventions represents a valuable and tenable strategy for the implementation of stratified medicine within Africa, especially for drugs that are highly utilized across the continent. These regulatory changes must first be driven by rigorous scientific efforts to bridge the disconnect that exists between the identification of African relevant genetic variants, and the functional validation of their potential clinical impact. Here we outline why we believe cellular hepatic models would be a useful tool to validate African relevant gene-drug interactions.Hepatocytes, accounting for 60–70% of the total liver cell population, play a central role in the biotransformation, intermediary and energetic metabolism of endogenous and exogenous xenobiotics, including pharmaceuticals . HepaticCYP450 enzymes are of relevance to pharmaceuticals as their induction/inhibition potential, genetic polymorphisms, epigenetic regulation and non-genetic host factors such as gender, age, disease(s), and polypharmacy can contribute to functional disparities. CYP450 enzymes are responsible for approximately 80% of hepatic Phase I metabolism, with 12 isoforms being responsible for ∼75% of Phase I oxidation reactions . In a liThe functional consequence of genetic polymorphisms leads to the classification of pharmacokinetic phenotypes as poor, intermediate, extensive/normal, and ultra-rapid metabolizers. Thus, contextualizing major genetic determinants of drug metabolism in different populations is essential to the provision of safe and efficacious pharmaceuticals . InvestmGiven the genetic diversity in Africa, executing precision medicine strategies could be considered beyond the capabilities of the healthcare infrastructure, in the predominantly developing nations which constitute the region. Yet, there are well defined examples of gene-drug pairs where ADRs could be minimized or resolved thereby significantly alleviating socio-economic impact.One example of such a gene-drug pair, for a non-communicable disease, is that of the breast cancer drug Tamoxifen and its key liver metabolizing gene. Tamoxifen is a selective estrogen receptor modulator, which relies on CYP2D6 to catalyze the formation of primary and secondary metabolites which have higher anti-estrogenic activity. The CYP2D6 gene locus is highly polymorphic, with the allelic variant CYP2D6*17 prevalent in 21.7% of the African population [14–34% in African sub-cohorts ]; yet itAnother concerning gene-drug pair is that of CYP2B6 and the antiretroviral Efavirenz. CYP2B6 variants, with a higher prevalence in the African population, include functionally deficient haplotypes CYP2B6*6 and CYP2B6*18 . In compHere we outlined two of the many well-established gene-drug pairs within the African context. Yet, data from the 1,000 Genomes Project indicates that SSA contains 25% more genetic diversity than the rest of the world (1000 Genomes Project Consortium 2015) rendering the absolute requirement for studies which validate the relationship(s) between host genetic and pharmaceutical interactions. Furthermore, since genes such as CYP2D6 and CYP2B6 are responsible for the metabolism of ∼27% of clinically approved drugs , it begsThe African Pharmacogenomics Consortium (APC) was launched in 2018, with the mandate to educate, build capacity, capability, governance, and technologies to promote the use of pharmacogenomics for the clinical benefit of African patients . HoweverSafety pharmacology informs risk-benefit relationships by assessing adverse effect liability and safety margins. Hepatotoxicity and aberrant xenobiotic metabolism are major contributors to post-marketing drug withdrawal, failure of investigational new drugs, and drug inefficacy. Population risk-benefit ratios, derived from clinical trial data, inform regulatory decisions despite knowing that patient-level responses will differ in terms of efficacy and adverse outcome risk. Importantly, as the contribution of an individual’s genetics to the risk-benefit relationship is better understood it is possible that reducing attrition rates during development, using the current testing paradigms, will not be synonymous with global improvement in patient outcomes .ex vivo tissue slices; 5) isolated perfused organs; 6) genetically engineered cells expressing metabolizing enzymes; 7) cell-free and in silico approaches; and 8) animal models  primary human hepatocytes (PHHs); 2) subcellular fractions including microsomes or S9 fractions; 3) cell lines; 4) l models . Howeverl models . The focPHHs are the “gold standard” or “historical standard” for modeling xenobiotic metabolism . PHHs exPHHs are however prone to rapidly declining metabolic function and dedifferentiate, within hours, in traditional monolayer cultures . The adoHowever, donor age, genetics and pathophysiology, as well as the impact of cryopreservation on attachment efficiency/viability and consequently their functional properties, are important factors to consider when using PHHs . Furtherin vitro hepatocyte model despite exhibiting reduced basal gene expression of Phase I and Phase II drug-metabolizing enzymes, as well as functional and phenotypic responses that are inconsistent with those of PHHs  such as HepG2 and HepB3, Fa2N4 cells, bipotent hepatic progenitors (HepaRG), and HepG2 clonal variants, HepG2/C3A . Due to  of PHHs .HepaRG cells can be differentiated to express numerous metabolizing enzymes and transporters at levels that are superior to those of other carcinoma lines . They arThe generation of immortalized PHH lines, with the capacity for self-renewal, is an interesting prospect for future research efforts as various immortalization strategies and use cases have been described . These lReprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is achieved by the stochastic overexpression of key stem cell transcription factors . SimilarSimilar to PHHs, iPSCs retain the original genetic complement of the individual from which they were derived, but with the added advantage of self-renewal. This characteristic further confers genome engineering capabilities to iPSCs . ConsequWhile immortalized cells are also amenable to genome engineering, the improved chromosomal stability and physiological relevance of iPSC derived HLCs lends a significant advantage relevant to the African context. Human iPSC-HLC models have been used to model the disruption of the ER in α1-antitrypsin deficiency , and urein vitro models from diverse genetic backgrounds, as donor demographics of PHHs are overwhelmingly in favor of Caucasian populations . Simila 1 other  only 35 r models .Bioengineered iPSC-HLCs derived from individuals of African ancestry, could provide an ideal platform to empirically assess the relationship between individual and cumulative African specific genetic variants and pharmacokinetic phenotypes within an isogenic background. While PHHs have an invaluable role to play in assessing inter-individual variation, and the validation of pharmacokinetic phenotypes, population stratification metrics need to be more stringently and singularly assessed. PHHs may remain the “gold standard” for determining pharmacokinetic phenotypes, however, access to the number of clinically relevant PHHs required to mirror the diversity in Africa is currently not feasible in terms of scalability, accessibility, and cost. Likewise, while the use of immortalized and carcinoma derived hepatic cell lines may present specific challenges for modeling pharmacokinetically relevant genotypes; the derivation of such lines from individuals of African ancestry would facilitate a readily accessible way for researchers to reshape the global underrepresentation of African genetics in science and improve the transferability of global search findings to an African relevant context. The necessity for these resources is perhaps most easily exemplified by the assessment of a few of our own iPSC lines where we focus on generating lines from donors of African origin. Here we identified a line which is both a poor metabolizer for CYP2B6 (CYP2B6*6/*18) as well as a reduced metabolizer for CYP2D6 (CYP2D6*1/*17), leading to almost complete ablation of Efavirenz metabolism and aberrant production of Tamoxifen’s potent bioactive metabolites respectively. This is not remarkable within the context of the prevalence in Africa but would be exceptionally rare globally as iPSC lines are predominantly derived from Caucasians.New scientific breakthroughs, and improved access to these technologies on the African continent, has positioned scientists to establish platforms which directly impact the burden of clinical healthcare. Access to enabling technologies including; genome engineering, iPSC differentiation, and malleable 3D biomimetic microenvironments, provides synthetic biology with tools to develop preclinical models with improved capacity for predictive extrapolation. While individualized treatments might remain an unrealistic goal in Africa, comprehensive profiling of genetic variants which impact pharmacokinetic/dynamic profiles would aid in the implementation of subpopulation stratification and subsequently improve healthcare strategies on the African continent. With the proposed landscape, independent, interdependent or sequentially applied models could be used to provide empirical evidence for the efficacy or inefficacy of marketed/prescribed drugs in clinical practice.in vitro pharmacovigilance platform that houses various hepatic models that recapitulate the xenobiotic metabolism of the liver to specifically link African-relevant genetic variants to efficacy/ADRs. These tools would support pharmacogenetic based decision-making and reduce the use of inadequate or harmful pharmaceutical interventions (To address these challenges, we propose an integrative ventions . While tventions , few natventions  which inOur research group, which successfully derived one of the first human iPSC lines on the African continent, has applied genome engineering strategies to edit iPSCs, and has been actively engaged in modeling iPSC-HLCs and other “disease-in-a-dish” models. Further to this, isolating PHHs from individuals of African descent, on the continent itself, would represent a significant milestone for the accessibility and utilization of genetically representative PHHs within the African research landscape. However, this will require key stakeholders from multidisciplinary backgrounds to collectively establish such an Afrocentric resource within the global pharmacovigilance arena. Solidifying the foundation of an African hepatic modeling platform will require concerted efforts to establish a new paradigm for preclinical and clinical research collaborations that collectively drive impact in healthcare. The convergence of a number of these traditionally siloed technologies and methodologies, within an established African research infrastructure, would support the feasibility of a hepatic modeling platform and serve as a launchpad for national and ultimately continent-wide initiatives.


Adipose tissue is a critical regulator of systemic lipid and energy metabolism, and studies have shown that the cellular composition of adipose tissue plays an important role in the tissue’s ability to function as a metabolic equilibrium hub. Adipose tissue is composed of diverse cell types and is comprised of adipocytes and stromal vascular cells, containing mesenchymal stem cells, preadipocytes, fibroblasts, pericytes, smooth muscle cells, endothelial cells, and immune cells. Importantly, various immune cell types have been implicated in several different aspects of adipose tissue biology, including tissue homeostasis, thermogenesis, and regulation of adipose tissue function. Discovering and understanding the role of adipose immune cells in different states, such as physiological processes or metabolic dysregulation, will lead to a greater comprehension of the full breadth of adipose-immune biology and the possibility of developing therapeutics for chronic metabolic conditions, including obesity and diabetes.Agueda-Oyarzabal et al. where they describe different immune cell population and how these cells interact with thermogenic adipocytes in the adipose tissue microenvironment. The authors detail the general roles and specific functions of macrophages, T cells, B cells, eosinophils, innate lymphoid cells (ILC), γδT cells, natural killer T (NKT) cells, monocytes, and mast cells in thermogenic adipose depots. The review also evaluates the crosstalk between immune cells and adipocytes that is essential to maintain thermogenic capacity of adipocytes.Adipose tissue hosts a diverse population of immune cells that includes T cells, B cells, macrophages, dendritic cells, neutrophil, and mast cells. Proper functioning of these immune cells are critical for adipose tissue function and dysfunction of immune cells in obesity can lead to inflammation and obesity-associated metabolic disease. Immune cells can also regulate obesity-related disorders by either facilitating or inhibiting adipose thermogenesis. Thermogenic adipose tissue such as the brown adipose tissue (BAT), dissipates energy as heat and has been ascribed to an anti-obesity function in mice and humans. A specialized type of adipocytes known as “beige adipocytes” in white adipose tissue function similar to brown adipocytes and numerous studies have pointed towards a protective role of beige adipocytes against obesity. The role of immune cells in thermogenesis is comprehensibly summarized in a review by Blaszkiewicz et al. build upon their prior studies and report an original work to show that a subset of cold-recruited myeloid lineage monocyte macrophages (Ly6c+CCR2+Cx3cr1+), labeled as cold-induced neuroimmune cells (CINCs) play a nerve-supporting role in the adipose tissue. They hypothesize that cold stress recruitment of CINCs produce brain derived neurotrophic factor (BDNF) to regulate neve innervation and neuro-adipose regulation of energy expenditure. Using RNA-seq, advanced confocal imaging in adipose tissue, and animal models, they comprehensibly demonstrate CINCs essential role in nerve survival and plasticity. Overall, their data reveal a unique role of immune cells in the maintenance of adipose innervation and metabolic function.Adipose tissue is densely innervated by peripheral nerves of the sympathetic nervous system (SNS). Upon activation of SNS such as cold exposure, peripheral nerves secrete norepinephrine that acts on adipocytes to activate thermogenesis and lipolysis. Immune cells in adipose tissues are also closely associated with nerves and are involved in degrading norepinephrine, thereby limiting adipocyte thermogenesis and lipolysis. The role of neuroimmune cells beyond catecholamine degradation is largely unknown and Lopez-Perez et al. show a phenotypic alteration of mast cells in omental white adipose tissue (o-WAT) of obese humans with type 2 diabetes (T2D). Using flow cytometry based approach, the authors simultaneously measure mast cell population and expression of biologically active mast cell surface receptors in the o-WATs and subcutaneous adipose tissues (s-WAT) of non-T2D, pre-T2D, and T2D obese patients. They report a significant decrease of mast cell receptors expression in the o-WATs of T2D patients, implicating a serious defect in mast cell function. This study suggest an important role of mast cells in o-WAT physiology and factors that lead to the defect in mast cell survival, activation, and secretory function could drive the pathophysiology of T2D. Depending on the location, excess adipose tissue can also lead to cosmetic and psychological burden. Particularly, deposition of excess fat in the submental region also known as “double chin” has spurred the use of fat reducing compounds for cosmetic purposes. A review article by Muskat et al. comprehensibly summarizes the biology and mechanism of the two fat reducing compounds, Deoxycholic Acid (DCA) and Phosphatidylcholine Acid (PDC). The authors detail induction of cell apoptosis, inflammation, and fibrosis by DCA and PDC. They further evaluate a role of these compounds on lipolysis and report that DCA does not induce lipolysis and PDC induces lipolysis through release of inflammatory cytokine, tumor necrosis factor-alpha (TNFα). Overall, different fat depots harbor diverse immune cells types that have specialized role in regulating adipose tissue physiology and pathophysiology. A better understanding of the roles of tissue-resident or recruited immune cells in maintaining adipose tissue homeostasis could inform us about how obesity leads to a dysfunctional adipose microenvironment.Adipose tissues can be found at different anatomical locations. The distribution of adipose tissue is of great importance in regards to obesity related co-morbidities. Insulin resistance often occurs when fat accumulates in intra-abdominal depots and is associated with a constellation of risk factors, in what is known as the metabolic syndrome. Mast cells residing in intra-abdominal depots such as the omental depot are generally considered to have deleterious role in adipose tissue. But recent studies have shown an important role of mast cells in adipose thermogenesis, adipogenesis, release of growth factors and bioactive molecules. Original research by In conclusion, this Research Topic aims to incorporate reviews and novel data in the immunometabolism field related to the exciting relationship between adipose tissue and immune cells in different physiological and pathophysiologic contexts, with a focus on how the immune cells can control or interfere with whole-body metabolism.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.We would like to thank all the authors who have submitted manuscripts to this Research Topic. PR is supported by R00DK114571 and Diabetes Research and Education Foundation (DREF) Grant # 501. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Glomerular parietal epithelial cells (PECs) have been increasingly recognized to have crucial functions. Lineage tracking in animal models showed the expression of a podocyte phenotype by PECs during normal glomerular growth and after acute podocyte injury, suggesting a reparative role of PECs. Conversely, activated PECs are speculated to be pathogenic and comprise extracapillary proliferation in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CrescGN). The reparative and pathogenic roles of PECs seem to represent two sides of PEC behavior directed by the local milieu and mediators. Recent studies suggest microRNA-193a (miR193a) is involved in the pathogenesis of FSGS and CrescGN. In a mouse model of primary FSGS, the induction of miR193a caused the downregulation of Wilms’ tumor protein, leading to the dedifferentiation of podocytes. On the other hand, the inhibition of miR193a resulted in reduced crescent lesions in a mouse model of CrescGN. Interestingly, in vitro studies report that the downregulation of miR193a induces trans-differentiation of PECs into a podocyte phenotype. This narrative review highlights the critical role of PEC behavior in health and during disease and its modulation by miR193a. Parietal epithelial cells (PECs) are resident kidney cells that line the inner Bowman’s capsule . They orThis narrative highlights the evidence of PEC behavior in glomerular diseases (FSGS and CrescGN) and its modulation by miR193a.PECs are attenuated squamous cells with a condensed nucleus and express specific markers such as paired box gene (PAX) 2, PAX8, annexin A3, Claudin-1/2/16, and Ubiquitin C-terminal hydrolase-L1 (UCH-L1) .Bariety et al.  used theKuppe et al.  have desPodocytes are lost in the urine under normal physiological conditions . The conPECs and podocytes share a common progenitor . Puellesl et al.  observedl et al. . Berger l et al.  showed rPodocyte regeneration was not observed in aging mice kidneys, which showed progressive loss of podocytes and the development of glomerulosclerosis . SimilarIn a mouse model of partial acute podocyte depletion, genetically labeled PECs were observed to give rise to differentiated cells resembling podocytes after four weeks . This fiWhile Eng and colleagues ,32 proviThe source of podocyte renewal during the recovery of human glomerular diseases is not confirmed to be PECs. Therefore, evidence on the reparative role of PECs after podocyte injury remains controversial. The PECs respond rapidly to extensive podocyte loss by activating themselves to migrate and secrete an extracellular matrix to seal the injured GBM. In an experimental mouse model, the severity of podocyte loss was correlated with the degree of proliferation of PECs . AlthougThe degree of PEC proliferation correlates with the severity of podocyte injury . The masCrescGN is characterized by extracapillary proliferation in the Bowman’s space in response to a breach in GBM and the resultant flow of inflammatory mediators, such as immune cells and phlogogenic plasma . ActivatAlthough activated PECs in both human FSGS and CrescGN show a similar staining pattern, CD44, claudin-1, CD24, and CD133 , the manPEC activation follows direct podocyte injury in FSGS and indirect podocyte loss in CrescGN ,36. MultMicroRNAs are small (approximately 20–25 nucleotides) non-coding RNAs with a predominant inhibitory role in gene expression as they destabilize messenger RNAs . MicroRNIn normal human kidneys, miR193a is predominantly expressed in PECs . miR193aIn in vitro studies, media containing vitamin D3, retinoic acid, and dexamethasone attenuated miR193a expression in PECs and reduced PEC proliferation. Similarly, the inhibition of miR193 by genetic knockdown in mice resulted in the decreased crescent formation and proteinuria in a mouse model of crescent GN . The mecWe previously showed that the downregulation of miR193a prevented podocyte dedifferentiation in a high glucose milieu  and enhaBringing together the above studies on PEC response modulation by miR193a, we summarize the following highlights:1. miR193a-induced podocyte injury is predominantly mediated through the downregulation of WT1 in podocytes, which is crucial for podocyte survival. Elevated miR193a exacerbates podocyte loss and that, in turn, causes excessive PEC activation. In addition, increased miR193a blocks PECs transitioning to podocytes. While increased miR193a in podocytes is observed in FSGS, the sequence of events leading to high miR193a is not yet known. Podocyte injury appears to drive PEC activation in both FSGS and experimental CrescGN, both of which are characterized by high miR193a in PECs limiting their transition to podocytes.2. The downregulation of miR193a  promotes the differentiation of PECs toward podocytes in vitro. The use of similar agents to downregulate miR193a, therefore, appears promising to improve podocyte renewal in diseased states, if successful strategies are employed to test such agents in clinical studies.The following research questions are unanswered at present: (i). Does miR193a decide PEC behavior in different forms of glomerular injury in vivo? (ii). What causes an increase in miR193a expression in primary FSGS and CrescGN? (iii). Does the enhancement of APOLIG0 protein expression reduce miR193a levels in vivo?While multiple aspects of the link between miR193a and PEC behavior in glomerular disease need to be explored, a crucial glaring question needs urgent attention—will the development of inhibitors of miR193a to treat kidney diseases be hindered by the promotion of cancer development in patients? Some of the future directions in this subject are discussed in PEC activation, besides podocyte loss, is a critical determinant of progressive glomerulosclerosis leading to chronic kidney disease. Activated PECs are the “pathogenic” side of PECs. On the contrary, PECs have a “reparative” role, i.e., they proliferate in response to podocyte loss and act as the source of podocyte renewal to sustain podocyte homeostasis. PECs are shown to transdifferentiate into a podocyte phenotype leading to podocyte gain during postnatal glomerular growth and to glomerular injury resolution after acute podocyte depletion in animal models. It remains to be studied if PECs are capable of postnatally replacing podocyte function after podocyte injury in humans. Therefore, research exploring the mediators upregulating the reparative role and downregulating the pathogenic role of the PECs seems promising in finding solutions to retard kidney disease progression. With the current progress in molecular research, microRNAs have gained considerable attention in regulating crucial cellular processes in common diseases. miR193a is expressed in PECs normally, probably because their phenotype is dependent on it. In vitro downregulation of miR193a leads to the loss of PEC markers and the expression of podocyte markers within PECs. High miR193a expression in PECs in experimental disease models favors their proliferation, and the inhibition of miR193a reduces PEC proliferation in vitro. The downregulation of miR193a and/or its downstream pathways might result in podocyte renewal and the prevention of progressive glomerulosclerosis. The modulation of the APOL1G0 and miR193a axis in the podocytes and PECs improves podocyte renewal from progenitor PECs in vitro. Further studies evaluating miR193a modulation on PEC behavior in vivo are needed to confirm the association in different forms of glomerular injury.


Nano-agents like gold nanoparticles, nano-diamonds, and carbon nanotubes work as excellent drug carriers while helping in intracellular tracking of delivered agents. To highlight the application of nanomaterials in therapy and diagnostic imaging (theragnostic), we invited articles related to this topic from established investigators. We received a total of seven manuscripts, including four original research manuscripts and three review articles, among of which six of them that were related to nanomaterials in theranostic applications were accepted and assembled as a special issue.Nanomedicine has been a growing field over the last couple of decades, and the development of new platforms for improving disease diagnosis, therapies, and drug formulations has been constantly evolving. The rapid clinical translation of the current Covid-19 mRNA vaccines is also in part due to the application of nanotechnology mediated gene delivery. Nanomaterials, while helping to develop targeted delivery of therapeutics, also serve as imaging agents/contrasts for monitoring the disease status Helichrysum odoratissimum as a potential nanoformulation for effective inhibition of Cutibacterium acnes, a commensal that causes acne in human skin, adhesion without showing any antibacterial activity . This serves as a useful formulation for preventing bacterial disease, and at the same time overcomes the possibility of developing a drug resistant bacterium, a common problem associated with the use of antibiotics for treating bacterial diseases. Similarly, Naqvi et al., has developed a polyethyleneimine (PEI) stabilized human serum albumin (HSA) nanoparticles for simultaneous delivery of a small molecule antioxidant sulforaphane (SF) and an antioxidant responsive SOD1 gene for improving the antioxidant property of cells . This type of delivery application will have a significant impact on the viability of cells in cell transplantation therapy, especially stem cell transplantation in cardiac therapy and β-cell transplantation in diabetes, by improving the survival of cells by enhancing the intracellular antioxidants level. In the same line of health care associated nanomaterial applications, the research manuscript by Zhang et al., has shown the preparation of a three-layered smart dressing biomimetic nanofiber membrane integrated with a microenvironment sensor . This novel dressing material for wound healing applications was prepared by cross-linking β-cyclodextrin-containing gelatin methacryloyl (GelMA + β-cd) within hydrogel. The integrated hydrogel in this biomimetic nanofiber helped wound healing by increasing the expression of vascular endothelial growth factor (VEGF) through hypoxia-inducible factor-1α (HIF-1α) to promote neovascularization.As shown in the figure , the oriIn general, nanomaterials synthesized at different conditions can result in particles with different shapes and sizes that are responsible for their distinct physical and chemical properties, which prove beneficial in a wide variety of applications . MultifuTang et al.). This system enhanced angiogenesis and osteogenesis by regulating macrophage recruitment and its polarization via Piezo1/YAP signaling axis. The use of this material in an orthopedic surgery resulted in recruiting M2 macrophages to the implant site and promoted angiogenesis. In addition, it also promoted osteogenic differentiation through increasing PDGF-BB and BMP-2 secretions and modulating Piezo1/YAP signaling via macrophage polarization and related cytokines levels. While the above four research manuscripts highlighted the importance of nanomaterials in healthcare related applications by research results, two other manuscripts extensively reviewed the nanotechnology in theranostic applications. One manuscript extensively discussed the treatment and imaging of Parkinson’s disease. The other manuscript discussed recent advances in exosomes; small nanosized vesicles secreted by the cells as their natural communication system, for drug and small RNA delivery applications in different diseases, including cancer.Three-dimensional (3D) printing is currently an emerging technology for making nanomaterial-based health care devices. Recently, 3D-printed microneedles were used for intradermal delivery of antiviral vaccines to achieve strong immune responses and for insulin delivery to achieve long-term functional effect. In this line, the manuscript by Tang et al., adapted a 3D printing using Ti2448 alloys for printing low stiffness plates for bone healing applications . This manuscript addressed the mechanism of disease development, and the list of diagnostics, therapeutics, and imaging strategies available for targeting PD using both preclinical and clinical imaging modalities. This review also briefly discussed the BBB related issues in therapeutic deliveries, and how antibody conjugated nanoparticles (SPION) can improve delivery across BBB.Parkinson’s disease (PD) is a common age-related neurodegenerative disorder. A progressive growth of patients with PD is increasing in several developed countries, leading to a tremendous healthcare burden. The aggregation of α-synuclein in the brain is the main cause for the neuronal dysfunction in this disease. There are no successful therapies available, and there are delivery- and toxicity-associated problems that limit the curative application of existing drugs. The blood brain barrier also postulates another major challenge in delivering effective doses of drugs for treating this disease. Nanotechnology, overcoming many of these barriers, has been explored in many drug delivery applications. Recently, several theranostic agents have been developed for treating the disease while monitoring their delivery by imaging. In this regard, a review manuscript by Zoey et al., has provided information on the use of various nanomaterials for theranostic applications in early diagnosis and targeting α-synuclein for improving cognitive functions in patients with PD . Exosomes are nanovesicles  secreted by cells loaded with cellular cargos as a natural communication system between cells. Based on the source cells, the loaded cellular cargos can be highly variable, which determine the biological properties of the exosomes. Recently, exosomes from various cells have been extensively evaluated for their biological functions in various diseases and applications in drug deliveries. Overall, this special issue provides a consolidated knowledge on the theranostic value of various nanomaterials and the associated technologies in healthcare applications. Although, nanotechnology has been extensively studied, the limited success of applications in the clinic is mainly due to the immune system and the associated adverse effect. Hence, recently a large amount of focus has been diverted towards soft nanomaterials (polymer nanoparticles and cell membrane vesicles), which possess flexibility to engineer to overcome immune system by immune evasion, and the exosomes with their inherent biomimetic effect. The development of biomimetic theranostic nanoparticles with selective immunomodulatory property will have tremendous role in various healthcare applications in the future.Finally, a review manuscript by Chen et al., extensively discussed the story around exosomes, a small membrane vesicle secreted by cells. This review mainly discusses about the biogenesis, biological functions, and loading therapeutic cargos and their applications in various diseases using exosomes (


Intrinsically disorderedproteins are ubiquitous throughout allknown proteomes, playing essential roles in all aspects of cellularand extracellular biochemistry. To understand their function, it isnecessary to determine their structural and dynamic behavior and todescribe the physical chemistry of their interaction trajectories.Nuclear magnetic resonance is perfectly adapted to this task, providingensemble averaged structural and dynamic parameters that report oneach assigned resonance in the molecule, unveiling otherwise inaccessibleinsight into the reaction kinetics and thermodynamics that are essentialfor function. In this review, we describe recent applications of NMR-basedapproaches to understanding the conformational energy landscape, thenature and time scales of local and long-range dynamics and how theydepend on the environment, even in the cell. Finally, we illustratethe ability of NMR to uncover the mechanistic basis of functionaldisordered molecular assemblies that are important for human health. IDPsthus access a vast conformational landscape that nevertheless encodesspecific biological activity.6 This conformationalheterogeneity endows IDPs with considerable advantages over theirfolded counterparts, for example, the ability to interact with multiplepartners, possibly simultaneously as in the case of hub-proteins.Combining transient and local disorder-to-order transitions with rapiddissociation rates allows efficient processing and provides the necessarylevel of multivalent, weak intermolecular binding to transiently formmembraneless organelles7 (another phenomenonwhose importance has revised our understanding of cell regulationand function). In general, although the potential benefits of conformationaldisorder are quite well discussed in the literature, we are stilldiscovering the true breadth of functional diversity encoded in IDPs.Unexpected discoveries regularly revolutionize our understandingof molecular biology. The remarkable observation that intrinsicallydisordered proteins are prevalent throughout all known proteomes representsone such example, forcing a reassessment of established approachesfor investigating biological function at the molecular level.9 terahertz,10 neutron,11 dielectric12 Mössbauer,13 and Raman14 spectroscopies can be usedto describe the characteristic time scales of protein motions. Time-resolvedX-ray diffraction techniques15 and X-rayfree electron lasers16 also provide simultaneousaccess to both high resolution structure and dynamics. Within thebroad panoply of physical techniques available to characterize biomoleculardynamics, nuclear magnetic resonance (NMR) spectroscopy occupies aunique place, providing atomic resolution information over an incrediblybroad range of motional time scales extending from tens of picosecondsto hours or even days .32 Progress in this direction has focused on the use of extensive explorationof conformational space, using for example stochastic sampling ofthe available degrees of freedom, and subsequent identification ofcombinations of conformers that when assembled into representativeensembles agree with experimental data and can describe the contoursof the Boltzmann ensemble.37 The success of such approaches is predicated on the ability to accuratelycalculate the expected value of experimental data for a given conformationor conformational sampling regime. The same end can be achieved viaensemble restrained molecular dynamics simulation,41 for example, by including experimentaldata into the force field via a target function applied over the entireensemble.51 The amount of detail concerning the conformational sampling of IDPsin solution that can be derived from all of these ensemble approachesdepends of course heavily on the extent of experimental data available.52Flexibility and dynamics not only define the physicalnature butalso the biological function of IDPs, and the two major challengesfacing interpretation of experimental data from IDPs are related tothese characteristics. The first concerns the accurate descriptionof the conformational space sampled by the protein. NMR reports ona population-weighted average over the ensemble of interconvertingstates sampled at equilibrium so that as long as the exchange ratesare fast on an NMR time scale, conformation-dependent parameters,such as chemical shift or scalar and dipolar couplings, report oninterconversion between a potentially immense number of conformers.In practice for NMR studies of proteins using 53 sensitive to local conformationaldynamics such as infrared,55 Raman,56 or neutron spectroscopy59 or to long-range interactionsusing single molecule fluorescence,69 electron paramagnetic resonance,72 and NMR paramagneticrelaxation spectroscopies,79 but by far the most powerful technique is the use of NMR spin relaxation.The advantage of the fast exchange regime,reporting on a population-weightedaverage over an ensemble of states that interconvert on time scalefaster than 100 μs, also highlights its key limitation thatmore precise information about the associated motional time scalesis not explicitly contained in this average. Knowledge of the timescales of diffusion and chain dynamics, of interconversion rates betweenlocally structured binding-competent and incompetent substates, andof transient contacts relating the conformational properties of distantregions of IDPs will all play an essential role in developing a deeperunderstanding of IDP reaction kinetics and thermodynamics. Understandingthe dynamic properties of IDPs complements Cartesian descriptionsof their exploration of conformational space, providing a new andessential dimension to our description of their functional behavior.In response to this challenge, time scales of conformational rearrangementsof IDPs have been investigated using a vast range of experimentaltechniques,85 have always accompanied advances in our understanding of the interpretationof NMR relaxation in terms of global and local molecular motions,demonstrating the synergy between these two atomic resolution techniques.Indeed, 15N and 13C NMR relaxation data haveoften been used to test and benchmark MD force fields and algorithms,91 establishing the accuracy of dynamic trajectories of soluble, foldedproteins.NMR spin relaxation probes the angular correlation functions ofrelaxation active mechanisms, typically dipole–dipole interactionsbetween neighboring nuclei, arising due to reorientation processesof macromolecules on time scales ranging from 10s of picoseconds to10s of nanoseconds or even slower. These time scales are also readilyaccessible to atomistic molecular dynamics (MD) simulation of fullysolvated proteins, rendering the combination of MD and NMR extremelypowerful. Advances in molecular simulation, in terms of accuracy offorce-fields or sampling of slower dynamic time scales,15N spin relaxation provides a remarkablysensitiveprobe of the motional time scales exhibited by IDPs, characterizingthe dynamic properties of bond vectors throughout the length of theunfolded protein.92 The physical interpretationof the dynamic time scales contributing to the quenching of the angularcorrelation function is however less straightforward than in the caseof folded proteins. The amount of information that can be extractedfrom spin relaxation is also limited by the efficiency with whichfast large-scale motions quench the angular correlation function. 15N spin relaxation measurements in unfolded proteins havenevertheless been measured extensively, leading to the detection ofextensive pico- and nanosecond motions, as well as clear correlationsbetween motional time scales and structural propensities detectedfrom chemical shifts and scalar and dipolar couplings.114118 Measured relaxation rates report on population-weighted averagesso that accurate simulation should account for fast motions occurringover the ensemble of states sampled by the protein. The value of relaxationrates associated with each substate depends on the nature of thisconformation, so that in principle it would be necessary to simulateeach of the substates and average the individual rates as a functionof their populations, or to simulate sufficiently long trajectoriesto sample all individual states. In the case of globular proteins,the identification and simulation of distinct conformational substatesthat are in fast exchange on the chemical shift time scale but thatexhibit distinct fast reorientational properties have indeed beenshown to significantly improve the description of the ensemble offast motions, as measured by the reproduction of experimental 15N relaxation rates.119 This demonstratesthe improved accuracy of dynamic information when considering theentire free-energy surface but also the interdependence of fast andslower motions in proteins. For IDPs, this potential interdependencehas an even greater importance and underlines the relevance of adequatesampling of the ensemble of conformational states.120Further insight into the actual physical origin of the motionalmodes and time scales giving rise to NMR spin relaxation can againbe derived from the combination of MD simulation with spin relaxationmeasurements.123 a more general application of these techniqueshas been hindered by the inability of state-of-the-art force fieldsto describe the dynamics of IDPs with acceptable accuracy.125 While the degrees of conformationalfreedom available to internuclear covalent bonds present in foldedproteins are mainly dictated by the immediate environment, and thereforeintraprotein interactions, for IDPs the solvent protein interactionstake on a far greater importance, so that an imbalance between potentialenergy terms reporting on protein–protein and protein–solventinteractions124 may result in inaccuratekinetic and thermodynamic behavior. The resolution of this question,and the development of force fields that can describe both foldedand unfolded proteins with equal accuracy,126 remains an important challenge.130 The availability of accurate and calibrated NMR relaxation ratesfrom proteins with well-described conformational behavior will undoubtedlycontribute to this important task.Despite major progress in the simulation of highlyflexible orunfolded proteins,136Beyond the fast exchangeregime, NMR relaxation experiments nolonger represent a population-weighted average of the reorientationalproperties of the exchanging species but report on motions occurringon time scales defined by the difference in chemical shifts of theexchanging subspecies, in the range of micro to milliseconds. In thisregime, NMR exchange spectroscopy is particularly powerful way toprobe the molecular mechanisms underlying the exchange contributions,providing information on the thermodynamics, free-energy landscape,and kinetics of the interconversion between the species.Finally, our understanding of the functional modes adoptedby IDPsis enriched by every physiologically relevant complex that is characterizedexperimentally. The functional interactome of IDPs is vast and potentiallyhighly diverse, and our experimental sampling of the interaction modesemployed by IDPs remains extremely punctual. Although specific modelsystems that are experimentally well-characterized provide usefulbench-marks, insight into the true diversity of the IDP interactomerequires more sampling, of more diverse systems, at atomic resolution.Exchange NMR, whether fast, intermediate, or slow, provides powerfultools to deliver this essential insight.The aim of this reviewis to describe recent developments of NMR-basedapproaches to understand the conformational dynamic behavior of IDPsin physiological, and even cellular environments, and to illustratethe insight that NMR offers to reveal the mechanistic basis of functionaldisordered assemblies that are important for human health. Part ofthe power of NMR spectroscopy lies in the use of combinatorial approacheswith structural techniques such as cryoEM and X-ray diffraction thatprovide the structural context within which the functional role ofIDRs can be best understood. Examples will also be shown of the abilityof NMR to characterize large-scale dynamics of complex biomolecularassemblies comprising highly disordered elements.2139 Characteristics such as charge and hydrophobicitydistribution of IDPs have been interpreted in terms of their rolein controlling physical parameters, for example, compactness and extendedness,140 and the ability of IDPs to participate in multivalentinteractions.144 Similarly, regulation of these degrees of freedom can be achievedby post-translationally modifying the chemical nature of the chain.148An accurate understanding of the conformational propertiesof IDPs,and intrinsically disordered regions (IDRs) of multidomain proteins,is of primordial importance. The dynamic behavior of IDPs is definedby the amino acid sequence, and the ability of the protein to interactvia, for example linear motifs, is encoded and controlled by the intrinsicconformational sampling. In addition, IDRs, often linking folded domains,define the free-energy landscape of the protein, providing the degreesof conformational freedom of the entire molecular assembly.Two recent studies described herein illustrate the importanceofa detailed consideration of the averaging properties of differentexperimental data types to understand the conformational nature ofIDRs. In particular, the combination of long-range and local transientstructure poses specific challenges to the analysis of disorderedproteins in terms of representative ensembles, and certain pitfallsmust be avoided to extract accurate structural information.79 Residual dipolar couplings (RDCs) depend on the average of the orientationsof the internuclear vector (I–S) with respect to the magnetic field,KIS describes physical constantssuch as the gyromagnetic ratioand the internuclear distance, and P2 (x) = (3x2 – 1)/2. Ina molecule of fixed shape, we can expand this average,αk refers to the orientation of the internuclearvectorwith respect to a traceless second rank tensor S thatdescribes the alignment properties of the molecule.Chemical shifts and scalar couplings present two important featuresthat directly impact their interpretation. First, they depend primarilyon the local structural environment of the observed spin, and second,if interconversion between the different states is much faster thanthe difference between the expectation values of the different statesin isolation, the measured NMR spectrum represents a weighted averageof the ensemble of states. Conversely, parameters whose experimentalvalues depend on time-dependent interactions, such as paramagneticrelaxation for example, require a more detailed consideration of theaveraging properties, as has been discussed.S can clearly vary significantlyover the ensemble such that proteins of different shape, and thereforedifferent alignment properties, but identical local sampling, wouldgive rise to very different RDCs:149 This reiterates the long-held observation thatto accurately describe local and long-range conformational sampling,it is necessary to respect both of these contributions to the averageover the ensemble of states.150In highlyflexible proteins, 152 and a genetic algorithm, to select representative subensembles ofconformers that in combination are in agreement with the experimentaldata. The sampling of the prior database is modified iteratively untilconvergence is achieved within the estimated uncertainty.37Theimportance of considering long-range order in the interpretationof RDCs was also illustrated in a recent study of the δ domainof RNA polymerase (δ−RNAP), where multiple NMR parametersand small angle scattering data were combined using the ensemble selectionapproach, ASTEROIDS, to compare the free energy landscape of differentforms of the protein. ASTEROIDS uses extensive conformational samplingdescribed in an initial prior database, broadly sampling amino-acidspecific statistical-coil distribution for the unfolded chain,153 The IDR is locally highly charged, with mainly acidic butalso basic stretches of amino acids. As in the case of a number ofacidic disordered domains in RNA-polymerase machinery, the acidicsequence has been suggested as an RNA mimic.154In the case of δ−RNAP, the90 amino acid C-terminalIDR follows the similarly sized folded domain.13C, 15N, and 1H backbone chemical shifts, paramagnetic relaxation enhancements(PREs), residual dipolar couplings (RDCs), and small-angle X-ray scatteringdata. PREs provide clear evidence of transient long-range order inthe IDP, with apparent contacts between regions exhibiting oppositecharges , and these RDCs were only correctly predictedwhen the long-range contact identified from the PREs was includedin the analysis.Experimental data used to describe the conformational samplingof the IDR included charges 2.155 Ana156 Interestingly, the loss oflong-range contacts induced by mutagenesis influences cell fitnessand transcription efficiency in vitro. While thecomplete knockout of the delta subunit makes transcription too fastand insensitive to regulation by initiating nucleoside triphosphates,the mutation disrupting long-range contacts has the opposite effect:it inhibits transcription from promoters that form unstable complexeswith RNA polymerase.Mutation of the cluster of basicamino acids to acidic residuesabrogates the long-range contacts, resulting in extinction of thecharacteristic PRE- and SAXS-derived evidence of compaction in thewild type protein, revealing a highly extended IDR in the absenceof the basic cluster, and a disappearance of the characteristic long-rangeRDC modulation. The combined analysis thus results in an accurate,integrated description of the ensemble of states sampled by both wild-typeand mutant protein in solution, providing insight into the impactof the electrostatic charge distribution on local and long-range conformationalbehavior.33.1158 The angular reorientation of such interactions can be describedin the time domain (correlation function C(τ)) or the frequency domain ). Protein backbonedynamics are typically characterized in solution using longitudinal(R1) and transverse (R2) autocorrelated 15N relaxation rates, heteronuclear 1H–15N cross-relaxation, and 15N longitudinal (ηz) and transverse (ηxy) cross-correlated dipole–dipole/CSA cross-relaxation (σNH).92 Theadvantage of measuring different rates lies in their distinct dependenceon different combinations of the angular spectral density functionat the characteristic Larmor frequencies defined by the spin system,ωN, ωH, ωH ±ωN.As introduced earlier, NMR relaxation occurs dueto angular fluctuations of relaxation-active interactions resultingin transitions and incoherent dephasing that relax the spin stateback to equilibrium.160 using reduced spectral density mapping164 to estimate J(0), J(ωN) and an approximate mean value at high frequencies <J(0.87ωH)> throughout the sequence.Alternatively,the correlation function of internal motional modes can be describedanalytically, in terms of geometric and temporal parameters , although it can be difficultto differentiate between these models on the basis of NMR relaxationrates alone. A simple and popular alternative is to use the model-freeapproach, where mathematical contributions to the autocorrelationfunction are parametrized. The approach is simply understood in thecase of internal modes in a folded protein,169 where it is possible to express the angular correlation functionasCO (t) is the correlation function for globalmotion, and a faster internal contribution, that is not associatedwith a specific motional mode, describes restricted motion relativeto the molecular frame:If enough measurements are available, thespectral density functions can be mapped from the different relaxationratesCI(t) is approximated to a singleexponential, the associated spectral density function can be describedase’ = (τR-1 + τe-1)−1τR describesthe overall rotational diffusionand S2 is the generalized order parameter. Extension168 to two internal components with distinct correlationtimes s′ = (τR–1 + τs–1)−1, τf′ = (τR–1 + τf–1)−1.If the internal correlation function kAk = 1, andThis formalism is commonly used to interpretrelaxation measuredin folded proteins, with the global contribution to the autocorrelationand spectral density functions assumed to be common for all sites.Although, due to their high flexibility, IDPs are not expected toexhibit a shared diffusion tensor for distinct regions in the chain,the same mathematical formalism can be used to model the spectraldensity functions of each site independently, assuming that the timescales of the component modes are sufficiently separated, and thatall the motions are isotropic:173 Alternatively, it is possible to describe the spectral density functionin terms of an analytical distribution of motions, of which the model-freeapproach represents one of the simplest manifestations.174 Here again, the complexity of the models makes differentiation difficult,although they have been successfully used to explain the dynamic behaviorof synthetic homopolymers,175 and surelyprovide a more physical representation of the complex dynamics offlexible proteins.103This formalism hasbeen diversely exploited for the interpretationof relaxation from partially denatured proteins and IDPs.114 rapidly quenching angular correlation and reducing the slowest sensitivetime scales to the nanosecond range (at room temperature and in freesolution). Nevertheless, the existence of segmental motions was suggestedfrom the bell-shaped dependence of transverse relaxation components, in chemically denatured and intrinsically disordered proteins,104 relating to stiffness or side chain bulkiness,176 and from 1H relaxometry.177 IDRs connected to folded domains have been shown to induce slowercomponents on the rotational diffusion properties of multidomain proteinsindicating the importance of local viscosity and drag on dynamic timescales.181 Faster time scales are expected to relate to more local dynamics,for example, of backbone dihedral angles, which may be important interms of local folding or binding;192 however, in general the physical origin of observed relaxation ratesremains weakly characterized.In highly dynamicmolecules such as IDPs, large-amplitude motionsoccur in the range of nanoseconds,3.2193 used an array of fixed correlation times (τk), distributed on alogarithmic scale, with variable amplitudes (Ak), that could also be zero, to analyze thespectral density function from 1H frequencies. This providesa grid of motional amplitudes corresponding to six characteristiccorrelation times for the entire protein, clearly delineating thefolded and unfolded domains, and revealing dominant time scales around1 ns in the unfolded domain.It is clear from 194 also studied the dynamics of a partly unfolded protein,the basic leucine-zipper region of GCN4. In this case, 15N R1, R2,and σNH, measuredat 600, 700, 800, and 900 MHz 1H frequency were analyzedby rearranging the measured relaxation rates using a modified spectraldensity mapping, and comparing these results to a model free analysisusing Gill et al.15N R1, R2, σNH, ηz, and ηxy measured at four magnetic field strengths. In a first step,autocorrelated and cross-correlated rates measured at each field strengthwere analyzed using reduced spectral density mapping at each magneticfield strength, confirming the self-consistency of the data, and theabsence of exchange contributions to R2. The data were then analyzed using 1 and θ), 4, 5 , or 6  parameters for all sites in themolecule, corresponding to 1, 2, or 3 contributions to the relaxation-activecorrelation function. The 3-component model was found to be justifiedthroughout the protein. Second, 10% of all data were removed fromeach data set, and their values predicted from the parameters determinedfrom the remaining data sets, again demonstrating that 3 componentsare essential to correctly predict experimental values. This impliesthat sufficient relaxation data have been measured to justify themore complex model.A similar study of the dynamic behavior of the 126amino acid C-terminaldisordered domain of Sendai virus nucleoprotein (NT), examined 197 and in cellulo and over a large range of temperatures (268–298K) :The study of NT, a disordered proteincontaining a short helical linear motif was extended to measure 8–298K) 3A.199 UpEk,a .Fitting to this function requires the determination of parametersdefining the relative amplitude of the three components at each temperatureand the effective temperature coefficients of the intermediate andslowest contribution (the fastest contribution around 50 ps showsinsignificant temperature dependence). Again, cross-validation byremoval of either 10% of all data, or data from each magnetic field,indicates that the analysis is satisfactorily overdetermined. It isworth pointing out that predictive cross-validation is not so commonin analysis of protein dynamics from NMR spin-relaxation but whenapplied shows a reassuring level of confidence in the data analysis.199The different temperature dependences of the three components aredescribed by temperature coefficients, or activation energies givenby τ3 with temperature, exhibits a smooth functionalong the sequence, reaching a maximum (20–25 kJ mol–1) in the center of the sequence. It was proposed that the slowestcontribution reports on chain or segmental dynamics. The reason thatslower motions are detected in the helix is that C(t) is not as efficiently quenched by the high amplitudefast motions occurring in the remaining unfolded part of the chain.The residual order left after the more restricted fast motions occurringin the helix allow for the detection of slower motion that has littleeffect on correlation functions from the less-structured parts ofthe chain. This is further supported by the analysis of data measuredusing protein constructs engineered to comprise 50, 75, or 126 aminoacids, revealing a clear dependence of the τ3 on the length of the peptide chain, as expected forchain dynamics considered using Rouse or Zimm models.203The simultaneous analysis of data fromall five temperatures 3B–F reve205 In this case, there is a discontinuity in activation energy betweenthe unfolded and helical regions, motivating the suggestion that thesecontributions report respectively on local fluctuations within Ramachandranwells and constrained internal dynamics or partial unfolding in thehelix.120The intermediate motion has a much flatter distribution overtheunfolded regions, and the apparent activation energies are in therange expected from studies of peptide backbone free energy landscapes.Although relaxation in IDPs is often thoughtto provide informationessentially concerning subnanosecond motions, the analysis shown hereclearly demonstrated that short, structured motifs in unfolded polymersare also dependent on slower, segmental or chain-like motions, orwhatever other motion finally quenches the angular correlation function.Most regions are not sensitive to these motions because of the extentof the faster motions, but if one can locally quench these, a greatdeal of insight can be derived from the resulting relaxation rates.We note that while the contribution of the slowest motion increasesat lower temperatures, as the fastest motion falls, the amplitudeof the intermediate motion systematically passes through a maximumat 288 K. This may provide us with information about the shape ofthe actual distribution of correlation times and their impact on thesampled correlation function.4.2209 This question is particularly relevant with respect to NMR in cellulo,216 where IDPs function in environments with molecular concentrationsreaching 400 g/L,219 very likely strongly affecting the timescales of IDP dynamics.222 The effect of local environmenton IDP function is also relevant for understanding the mechanisticrole of IDPs in membraneless organelles.225 IDPs are subjected to extreme solvent accessibility compared tofolded proteins, suggesting that the physiological environment incomplex multicomponent environments will very likely strongly influencedynamic modes and time scales. Single molecule fluorescence techniqueshave provided unique insight into the importance of so-called internaland solvent friction on IDP dynamics and partially folded or destabilizedprotein states as well as on the kinetics of protein folding.227 These approaches have been usedto investigate the dynamics of IDPs229 and proteinfunction230 in the cellular environment.Although significant progress has thusbeen made over recent years in our understanding of the informationprovided by NMR relaxation studies of IDPs, it remained unclear howto interpret data measured in more complex, and more specificallyin the more crowded, physiological environments in which they function.in cellulo, indicating changes in both population and exchangerates as a function of the cellular milieu, and a dependence on weak,so-called quinary231 interactions betweenthe protein of interest and diverse other molecules constituting theintracellular matrix.234 NMR was also used to describe the impactof the cellular milieu on protein dynamics, from small globular proteinsto IDPs.238 In a detailed study, Theillet and co-workers compared the influenceof different viscogens on the dynamics of α-synuclein, with 15N relaxation measurements made in mammalian cells, revealingchanges in dynamics of the termini of the protein, presumably associatedwith crowding-induced compaction or inter- and intramolecular interactions.The extent of changes appeared to be more pronounced in cellulo, suggesting additional impact of intermolecular interactions onthe relative deceleration of the NH-backbone fluctuations.198 In the context of these examples, and the growingbody of experimental data,244 a physical framework that incorporates the effects of molecularcrowding on the dynamics of the protein would provide a welcome toolallowing quantitative interpretation of NMR relaxation measured underphysiological conditions.Similarly, NMR spectroscopy has been used to investigate modulationof the folding/unfolding equilibrium of globular proteins 207 This calibrationallowed the dynamics of two IDPs to be mapped as a function of environmentalconditions, including both viscosity and temperature. The two IDPsexhibit distinct physical properties, comprising both partially foldedand highly flexible elements. Local, or nanoviscosity was gauged bymeasuring 1H longitudinal relaxation of water,157 which, at the high magnetic fields used here,is expected to be dominated by rotational diffusion of the water molecules.247 The overall dependences of the nanoviscosity of the solvent andsolute on the concentration of viscogen show similar features, withthe intermediate and slow correlation times of the backbone of theprotein, and the 1H R1 both deviating from thelinear regime in the range of 200 mg/mL , suggesting that the observation may be general. The observeddifferences in effective friction coefficients may be related to observationsmade by Schuler and co-workers that translational diffusion slowsdown considerably more than rotational diffusion of the IDP prothymosinα inside crowded cells, suggesting very different length scalesand susceptibilities to crowding.229Recent work further addressed thischallenge by measuring dynamicsof IDPs as a function of environmental complexity. An extensive setof multifield NMR relaxation rates were measured over a broad rangeof conditions, using inert crowding agents to systematically modifyviscosity, as well as temperature 4.207 Thi00 mg/mL 4. Nevertτk) to the nanoviscosity of the solvent:C) = (ηC – η0)/η0 = /R1,0, and R1,0 andη0 are the longitudinal relaxation rate of waterand the viscosity in the absence of viscogen, R1,C is the longitudinal relaxation rate, ηC is the viscosity, and τ′k,∞ is a prefactorrepresenting the correlation time at infinite dilution and temperature. εk is the residue-specificfriction coefficient relative to ηC of intermediate or slow motions. The model turnsout to be robust and remarkably transferable in vitro. For example, once sequence-specific friction coefficients havebeen determined as a function of concentration for a particular protein,highly sensitive dynamic probes such as a complete set of 15N relaxation rates measured in very different crowding conditionsare predicted with very high accuracy, simply on the basis of themeasurement of the water R1 . The problem of reproducing experimentalrelaxation rates from IDPs using MD simulation is illustrated in 258Spin relaxation remains the most powerful NMR observable tocharacterize dynamic time scales at a sequence specific level, andreproduction of experimental values is often the most challengingfor MD simulation. As described earlier, assuming conformational exchangethat is fast on the chemical shift (and relaxation rate) time scale,experimentally observed rates derive from a population-weighted averageover individual relaxation occurring within the different states sampledup to the micro- to millisecond range, such that ⟨τeff ≪ tmax,259 where tmax is themaximal sampled time of the angular correlation function. To addressthis problem, the following procedure was adopted: The entire trajectory,or multiple distinct trajectories nucleated from different conformations,are divided into subtrajectories of 100 ns, from which correlationfunctions Ci(τ) (and rates Ri) are calculated and combined in an ensemble average that explicitlymimics the actual heterogeneous conformational origin of the measuredrelaxation. The maximum length of each subtrajectory is dictated accordingto the experimental analysis described above for the studies of twoIDPs, NT and MKK4. At T = 298 K, the slowest contributionto the rotational correlation function detected by experimental spinrelaxation (see above) is approximately 5 ns, so that the dynamicreorientations occurring in each distinct substate can be reasonablysampled using a sampling window of 100 ns (tmax= 50 ns). The ABSURD (average block selection using relaxationdata) approach then estimates the relative weights or segments of Ci(τ)with respect to a single experimental relaxation rate, compiling anensemble of subtrajectories that interchange on time scales significantlyslower than the correlation time limit (100 ns) and significantlyfaster than the chemical shift time scale (100s of μs).125 In this way, a representative ensemble of time-dependenttrajectories is identified, thereby extending the concept of conformationallyaveraged ensemble-descriptions into the time dimension. Optimizationagainst a unique relaxation rate at a single field identifies an ensembleof trajectories that systematically improves agreement with a broadset of rates, sensitive to motions occurring on a range of time scales  and global (SAXS) conformational sampling properties.Analysis of these trajectories indicates that theorigin of thediscrepancy derived from the over-representation of rare events, suchas long-range contacts, whose frequency is poorly sampled, leadingto statistical instability because the sampled correlation time doesnot fulfill the necessary criterion  fields) 6, as wel260 it is challenging to make this observation for IDPs.120The fact that the ensemble of trajectories improves reproductionof “passive” dynamic reporters highlights the importanceof correctly sampling the free energy landscape of the IDP in solution,and illustrates the complex interdependence of motions occurring ontime scales varying over many orders of magnitude. While it has previouslybeen shown that simulating motions occurring in distinct substatesimproves reproduction of relaxation in folded proteins,5.2257 This deconvolutionof the angular components allowed the identification of locally correlatedand segmental motions along the chain. The advantage of such an approachwas exemplified in a comparison of temperature dependent 15N relaxation measured on Sendai virus NT, and compared to relaxationcalculated from average correlation functions derived using differentforce fields.261 This allowed the identificationof the best force field over a range of temperatures  or slow exchange, that in principle allows forsimultaneous detection of both environments.269 Electrostatic and hydrophobicinteractions are thought to dominate these weak interactions, andthese are shown to differ largely between amino acid types. The authorsshow that these interactions can be parametrized and the binding profileof a given IDP can be accurately predicted using a simple mathematicalmodel. This method also has the considerable advantage that transverserelaxation rates are impacted by motions occurring on time scalesthat are normally difficult to access by solution state NMR, alsoproviding insight into the intrinsic dynamics of folded proteins.270The former regimehas been elegantly exploited by Brüschweileret al. to investigate the binding modes of different amino acids presentin disordered proteins by measuring the impact of aqueous colloidaldispersions of anionic silica nanoparticles on the transverse relaxationrates of IDPs.R1ρ),135 Carr–Purcell–Meiboom–Gill(CPMG) relaxation dispersion,136 chemical exchange saturation transfer (CEST),272 and zz-exchange274 provide information about exchange processes from the tens of microsecondsto the subsecond range.Beyond the fast exchange limit, intermediateexchange, occurringon time scales that are comparable to ΔΔω, leadsto line-broadening of the observable peaks 1. This l6.1182 whostudied the interaction between the KIX domain of CREB binding proteinand the phosphorylated form of kinase inducible activation domain(pKID). 15N CPMG measurements in the presence of substoichiometricadmixtures of KIX provided evidence for weak binding between pKIDand KIX, and allowed the authors to propose a model of the bindingtrajectory according to a three-site exchange model, describing bindingvia a partially folded encounter complex. This approach has been furtherexploited, using a combination of 1H, 13C, and 15N CPMG, to map the interaction trajectory of Sendai NT uponbinding to the C-terminal domain of the phosphoprotein (PX).191 While 1H and 15N amidechemical shifts are commonly used as probes to map interaction interfaces, 13C backbone chemical shifts are more sensitive to secondarystructure. 1H, 13C, and 15N CPMG,measured at substoichiometric admixtures of PX, was used to map theconformational transitions along the interaction trajectory of thepartially formed helical motif  of the germ granule protein Ddx4,discovering a slowly exchanging excited state with increased intermolecularcontacts.283To achieve adeeper understanding of the effects of crowding onthe thermodynamics and kinetics of reactions involving IDPs and theirpartners, a more detailed, residue-specific picture is required, forexample, using relaxation and exchange measurements in crowded environmentsand living cells. Kay and co-workers already performed 6.268 In thiscase, 15N R1ρ and chemical shift titrationconfirmed that the interaction was in fast exchange, allowing an estimateof the intrinsic individual dissociation constant of a single siteof around 8 mM. The presence of multiple motifs in a single proteinclearly illustrated the effect of multivalency on the apparent affinity,which decreased with increasing multivalency. Finally, assignmentof both free and bound forms of Nup153 demonstrated a complete absenceof backbone conformational transition upon binding, with the disordereddomain maintaining a high level of plasticity in the complex. On thebasis of these results, a model was proposed of rapid passage, assuredby the quasi continuum of NTR-binding sites present throughout thepore, and the fast on and off rates that are maintained by multivalentultraweak binding throughout this continuum. Related results werealso found for other nucleoporins,285 suggestingthat the mechanism may be general.It is becoming increasingly clear that not allIDPs fold upon binding to their partners, even locally. The nuclearpore is filled with proteins (FG-nucleoporins) comprising extremelylong IDRs, that are decorated with phenylalanine-glycine (FG) motifs,that control transition between the cytoplasm and the nucleoplasm.Larger proteins can only pass the filter when bound to nuclear transportreceptors (NTRs). Despite the high selectivity of the filter, transportacross the pore is extremely fast. The crucial interaction betweenNTRs and FG motifs was recently investigated using NMR, revealingweak chemical shift perturbations in the nucleoporin Nup153 in thepresence of a series of NTRs.286 Paramyxoviral phosphoproteins (P) are essentialcofactors of the replication complex: they are tetrameric and allcomprise long IDRs that are hundreds of amino acids in length andwhose function remains largely unknown.287 N has a folded domain that encapsidates the viral genome, protectingit from the host immune system, and a disordered C-terminal domain.ASTEROIDS analysis of the 304 amino acid IDR of P from MeV identifiesshort helical elements in the N-terminal domain, and an additionalfourth helix 150 amino acids downstream of this (α4), adjacent to a highly acidic strand. The N-terminal helices bindtightly to N, maintaining it in its monomeric form prior to encapsidationof the RNA genome. The 90 kDa NP complex was investigated using NMR,including over 450 intrinsically disordered residues, identifyingthe known N-terminal chaperone binding site, but also a second, previouslyunknown binding site positioned at the fourth helical element, α4  and theintrinsically disordered phosphoprotein of Measles virus (MeV).ment, α4 9. 15N CP6.3288 and positive sense293 RNA viruses exhibiting extensive disorder. A recent descriptionof the nucleoprotein of SARS-CoV-2, involved in protection of theviral genome and regulation of gene transcription, revealed that theflexible central region undergoes a disorder to order transition,folding around the N-terminal domain of its viral partner nsp3 andinducing a collapse of the remainder of the protein that impacts itsability to bind RNA.294Disordered domains are thoughtto play a role in the replication of numerous single strand RNA viruses,with components of the replication machinery from both negative15N CEST experiments, revealing open form chemical shifts thatare essentially identical to the isolated domains in free solutionand determine the exchange rate to be around 20 s–1.295 The closed form is stabilized byan interdomain salt bridge296 while inthe open form the linker connecting the two domains becomes highlydynamic and the two domains evolve freely. The host transcriptionfactor ANP32a was identified as an essential cofactor for the adaptationof the viral polymerase,297 suggestinga direct interaction between the two proteins. ANP32a has a highlyacidic, intrinsically disordered domain whose length varies betweenspecies, with the avian form containing a 33 amino acid insert, comprisinga unique hydrophobic hexapeptide and a repeat of the first 27 acidicamino acids. Somehow the absence of this insert in mammals is compensatedby a single E627 K mutation of the avian polymerase, allowing cross-speciesinfection. It was therefore important to investigate the complexesbetween these two highly flexible proteins.InfluenzaA represents another example where extreme disorder appears to playan essential role in viral function. To efficiently replicate in humancells, avian influenza polymerase undergoes host adaptation, withadaptive mutants (in particular E627 K) localized on two C-terminal(627 and NLS) domains of the PB2 polymerase subunit. This region ofthe protein shows remarkable behavior in solution, populating an equilibriumbetween open and closed conformations that can be characterized using 298 The intrinsic KD measured from the side of ANP32a is more than1 order of magnitude lower than the KD measured from the side of 627 due to the multiple interaction siteson ANP32a dispersed along the IDR visiting the same sites on 627-NLS.To characterize the dynamic ensembles, a series of eight cysteinemutants of the avian and human adapted forms of 627-NLS were made,and PREs measured on ANP32a. In the fast exchange regime, these dataprovide a sensitive map of the population-weighted proximity of thetwo proteins over the dynamic assembly and were used to develop anensemble description of the human and avian complexes using the ASTEROIDSensemble approach.Here again, theIDR mediates the interaction, with a polyvalentinteraction between the acidic tail of ANP32a and the positively chargedsurface of the 627 domain.This comparison identifies clear distinctionsbetween the bindingmodes exploited in the two complexes 10, as sh299 Fluorescencespectroscopy reveals affinities in the picomolar range, while NMRand smFRET reveal that the proteins remain dynamic within the complex,implying a high level of dynamic polyvalency and possible formationof transient ternary complexes.300 Thepresence of dynamics in the bound state of IDRs was also characterizedin two recent studies of the disordered domain of kinases MKK7,301 MKK4303 in complex with JNK1and p38α. CEST, CPMG, and spin relaxation were measured as afunction of stoichiometric ratio, suggesting that the bound stateof MKK7, and the kinase specificity regions flanking the main interactionsite of MKK4, both exhibited additional dynamics in the bound state,in the former case on the micro to millisecond time scale and thelatter on relaxation-active ps-ns time scales. Similar data were usedto investigate the interaction between Artemis and the DNA bindingdomain of ligase IV, in this case identifying a single step bindinginteraction.304It is perhaps not surprising that electrostatic interactionsinlow complexity IDPs can be responsible for highly multivalent interactions.This was clearly demonstrated by a combination of smFRET and NMR spectroscopy,together with coarse grained MD simulation, to investigate the complexbetween two IDPs, the strongly basic histone H1 and the highly negativelycharged prothymosin-α.7Over the course of this review, we have demonstrated the uniqueinsight that NMR offers concerning the structure, dynamics and interactionsof IDPs at atomic resolution not only in reduced systems comprisingisolated proteins but also in the context of more complex molecularenvironments that are relevant to physiological function. In particular,we have drawn attention to the importance of describing the ensembleand time-averaging processes that govern interpretation of NMR parameters,and the remarkable insight that this can provide concerning the functionalmodes exploited by such highly dynamic systems. The power of NMR resultsin part from analytical understanding of the ensemble and time-averagingprocesses occurring on time scales covering orders of magnitude frompico- to milliseconds that remains one of its unique advantages forstudying flexible molecules. In addition to providing unique new insightinto the relationship between protein flexibility and function, thecombination of atomic resolution characterization of essential dynamicprocesses from NMR with complementary structural and dynamic probesthat can be measured on similar sample preparations ensures an excitingfuture for NMR as an integral tool for the investigation of increasinglycomplex biological systems.