SWDs are generally associated with sudden and brief non-convulsive epileptic events mostly generating impairment of consciousness and correlating with attention and learning as well as cognitive deficits. To date, SWDs are known to arise from locally restricted imbalances of excitation and inhibition in the deep layers of the primary somatosensory cortex. SWDs propagate to the mostly GABAergic nucleus reticularis thalami (NRT) and the somatosensory thalamic nuclei that project back to the cortex, leading to the typical generalized spike and wave oscillations. Given their shared anatomical basis, SWDs have been originally considered the pathological transition of 11–16 Hz bursts of neural oscillatory activity  occurring during Non-Rapid Eye Movement (NREM) sleep, but more recent research revealed fundamental functional differences between sleep spindles and SWDs, suggesting the latter could be more closely related to the slow (<1 Hz) oscillations alternating active (Up) and silent (Down) cortical activity and concomitantly occurring during NREM. Indeed, several lines of evidence support the fact that SWDs impair sleep architecture as well as sleep/wake cycles and sleep pressure, which, in turn, affect seizure circadian frequency and distribution. Given the accumulating evidence on the role of astroglia in the field of epilepsy in the modulation of excitation and inhibition in the brain as well as on the development of aberrant synchronous network activity, we aim at pointing at putative contributions of astrocytes to the physiology of slow-wave sleep and to the pathology of SWDs. Particularly, we will address the astroglial functions known to be involved in the control of network excitability and synchronicity and so far mainly addressed in the context of convulsive seizures, namely (i) interstitial fluid homeostasis, (ii) K Epilepsy is a highly heterogeneous neurological condition characterized by enduring predisposition to unpredictable pathological discharge of rhythmic activity in the brain networks, which is commonly referred as seizure activity . In virtpetit mal seizures  as the rhythmogenic cortico-thalamo-cortical network mechanism of SWDs , 26x vivulations  since dithe SWDs  (25). Ththe SWDs –78. Morelization . AdditioAfter more than three decades of accumulating evidence, nowadays it is widely established that astroglia constitute a ubiquitous non-neuronal communication system in the brain involved in virtually every physiological and pathological scenario of the central nervous system –82. Not + concentration. Their high K+ permeability mediated by inwardly-rectifying Kir and two-pore-domain K2P channels, Na+/K+ pumps and Na+/K+/Cl− transporters associated with their extensive GJ coupling enables them to uptake and redistribute excessive extracellular K+ resulting from neuronal firing , astroglial networks provide a perfect spatial buffering for neural activity  97–99).–99.97–99l firing –103. Astgulation  as well out mice , 106. Gaout mice  as well out mice , 109 in rneurons . In vivoseizures . Interesthy rats . Neverth+ uptake is associated with cellular swelling due to Na+/K+-pump dependent water influx  and oleamide  are specific Cx43 blockers  clearance under physiological conditions, thus counteracting network priming through aberrant shifts in the E/I balance possibly leading to network synchronization. Astrocytes rely on their extensive GJ coupling enabling effective spatial ionic, osmotic, and functional buffering. GJ hemichannels as well as pannexin-1 channels may be responsible for augmented synchronous activity through ATP and glutamate release and following Ca2spreading throughout the astroglial network. So far, we are still missing evidence for linking the astroglial fine-tuning of the extracellular ion and transmitter homeostasis to SWDs. However, as it is the case for other kinds of epileptiform activity, their role in regulating such network excitability is very likely.stargazer mice, astroglial GABA transporter GAT-1 malfunction leads to increased GABA levels in the thalamus resulting in altered tonic inhibition of GABAA receptors on the thalamo-cortical neurons  released by thalamic neurons promotes phase-specific long-term depression of neuronal excitability in the NRT as well as in the thalamus itself and thus possibly contributing to thalamocortical synchronization and the altered dynamics of T-type Cac nuclei . Interesc nuclei . Moreovec nuclei –216. Thic nuclei , 218 andc nuclei , 31] NPYc nuclei . Notablyc nuclei . Since ac nuclei  and relec nuclei  NPY and c nuclei , 223, onAlterations of astroglial neurometabolic coupling and contribution to the glutamine-glutamate-GABA cycle may be at the basis of SWDs, possibly through enhanced metabolism and glutamate presentation to cortical neurons. Moreover, astroglial control of extracellular neurotransmitter level, based on the expression of glutamate and GABA transporters  and receptors (both metabotropic and ionotropic) and direct and indirect release of glutamate and GABA, plays a fundamental role in maintaining the E/I balance in the cortex, thalamus and NRT. Astroglial ATP release and subsequent adenosine production seem to have context-dependent effects on neural excitability, but generally in line with observations derived from convulsive seizures pointing at an antiepileptic and proepileptic role of A1 and A2 receptors, respectively. Shifts in the E/I toward inhibition in the thalamus  and toward excitation in the NRT and cortex have a pro-epileptic effect on SWDs. Unexpected net outcomes of pharmacological or genetic manipulation may be due to differential impact on different key nodes of the cortico-thalamo-cortical network and/or to astrocytic ability to both preserve and reverse the sign of the input signal.2+ oscillations are one of the most studied indicators of astroglial activity and information coding mechanism at the core of the astroglial signaling cascade resulting, among others, in gliotransmission   terms. In particular, research on the contribution of astroglial Ca2+ signaling in seizure phenotype has not yet provided causative links to the SWD pathophysiology. Nevertheless, in the following paragraph we include some observations that encourage further research on the topic.Intracellular Casmission . In the  signals , 225. Co balance –234. Notsy (TLE) . Moreovemination . However2+ oscillations in absence of neuronal input and induce glutamate release and NMDA-receptor mediated long lasting inward currents in thalamocortical neurons as studied in acute brain slice preparations  anti-epileptic roles of Ca2+signaling. Notably, astroglial Ca2+signaling may also contribute to seizure suppression. To which extent this applies to SWDs is still unclear.Astroglial proliferation and morphological, biochemical, and functional changes associated with epilepsy as well as with other neurodegenerative diseases are commonly referred to as reactive astrogliosis –248. Their4.1, AQP4, connexins, and glutamine synthetase as well as upregulation of adenosine kinase  reduces SWDs in WAG/Rij rats and inhibits their LPS-induced worsening  occurring during stage II NREM sleep  272, 28, 287. Thzeitgeber time (ZT) 0) and from dark to light (ZT13) altered both NREM and REM duration without affecting sleep/wake cycles and the sole seizure induction at ZT13 induced increased levels of IL-1 and increased NREM sleep specifically   as well  neurons . WAG/Rij neurons . The res neurons , 342.+ concentration during sleep  flow is responsible for waste and interstitial fluid clearance during sleep  and inwa shuttle . Sleep d shuttle  in additeceptors .In vivo characterization of Ca2+ signaling in both rat cortical astrocytes and neurons revealed that astroglial synchronized activity reliably precedes neuronal oscillations and that both astrocyte uncoupling and intracellular Ca2+ chelation reduced the fraction of astrocytes and neurons involved in the cortical slow waves. Remarkably, neurons closer to active astrocytes were more likely involved in the oscillations  and that increased activity in the thalamus specifically preceded these signals (3) phosphatase, and therefore displaying reduced IP3-dependent Ca2+ activity, spent more time in REM sleep and revealed more transitions to REM sleep from passive wakefulness  increased astroglial synaptic vesicle mobility .Impairment of astroglial exocytosis and gliotransmission using cell-specific expression of dnSNARE results in reduced tonic A1R adenosinergic signaling, altered sleep homeostasis and reduced slow-wave power, and reduced sleep pressure in mice , as confllations . Recentl signals . Convers signals . Moreoveefulness . In linearousals . RemarkaEM sleep , thus suEM sleep . Furthermobility , thus suA receptors as suggested by previous studies on sleep-deprived mice   , 399 ande (iNOS) , 401. NOe (iNOS) , 403 bute (iNOS) , thus suAstroglial homeostatic control of the extracellular space and synaptic transmission through K+clearance mediates widespread neuronal hyperpolarization and decreased firing activity during sleep and impacts NREM sleep architecture. Astroglial regulation of the extracellular volume controls both waste and cerebrospinal fluid clearance essential for correct functioning of the brain and relies on AQP4-mediated water influx and GJ coupling. Adenosine released by astrocytes governs sleep homeostasis, sleep pressure and slow-wave power and astroglial gliotransmission contributes to neuronal slow oscillations in the cortex. Moreover, artificial lowering of astroglial Ca2+oscillations and connectivity leads to reduced slow-wave power and relative shortening and fragmentation of NREM sleep. Finally, astroglial TNFα, IL-1β, and NO are likely to impact sleep architecture by selective increase of slow-wave sleep. These findings support the fact that astroglia play a fundamental role in the physiology of NREM sleep and therefore represent a promising target to study pathophysiological alterations inducing or sustaining the abnormal recruitment of the cortico-thalamo-cortical network during SWDs.From a clinical, social and human point of view, epilepsy is probably one of the most heterogeneous neurological diseases. This variability partially relies on the fact that epilepsy may originate from a plethora of different conditions, among others traumatic injury, stroke, CNS infections or inflammation, brain tumor, genetic predisposition, and drug or alcohol abuse. In addition, in six out of 10 epilepsy cases the pathological origin is unknown. Yet, from a scientific point of view, epilepsy can ultimately be reduced to a local imbalance of excitation and inhibition and altered synchrony and functioning of neural networks in the brain. The heterogeneity of pathological outcomes associated with epilepsy arises from the variety and complexity of functions carried out by the human brain and the multiple layers of fine-tuning that each of them requires for reliable physiological functioning of the electrical activity in neural circuits. Given the existence of common molecular and cellular mechanisms at the basis of epilepsy and their nature as pathological transitions of altered physiological processes, both epilepsy research and clinical treatment benefit from the understanding of the inner functioning of neural networks.Since spike and slow-wave discharges (SWDs) share some key anatomical and functional physiological brain oscillations naturally occurring during slow-wave sleep, absence seizure research could advance our understanding of both epilepsy and healthy brain mechanisms. In this review, we collected evidence supporting the functional and mechanistic relationship between slow-wave sleep and SWDs, thus providing insights into network alterations that contribute to the pathology of SWDs. Moreover, proving and characterizing the interdependency between epilepsy, sleep architecture and sleep/wake cycles possess an undeniable therapeutic value, since sleep is a pre-existing condition affecting any treatment outcome and efficacy.2+ signaling and gliotransmission as well as cytokine release are hallmarks of astroglial function for physiological brain performance and were addressed in the context of SWDs and sleep research. Please note that it is insufficient and underestimating of the system complexity to label the astrocytic contribution to neural homeostasis as exclusively anti- or pro-epileptic. Many astroglial mechanisms may be beneficial or detrimental with respect to different forms of epilepsy, not to mention different network connectivities and states. Current research on astroglial contribution to epileptic brain functioning mostly relies on studies focused on convulsive seizures, possibly due to their lower remission rate and their clinical symptoms which appear more obvious and life threatening. Nevertheless, some clues suggest the mechanisms governing network excitability and synchrony may have a role in SWDs, too. This work was not intended to be and is far from being comprehensive neither of the role of astroglia in epilepsy nor of their contribution to sleep homeostasis and architecture but provides with significant associations in the tripartite synapse engaging astroglia, epilepsy and sleep in the context of the pathophysiology of cortico-thalamo-cortical oscillations. Understanding how astroglia contribute to the mechanisms underlying slow-wave sleep and how these are altered in pathology could possibly shine light on new therapeutical targets for a plethora of epileptic forms displaying SWDs, among which absence epilepsy, a condition that still affects 50 million people worldwide and is pharmacoresistant in almost one third of those.We focused our attention on the role of astrocytes in the physiology of sleep and in their putative pathophysiological contribution to SWDs. Astroglial control on extracellular homeostasis in terms of ionic composition, volume regulation and transmitter clearance, astroglial connectivity, CaDG screened the literature, conceptualized the review focus, wrote the first draft, designed and realized the figures, and finalized the manuscript. AS and FK contributed to figure conceptualization, reviewed and finalized the manuscript. All authors approved on the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The adult human heart can only adapt to heart diseases by starting a myocardial remodeling process to compensate for the loss of functional cardiomyocytes, which ultimately develop into heart failure. In recent decades, the evolution of new strategies to regenerate the injured myocardium based on cellular reprogramming represents a revolutionary new paradigm for cardiac repair by targeting some key signaling molecules governing cardiac cell fate plasticity. While the indirect reprogramming routes require an in vitro engineered 3D tissue to be transplanted in vivo, the direct cardiac reprogramming would allow the administration of reprogramming factors directly in situ, thus holding great potential as in vivo treatment for clinical applications. In this framework, cellular reprogramming in partnership with nanotechnologies and bioengineering will offer new perspectives in the field of cardiovascular research for disease modeling, drug screening, and tissue engineering applications. In this review, we will summarize the recent progress in developing innovative therapeutic strategies based on manipulating cardiac cell fate plasticity in combination with bioengineering and nanotechnology-based approaches for targeting the failing heart. Lacking any regenerative potential , the aduIn healthy adult hearts, cardiomyocytes account for approximately 30% of all cells, whereas the remaining cell types are non-cardiomyocytes, such as immune cells, vascular endothelial cells, and fibroblasts, with the latter comprising ~11% of the total heart resident cell population . Since cMounting evidence suggests that cardiac regeneration can be mainly achieved by reprogramming non-cardiomyocytes into cardiomyocytes, differentiating pluripotent stem cells into cardiomyocytes, and re-activating the proliferation of pre-existing cardiomyocytes . These aPossible therapeutic interventions would greatly benefit from a cell-specific targeting to foster the shift from the systemic to the cellular setting, and in this context, the application of nanotechnologies for tissue engineering and drug delivery holds great potentiality. This has inspired the development of new treatment strategies aimed at the identification of crucial players involved in cell fate plasticity and their specific nano-targeting for cardiac regeneration ,7.Since the latest findings in inducing cardiomyocyte cell cycle re-entry have been already extensively reviewed elsewhere in this special issue ,9, here Since the discovery of cellular reprogramming and induced Pluripotent Stem Cells (iPSCs) generation in 2007 , novel pHuman iPSCs (hiPSCs) are similar to human embryonic stem cells (hESCs) in morphology, gene expression profile, epigenetic state, and differentiation potential, as they can differentiate into cells of all three embryonic germ layers, including cardiomyocytes . PatientAlthough hiPSCs undoubtedly represent an exceptional technology for in vitro diseases modeling, their clinical translation is still considerably hampered by the lack of scalable differentiation protocols and the potential tumorigenic activity. Conversely, the possibility to directly reprogram somatic cells into induced cardiomyocytes (iCMs) without passing through a pluripotent state, the so-called Direct Cardiac Reprogramming (DCR), has become a promising strategy to produce functional cells in vivo for therapeutic purposes, and significant progress has been made over the last decade in identifying the optimal DCR protocol as well as the underlying molecular pathways . CFs areRegardless of the reprogramming strategy, the acquisition of a new phenotype passes through the modification of gene regulatory networks of the resident cell type to fit the characteristics of the incoming target cell, in terms of cell behavior, proliferation, and metabolic rate . These siPSCs are generated by forcing the expression of specific transcription factors including OCT4, SOX2, c-MYC, and KLF4  in combiForerunner studies on animal models for MI have already demonstrated that transplantation of iPSC-derived CMs (iPSC-CMs) successfully improved the cardiac phenotype and attenuated cardiac remodeling ,31,32,33Two principal strategies have been used to promote hiPSC differentiation into cardiomyocytes. Initial protocols were based on the generation of single iPSCs suspension cultures which spontaneously aggregate and form embryoid bodies (EBs), tridimensional (3D) structures in which cells differentiated into the three germ layers mimicking embryogenesis . FollowiSubsequently, simplified procedures based on monolayer cell cultures (2D) in place of EB formations have been established. These differentiation protocols enabled the derivation of cardiac progenitor cells (CPCs) from hiPSCs, followed by a second differentiation step to promote the maturation of different cardiovascular cell subtypes ,40. The Unfortunately, differentiation approaches reported so far have limitations in generating immature CMs resembling fetal cells in their function, morphology, and electrophysiology . AdditioSeveral approaches were attempted to overcome the issue of hiPSC-CMs immaturity, such as the long-term culture ,47, the Among the methods developed to increase the maturation of iPSC-CMs, the modulation of extracellular matrix (ECM) surrounding hiPSC-CMs provided great results. One example is the so-called “matrix sandwich” method, in which confluent iPSCs are covered with a matrix of Matrigel mixed with a combination of specific growth factors and cytokines to induce cardiac differentiation , as wellDespite a large effort for protocol improvements, the main limitation of 2D cultures remains the inability to mimic the in vivo heart complexity, either in terms of spatial architecture or of multi-cellular interactions, which have been demonstrated to be crucial for CMs maturation in vivo ,59,60. TNon-scaffold-based 3D systems are typically hiPSC-derived spheroids  and hiPSThe presence of different cardiac cell types offers the opportunity to model those interactions which may be decisive to establish a pathological phenotype , making Patient-specific hiPSC-based cardiac spheroids and organoids recreate the 3D cardiac and vascular networks in which external environmental stimuli influence cardiac tissue development and maintenance. The multiple cell cultures of cardiac microvascular ECs together with human CFs in the formation of cardiac spheroids also resulted in an increased maturation of hiPSC-CMs, as demonstrated by a better tissue organization and synchronous beating of hiPSC-CMs, and formation of blood capillary-like networks . With thA recent characterization of hiPSC-CMs in tri-culture revealed improved microvasculature and increased contraction rate in 3D microtissue spheroids when compared with control 3D spheroids .3D human cardiac organoids have been used for the screening of a panel of environmental toxins, assessing organoid beating activity and viability . In anotHowever, the self-assembly process is a random procedure that results in heterogenous organoids in terms of cell composition, size, and shape, which limit the application of this method to regenerative medicine .Scaffold-based 3D systems rely on combining cells and biocompatible scaffolds to recreate a functionally native tissue, recapitulating the exact cellular composition and ECM structure. The final purpose of such tissue engineering (TE) methodologies is of high clinical relevance, as they aim at replacing the diseased heart tissue ,74. Scaffolds are responsible for the structural support of the seeded cells, while also affecting functional aspects of cell behavior  . Indeed,The excellent biological activity and the fact that many of them are naturally present in the human body make the group of natural scaffolds the most popular among the backbone used to date. However, their shortcomings are their low mechanical strength, the potential to induce immune responses, and batch-to-batch variability . SynthetA valuable 3D heart model has been generated by combining hiPSC-derived CPCs and mouse decellularized heart matrix . This stAnother approach relies on loading the desired cells into polymers to generate sheets of spontaneously beating CMs, the so-called “heart-on-chip” tissue, mimicking the human myocardium . This meCell-based cardiac patches have been designed to increase the survival ratio of the embedded cells and to ensure cellular retention. Human cardiac muscle patches have been generated by suspending hiPSC-CMs, smooth muscle cells, and ECs in a fibrin scaffold. Transplantation of this patch in a porcine model of MI resulted in significantly reduced infarct size and improved cardiac function associated with a reduction in left ventricular wall stress . Human-eVery recently, Zhu et al. presented an innovative injectable patch that will revolutionize the delivery of cardiac patches, which usually needs a traumatic open-chest surgery . They deIn recent years, 3D bio-printing has also been used to recreate functional hiPSC-based cardiac tissues ,83,84. TThe proper modeling of the myocardium should also include electrical  and mechThe combination of both electric and mechanical stimulation was even more effective in the creation of advanced hiPSC-derived cardiac microtissues . MoreoveOne of the main challenges in the use of polymeric scaffolds is that the majority of polymeric materials used for tissue engineering are electrically insulated at biologically relevant frequencies  and thusThe combination of 3D scaffolds and hiPSC technology might be useful to establish new approaches for the treatment of a variety of cardiovascular-related defects, such as to produce vessels and/or valvular constructs  or to maThese pioneering studies have yielded promising preclinical results that must be implemented before hiPSC-derived cardiac cells can be projected towards a safe and effective translation in the clinical setting. Major concerns are related to the possible chromosomal aberrations , which cAfter MI, CFs are activated and recruited to the injured site to form scar tissue replacing the necrotic heart muscle. Therefore, reprogramming these abundant cell populations into functional CMs would be an ideal strategy for heart repair in response to ischemic injury . The proDirect reprogramming has been achieved for several human and mouse cell types by the forced expression of transcription factors and/or non-coding RNAs or through the delivery of small molecules modulating crucial pathways . NonetheSince the first report of the conversion of mouse embryonic fibroblasts (MEFs) into myoblasts by forced expression of the myoblast determination protein 1 (MYOD) , severalReprogramming requires the inhibition of the fibroblast signatures with the parallel enhancement of cardiac fate, which involves a drastic chromatin remodeling occurring in the starting fibroblast to overcome existing epigenetic barriers and to acquire the CM-like chromatin pattern . The sucLineage-specific pioneer transcription factors (TFs), that bind and open closed chromatin to enable the binding of other canonical TFs , are typThese studies demonstrated that reprogramming factors are not equally important, and their stoichiometry is crucial for the good progress of DCR . DespiteModification of the GMT combination by the addition of other TFs proved to be more effective also for human DCR, like the overexpression of GMT with Mesoderm Posterior BHLH Transcription Factor 1 (MESP1) and Myocardin (MYOCD)  or the cIn the last two decades, different studies made important advances in identifying microRNAs (miRs), a group of small non-coding RNAs, as pivotal players in regulation of cell fate plasticity . miRs haChanges in chromatin accessibility, which may decrease, increase or be transiently reconfigured, mainly occur in regions distal to the transcription start sites of specific loci  and are Increased H3K4me3 levels at cardiac genes are also observed upon the knockdown of the Polycomb Repressive Complex 1 (PRC1) component B cell-specific Moloney murine leukemia virus Integration site 1 (BMI1), whose downregulation facilitates the removal of the transcriptionally repressive mark Histone H2A Lysine 119 ubiquitination (H2AK119ub), increasing the reprogramming efficiency ,127,128.Knockdown of EZH2 also significantly increased human iCM reprogramming efficiency, leading to cardiac gene activation and repression of collagen and extracellular matrix genes. Furthermore, EZH2 inhibitors targeting its catalytic activity also promote human iCM reprogramming, suggesting that EZH2 may restrain cardiac conversion through H3K27me3-mediated gene repression . Very recently, Garry et al. demonstrated that overexpression of the histone reader PHD Finger Protein 7 (PHF7) in mouse tail-tip fibroblasts (TTFs) generated a 10-fold increase of iCM with respect to AGHMT alone . PHF7 wiThe epigenetic repatterning and the consequent remodeling of gene regulatory networks governing cell fate switches depend on the coordinated action of different signaling pathways, whose manipulation may dramatically enhance DCR efficiency. Several approaches in DCR adopted the addition of small molecules inhibiting fibroblast signatures and enhancing cardiac fate. The main signaling pathways and their targeting molecules are summarized in The Transforming Growth Factor-β (TGF- β) pathway is one of the main signaling pathways active in fibroblasts, participating in the cell regulatory network through synergistic and antagonistic interactions with many other signaling routes ,132. TheThe negative regulation of JAK/STAT and NOTCH pathways was shown to improve DCR efficiency ,135,137.These reports indicate that a prerequisite for reprogramming is the overcoming of possible related barriers, such as fibrosis with the associated gene network, highly expressed in postnatal and adult fibroblasts compared with embryonic fibroblasts. Considering the relationship between fibrosis and inflammation, we can assume that anti-inflammation may represent an additional potential target for lineage conversions, especially from a clinical translation perspective . Indeed,Together with the repression of the fibroblast gene network, it is essential to molecular booster networks involved in the promotion of the cardiac cell phenotype. The WNT/β-catenin signaling during embryonic development presents a biphasic role, being activated at early stages to induce the expression of mesendodermal markers such as Brachyury and Eomesodermin that must be inhibited at later stages to drive cardiac-lineage specification . β-catenOriginally, compounds were added to TFs or miRs cocktails to cooperatively enhance the effectiveness of the DCR protocol. Nevertheless, recent studies in mice and humans revealed the possibility of replacing the overexpression of exogenous genes and non-coding RNAs with a mixture of chemical compounds capable of inducing DCR ,128,141.In 2015, Fu et al. achieved the first full chemical-induced DCR (CiDCR) of fibroblast into iCM with the cocktail CRFVPT  in mice . They opShortly after, a similar CiDCR protocol based on a combination of nine compounds in part overlapping the cocktail used for mouse cells  was suffThe vast pool of CFs could serve as an endogenous source of new cardiomyocytes for regenerative therapy. In 2012, Qian et al. revealed that the injection of a retrovirus (ReV) encoding individual GMT factors in peri-infarcted areas of mouse hearts was able to reprogram resident CFs into iCMs . By usinConcurrently with Qian et al. , Song etSince the use of multiple viral vectors encoding individual factors could decrease the reprogramming efficiency, several groups have focused on the use of polycistronic vectors encoding different splicing orders of GMT factors into infarcted mouse hearts, like the injection of a 2A-polycistronic ReV vector encoding TMG factors, which induced a twofold increase in the number of mature iCMs compared to the three single vectors , injectiAs integrative viral vectors still present major concerns regarding the possible mutagenetic effects, some groups starting to use non-integrative viral vectors, such as Sendai virus (SeV) vectors. While AdV and LeV induced equivalent expression levels of GMT factors and had a similar transdifferentiation capacity compared to ReV polycistronic vectors , SeV polThe improvement in several cardiac function parameters was also reported upon transduction of LeV vectors overexpressing miR combo to reprogram resident CFs into CMs in vivo in infarcted mouse hearts ,150, as Finally, the administration of CRFVPT cocktail with the addition of Rolipram, a selective phosphodiesterase-4 inhibitor, reduced fibrotic area and significantly improved heart functions in post-MI mouse models . NeverthCollectively, these studies not only demonstrated that DCR could be an effective strategy for cardiac regenerative interventions, but also indicated that the heart microenvironment can be more favorable to cardiac reprogramming than the in vitro culture conditions in terms of the efficiency and maturation of mouse iCMs ,142,144.Indeed, the local environment could have important influence on the efficiency of fibroblast direct reprogramming to cardiomyocytes . One of Other aspects of the post-infarct microenvironment in the heart that could influence the reprogramming of activated fibroblasts are pro-inflammatory and pro-fibrotic cytokines released following the myocardial injury  to recruAs previously mentioned, the best-known barrier to DCR are pro-fibrotic signals, whose inhibition promoted CFs reprogramming not only in vitro but also in vivo , as demoFinally, mechanical properties of the damaged myocardium could also contribute to iCM reprogramming, as scar area is stiffer than the healthy myocardium due to ECM accumulation and fibrosis, and cells modify their gene expression pattern depending on the rigidity of the underlying matrix. This process was guided by two transcriptional co-activators, the Yes-Associated Protein 1 (YAP) and WW Domain Containing Transcription Regulator 1 (TAZ) , which mThe possibility to regenerate the heart through DCR represents a fascinating as well as promising perspective. As highlighted, a great deal of effort has been devoted to the dissection of the DCR process to set an effective protocol based on the use of different agents  . Given tIn general, nanotechnology-based systems could more efficiently deliver, in a target-specific way, reprogramming agents with high molecular weight such as nucleic acids or allow the simultaneous delivery of compounds with different physical and chemical properties. In addition, the desired reprogramming agents could be delivered to the identified cellular and tissue targets adjusting chemical, spatial and temporal variables, which have been shown to be significantly involved in the reprogramming efficiency. Finally, the targeted delivery of the reprogramming agents could significantly reduce cellular and systemic toxicity of the potentially harmful ones, facilitating its transition to a clinical setting . Indeed,The first response to concerns related to the use of potentially harmful genome-integrating viral vectors to deliver the reprogramming transcription factors led to the development of nonviral delivery systems such as nanoparticles. Even if not in the DCR setting, the use of a nanoparticle-based delivery system for the iPSC reprogramming factors led to higher transcription rates and, more interestingly, could be serially repeated to optimize reprogramming efficiency .Similarly, to overcome the challenges related to mRNA transfection to reprogram mouse CFs to iCMs, an mRNA transfection system obtained by fusing polyarginine with the lipofectamine complex was used. This system led to efficient transfection with low toxicity, which allowed for multiple transfections of the GMT mRNAs over a two-week period. Interestingly, such iCMs showed increased expression of cardiomyocyte marker genes . Recently, the cardiac reprogramming factors were successfully delivered using cationic gold nanoparticles loaded with GMT in both human and mouse somatic cells. This approach generated functional iCMs from MEFs in vitro and from CFs in vivo in mouse models of MI, resulting in low cytotoxicity and efficient in vivo DCR with a reduced infarct size and improved cardiac function . Very recently, a miR combo cargo was loaded in polyethyleneimine coated nitrogen-enriched carbon dots for cardiac reprogramming. This approach also led to negligible toxicity and efficient DCR of CFs to iCMs with functional recovery in mouse infarcted hearts, without any genomic integration . A very interesting approach led to the development of a non-viral biomimetic system obtained by coating FH peptide-modified neutrophil-mimicking membranes on silicon nanoparticles, which were loaded with miR Combo. Such nanoparticles used the natural inflammation-homing ability of neutrophil membrane protein and FH peptide’s high affinity to tenascin-C (TN-C) produced by CFs, to sequentially target CFs in the injured heart and deliver their cargo leading to efficient reprogramming and in improved cardiac function .cycle in vitro. Poly-lactic-co-glycolic acid nanoparticles were engineered to effectively release their cargo up to 4 weeks. The intramyocardial injection in mouse or pig models of MI of such nanocarriers was able to decrease cardiomyocyte apoptosis and increase angiogenesis thus reducing the infarct size and left ventricular remodeling, preserving cardiac function [Concerning the targeted delivery of chemical compounds, two interesting experiences have been performed on nanotechnology-based approach. One of these approaches was conceived to deliver Forskolin and Repsox, which have previously been used in a reprogramming cocktail ,141, usifunction .Very recently, an interesting proof-of principle experience encompassing many of the advantages of a nanotechnology-based approach to DCR has been performed. A small molecule inhibitor of GATA4-NKX2-5 loaded polymeric biocompatible elastomer, poly (glycerol sebacate) (PGS), coupled with collagen type I, was used to engineer a patch to be applied to the infarcted myocardium. Moreover, in the patch, a chemical agent was incorporated to improve cell conductivity and facilitate cell signaling. Even if in vitro experiments, this approach led to myoblasts attachment and proliferation to the patch meeting the mechanical, conductive, and biological needs necessary for a cardiac regenerative therapy . In the last decades, the evolution of new strategies to regenerate the injured myocardium based on cellular reprogramming represented a revolutionary new paradigm, providing a unique and efficient way to generate cell types of interest for cardiac repair by intervening on the plasticity of cell fate . While tA major obstacle for DCR is the low conversion efficiency and more efforts are necessary to gain insight on molecular mechanisms governing this process, not only to accelerate the optimization of reprogramming for upcoming clinical application but also to better understand the biology of fibroblast plasticity, in order to identify new potential reprogramming factors. The targeted delivery of reprogramming factors is also a major issue. The use of LeV or ReV delivery of genetic factors that induce fibroblast reprogramming has been previously linked to carcinogenesis  and the The precise cardiac targeting for the delivery of therapeutics may be achieved mainly through nanotechnology-based systems. Nanocarriers would allow the delivery of reprogramming cargoes to the tissue targets adjusting chemical, spatial and temporal variables to reduce possible cellular and systemic toxicity. However, new targeting methods must be further explored to generate innovative delivery strategies to overcome the low targeting capability and treatment efficacy of current ones.3D cardiac patches are a promising method in cardiac repair and can be either cellular or non-cellular. Cellular patches are generated through the seeding of different live cells into various 3D scaffolds and the future interdisciplinary cooperation between bioengineering and iPSC technology will be crucial in this research area. Compared with 3D cellular cardiac patches, non-cellular patches generated by seeding different cell derivatives into various 3D scaffolds, may have better stability, biocompatibility, modifiability, and low tumorigenicity and immunogenicity. 3D bioprinting technology has been widely utilized in cardiac repair by integrating biomaterials with different cell types to precisely pattern a cardiac structure . HoweverFinally, understanding the long-term consequences of novel cardiac regeneration strategies, particularly in large animal pre-clinical studies, is a mandatory step toward clinical translation. Overcoming these clinical barriers will undoubtedly begin a new era in the treatment of human heart disease.


Early life trauma has a negative impact on the developing brain, and this can lead to a wide range of mental illnesses later in life. Childhood trauma is associated with increased psychotic symptoms and negative emotions such as depressive, anxiety, and stress symptoms in adulthood. Childhood trauma has also been shown to influence sub-clinical ‘schizotypy’ characteristics of psychosis in the general population. As it has been reported that mental health outcomes after early life trauma exposure are influenced by gender, the current study aimed to investigate the gender differences in the relationship between childhood trauma, schizotypy and negative emotions. Sixty-one non-clinical participants (33 men and 28 women) aged between 18 and 45 completed self-report questionnaires to measure early life trauma, schizotypy and negative emotions. Despite similar levels of childhood trauma in men and women, early life trauma in women was associated with increased schizotypy personality characteristics (Cognitive Disorganisation) and increased depression, anxiety and stress later in life, but no correlations were observed in men. Our findings suggest that the sociocultural and biological processes affected by early life adversities may differ between the genders. Women may be more vulnerable to the influence of childhood trauma, which may be associated with increased psychopathology later in life. Research shows that schizophrenia symptoms and the course of illness differ between men and women; for instance, men have a higher prevalence of negative symptoms, lower functioning and an earlier symptom onset of three-to-five years compared to women . In contOne gender-related factor underlying the development of schizophrenia symptomatology could be early life trauma. A higher prevalence of childhood trauma has been observed in first-episode psychosis patients compared to healthy controls, and it has been suggested that psychosis may result from trauma experienced in childhood . ChildhoWhile gender differences in schizophrenia symptomatology have been previously observed , researcA review of 25 studies investigating childhood trauma and schizotypy observed that all studies found an association between schizotypy and at least one type of trauma, mainly physical abuse, sexual abuse or neglect . Two stuWe aimed to explore gender differences in the relationship between schizotypy and trauma outside the context of schizotypal personality disorder using a cross-sectional correlational approach. As depression and anxiety are expressed at higher rates in females than males in the general population, and because women with schizophrenia present more with mood symptoms rather than positive and negative symptoms, we also investigated the association between childhood trauma and depression, anxiety and stress in both genders, which has not been explored alongside schizotypy.Sixty-one non-clinical participants aged between 18 and 45 responded to advertisements and met the inclusion criteria of being fluent English language speakers. Participants were excluded if they met diagnostic criteria for a psychotic or affective disorder  screening module criteria), a history of stroke, severe head injury, loss of consciousness or epilepsy or any significant visual impairment, other than refractive.All participants completed the Oxford-Liverpool Inventory of Feelings and Experiences (O-LIFE), a 104-item self-report questionnaire developed to measure schizotypy in non-clinical individuals . The O-LThe Depression Anxiety Stress Scale (DASS) was used to measure the negative emotional states of depression, anxiety and tension/stress over the past week . The shoThe Childhood Trauma Questionnaire-Short Form (CTQ-SF) was included to assess early life stress/adversity . The shoAs the current study investigates gender differences, it is important to note that strong measurement invariance across gender has been observed for the O-LIFE , DASS-21p ≤ 0.005 for the Mann–Whitney U-tests to account for 11 comparisons and p ≤ 0.002 for the Spearman’s rho correlations to account for 30 correlations in men and women. A correlation heatmap was generated using GraphPad Prism version 9.1.2.Data analyses were performed using IBM SPSS version 26. As variables violated the assumptions of normality, Mann–Whitney U-tests were conducted to compare gender differences. Spearman’s rho correlations between childhood trauma, schizotypy and negative emotion were conducted separately for men and women. Bonferroni corrections were made to account for multiple comparisons; Mean and median age, trauma, schizotypy and negative emotion measures are described in Looking at the correlation heatmap , the corp = 0.002; 95% CI, 0.21–0.78) and physical neglect . With regards to negative emotion in women, emotional abuse positively correlated with depression  and stress . Physical neglect was also significantly positively correlated with depression  and stress . Sexual abuse also positively correlated with anxiety .In women, Cognitive Disorganisation was significantly positively correlated with sexual abuse (rho = 0.56, Our study found that the Cognitive Disorganisation schizotypy factor, which captures poor attention, concentration, decision-making and social cognition, was positively correlated with physical neglect and sexual abuse in women, highlighting that schizotypy is not associated with a single type of trauma. This is consistent with the increased self-reports of memory disorganisation  and poorOf the two observed significant relationships in our study, Cognitive Disorganisation was most strongly correlated with physical neglect, consistent with schizophrenia research showing the most robust relationship between physical neglect in childhood and functional and social impairment . The CogWhile childhood trauma was associated with schizotypy in women, this was not observed in men. This is consistent with Toutountzidis et al. , but notUnexpectedly, depression, anxiety and stress levels did not significantly differ between men and women. However, trauma positively correlated with depression, anxiety, and stress in women but not in men, consistent with previous observations of a relationship between higher CTQ scores and depressive symptoms in women during early psychosis ,9. In woThe trauma–mood relationship observed in women but not in men could also potentially be explained by the different coping mechanisms employed by men and women. Cognitive emotion regulation strategies have been shown to mediate the relationship between childhood trauma and depression and anxiety in adulthood across both genders . EmotionIncreased HPA axis activity has been reported in both schizophrenia  and deprBiologically, our significant findings in women but not in men may be explained by sex differences in the HPA axis. The HPA axis has been shown to be more reactive to stress in females compared to males . This maSex differences in HPA axis function is demonstrated in both animal and clinical studies. Findings from animal studies have been consistent, showing higher glucocorticoid levels observed in females after stimulation of the HPA axis . The intThe findings of this study should be interpreted within the context of several limitations. Firstly, as individuals are likely to experience more than one type of trauma, the trauma types are likely intercorrelated, making it difficult to draw conclusions about the impact of specific types of trauma on schizotypy and negative emotion. Future research should investigate the effect of cumulative trauma, of both single and multiple trauma types across childhood and into adulthood. Our reported trauma scores were also relatively low, and our research needs to be replicated in a larger, broader, non-clinical sample with more individuals who have experienced childhood trauma.Our study demonstrates the relationship between childhood trauma, disorganised schizotypy and negative emotion in women. This highlights the importance for individuals identified as high risk for psychosis with childhood trauma experiences to receive early interventions, such as trauma-focused cognitive–behavioural therapy, to address cognitive distortions related to the trauma. Our findings also indicate increased vulnerability in women to adverse childhood events and suggest that the sociocultural and biological mechanisms affected by early life adversities may differ between genders. This contributes to a growing body of work highlighting the importance of early life trauma in both schizotypy and mood disorders, and supports the development of gender-specific interventions to strengthen resilience.


Traumatic injury of the spinal cord (SCI) is a devastating neurological condition often leading to severe dysfunctions, therefore an improvement in clinical treatment for SCI patients is urgently needed. The potential benefits of transplantation of various cell types into the injured spinal cord have been intensively investigated in preclinical SCI models and clinical trials. Despite the many challenges that are still ahead, cell transplantation alone or in combination with other factors, such as artificial matrices, seems to be the most promising perspective. Here, we reviewed recent advances in cell-based experimental strategies supporting or restoring the function of the injured spinal cord with a particular focus on the regenerative mechanisms that could define their clinical translation. Only in the United States, the annual incidence of SCI is estimated at approximately 54 cases per million people  with an average annual cost of healthcare for SCI patients of 76,327 USD per year . These data clearly indicate that SCI is not only a serious health condition that significantly reduces the quality of life, for both patients and their families, but also an important socio-economic problem, therefore an improvement in clinical treatment for SCI patients is urgently needed.Spinal cord injury is a devastating neurological condition, leading to many severe deficits such as paralysis, sensory abolition, and respiratory, circulatory, and digestive system dysfunctions that usually result in permanent disability. The estimated global SCI incidence reaches 40 to 80 cases per million people and gradually increases with the rate of car or extreme sports accidents and in total. Costs of spinal cord injury are higher than those of comparable conditions such as dementia, multiple sclerosis, and cerebral palsy  ,15 or byHowever, the complexity and irreversible nature of the mechanisms involved in nerve tissue damage make it difficult to design an effective SCI pharmacotherapy. Therefore, exercise-based movement training coupled, or not, with spinal cord epidural electrostimulation remains the most efficient therapeutic procedure routinely used in SCI clinics. Experimental data from animal models show that its beneficial effect is mainly mediated by increasing the production of neurotrophins BDNF  and BDNFRecently, the development of cell-based regenerative approaches in medicine has revived the debate within the field and opened novel perspectives in SCI treatment. A wide range of cell types has been examined in animal models of SCI, including fully differentiated cells such as Schwann cells (SCs) , olfactoSince whole-cell administration may likely be associated with certain clinical risks such as low cell survival rate, graft rejection, limited or improper differentiation, or tumor formation, novel approaches employing cell-derived structures or substances have recently been proposed. Among them, a very promising strategy employs small extracellular vesicles (sEVs), naturally released by cells to communicate with each other. sEVs can deliver many potentially neuroprotective molecules (such as miRNAs and proteins) directly to host cells of the injured spinal cord, affecting their activity and fate .In this review, we discuss the recent achievements in exogenous cell-based restorative approaches that have been experimentally proven to be beneficial in rodent models of SCI with a special focus on their mechanisms of action in preventing diverse unfavorable changes caused by spinal cord trauma. The mechanism of action of exogenous cells in SCI treatment is mainly based on the modulation of the injured tissue microenvironment and/or replacing lost cells. Therefore, the greatest experimental efforts are mainly directed towards the inhibition of the inflammatory reaction that occurs after SCI, inhibition of neuron death, supporting axonal regeneration as well as rebuilding and/or activating surviving spinal cord neuronal networks.Inflammation is a natural immune response to tissue injury leading to control, clearance, repair, and eventually restoration of homeostasis. Upon spinal cord injury, neurons and other damaged cells release a plethora of inflammatory mediators activating various cell types including microglia, astrocytes, and endothelial cells. This further contributes to neuronal damage by initiating an inflammatory cascade in both the spinal cord and the immune system . TheAlthough inflammation and gliosis are hallmarks of the CNS pathology and targets of certain well-known anti-inflammatory drugs that could be potentially used in clinical SCI treatment, the appreciation of the beneficial side of neuroinflammation has initiated an extensive debate on the actual role of these processes in neurological disorders. Evidence that comes from the genetic or pharmacological inactivation of microglia/macrophages sheds light on the critical role of the timing and extent of microglial activation, distinct roles of different populations, and the beneficial role of inflammation in the regenerative phase of the post-injury response. Recent reports indicate that the response of microglial cells to the changes in CNS homeostasis is not uniform, and rather microglia and macrophages adopt a spectrum of phenotypes and functions, from pro-inflammatory to anti-inflammatory . SooIt has been reported that the modulation of macrophage polarization could be achieved not only by grafting whole cells but also by the administration of a cell-conditioned medium. Both endothelial progenitor- and neural stem cell-conditioned media successfully mediated an anti-inflammatory effect and reduced the number of inflammatory macrophages in rodents with injured spinal cords ,62. NSC-At the molecular level, the immune reaction after SCI is a cascade of events driven by changes in the spinal cord environment, dependent on diverse groups of molecules, mainly chemokines and cytokines. They are involved in complex signaling pathways either activating or disabling immune cell function. Cytokine concentration can also be modified indirectly by regulating signaling pathways effectors, for example, nuclear factors. Transplantation of the human spinal cord immortalized neural stem cells into rats subjected to SCI resulted in the inhibition of NF-κB production and, as a result, modulation of the pro-inflammatory response . In the Apoptosis, as a result of spinal cord injury, affects several groups of cells, including neurons, oligodendrocytes, microglia, and astrocytes . PreventAnother proposed mechanism of the anti-apoptotic action of transplanted cells may be related to mitochondrial transfer from MSCs to the affected motor neurons in the injured spinal cord. Such organelle internalization, shown in vitro and in vivo, improved the bioenergetics of neurons, which could lead to the blockage of apoptotic pathways followed by the facilitation of locomotor recovery .Novel approaches in SCI treatment that are currently being tested in animal models employ the application of non-coding, single-stranded short fragments of RNA (microRNAs) that regulate the expression of many important genes involved in apoptosis. The global miRNA expression profile changes after SCI, showing a downregulation of miR-124, -138, -235-3p, -137, and -30b-3p and an upregulation of miR-1, -15b, -34, and -145. Potential targets of those mRNAs are genes related to apoptosis ,90. EngrVascular disruption is one of the traumatic consequences of spinal cord injury. Due to the alterations of microvessel structures, the blood flow changes and the blood–spinal cord barrier breaks down, which enables enhanced infiltration of inflammatory cells and intraparenchymal hemorrhages. Ultimately, both endothelial and neurons die due to ischemia ,96 and vAngiogenesis, measured by the increasing density of blood vessels, is stimulated via the bioavailability of angiogenic factors such as vascular endothelial growth factor (VEGF). Therapeutic approaches for SCI therefore mainly focus on enhancing the VEGF local concentration to support angiogenesis and restore vascular perfusion . HoweverSuch beneficial effects can be reached by other strategies, among which there is the transplantation of angiogenic growth factor-producing cells. Promising results were obtained when engrafting olfactory ensheathing cells alone or in co-transplantation with adipose-derived stromal cells, which secrete proteins involved in VEGF pathways . HoweverAngiogenesis can be modulated with the aid of synthetic and biomaterials, which are applied in the injury site . There aDuring spinal cord injury, high-power mechanical forces often cause nerve tissue damage via crushing or tearing. It usually results in the disruption of axons running along the spinal cord. Mechanical damage often leads to secondary injuries via so-called Wallerian degeneration, which is defined as a progressive anterograde disintegration of axons and accompanying demyelination after an injury to the proximal axon or cell body . On the To remove the already formed scar, chondroitinase ABC, the enzyme digesting chondroitin sulfate and the main component of the glial scar, may be employed . DegradaThe enzymatic approach produces particularly good results in combination with other procedures, such as simultaneous peripheral nerve transplantation , hyperbaHowever, it was shown that chondroitinase ABC loses its enzymatic activity very quickly at body temperature, which significantly limits its application in human SCI therapy . TherefoBMP), and the decrease in its expression by shRNA abolished GDAsBMP-induced axon outgrowth in vitro. Furthermore, transplantation of POSTN-deficient GDAsBMP into the injured rat spinal cord resulted in lower axonal regeneration. On the other hand, the addition of recombinant periostin to neurons in vitro abolished the adverse effects of glial scar molecules and increased neurite extension via the influence on FAK and Akt pathways [The presence of astrocytes within the affected tissue might also be, however, beneficial to the regenerating axons. Regeneration of the injured axons is very often observed along “bridges” created by endogenous astrocytes , thus glpathways .Axonal regeneration could be also promoted indirectly. It has been shown that intraspinal transplantation of oligodendrogenic neural progenitor cells in nude rats after spinal cord injury led to the functional recovery of injured animals measured by BBB scores and Catwalk analysis of locomotor functions. In this case, transplanted cells mainly differentiated into oligodendrocytes, which promote tissue sparing and axon remyelination . NPC canMolecules instructive for signaling pathways involved in axon growth, such as microRNAs, could also be employed to modulate axon survival after injury. Intravenous application of exosomes delivered from stem cells expressing miR-133b in rats after compression spinal cord injury led to an increase in axon regeneration and improvement of their locomotor ability . The obsMyelin is a lipid-rich structure produced and wrapped around axons to protect them and enable fast impulse propagation . SCI affSpontaneous myelin regenerative events often occur after SCI, indicating that the spinal cord attempts to repair itself. In rodent models of experimental SCI, oligodendrocyte progenitors repopulate the affected area and remyelinate intact demyelinated axons . After sUnfortunately, spontaneous remyelination in the CNS often fails or is incomplete, and non-myelinated axons can be observed months after SCI in rat models ,150,151.Several potentially therapeutic strategies for enhancing remyelination in SCI treatment have been already experimentally examined. One of the promising approaches is a direct method of replacing oligodendrocytes lost during injury by grafting OPCs of different origins. OPCs derived from human embryonic stem cells intraspinally grafted in rats 7 days after spinal cord contusion survived in the spinal cord tissue, redistributed over short distances, and differentiated into oligodendrocytes, successfully enhancing remyelination. Functional improvement manifested as longer forelimb stride length, increased forelimb step range, and a higher number of passed-perpendicular steps as measured by kinematic analysis. What is more, rats exhibited motor behavioral recovery up to 4 months after transplantation with no significant toxicities observed ,154. It Positive effects were observed after the experimental grafting of SCs into the injured spinal cord as well. Given the fact that autologous cells are difficult to obtain, SCs can be derived from other sources, including bone marrow and skin-precursor cells ,163. BetNeural necrosis and apoptosis destroy the innervation derived from the higher supraspinal structures that activate and modulate the activity of spinal cord networks located below the injury site. Thus, the reorganization of local neuronal circuits through the repopulation of damaged tissue with the newly generated neurons is essential for the restoration of lost function.A range of cell types has been experimentally examined as a potential source of neurons to replace the lost populations. Intraspinal transplantation of motoneuron-like cells, pre-differentiated from human adipose-derived stem cells in vitro, has been reported to significantly improve locomotor abilities in mice after crushing SCI . TransplAn optimistic notion that certain lost spinal cord connections could be successfully replaced has been recently strongly supported by results of pre-clinical primate experiments showing that human spinal cord-derived multipotent NPCs transplanted at 4 weeks after cervical hemisection in rhesus monkeys gradually maturated and improved forelimb function . Human cSCI very often leads to the disruption of supraspinal GABAergic inhibitory projections, which innervate interneurons in I-III lamina of the spinal cord dorsal horn , leadinglocus coeruleus and locus subcoeruleus (noradrenergic neurons) [n. raphe pallidus, n. raphe obscurus, n. raphe magnus [Locus ceruleus [2 [7 [Many reports contribute to building the growing body of evidence that both noradrenergic ,177,178 neurons) ,189 and e magnus , and theceruleus  or serotceruleus ,192 tranceruleus ,193,194.uleus [2 ,195 and us [2 [7  serotonius [2 [7 . RecentlIn recent years, extensive experimental work has been devoted to understanding the mechanisms underlying neuroprotective or regenerative outcomes of cell transplantation in neurological conditions such as spinal cord injury. Despite the advances in uncovering the regenerative mechanisms triggered by exogenous cells, certain long-lasting issues still need to be resolved. Reviewing the papers listed here, we focused our attention on the cell-based approaches that ensure significant functional regeneration of the injured spinal cord and provide some facilitation in motor function recovery. Although several methods (like BBB or BMS) were created to achieve a level of standardization of locomotor performance evaluation, they might be not sufficient to assess certain important changes in the locomotor pattern, such as intra- and interlimb coordination and their relation to locomotor speed, particularly in the chronic stage of injury in long-lasting experiments. The majority of attempts reported to date show very slight improvement of locomotor function, particularly after total spinal cord transection/complete injury or severe spinal cord contusion. In many cases, the locomotor improvement is limited to better movement in individual joints without any better limb coordination or plantar stepping and body weight support. Since the majority of SCI patients suffer from severe complete or incomplete loss of function as a consequence of cervical spinal segments injury, it seems that more efforts are still required to adapt the experimental situation to clinical needs.As shown above, an increasing number of data from experimental and pre-clinical studies has been generated to support the notion that exogenous cell transplantation promotes repair in the SCI and could be the most promising translational pathway. Nevertheless, the majority of pre-clinical cell-based studies were performed with rodent models and some of them have already reached early-phase human clinical trials. However, the current revision of the registered trials revealed that the overwhelming majority of them remain at an early stage . Ninety-Although different aspects of SCI pathology and many of their overcoming mechanisms via cell-based treatment have been experimentally examined and successfully assessed in animal models, despite the initial enthusiasm it sparked, their implementation to routine clinical practice is notably limited to date. Since the chronic treatment of spinal cord injury is particularly challenging, it became evident that the capacity of these approaches to facilitate regeneration and achieve meaningful functional recovery is rather modest when considered individually. Therefore, it is now widely postulated that combined therapeutic strategies targeting different aspects of SCI pathology and acting complementary could likely offer more satisfactory functional outcomes and should be clinically tested.


Action of the FPC protects forks from collapse at both programmed and accidental protein barriers, thus promoting genome integrity. In addition, FPC stimulates the DNA replication checkpoint and regulates topological transitions near the replication fork. Eukaryotic cells have been proposed to employ physiological programmed fork pausing for various purposes, such as maintaining copy number at repetitive loci, precluding replication-transcription encounters, regulating kinetochore assembly, or controlling gene conversion events during mating-type switching. Here we review the growing number of approaches used to study replication pausing in vivo and in vitro as well as the characterization of additional factors recently reported to modulate fork pausing in different systems. Specifically, we focus on the positive role of topoisomerases in fork pausing. We describe a model where replisome progression is inherently cautious, which ensures general preservation of fork stability and genome integrity but can also carry out specialized functions at certain loci. Furthermore, we highlight classical and novel outstanding questions in the field and propose venues for addressing them. Given how little is known about replisome pausing at protein barriers in human cells more studies are required to address how conserved these mechanisms are.During nuclear DNA replication multiprotein replisome machines have to jointly traverse and duplicate the total length of each chromosome during each cell cycle. At certain genomic locations replisomes encounter tight DNA-protein complexes and slow down. This fork pausing is an active process involving recognition of a protein barrier by the approaching replisome  Saccharomyces cerevisiae, where the understanding of DNA replication mechanisms is most complete. We discuss pausing at RFBs from various perspectives: detection methods, diversity, regulators, proposed physiological roles, and finish by summarizing emerging models and outstanding questions in the field.In order to duplicate chromosomes, replicative polymerases have to access each base in the DNA. This requires removing DNA-binding proteins, resolving topological constraints and melting the DNA double helix step-by-step along the whole length of each chromosome. A chromosome’s features vary along its length. Accordingly, it is not surprising that replication elongation rates are variable and that forks tend to slow down at regions where the bases are difficult to access, due to DNA secondary structures , The pioneering work of MRC1 gene function in yeast . This targeted approach revealed an estimated total of ∼1,400 RFBs in the yeast genome , telomeres and inactive replication origins  was repurposed as a protein (or protein-covered R-loop) replisome barrier. dCas9 efficiently blocks the yeast replisome in vivo  and all d yeast; ]. FutureHML/HMR silencers). Barriers also vary in their efficiency (% of blocked forks) and strength (time the replisome spends on the barrier), with CEN barriers being transient  or tranay  screensn levels .via covalent bonds. It is documented that high levels of Top1 are stably and covalently attached at Fob1 rDNA barriers  may promote fork arrest through mechanisms independent of their DNA-binding affinity, it seems likely that strong DNA binding in and of itself can cause fork blockage. Indeed, the most effective blocks so far characterized consist of arrays of binding sites for high-affinity DNA-binding proteins, such as yeast Rap1 or the bacterial LacI repressor . HoweverMRC1 lead to separation of nascent DNA signal (BrdU) and CMG helicase components in cells challenged with HU  during mitosis . In yeassof mutants specifically deficient in pausing but proficient in other functions (Thus replication pausing, and the FPC as its main executive, carries out many different roles in yeast cells. It is worth noting here that the essential role of the FPC in pausing makes it tempting to study the cellular consequences of pausing loss by simply inactivating one or both of its components. However, interpretation of observations in FPC null mutants may be confounded by the multiple additional roles of the FPC in checkpoint, fork rotation, and sister chromatid cohesion functions . Using tunctions  will be Given recent advances in cryoEM-derived structures of replisome complexes , we antiin vitro reconstitution experiments defined a minimal set of proteins required to elicit pausing at linear non-chromatinized DNA substrates (in vitro studies, either in bulk solution or at the single-molecule level, are likely to explore the role of additional replisome-associated factors, post-translational modifications, and nucleosomes.Recent bstrates . Given tsof mutants will help to clarify the physiological roles of pausing and barriers. To address understudied barriers, we anticipate the development of systems to stall replication forks directly via a transcribing RNA polymerase complex  to inveequences . Such stGiven TIMELESS-TIPIN’s pro-oncogenic role  and degrDiscovered more than three decades ago, replication fork pausing still poses many unresolved questions as to mechanisms and physiological roles. However, as new approaches to measure pausing are devised, additional pausing factors identified, regulated systems engineered and recombinant minimal systems reconstituted, the field advances. Topoisomerases were recently found amongst the positive regulators of pausing, which establishes a novel link between replisome progression and topological transitions at the fork. The relation between torsional stress and chromatin resistance to replisome progression will be an important venue for future research.MS and DS reviewed literature, designed sections of the manuscript, and wrote and revised first draft. MS made figures. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The COVID-19 pandemic has exerted a significant toll on the lives of people who use opioids (PWUOs). At the same time, more flexible regulations around provision of opioid use disorder (OUD) services have led to new opportunities for facilitating access to services for PWUOs. In the current scoping review, we describe new services and service modifications implemented by treatment and harm reduction programs serving PWUO, and discuss implications for policy and practice.Literature searches were conducted within PubMed, LitCovid, Embase, and PsycInfo for English-language studies published in 2020 that describe a particular program, service, or intervention aimed at facilitating access to OUD treatment and/or harm reduction services during the COVID-19 pandemic. Abstracts were independently screened by two reviewers. Relevant studies were reviewed in full and those that met inclusion criteria underwent final data extraction and synthesis (n = 25). We used a narrative synthesis approach to identify major themes around key service modifications and innovations implemented across programs serving PWUO.Reviewed OUD treatment and harm reduction services spanned five continents and a range of settings from substance use treatment to street outreach programs. Innovative service modifications to adapt to COVID-19 circumstances primarily involved expanded use of telehealth services , increased take-home medication allowances for methadone and buprenorphine, expanded uptake of long-acting opioid medications , home delivery of services , outreach and makeshift services for delivering MOUD and naloxone, and provision of a safe supply of opioids.The COVID-19 pandemic has posed multiple challenges for PWUOs, while simultaneously accelerating innovations in policies, care models, and technologies to lower thresholds for life-saving treatment and harm reduction services. Such innovations highlight novel patient-centered and feasible approaches to mitigating OUD related harms. Further studies are needed to assess the long-term impact of these approaches and inform policies that improve access to care for PWUOs. The coronavirus disease 2019 (COVID-19) pandemic has exerted a significant toll on the lives of people who use opioids (PWUOs). Rising overdose deaths since the COVID-19 pandemic have been reported in the U.S.  and EuroGiven these circumstances, easing access to life-saving opioid use disorder (OUD) services has become more urgent than ever. Medications for opioid use disorder (MOUD), including methadone, buprenorphine, and extended-release naltrexone, are highly effective at reducing overdose risk , 12 and Access to OUD services has become especially precarious during the COVID-19 pandemic, as service closures, lower staff availability, and concerns around COVID-19 transmission have led to reduced service availability or utilization at a time when these services are needed most , 19. At In the current study, we conducted a scoping review of articles published in 2020 to understand how OUD treatment and harm reduction programs globally have innovated and adapted their services to COVID-19 circumstances and identify ongoing gaps in delivering services for PWUOs. To this end, the objective of this review was to: (1) identify characteristics of programs that adapted OUD services during the pandemic; (2) describe innovative services or service modifications that emerged as a result of COVID-19 circumstances; and (3) discuss implications for policy and practice responses to meet the needs of PWUO during and beyond the current COVID-19 pandemic.We conducted this review in accordance with the Preferred Reporting Items of Systematic Reviews and Meta-Analyses for Scoping Review (PRISMA-ScR) checklist  Appendi: Table 3We sought peer-reviewed English language articles published in 2020 that described either new programs or adaptations of existing programs to deliver evidence-based treatment with MOUD  or other overdose prevention and harm reduction services for PWUO under COVID-19 emergency conditions. We limited our search to articles that described particular programs/interventions and not those that offered aggregate data on service modifications from multiple providers or programs.We consulted with a librarian at the New York University Health Sciences Library to develop the search approach and extraction process. We adopted a broad search strategy to identify peer-reviewed literature from multiple electronic databases in November 2020. A second search was conducted in February 2021 to include articles published between November and December of 2020. Electronic database searches included PubMed , Embase , PsycInfo , and LitCovid, a curated literature hub that tracks up-to-date scientific information about the 2019 novel Coronavirus in PubMed , 26  AND (COVID* OR COVID-19 OR sars-cov-2 OR coronavirus OR pandemic OR quarantine).Initial database search results were imported into Endnote, and subsequently uploaded into Covidence, a subscription-based systematic review tool . Upon imProgram characteristics collected from articles that met inclusion criteria included: author and month of publication; location of program ; care setting ; name of program/managing organization; managing organization type ; article study design ; whether programs offered OUD treatment, harm reduction services , or both; and types of services/medications offered . We also collected information related to service modifications during the COVID-19 pandemic including: whether the program was newly created or a modification of an existing program prior to the pandemic; unique strategies and services implemented to meet patient needs during the COVID-19 pandemic; any reported outcomes of the program(s); key obstacles and facilitators encountered; and lessons reported for OUD service policies and practices.We used a narrative synthesis approach  to iteraOur search resulted in 634 total articles, out of which 49 went through full-text extraction, and 25 met final inclusion criteria for this review Fig. .Fig. 1SeDescriptive characteristics of programs in the 25 articles included in the review are summarized in Table OUD treatment and harm reduction programs employed a range of strategies to adapt their services to the changing circumstances of COVID-19 and ensure services reached people in need. Detailed descriptions of strategies adopted by each program are summarized in Table The most common innovation centered on the provision of telemedicine services using telephone or online platforms. Telemedicine services were often instituted to initiate or continue treatment with buprenorphine while minimizing in-person patient-physician encounters. Telemedicine-based buprenorphine was implemented across a range of settings, from nation-wide . In theIn addition to buprenorphine prescribing, telehealth platforms were also used to deliver adjunct psychosocial services to OUD patients. For example, one outpatient MOUD treatment clinic in Rhode Island used telehealth to continue delivering counseling sessions to MOUD patients . AnotheMultiple programs in the U.S., Canada, India, Spain, Italy and England instituted longer take-home policies for dispensed opioid agonist medications . This strategy was adopted by substance use treatment clinics . In OhiMultiple programs acknowledged that offering telehealth or home delivery services was a necessary, yet insufficient approach for reaching patients experiencing unstable housing or lacking mobile device ownership. Thus, many programs described the creation of makeshift services or outreach programs to expand the delivery of OUD services during the pandemic. In a Boston-based program , mobileIn British Columbia, one program described ‘pandemic withdrawal management’ practices that were implemented in response to provincial Risk Mitigation Guidance that allowed the prescribing of a safe supply of pharmaceutical-grade alternatives to illicit substances, including hydromorphone, stimulants, alcohol, benzodiazepines, and nicotine. Such practices were described to help manage withdrawal symptoms among PWUO by providing a known quality, non-adulterated, pharmaceutical grade alternative to the increasingly toxic illicit drug supply while allowing people to adhere to physical distancing guidelines . Such pOUD treatment and harm reduction programs experienced several obstacles throughout the process of adapting their services to COVID-19 circumstances. One commonly reported obstacle included lack of financial resources to support delivery of OUD services. Duncan et al., reported how COVID-19-related costs overwhelmed limited jail healthcare resources, straining the provision of OUD treatment . Marcus Despite these obstacles, several programs described innovations ensuring continuity of care. First and foremost, most programs acknowledged the critical role of relaxed regulations around MOUD and harm reduction as well as generally greater leniency around OUD service provision that allowed them to provide lower threshold services under COVID-19 conditions , 46, 48.In the current scoping review, we identified innovative policy and program-level efforts that rapidly adapted OUD treatment and harm reduction services to ensure continuous care for PWUOs during the pandemic. Studies covered by this preliminary review were primarily descriptive, highlighting the feasibility of offering telehealth services, increased methadone take-home doses, home delivery of MOUD and naloxone, and prescribing a safe supply of opioid medications for PWUO to reduce risk of overdose from illicit opioids. Strategies to stem disparities in MOUD access for hard-to-reach populations included the provision of long-acting formulations of buprenorphine in place of sublingual buprenorphine in clinics, correctional facilities, and patients’ residences, creation of makeshift services to dispense methadone in emergency shelters, and provision of mobile devices or sanitized phone booths in underserved communities to facilitate engagement with telehealth services. These novel adaptations and experiences of the aforementioned programs under the COVID-19 emergency contribute important practice and policy lessons for the delivery of OUD services that are summarized below:A substantial literature has highlighted the tremendous burden that opioid agonist patients have historically experienced attending frequent in-person clinic visits –59. ThisDespite these promising findings, provider and administrator surveys during the pandemic and prior disasters suggest mixed sentiment to easing restrictions on telemedicine-based visits or extension of methadone take-home doses , 63, 64.Financial and logistical barriers persisted in some patients’ ability to access medications during the pandemic, as described by Castillo et al. . In caseArticles described in our review highlight the general safety and feasibility of telemedicine and align with studies prior to the COVID-19 pandemic demonstrating clinical utility among patients receiving treatment for nicotine or alcohol use disorders,  or bupreAdditional studies are needed to identify systems-, provider-, and patient-level factors influencing the delivery of telehealth services, and exploring which patient sub-groups would benefit from telephone-, video-, and/or in-person encounters with their providers. This includes better understanding and providing guidance around which patients are at risk of harm from relaxed medication restrictions, if any, and whether and how such risks should inform decisions around appropriate use of telehealth or take-home schedules for medications such as methadone. In addition, more work is needed to assess the role of integrated care models in easing patient access to specialty care  or social services during emergencies.Finally, reviewed articles contributed some important insights around the delivery of harm reduction services during the pandemic. Mailing or coordinating in-person pick-up of Naloxone as described by Nordeck  and CourThe provision of safe supply of opioids in British Columbia for PWUOs described by MacKinnon and colleagues  resultedOur review is subject to multiple limitations, including being confined to studies published in English in 2020, those describing specific OUD treatment or harm reduction program modifications during the pandemic, and excluding the grey literature. Thus, it is likely that many innovations from non-English speaking or non-academic settings, or which were published in 2021 were not assessed. Furthermore, programs described in this review often lacked long-term health outcomes and require subsequent prospective and retrospective analyses to clarify their safety and clinical impact over time. While most articles claimed program adaptations were generally well-received by OUD patients and staff, robust and longer-term studies are needed to rigorously identify programmatic and policy modifications that optimize uptake of MOUD and harm reduction services and improve patient outcomes.The COVID-19 pandemic has posed unprecedented challenges globally for PWUOs. Nevertheless, it has also accelerated the development and scalability of innovations and transformed policies, care models, and technologies to lower thresholds for life-saving treatment and harm reduction services. The pandemic has further confirmed the need for additional studies, revision of ethical and legal frameworks, and additional patient- and provider-driven innovations around the provision of OUD services. As we continue to work to confine the ongoing overdose and COVID-19 crises, we hope that lessons learned from the rapid innovations in OUD services will bring lasting changes and improvements in the delivery of effective and humane care.


Disparities in fruit and vegetable intake (FVI) and diet-related diseases exist among low-income and racial/ethnic minority populations ,2,3,4. IDi Noia and Gellerman upheld the Veggie Meter using resonance Raman spectrometry as a non-invasive, objective biomarker approach to measure FVI for assessing dermal carotenoids among low-income populations . HoweverDietary recalls assess FVI by capturing a variety of self-reported foods . Recall Screeners are frequently employed in intervention studies that target FVI, since they are brief, inexpensive, and assess defined variables of interest . ScreeneFVI is an indicator of a behavior and, if interpreted out of context, can be used to place blame on the individual for poor dietary choices. Pairing FVI with other known data sources  can shift the conversation to focus on systematic and external barriers that influence dietary patterns among populations.Sociodemographic categories also may not reflect actual identities. In the U.S., FVI measures are typically validated in English, and should be expanded to other languages to ensure the relevance of translations. Questions based upon sex/gender customarily ask about the female and male genders, and do not extend to non-binary, third gender, and/or self-described options. Racial/ethnic groups are typically limited to five categories specified in the 1997 Office of Management and Budget (OMB) standards on race and ethnicity, and do not address the heterogeneity among groups in responsive and inclusive ways [FVI and related data are potentially powerful tools to illuminate systemic inequities, but they are only as useful as their applicability across populations. Measures integrating diversity  could ensure the inclusion of the variety/types of FVs consumed to reflect the intake of diverse populations. Measures integrating equity  could measure factors that drive FVI. Measures integrating inclusion  could address sociodemographic variables and languages with which people identify. The ability to understand the effectiveness of interventions in eliminating disparities in FVI is dependent upon measures adequately representing dietary patterns and influencing factors, but this is not feasible without incorporating DEI.


Nanogels in biomedical field are promising and innovative materials as dispersions of hydrogel nanoparticles based on crosslinked polymeric networks that have been called as next generation drug delivery systems due to their relatively high drug encapsulation capacity, uniformity, tunable size, ease of preparation, minimal toxicity, stability in the presence of serum, and stimuli responsiveness. Nanogels show a great potential in chemotherapy, diagnosis, organ targeting and delivery of bioactive substances. The main subjects reviewed in this article concentrates on: (i) Nanogel assimilation in the nanomedicine domain; (ii) Features and advantages of nanogels, the main characteristics, such as: swelling capacity, stimuli sensitivity, the great surface area, functionalization, bioconjugation and encapsulation of bioactive substances, which are taken into account in designing the structures according to the application; some data on the advantages and limitations of the preparation techniques; (iii) Recent progress in nanogels as a carrier of genetic material, protein and vaccine. The majority of the scientific literature presents the multivalency potential of bioconjugated nanogels in various conditions. Today’s research focuses over the overcoming of the restrictions imposed by cost, some medical requirements and technological issues, for nanogels’ commercial scale production and their integration as a new platform in biomedicine. Taking into account the recent necessities for drastic improvements of current therapies and diagnostic investigations  crosslinked or physically crosslinked with non-covalent bonds by hydrogen bonds, electrostatic and hydrophobic interactions. The great capacity of absorbing water is attributed to the presence of hydrophilic functional groups, such as –OH, –CONH–, –CONH2– and –SO3H, along the macromolecular chains in the polymer structure.Usually the authors describe nanogel in terms of crosslinked polymer chains with size up to 100 nm  a hydrophilic polymer network by chemically crosslinking poly(ethylene glycol) and (2) poly(ethyleneimine) for antisense oligonucleotides delivery  in the field of nanogels as versatile nanocarriers for: intracellular delivery of genetic material, specific targeted protein delivery and vaccine delivery.Nanogels are promising and innovative biomedical systems as dispersions of hydrogel nanoparticles formed from physically or chemically crosslinked polymeric networks that have been called as next generation of drug delivery systems due to their relatively high drug encapsulation capacity, diversity of drugs that can be encapsulated, uniformity, tunable size, ease of preparation, minimal toxicity, stability in the presence of serum, and responsiveness to external stimuli , poly(lactic)–poly(glycolic) copolymers, polyacrylates and polymethacrylates, poly(e-caprolactone), are some typically synthetic polymers for nanogel preparation. They are mostly spherical particles but the current advancement in synthetic strategies allows for the fabrication of nanogels of different shapes    of bioactive substances   when it turns hydrophobic and removes the water content. As a consequence, the size of the nanogel particles decreases and the therapeutic agent is released via temperature triggered stimulus  because of its biocompatibility. In the LCST domain (32–34 °C), this structure displays coil to globule conformational changes and vice-versa associated with reversible formation and breakage of the hydrogen bond interaction between the polymer and water molecules  and poly(N-isopropylacrylamide) macromolecular chains showed a faster increase in the hydrophilicity and LCST at all the pH values, mainly at pH lower than 5   determine the nanodroplet dimensions, which will finally assure the nanogel particles’ size, size distribution and the stability  and reversible addition fragmentation chain transfer (RAFT). Other researches used ATRP in an inverse microemulsion for the synthesis of stable cross-linked nanogels of water-solublepolymers  nanogel in the presence of an amphiphilic macro RAFT agent, was used by Dorwal  and Yan Amphiphilic copolymers in solution have the property of self-assembling through the functional groups along the macromolecular chains. By locking this assembly, they can obtain covalently crosslinked networks through crosslinking reaction of the polymeric precursors derivatized with polymerizable groups. Thus, a pioneer paper of Edman et al.  reportedStarting from polymer precursors, assembled and crosslinked to obtain nanogels, it is possible to tune the particle size by varying polymer concentration and utilizing the LCST behavior of polymers , presence or click chemistry and photo-induced crosslinking provide methods to synthesize nanogels starting from polymer precursor.In Disulfide bonds are found in natural peptides and proteins, assuring the rigidity and structural stability under certain conditions. Also, disulfide can reversibly reduce to thiol, as a function of thiol concentration of the environmental. Thiol-disulfide exchange is the principal reaction by which disulfide bonds are formed and rearranged in a protein and is a way for the preparation of recyclable cross-linking of micelles  groups to thiols which further exchange with remaining PDS groups, while nanogels are obtained , preventing the leakage of the load encapsulated drug and enhancing the therapeutic efficacy. On the other hand, traditional covalent crosslinking usually involves crosslinking agents, which may cause unwanted toxic effects and damage the entrapped delicate substances, such as cells, proteins, etc. Therefore, more and more biocompatible reactions are necessary in the biomedical field.In conclusion, the formation of nanogels by covalent crosslinking reactions induces the stability in a complex and unkind environment  -b-poly(methacrylic acid)  nanodevices -bl-poly(propylene glycol)-bl-poly(ethylene glycol) and poly(ethylene glycol) were investigated. The hydrophobic poly(propylene glycol)-rich nanodomains are suitable for incorporation by physical entrapment of hydrophobic drugs, such as doxorubicin up to 9.8% –surfactant complexes leads to the network collapse revealed by the decrease of the particle size from ca. 300 nm to ca. 50 nm. Due to their crosslinked architecture, the poly(ethylene oxide)-cl-PEI-based complexes are resistant to the changes in the environmental characteristics, such as pH and salt concentration. Poorly soluble biologically active molecules, retinoic acid (RA) and indomethacin, are also immobilized in this network. The nanogels loaded with RA, for example, form stable aqueous dispersions at physiological pH, recommended in pharmaceutical formulation.Bronich et al.  studied Moreover, the nanogel formulations can be chemically modified to incorporate various ligands for targeted drug delivery or triggered drug release. The drug release from nanogel-drug conjugates is considerably extended and can be controlled by ligand stability. Preclinical studies suggest that nanogels can be used to efficiently deliver biopharmaceuticals even in cells, as well as to increase drug delivery across cellular barriers.Over the years, various review articles have reported the rapid development of nanogels as carriers for biomedical applications , plasmid DNA (pDNA), siRNAs and micro RNAs (miRNAs) used in targeted inhibition of specific mRNA sequences has emerged as one of the most promising method to treat and diagnose numerous diseases like cancer, neurodegenerative disorders and viral infections . The study results indicated that biocompatible and biodegradable core–shell nanogels containing PEI shells and compact gelatin cores can be employed as a new class of delivery carrier for siRNA therapeutic agents.The PEI/gelatin nanogels had spherical shape with an average diameter of 200 ± 40 nm, reduced toxicity (four-times) compared to the native PEI and were able to effectively deliver the siRNA into HeLa cell. Furthermore, the gelatin/PEI nanocarriers exhibited a considerable gene silencing effect through efficient delivery of siRNA, process that was considerably higher than that of the commercial available transfection agent, LipofectamineSimilarly, Park et al.  synthesiDispenza et al.  reportedRecent advances in medicine have highlighted the promising capacity of many proteins and peptides to be employed as therapeutic agents  P(HEMA-co-MAA) nanogels functionalized with mannan. Nanogels bioconjugation on the surface with mannan was designed with the purpose to enhance M cell uptake and target C-type lectin receptors (CLRs) on antigen-presenting cells (APC) by mimicking carbohydrate moieties found on the surface of pathogens. The bioconjugated nanogels demonstrated not only pH-sensitive properties but also ensured an enhanced entrapping and protecting of the loaded material at low pH values, and initiated protein release after switching to intestinal pH values. Surface functionalization with mannan led to an enhanced uptake by macrophages as well as increasing the expression of relevant costimulatory molecules. These results indicate that the surface conjugation of P(HEMA-co-MAA) nanogels with mannan as carbohydrate moieties to provide “pathogen-like” features is a promising approach to prepare a more efficacious oral vaccine system.Durán-Lobato et al.  develope+ effector T cell activation and mucosal immunity  (PEG)-like polymer block based on methoxy triethylene glycol methacrylate (mTEGMA) and a hydrophobic polymer block based on pentafluorophenyl methacrylate (PFPMA). Immunization studies on mice have highlighted that imidazoquinoline-bioconjugated nanogels were more potent at inducing T-cell responses and antibody responses against tuberculosis antigen when compared with soluble IMDQ. This approach demonstrates the potential of these IMDQ nanogels to be used as adjuvants for vaccination , bioconjugation and encapsulation of bioactive substances and methods of preparation, too. A special section is dedicated to nanogel carriers with promising applications in the leading areas of the biomedical research, such as: intracellular delivery of genetic material, specific targeted protein delivery and vaccine delivery.It is obvious that researchers focus primarily on finding new nanogel structures with more and more capabilities: improved design to upload/release bioactive substances over a specified period of time and targeting properties to enable highly selective uptake into the desired organs.While nanogel concept registered a noteworthy evolution, an urgent necessitate for relevant clinical data and a substantial number of unsolved issues regarding their pharmacodynamics, metabolism and pharmacokinetics, still need to be overcome, before nanogels can completely make the transition from clinical trial to current clinical application. At that point in the near future, nanogels as bioactives’ delivery carriers would improve the efficiency of medical care and benefit of the patients.