Rationale: Of late, evidence emerges that the pathophysiology of psychiatric diseases and their affiliated symptomatologies are at least partly contributable to inflammatory processes. Also in alcohol use disorders (AUD), this interaction is strongly apparent, with severely immunogenic liver cirrhosis being one of the most critical sequelae of chronic abusive drinking. This somatic immune system activation negatively impacts brain functioning, and additionally, alcohol abuse appears to have a direct detrimental effect on the brain by actively stimulating its immune cells and responses. As cognitive decline majorly contributes to AUD’s debility, it is important to know to what extent impairment of cognitive functioning is due to these (neuro-)inflammatory aberrations.Method: We hereby summarize the current existing literature on the interplay between AUD, inflammation, and cognition in a systematic review according to the PRISMA-P guidelines for the systematic review.Main findings: Although literature on the role of inflammation in alcohol use-related cognitive deficiency remains scarce, current findings indicate that pro-inflammatory processes indeed result in exacerbation of several domains of cognitive deterioration. Interestingly, microglia, the immune cells of the brain, appear to exert initial compensatory neuroprotective functionalities upon acute ethanol exposure while chronic alcohol intake seems to attenuate these responses and overall microglial activity.Conclusion: As these results indicate inflammation to be of importance in cognitive impairment following alcohol consumption and might as such provide alternate therapeutic avenues, a considerable increase in research efforts in this domain is urgently required. Alcohol is the most commonly (ab)used substance worldwide with high-risk drinking occurring in up to 30% in Western populations . On a glAUD is associated to different cognitive deficits, such as abnormalities in working memory, attention, and executive functions like response inhibition . As thorThe specific etiopathogenesis of these AUD-related cognitive impairments is rather complex, as it may be an inherent pre-morbid trait vulnerability, but may also result from alcohol-related brain damage (ARBD). As alcohol has major neurotoxic effects, its abuse is related to mild brain atrophy that seems mostly driven by white matter loss and changes in cortical neuronal dendritic arborization . ARBD caAs changes in immune system activation may result in cytotoxic effects, thereby impacting neurotransmission, neuroendocrine function, and neural plasticity , 20, neuThese findings are supported by animal studies linking inflammation to alcohol disorders and related cognitive dysfunctioning. For instance, elevated levels of the pro-inflammatory cytokines interleukin 6 (IL-6), IL-1b, and tumor necrosis factor alfa (TNF-a) have been found in the rodent brain after chronic alcohol consumption , 22.Moreover, Marshall et al.  demonstrWith this systematic review, we document the influence of AUD-related inflammation on decreased cognitive functioning observed in AUD patients.This systematic review was conducted and reported according to the PRISMA-P  guideline . We aimeOf note, the latest edition of DSM (V) refers to the disorder of being dependent on alcohol consumption as “alcohol use disorder, AUD.” However, this terminology is rather recent, and literature predating this overarching term mostly refers to this or similar conditions with “alcohol dependence” or related expressions. In order to avoid rephrasing bias in this work, we opted to retain the terminology as applied in the original paper. As such, terminology applied throughout this review remains somewhat heterogeneous in its definition of alcohol consumption disorders.The PubMed database search resulted in an initial 243 records. Following removal of duplicates and screening of abstracts for relevance, 28 records remained. Eligibility of these 28 records was assessed by detailed evaluation of the full texts. Eight papers were deemed not relevant, and three papers concerned preclinical research, resulting in a final selection of 17 papers included in the current review.Alcohol abuse has been associated with cognitive impairment in humans, mainly by deleterious effects on memory function and on executive functions including cognitive flexibility and response inhibition . In addipost hoc exploratory analyses), these findings need to be confirmed approached with caution. The authors also aimed to find associations with cognitive functioning, and in line with the findings of Felipo et al. , Yen et al.  looked fo et al.  results o et al. . MoreoveWilhelm et al.  investigMore indirect findings come from Duivis et al. , who demBoyer et al.  found a Although only a handful of studies looked into the associations between cytokines and cognitive dysfunctioning in alcohol-dependent patients, early results suggest impairments  are associate to increases in pro-inflammatory markers and reductions in anti-inflammatory cytokines. Alcohol abstinence may partially remediate some of these deficits as mirrored by normalization of cytokines.via PET tracer imaging in newly abstinent (< 1 month) alcohol-dependent patients (n = 9). In contrast to their expectations, they found less microglial activation than healthy controls, which they suggested to be the result of a potential loss of microglia cells. Although microglia appeared to be less activated in patients, tracer binding strongly correlated with delayed verbal memory, pointing toward poor memory performance in those patients with low microglia activation. This would intuitively contrast with the report of Marshall et al. (M2 phenotype) rather than a neurotoxic (M1) profile in response to ethanol and that chronic alcohol abuse decreases the number of these safeguarders.Kalk et al.  recentlyl et al. , who shoThe literature investigating the impact of alcohol use and abstinence on glial activation patterns and their functionality is too scarce to be informative. However, the little preliminary data that are available suggest this to be a research avenue of interest for future studies focusing on the role of inflammation on cognitive deterioration by alcohol use.Many of the articles included in this review pointed toward an important role of the liver function and gut permeability and its relation with alcohol (ab) use and subsequent inflammatory processes.via the liver  is the most prevalent type of chronic liver disease worldwide. ALD is accompanied by an inflammatory presentation characterized by increases in pro-inflammatory cytokines and chemokines , 36. Alche liver , 8.postmortem findings suggest that a pro-inflammatory state, abnormal astrocyte, and microglial activation also seem to be involved  implies the deterioration of brain functioning arising from acute and chronic liver failure as a result of chronic alcohol abuse , 37. Thiinvolved . Dennis involved  investigHE has also been shown to increase plasma IL-6 levels , but it Furthermore, the cognitive impairments inversely related to levels of the anti-inflammatory cytokine IL-10 in AD patients as described by Leclercq et al.  appearedSo, although associations between alcohol-induced immune dysregulations and cognitive dysfunctioning in the absence of liver pathology have been shown, affected liver functioning may further fuel a pro-inflammatory state peripherally and in the CNS, thereby negatively impacting cognitive functioning in AUD patients.via PET imaging an increase in glial cell activation in patients with HE, Kalk et al.  pathway. This inflammatory degradation process catabolizes the essential amino acid tryptophan to kynurenine and its metabolites. It mainly occurs in microglia and astrocytes and is activated by pro-inflammatory cytokines . SeveralThe relationship between immune dysregulation and cognitive deficits in AUD patients seems to be further modulated by hepatic and alcohol use–induced gastro-intestinal pathologies , 39, altAlthough we attempted to reflect on the existing literature as accurately as possible, this review contains several study- and review design–inherent limitations.First, the DSM-V categorizes all alcohol-related disorders under the same flag of “AUD.” However, as some literature described in this review predates this novel classification, investigated populations over the included studies are often differentially defined. As such, this literature overview might encompass a heterogeneous population with differing levels of alcohol abuse as papers scrutinize “alcohol dependence” or “chronic alcohol abuse” while data were piled in this work as hallmarking the whole of AUD. Second, in addition to “classical” cognitive functions like working memory and processing speed, alcohol dependence is dependent on more complex cognitive reasonings like the sociocognitive ability to infer other’s thoughts (theory of mind). Although we did not ambition to exclude papers on the relation of inflammation with these more complex cognitions, no works were found on this topic. Likewise, although extensive literature is available on the considerable role of different domains of cognitive impairment on patient prognosis, therapeutic outcome, relapse rate,… [for review see Ref. ], no repLastly, while extensive preclinical literature provides strong evidence for this three-way interaction between AUD, its cognitive impairments, and immune system aberrations, the actual number of studies investigating it is surprisingly low. This evidently renders making hard statements on the nature of the interactions somewhat difficult. However, this review aimed at providing sufficient indications that addressing this research avenue more in depth might vastly elucidate inflammatory pathways to be of importance in cognitive deficits marking AUD.VC and MM performed literature search and wrote the article. MD and GD acted as advisory board on included papers and reviewed 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.


For more than three decades, Botulinum neurotoxin (BoNT) has been used to treat a variety of clinical conditions such as spastic or dystonic disorders by inducing a temporary paralysis of the injected muscle as the desired clinical effect. BoNT is known to primarily act at the neuromuscular junction resulting in a biochemical denervation of the treated muscle. However, recent evidence suggests that BoNT’s pharmacological properties may not only be limited to local muscular denervation at the injection site but may also include additional central effects. In this review, we report and discuss the current evidence for BoNT’s central effects based on clinical observations, neurophysiological investigations and neuroimaging studies in humans. Collectively, these data strongly point to indirect mechanisms via changes to sensory afferents that may be primarily responsible for the marked plastic effects of BoNT on the central nervous system. Importantly, BoNT-related central effects and consecutive modulation and/or reorganization of the brain may not solely be considered “side-effects” but rather an additional therapeutic impact responsible for a number of clinical observations that cannot be explained by merely peripheral actions. Botulinum neurotoxin type A (BoNT) is used in a variety of clinical conditions such as spastic or dystonic disorders . BoNT reSeveral early animal studies provide evidence for a retrograde transport of BoNT, similar to the well-known retrograde transport of tetanus toxin ,4, but tIn this review, we focus on the main human experimental and clinical studies providing information on the central action of BoNT. Central effects are discussed based on clinical observations as well as neurophysiological and imaging studies.Experience from clinical routine already suggests that BoNT-related muscle weakening may not exclusively be the consequence of BoNT’s action at the neuromuscular junction, but also at the spinal and supraspinal levels. In dystonia, clinical improvement without or with little weakness is frequently observed, whereas other patients do not improve despite relevant muscle weakness ,11, thusIn summary, there is unequivocal clinical evidence for an (indirect) central effect of BoNT, an observation that is further underscored by numerous human electrophysiological studies.A large number of clinical neurophysiological studies provide detailed evidence for distinct BoNT effects within spinal cord circuitries, the brainstem and the sensorimotor cortex.Trompetto et al. recognized that BoNT acts differently on extra- and intrafusal muscle spindles as measured by maximal M-wave and maximal voluntary contraction  as well as the tonic vibration reflex . In this study of patients with focal hand dystonia (FHD), BoNT induced a persistent clinical benefit even though indicators of extrafusal chemodenervation had fully recovered. This suggests that changes in Ia-afferences may have resulted in (indirect) central effects . F-wave Brainstem excitability can be experimentally studied using the blink reflex and its variations, or brainstem auditory evoked potentials. Several studies did not reveal any modulation of hyperexcitable brainstem pathways in patients with blepharospasm following BoNT injections into the orbicularis oculi muscle ,33,34. FModulation of Ia-afferences and the stretch reflex loop were considered responsible for the increase in somatosensory evoked potentials following BoNT injection in patients with cerebral palsy ,41  in order to investigate possible changes in cortical activation patterns following BoNT therapy. In an early study by Ceballos-Baumann and colleagues in patients with FHD, BoNT improved writing but failed to improve the associated reductions of the regional cerebral blood flow  of the primary motor cortex [Early imaging studies made use of Hr cortex . On the r cortex . This str cortex .The largest neuroimaging evidence for the central effects of BoNT comes from functional magnetic resonance imaging (fMRI) studies. Several studies reported a decreased, and in part more lateralized, activation pattern in contra- and ipsilateral cortical motor areas following BoNT therapy in patients with spastic hemiparesis due to stroke ,60,61. HIn dystonia, cerebral activation patterns are not confounded by stroke-related parenchymal injury. Here, BoNT treatment was associated with widespread changes on fMRI-measured activation  within several brain regions, such as the bilateral primary and secondary somatosensory cortex, the bilateral supplementary motor area, the contralateral primary motor cortex and the cerebellum in drug-naïve CD patients . Hence, Resting-state fMRI allows the investigation of distinct brain networks and the interaction between different brain areas at rest . This poFew studies exist that explicitly investigated structural changes of the brain due to BoNT therapy in focal dystonia patients. Two studies found white matter abnormalities in the basal ganglia regions in patients with CD and FHD, using diffusion tensor imaging ,80. ThesAll the studies presented here used BoNT type A. Hence, no conclusions can be made regarding other types of BoNT such as type B, which is also commercially used. However, as all types of BoNT act at the cholinergic presynapse, blocking acetylcholine release at the neuromuscular junction, the indirect central effect should not significantly differ between different types of BoNT. Furthermore, patients were investigated in a clinical setting, not reporting or differentiating between dosage, injected muscles or time since last BoNT injection. However, for baseline investigations, patients were studied at least 12 weeks since their last BoNT treatment, when the clinical effect of BoNT should have been minimized. They were usually investigated around 4 weeks following BoNT injections. However, as reported in our last study , drug-naBoNT primarily acts at the neuromuscular junction, resulting in a biochemical denervation and muscle weakness of the injected muscle, a mechanism which undoubtedly constitutes the main action and cause for the reliable clinical effect of BoNT in several neurological disorders. Nevertheless, beside its peripheral action, strong clinical, neurophysiological and neuroimaging evidence exists indicating additional BoNT-related central effects. It is somewhat limiting, however, that all these latter investigations were not sufficiently capable to differentiate between direct actions of BoNT on the CNS and indirect effects due to the modulation of the afferent sensory input to the spinal cord and brain. However, the current literature suggests that indirect effects of BoNT on the brain may be more prominent. Here, changes to the afferent input are thought to result in short- and long-term plastic changes to the CNS, as assessed by functional and structural methods. This modulation or reorganization of the brain may itself have an additional therapeutic effect. It may potentially be responsible for the long-lasting clinical effect of BoNT or its effect in non-treated muscles. It will be interesting to see whether other neurophysiological methods such as transcranial magnetic or direct current stimulation will provide novel insight into BoNT’s plasticity effects and whether their clinical application may help to improve and prolong the (positive) central plastic effects.


Drosophila has become a valuable model to study epidermal wound healing because of the comprehensive genetic toolkit available in this organism and the similarities of wound healing processes between Drosophila and vertebrates. Other reviews in this Special Issue cover wound healing assays and pathways in Drosophila embryos, pupae and adults, as well as regenerative processes that occur in tissues such as imaginal discs and the gut. In this review, we will focus on the molecular/genetic control of wound-induced cellular processes such as inflammation, cell migration and epithelial cell-cell fusion in Drosophila larvae. We will give a brief overview of the three wounding assays, pinch, puncture, and laser ablation, and the cellular responses that ensue following wounding. We will highlight the actin regulators, signaling pathways and transcriptional mediators found so far to be involved in larval epidermal wound closure and what is known about how they act. We will also discuss wound-induced epidermal cell-cell fusion and possible directions for future research in this exciting system.For animals, injury is inevitable. Because of this, organisms possess efficient wound healing mechanisms that can repair damaged tissues. However, the molecular and genetic mechanisms by which epidermal repair is accomplished remain poorly defined.  Drosophila has a long history of fundamental discovery related to developmental biology . During DC, two apposed epidermal sheets migrate dorsally and close a dorsal gap to form a continuous epidermal layer around the developing embryo  in Drosophila, is also required for epidermal wound closure. Profilin protein levels increase surrounding the wound throughout and following active closure. Loss of chic reduces actin polymerization around the wound (Drosophila yorkie (yki), a YAP-like transcriptional activator, and scalloped (sd), a TEA Domain family protein-like transcription factor (TEAD), common transcriptional mediators of the Hippo pathway, also cause reduced actin polymerization around the wound edge and wound closure defects , translocates to the wound margin a few hours after wounding and is required for both wound closure and up-regulation of filamentous actin around the wound margin , is also required for wound closure  exhibit wound closure defects when targeted by RNAi in the larval epidermis  blocks puncture wound closure  was originally implicated in DC , JNK kinase kinase , JNK kinase , JNK (basket), DJun (Jun-related antigen) and DFos (kayak), all led to a substantial impairment of wound closure (JNK is a member of the mitogen activated protein kinase (MAPK) family, and plays important roles during development, physiology and disease . JNK , the transcriptional activator of the Hippo pathway, controls organ size and tissue regeneration through its well-characterized roles in balancing apoptosis , which is also important for normal wound closure , which encodes a critical adaptor protein for Hippo activation, was conditionally knockout during heart regeneration in mice  transcription factor are activated slightly later and further away from the wound center than JNK signaling  and the full suite of downstream functional targets remains to be identified. In addition, how the epigenetic changes observed upon wounding couple to wound-induced transcriptional regulation will be an intriguing topic to pursue.Drosophila. Although the function of cell-cell fusion during wound healing in larval and pupa is still unclear, cell-cell fusion appears to be critical for epidermal wound healing in adult flies , integrin-linked kinase (ILK), or β-integrin itself in the larval epidermis resulted in multinucleate epidermal cells even without wounding , which encodes an upstream kinase of the JNK signaling cascade, also increased wound-induced cell fusion  signaling (JNK affects cuticle detachment, migration efficiency, and abolishes cell shape changes in both leading edge and follower cells (Although certain genes and signaling pathways are crucial for larval wound healing, many open questions remain. One of the fundamental questions, for any repair process, ishow does the tissue first sense the wound and initiate appropriate wound responses? JNK signaling is rapidly turned on after wounding  and lossThe VEGFR/Pvr and its ligand Pvf1 are also reasonable candidates for an early signal. Pvf1 becomes accessible to its receptor following wounding and epidermal Pvr is required for closure , suggestPhysical force has long been proposed as a wound-induced signal . This waLarval epidermal wound healing is a form of collective migration because the epidermal cells involved migrate as a sheet, maintaining their connections to each other even as they heal the wound. Because of this, the responses of leading edge cells and follower cells need to be coordinated in order to close a wound. There is evidence already for distinct cellular responses of wound-edge and follower cells (How are different signaling pathways activated in the leading cells or the followers? Wound-induced JNK signaling activation forms a gradient, with the cells closest to the wound showing the highest levels of JNK activation . One posAnother model is that different pathways are activated in different spatiotemporal domains and control responses appropriate to those times and locations. There is already some evidence for this-JAK/STAT signaling is activated farther away from the wound and later after wounding than JNK is  and how Drosophila embryogenesis (Larval wound healing serves as a powerful screening platform to identify genes that are required for wound healing (Larval epidermal wound healing is a powerful platform to identify genes that are required for postembryonic wound healing. Many signaling pathways and their functions have been identified in this system. Although there are still many remaining questions to be explored, the knowledge gained from this system is likely to have implications in tissue repair and regeneration in other organisms since wound healing is highly conserved in metazoans.


The main treatment available for idiopathic macular holes is represented by pars plana vitrectomy with internal limiting membrane peeling. However, late-stage macular holes are affected by a higher risk of surgical failure. Although adjuvant techniques can be employed, a satisfactory functional recovery is difficult to achieve in refractory macular holes. Given their neuroprotective and antiapoptotic properties, mesenchymal stem cells (MSCs) may represent an appealing approach to treat these extreme cases. The purpose of this review is to highlight the findings regarding healing mechanisms exerted by mesenchymal stem cells and preliminary application in cases of refractory macular holes. When compared with MSCs, MSC-derived exosomes may represent a feasible alternative, given their reduced risk of undesired proliferation and easiness of use. A macular hole is an anatomic gap in the retina occurring at the level of the fovea. The majority of macular holes is idiopathic, although they can be associated with highly myopic eyes or ocular trauma, especially in the elderly .The general prevalence of full-thickness idiopathic macular holes found in the Beaver Dam Study is 0.3%, with rates increasing to 0.8% in people with 75 or more years of age . The genOccasionally, macular holes are associated with retinal detachments, leading to severe visual impairment, especially in elderly people with elevated myopia and in the presence of a posterior staphyloma , 4.The typical progression of macular holes is characterized by a series of stages  over a period of weeks, featuring retinal defects which in severe cases involve the macular region in all its thickness . In the As a treatment for macular holes, Kelly et al. introduced in 1991 the use of pars plana vitrectomy, with removal of epiretinal membranes and introduction of long-acting gas tamponades . Internaμm) and late-diagnosed macular holes (>6 months) are affected by low success rates [Other several advances in this surgical field allowed more satisfactory outcomes with a 98% closure rate of early-onset macular holes . Howeverss rates , particuss rates . In factss rates . Moreovess rates .As of today, the goal of the surgical procedure focuses on the resolution of vitreal or epiretinal tangential tractions on the central retina and macular hole's margins, achieving its closure with good anatomical results in more than 90% of eyes with full-thickness macular holes –20. EvenRetinal degenerative diseases can be characterized by loss of cellular elements, such as retinal ganglion cells (RGC), retinal pigment epithelium (RPE) cells, and photoreceptors, and the following visual impairment is typically irreversible, since retinal cells lack self-repair capability. Several cell-based treatments have been proposed and evaluated to overcome these limits.Cell therapy represents an appealing alternative to obtain regeneration of damaged retina, potentially improving functional outcomes also in surgery of macular hole. Transplantation of retinal cells has been considered as a potential treatment for retinal degenerative diseases, particularly in late stages associated with severe cell damage. This approach aims at replacing lost retinal cells using stem cells, progenitor cells, and mature neural retinal cells with potential applications in a wide range of retinal degenerative conditions.The purpose of this review is to highlight perspectives of cell-based treatment for refractory macular holes, focusing particularly on the current evidence regarding the application of autologous mesenchymal stem cells (MSCs) since they overcome the ethical and safety problems of embryonic and induced pluripotent stem cells . In addition, mesenchymal stem cells can be easily extracted from different human tissues and possess neuroprotective and immunomodulatory properties.Appealing anatomic and functional outcomes have been reported in animal models of retinal degeneration by various study groups evaluating the transplant of RPE cells, retinal progenitor cells, photoreceptor precursors, or full-thickness retinal sheets –44. HoweIn fact, it is possible to obtain a virtually unlimited source of different retinal cells  from the expansion of pluripotent stem cells, both embryonic and iPSCs, as shown in numerous studies –53.Several stem-cell types can differentiate into photoreceptors in preclinical models of human retinal degenerative disease, including bone marrow-derived hematopoietic stem cells, bone marrow-derived MSCs, neural progenitor cells (NPCs), and forebrain-derived progenitor cells . Carr etMesenchymal stem cells possess the ability of self-renewal and unlimited differentiation. Moreover, it is possible to retrieve MSCs from several human tissues . Given their well-known properties and the possibility to isolate MSCs with ease, this cell type represents a promising candidate for the treatment of retinal degenerative diseases . In factMSCs derived from both bone marrow and adipose tissue have been injected into the subretinal space of animal models of retinal degeneration with significant improvements in terms of visual function and RGC density, although for a short period –68. OtheIt is indicated that MSCs can express a variety of factors which could protect the injured retina, such as NGF, CNTF, BDNF, bFGF, NTFs, and IGF1 , 72, 73.NCT01518127, NCT01068561, and NCT01518842) [NCT01560715), a quality-of-life improvement in patients affected by retinitis pigmentosa was observed, although not sustained at 12 months after MSCs injection [The intravitreal use of CD34 + BMMSCs in patients with age-related macular degeneration has been evaluated in three phase I clinical trials, with improved visual acuity over a 12-month follow-up (1518842) , 79. In njection –82.NCT01920867), with improvements in terms of visual acuity and visual field parameters [However, in a study employing CD34 + BMSCs intravitreal injections (with 3.4 × 106 cells in a 0.1 ml suspension), no improvement in visual function was observed in 6 patients affected by AMD, even if no complications occurred , 82. In rameters –86.NCT02024269), leading to the withdrawal of the study [Severe complications and vision loss, related probably to stem-cell preparation, have been observed in a clinical trial adopting adipose-derived MSCs  in comparison with control eyes (p=0.001), with more evident differences at early postoperative time [Encouraged by these positive findings, Xuqian et al. injected in vitro adipose-derived MSC cultures into rabbits with retinal holes undergoing vitrectomy, evaluating the effects with optical coherence tomography and immunohistochemical analysis. OCT images of treated eyes day showed reattachment of the everted hole edges at the second day after surgery with onset of healing tissue at the 4ive time .To confirm the effects of adipose-derived MSCs, additional cell injections were performed in refractory retinal holes after surgery in the control group, with clear observation of a healing process similar to the one occurred in the treatment group.The histological analysis by Xuqian et al. confirmed that healing tissue cells were different in the retinal holes of the treated group compared to the control group. In fact, control eyes presented a proliferation of collagenous fibrotic tissue, while the retina of treated eyes featured mesenchymal tissue with photoreceptor- and bipolar-like cells. However, it was observed that these cells were not originated from injected MSCs since the transplanted donor cells did not survive abundantly in the healing tissue. Xuqian assumed that the healing process in the transplantation group was promoted by MSCs expression of cytokines with antiapoptotic functions and related to extracellular matrix development, such as intercellular adhesive factors, with better adhesion of the retina to the RPE.A recent case series of 7 human patients affected by refractory and late-stage macular holes evaluated effects on visual function and safety of MSC suspension and MSC-Exos in combination with pars plana vitrectomy and gas tamponade (SF6 or air in patients receiving exosomes) or heavy silicon oil (in patients receiving cell suspension). Silicon oil was employed in order to limit the dispersion of the cell suspension, and patients were instructed to remain in supine position for 1 day. Silicon oil was then removed with surgery in the two patients receiving the cell suspension .µg of MSC-Exos via intravitreal injection in association with an air tamponade (in accordance with an improvement in BCVA from 20/200 to 20/50).BCVA improvements were observed in five patients presenting hole closure after surgery, with a mean postoperative visual acuity of 20/110 (ranging from 20/160 to 20/50). The complete hole closure was observed at 19 days after surgery, by average . The only patient who did not experience improvements in visual acuity was a woman with a 4-year history of macular hole, which was already treated with previous unsuccessful surgery. Nevertheless, she presented hole closure at 1 month after pars plana vitrectomy with intravitreal MSC-Exos, even if her BCVA remained unchanged. Only one of the treated eyes did not presented a macular hole closure after pars plana vitrectomy in combination with phacoemulsification and intravitreal injection of MSC-Exos, but its BCVA improved slightly (from 20/200 to 20/160) probably due to cataract removal. Among all the patients included in the case series, reduction in central scotoma was observed, with significant improvements in one patient receiving 20 μg of MSC-Exos presented signs of inflammation in the anterior chamber, which was resolved 3 days after application of steroid eyedrops. Thereafter, MSC-Exos dose was reduced to 20 μg without observation of other cases of significant inflammation. All patients were subjected to a follow-up period lasting between 0.5 and 3 years, without evidence of side effects related to MSCs and MSC-Exos and risk of teratoma development. However, limitations of this preliminary study lie in the small number of patients and the absence of a comparison with a control group, preventing the assessment of the real therapeutic effects of MSCs and MSC-Exos.In regard to adverse reactions, formation of fibrotic epiretinal membrane due to proliferation of injected cells occurred in one patient receiving MSCs in association with pars plana vitrectomy and heavy silicon oil endotamponade. This fibrotic membrane was then removed through an additional surgical procedure, and the pathological analysis revealed the proliferation of fibroblast-like cells. In the following 6 months, no recurrence of fibrotic membrane was observed, and a decrease in central scotoma was reported. Moreover, the first patient who underwent treatment with 50 Adjuvant surgical strategies, such as ILM inverted flap and ILM insertion, have been adopted to improve outcomes in refractory and large macular holes with successful anatomical and visual outcomes , 92, 93.However, these techniques are not without potential problems, such the risk of direct damage to RPE. Moreover, the heterogeneous characteristics of late stages and refractory macular holes may prevent the actual assessment of the effects of adjuvant procedures on postoperative results. Among the few studies comparing different adjuvant techniques, the analysis by Park et al. concluded that the inverted flap technique resulted in better recovery of photoreceptor layers with better functional outcomes, when compared with ILM insertion .Stem-cell therapy has emerged as a novel candidate approach for the treatment of retinal degenerative diseases, given their proven effects of neurogenesis, cell replacement, reduced apoptosis, and modulation of inflammation and immune responses , 96. MSCIn case of a macular hole, it is advisable to achieve the contact between the retinal neural epithelium and the RPE, in order to obtain hole closure . ThereafXuqian et al. in their analysis on animal models concluded that MSCs promote hole closure rate with accelerated healing. Morphological histochemical and OCT observations showed significant differences between repair tissues in eyes receiving MSCs and the reference group, which presented a typical fibrotic scarring .Previous studies have demonstrated that MSCs promote the expression of adhesive factors  and extrAccording to these findings, a possible rationale for future stem-cell therapy may be developed, and failure of macular hole surgery may be explained by long-term detachment of neuroretina from the RPE layer.In comparison to gene therapy, MSCs provide multiple rescue pathways with a plurality of combined effects. Moreover, in a previous paper published by our group, we observed that the presence of a human retinal pigment epithelial cell-line supernatant helped cells preserve the typical MSC morphology . TherefoIt has been reported that up to 44% of large macular holes remain open after vitrectomy with ILM removal  and repeIn the only available pilot study, macular hole closure was achieved in six of seven patients who underwent MSC or MSC-Exo therapy, with five patients showing improvements in BCVA. These results suggest that MSCs and MSC-Exos may contribute to hole closure and visual function recovery . MSCs anCompared with cell suspensions, exosomes are easier to employ, presenting lower immunogenicity and risk of proliferation. It was reported that MSC-Exos have similar protective effects towards the retina after laser-induced lesions and experimental model of uveitis , 105.Since MSC-Exos do not tend to disperse in the vitreal cavity after injection, heavy silicon tamponades and patient positioning may not be relevant to promote their effects. However, the mechanism of MSC-Exo therapy is still not fully understood. It has been suggested that miRNAs carried by MSC-Exos promote neurogenesis and functional recovery .Another issue that must be addressed in larger trials is toxicity of MSCs or MSC-Exo in the treatment of retinal diseases. Among the existing clinical trials, three patients were affected by severe bilateral vision loss after receiving intravitreal injections of autologous adipose tissue derived stem cells, due to severe vitreoretinal proliferation  . However, MSCs application at the retinal level can lead to unwanted differentiation of transplanted cells with onset of vitreoretinal proliferation, and the presence of preexisting tumors whose growth may be promoted by immunosuppression provided by MSCs represents an important contraindication . Autolog


Chronic inflammatory demyelinating polyneuropathy (CIDP) is a kind of autoimmune-mediated inflammation and demyelinating disease. The etiology is mainly related to autoimmune dysfunction. The conventional treatments of CIDP have relied on immunomodulation and inhibition therapies such as adrenal cortex hormone, intravenous immunoglobulin (IVIg) and plasma exchange. Hematopoietic stem cell transplantation (HSCT) is known as a novel therapy for autoimmune disorders, which provides the chance to cure CIDP. More than 70 patients with refractory CIDP have received HSCT. The clinical symptoms and electrophysiological examination results of most patients have been improved. However, the treatment still has risks. This review describes the pathogenesis of CIDP and the current conventional treatments, and highlights the application of HSCT in CIDP, including its efficacy and safety. CIDP is mainly a group of clinical syndromes. The course of the disease is chronic progression or recurrence lasting more than 8 weeks. It often manifested as symmetrical weakness of the proximal or distal limbs, loss of sensation and tendon reflexes. The main diagnostic methods include neurophysiological test, cerebrospinal fluid and pathological examination. Most patients often have cerebrospinal fluid protein-cell separation. Neuroelectrophysiological test suggests demyelinating lesions. Nerve biopsy shows segmental demyelination and regeneration, even the formation of “onion bulbs-like” changes. A retrospective study by Karine et al. The pathogenesis is still not very clear. Previous clinical case reports show that the onset of CIDP may be related to the induction of specific infections such as hepatitis B virus, EB virus and hepatitis C virus etc. The main targets of antibodies in CIDP are antigens on myelin and Schwann cells Infiltration of T lymphocytes Macrophages are also the main inflammatory cells and form colonies around blood vessels in nerves Current treatments that have been validated by clinical randomized controlled trials include corticosteroid, plasma exchange and IVIg. Regarding the study of corticosteroid therapy, in a prospective cohort study in 2012 The clinical effect of plasma exchange was confirmed in early clinical trials in 1986 In 2001, Mendell et al. Apart from the above-mentioned first-line treatments, other immunosuppressive and regulatory drugs have also been reported for clinical use and research, such as azathioprine. A clinical randomized trial in 1985 The clinical use of interferon (IFN) has been reported Although there are so many immunosuppressive therapies, they cannot induce long-term, drug-free and symptom-free remission, some patients with CIDP still relapse, even leading to disability and death. Therefore, patients may choose hematopoietic stem cell transplantation as a better treatment option.Hematopoietic stem cell transplantation has been widely used to treat refractory autoimmune diseases, aiming at eradicating autoreactive immune cells and rebuilding a new immune system that is self-tolerant. Relief of clinical symptoms of autoimmune diseases after hematopoietic stem cell transplantation is the result of immune system reconstruction From 2002 to 2018, there are about 70 cases of refractory CIDP treated by HSCT worldwide. In general, the relapse frequency of the patients has decreased with different degrees. The electrophysiological examination results have been significantly improved. The symptoms have diminished, the progress of the disease has been effectively controlled, and some patients have stopped immune regulation or inhibition therapy after HSCT.The earliest reported treatment of CIDP with hematopoietic stem cell transplantation was in 2002. Vermeulen et al. In 2007, Oyama In 2016, R. Kotas From 2002 till now, another 66 patients with refractory CIDP received HSCT treatment. These patients needed to fulfil the definite, probable, or possible EFNS/PNS criteria for CIDP. They were incomplete response or relapse after first-line treatment, and non-response to at least one second-line immunosuppressive drug . After HSCT, most of them have achieved good clinical efficacy. See Table Electrophysiological examination is an important method to evaluate the efficacy of HSCT in patients with CIDP. Specific observation indexes include compound muscle action potential (CMAP), sensory nerve action potential (SNAP) and nerve conduction velocity of limbs. The increase in amplitude of action potential and conduction velocity often indicate remission of the disease.Vermeulen et al. Remenyi et al. In 2009, Mahdi-Rogers et al. In 2013, Press et al. Safety-related indicators of HSCT therapy include adverse reactions, toxic effects and transplant-related mortality (TRM). Adverse reactions and toxic effects often include early adverse reactions and toxic effects (within 100 days after transplantation) and late adverse reactions and toxic effects (after 100 days of transplantation). The former may be related to allergic reaction and side effect induced by immunosuppressive drugs and opportunistic infection after immunosuppression. Common symptoms include fever, infection, abnormal liver function, etc. Bacterial or fungal infection occurs early after HSCT in neutropenia, while long-term hypolymphemia can reactivate latent viruses and other opportunistic infections, thus endangering patients until their immune systems are rebuilt Compared with autologous hematopoietic stem cell transplantation, allogeneic hematopoietic stem cell transplantation may bring more complications and higher mortality Transplant-related mortality is the most important problem in HSCT. In 2010 Gualandi et al. CIDP is an autoimmune disease, which is currently considered to be related to cellular and humoral immune abnormalities. Patients are often characterized by loss of sensation and weakness of limbs. Severe cases may even lead to disability or even death. Conventional treatments include immunosuppression and immune regulation, but some patients still have no response to such treatments, so HSCT is used. So far, more than 70 people have been treated with HSCT. HSCT is the only therapy that can induce long-term, asymptomatic remission in refractory autoimmune diseases and a possible new way to solve the problem of autoimmune regulation abnormalities, so that it provides a new approach and method for refractory CIDP. Judging from the effect of current preclinical tests on CIDP, although large-scale clinical RCT tests are lacking, and cases of relapse and complications related to HSCT have been reported, HSCT is safe and feasible in general. The symptoms and neurological score of most patients improved and a longer-term remission period was obtained. The complications after HSCT include toxic and side effects of conditioning drugs, virus and bacterial infection caused by hypo immunity after transplantation, but most of them can be improved by corresponding treatments. In conclusion, HSCT is a viable treatment option for refractory CIDP.


Postmortem studies in patients with schizophrenia (SCZ) have revealed deficits in myelination, abnormalities in myelin gene expression and altered numbers of oligodendrocytes in the brain. However, gaining mechanistic insight into oligodendrocyte (OL) dysfunction and its contribution to SCZ has been challenging because of technical hurdles. The advent of individual patient-derived human-induced pluripotent stem cells (hiPSCs), combined with the generation of in principle any neuronal and glial cell type, including OLs and oligodendrocyte precursor cells (OPCs), holds great potential for understanding the molecular basis of the aetiopathogenesis of genetically complex psychiatric diseases such as SCZ and could pave the way towards personalized medicine. The development of neuronal and glial co-culture systems now appears to enable the in vitro study of SCZ-relevant neurobiological endophenotypes, including OL dysfunction and myelination, with unprecedented construct validity. Nonetheless, the meaningful stratification of patients before the subsequent functional analyses of patient-derived cell systems still represents an important bottleneck. Here, to improve the predictive power of ex vivo disease modelling we propose using hiPSC technology to focus on representatives of patient subgroups stratified for genomic and/or phenomic features and neurobiological cell systems. Therefore, this review will outline the evidence for the involvement of OPCs/OLs in SCZ in the context of their proposed functions, including myelination and axon support, the implications for hiPSC-based cellular disease modelling and potential strategies for patient selection. This burden was quantified in the Global Burden of Disease Study 2013, which found a remarkably high number of disability-adjusted life years (DALY) and years lived with disability (YLD) in SCZ compared with other medical conditions.7 The clinical features of SCZ have been subdivided into positive, negative and cognitive symptoms. Positive symptoms are likely to be associated with a hyper-dopaminergic state. Neuroleptics that act mainly at the dopamine receptor 2 (DRD2), which is highly expressed in the basal ganglia, are effective in reducing positive symptoms in many patients.8 An increasing body of evidence indicates that negative and cognitive symptoms are functionally associated with an excitation/inhibition dysbalance in the function of glutamatergic and ɣ-aminobutyric acid (GABA)ergic synapses. This dysbalance is proposed to eventually lead to a disconnection of critical cortico-cortical and cortico-subcortical projection systems, although the underlying mechanisms are not fully understood.9 No effective pharmacological treatment is available for negative and cognitive symptoms.8Schizophrenia (SCZ) is a severe, disabling neuropsychiatric disorder with a lifetime prevalence of 0.3%–2.3%.10 The lack of human neurobiological test systems to study the consequences of genetic and pharmacological perturbation, for example, has been a major limitation in the field of psychiatry to date. Research has been mainly restricted to peripheral tissues, such as blood, correlative imaging studies, genetics and molecular and histological analyses of postmortem brain samples. Taken together, these approaches have revealed strong evidence for a synaptic dysfunction. In addition, current evidence of altered white matter (WM) structures and reduced myelin gene expression in SCZ suggests that dysfunctional oligodendrocyte precursor cells (OPCs) and/or oligodendrocytes (OLs) may also contribute to the dysconnectivity of brain regions seen in this disorder.14 Moreover, despite compelling advances in the understanding of genetic factors that contribute to the risk for SCZ, little is still known about the final mechanistic consequences of such risk factors in the biological systems involved in the genesis of SCZ.17So far, all attempts to target the synapse-associated glutamatergic system have failed and thus other mechanisms need to be explored to identify novel treatment options.20 These systems hold promise for comparing cellular phenotypes in patients and healthy controls, whereas keeping the individual genomic risk background of each individual. Likewise, such cellular models have some characteristics that make them especially well-suited to assessing how pharmacological treatments may restore affected pathways in patient-specific cell types.22The unavailability of proper model systems to accurately assess the functional consequences for and contribution of dedicated molecular and cellular signatures to these disorders is one possible reason for the lack of biological insight. Animal models, especially inbred genetic mouse models, are well-suited to characterize the molecular and phenotypic  impact of one or a few risk genes but cannot capture the complex genetic risk architecture of SCZ. However, recent developments in the methodologies for the induction of pluripotent stem cells and their differentiation into neuronal and glial cell types have paved the way for the generation of patient-specific cellular systems.This review aims to (i) revisit dysfunctions of OPCs/OLs in SCZ, including and beyond myelination, and (ii) discuss strategies for patient selection towards stem-cell generation and cellular modelling as a pre-requisite to target OPC/OL deficits with pharmacological approaches.23 The hypothesis that SCZ is not a neurodegenerative but a neurodevelopmental disease that alters brains circuits was proposed nearly 40 years ago25, but was recently rediscovered and is gaining increasing influence.29Puberty and adolescence are vulnerable periods of brain development and are characterized by a multitude of psychosocial challenges related to work and interpersonal relationships. Adolescence coincides with the average age of onset of affective and non-affective psychoses.31 Postmortem analyses of human brain tissues have revealed that myelination follows a defined chronological and topographical order along the caudal to rostral axis.33 The fact that associative cortical areas such as the prefrontal cortex are among the latest to become myelinated, i.e. not until early adulthood, and are generally less myelinated has potential implications for SCZ.14 The developmental aspects of myelination that may be affected in SCZ remain largely unclear, however, because most studies have been performed in adult postmortem samples.34 Defects in myelination during development could be caused by a failed or delayed differentiation of the OPC lineage and/or by failed or delayed OL maturation. On the other hand, myelin deficits in SCZ could also reflect failures in the adaptive mechanism termed ‘myelin plasticity’. It has been known for a long time that electrical activity stimulates myelination and that many structural aspects of myelin-axon interactions can modulate for example conduction velocity, including myelination per se; myelin thickness; the spacing and size of the nodes of Ranvier and the clustering of ion channels.35 It has recently been established that several of these mechanisms are plastic and likely to contribute to the overall plasticity of the brain.36 In addition, segmented or partial myelination and the pattern and relationship of myelination by individual OLs of functionally distinct or potentially synchronized axons (coupled myelin bundles) have been mentioned as being important, as yet unanswered questions in the field that add additional opportunities for remodelling and plasticity.37 Finally, disturbances in myelination impair connectivity and coherence between brain regions by causing improper spike timing and desynchronized signal processing.39 Thus, the consequences of deficits in developmental and adaptive myelination align well with the prevailing hypothesis of schizophrenia as a ‘dysconnectivity disease’.40 More recently, OLs have been proven to metabolically support axons,42 an essential biological mechanism that is important for brain function and that can be uncoupled from myelination. Myelination coincides with the maturation of brain networks, which is mainly driven by the integration of interneurons into local cortical microcircuits. It has thus been hypothesized that, in addition to optimizing action potential fidelity, the segmented myelination of fast-spiking parvalbumin-positive (Parv+) interneurons may help to cope with the high energy demands of these cells.43 Strikingly, in the cortex of rodents most myelinated axons of interneurons belong to the Parv+ subtype.44 The suggestion that dysfunctions of Parv+ interneurons are associated with SCZ also led to the idea that failure of both processes, i.e. optimizing of action potential fidelity and metabolic support, may together precipitate the network dysfunctions associated with SCZ.45 However, this view was recently challenged because the most comprehensive analysis of the contribution of cell type-associated SCZ risk variants so far did not point to an involvement of Parv+ interneurons in the aetiology of SCZ, but rather highlighted the role of reelin-positive cortical interneurons.46 Also, neither OPC- nor OL-specific gene sets were enriched in SCZ risk genes identified in genome-wide association studies (GWASs).46 Although these observations cannot be considered as definitive, they suggest that OL dysfunctions in SCZ may rather be a secondary consequence of genetic risk factors operating primarily in glutamatergic neurons. This does not, however, exclude ‘myelin plasticity’ as a potential target for research into treatment options. On the other hand, a study where glial precursors isolated from childhood-onset SCZ patients were transplanted into mice also provided evidence for a direct and ‘primary’ contribution of OLs to SCZ47 (see below for details).An important process that takes place during early postnatal human brain development is myelination. The development of WM tracts occurs at a high rate in the first years of childhood and continues during puberty and even young adulthood.34 In short, in vivo brain imaging studies revealed decreased fractional anisotropy as a sign of impaired WM tract integrity,49 a lower myelin water fraction in the WM of the frontal lobe and corpus callosum50 and functional dysconnectivity in relevant neuronal networks,51 which is supported by electrophysiological measurements.40 Studies in SCZ patients that combined imaging and neurocognitive testing revealed associations between impaired cognitive performance and decreased fractional anisotropy,53 disturbed connectivity55 and reduced volume of specific brain areas (hippocampus and nucleus accumbens).56 Furthermore, histopathological findings in postmortem brain tissue indicated a decreased number of OLs in the dorsolateral prefrontal cortex (DLPFC) and cornu ammonis (CA) 4 region of the anterior and posterior hippocampus,59 and reduced OL number was correlated with cognitive dysfunction.60 Moreover, a reduced density of perineuronal OLs,61 lower myelin basic protein (MBP) immunohistochemical staining intensities62 and damaged myelin sheaths and apoptosis of OLs64 have also been documented in light microscopy and electron microscopy studies. Transcriptomic studies in postmortem tissues reported reduced expression of myelin- and OL-related genes, such as MAG and MBP, in several relevant brain regions,66 and myelin-associated proteins have been shown to be decreased in proteomic studies.68A series of studies with different techniques provided much evidence for disturbed OL function and myelin deficits in schizophrenia, which has been reviewed in detail elsewhere.47Taken together, accumulating evidence suggests that disturbances of OL function and myelination likely contribute to the dysconnectivity of brain regions and cognitive impairments seen in SCZ. Whether the deficits in OL functions and their impact on cognition is a primary causative mechanism or a secondary consequence of neuronal and synaptic alterations, or both, is still unclear, although evidence exists for both options.loci have been unequivocally identified after controlling for multiple testing and for sources of confounding, such as population stratification.69 Likewise, these studies have provided compelling evidence of the polygenic architecture of this disorder. Polygenic risk scores (PRSs) can be calculated for everyone and summarize in a single risk score the effects of many single nucleotide polymorphisms (SNPs) under an additive model. PRSs have shown an excellent replicability across independent samples of SCZ patients, although they lack predictive value as regards disease risk.70GWASs and exome sequencing approaches have provided outstanding and solid results regarding the role of common and rare genetic variations in psychiatric disorders, such as SCZ. In SCZ, an increasing number (so far around 150) of genetic risk loci associated with SCZ contain at least one expression quantitative trait locus (eQTL) for a gene within 1 Mb. Active brain enhancers are enriched in these associations, highlighting the regulatory nature of such associations with SCZ risk. Further analysis revealed that several risk SNPs are associated with genes of known regulatory function in neurons and also with genes relevant for glial cells and OPCs/OLs.73 SCZ risk variants are enriched within genomic regions that are marked by histone H3-K4 methylation (H3K4me3), which points to the impact of processes related to the control of gene expression.73 Pathway analyses have shown the aggregation of risk variants in pathways related to neuronal signalling, postsynaptic density, FMRP-bound transcripts and mitochondrial and glial function.75 Interestingly, Duncan et al. revealed a stronger association of an expert-curated glia-OL pathway (comprising 52 genes) with SCZ than with bipolar disorder.74 Worth mentioning in this context is the fact that, numerically, most pathway analyses underline the best evidence of disturbed neuronal function in SCZ.An important proportion of 46 In contrast to the previous, above mentioned pathway analyses, which relied on lists of expert curated cell type markers,75 Skene et al.46 computed cell type-specific gene expression metrics on the basis of scRNAseq and snRNAseq data. Moreover, they weighted risk gene contributions for genetic association probabilities on the basis of GWAS data. With this approach, the group made several observations with respect to brain cell types associated with SCZ: (i) cell-type profiles from mature  but not from immature cell types  were associated with SCZ; (ii) a dedicated set of mature neuronal cell types  showed greater association with SCZ than any other neuronal and glial cell type; and (iii) human, but not mouse, reference mRNA cell type profiles indicated that OPCs and OLs also showed a moderate enrichment. These observations likely helped to shape the current hypotheses on SCZ aetiology, which highlight late cortical developmental processes, including microcircuit maturation (‘micro-connectivity’), rather than early neurodevelopmental processes. Moreover, the association of gene sets from cortical and hippocampal projection neurons support the idea that the connectivity between brain regions (‘macro-connectivity’) may also be critical. Because both ‘micro-connectivity’ and ‘macro-connectivity’ essentially depend on trophic support and myelination (see above), it appears possible that the ‘intermediate’ enrichment of OPC-/OL-specific gene sets further supports these assumptions. Although the analyses by Skene et al.46 made use of the most comprehensive data sets available so far, the conclusions of their study are not definite because of some technical limitations: (i) non-cell type-specific gene sets may operate to a variable, disease-relevant extent in different cell types ; (ii) so far, human brain region- and cell type-specific RNA reference profiles are only available as nuclear transcriptomes, which generates a bias on transcripts involved in cellular processes, such as those associated with dendrites and/or synapses ; and (iii) although scRNAseq and snRNAseq data sets offer an unprecedented cell type definition and resolution, in general the depth of sequencing per individual transcriptome is low and low abundance transcripts coding for transcription factors or signalling components, for example, may be underrepresented. Besides these limitations, the above findings give the impression that common risk variants of SCZ primarily target dysfunctions of striatal and cortical neurons, which may cooperate with a minor fraction of risk factors operating in OPCs/OLs to disturb the local and remote connectivity of brain networks  and single nucleus (sn) RNAseq analyses from mouse and human brain tissues, which provide dramatically enhanced cell-type resolution.rks Fig. .Fig. 1Pr78On the phenomics side, the use of a case-control condition as a target phenotype has inherent limitations, including patient heterogeneity and overlap of the symptom spectrum. A potentially more promising strategy might be to use alternative or additional (sub)phenotypes within these patients that may be closer to the aetiological roots of psychotic disorders.79 and an attractive but still mainly untapped target for drug development.29 Several aspects of cognition, including working memory, attention and executive performance, are affected in SCZ and are already detectable in early-onset SCZ and high-risk individuals.80 Moreover, cognitive impairments that occur during the first episode are very stable and improvements or deteriorations are hardly detectable in SCZ patients even after 10 years.82 Besides the clinical aspects of cognitive impairment as one of the main symptoms in SCZ, there is also a genetic relationship between cognition and SCZ risk genes. Thus, an increased PRS for SCZ is associated with reduced spatial visualization (measured by a block design test) in a cohort of patients, relatives and healthy controls.83 Assessment of cognitive abilities with the Brief Assessment of Cognition in Schizophrenia (BACS) also showed an association of PRS with cognitive performance in both SCZ patients and healthy controls.84 Besides a general association between increased polygenic risk for SCZ and impaired cognition, a large study identified 21 independent SCZ risk loci that influence cognitive functioning.85 Furthermore, cognitive performance was found to be slightly worse in adult healthy carriers of risk copy number variations that are associated with SCZ than in healthy non-carriers.87 On the functional level, a higher PRS is associated with hypoactivity in the prefrontal cortex during working memory tests in healthy controls.89 Most recently, the largest GWAS on human intelligence performed so far identified hundreds of genes involved in this trait.90 The same study reported a negative genetic correlation between intelligence and SCZ . Remarkably, most prominent gene sets associated with cognition are enriched in medium spiny neurons and hippocampal projection neurons.90Cognitive impairments are a key aspect of SCZIn summary, this body of clinical and genetic evidence suggests that cognitive dysfunction in SCZ is an integral part of the disorder rather than a consequence of it. Therefore, using cognition as a stratification criterion for patient selection has the potential to capture key elements in the genetic/molecular architecture of SCZ that are measurable in cellular systems. The results of the aforementioned studies based on SCZ PRS and cognition further support this notion. However, the amount of variation in cognition explained by the SCZ PRS is very modest, which strongly advises against using it as the only patient selection criterion. Although the SCZ PRS is currently the best measure of genetic risk available, the score alone may not yet allow a proper stratification of patient subgroups for decisions regarding iPSC generation and follow-up analyses.93 These findings were recently verified by the largest imaging study conducted so far, which analysed 29 independent and international imaging studies that included a total of 4322 individuals with SCZ.94 Moreover, individuals at high risk for psychosis already display altered WM integrity similar to the pattern of first-episode patients.95 Systematic reviews and meta-analyses substantiate a pattern of WM impairments and structural dysconnectivity in patients with early-onset SCZ, drug-naive patients and clinical high-risk individuals96 and link disturbed WM in first-episode SCZ to cognitive deficits.97 A pioneering study identified a strong association between two risk SNPs in genes important for OL function (MAG and OLIG2), the integrity of WM tracts and cognitive performance.98 The use of genome-wide analyses will likely provide stronger confidence about which genetic factors and pathways may link WM deficits with cognitive impairments in SCZ. In terms of identifying novel drug targets, understanding the biological pathways that modulate the course of the disease over time will provide additional opportunities beyond current genetic approaches.99Patients with early-onset and first-episode SCZ already display widely distributed reduced WM homogeneity, as indicated by a substantial reduction of fractional anisotropy in most brain areas, including several fibre tracts; this reduced WM homogeneity has been associated with disturbed higher order cognitive function.Accumulating evidence suggests that WM pathology and functional dysconnectivity are key aspects of SCZ pathophysiology that contribute to cognitive impairments. Thus, the combined use of phenotypes related to cognition and neuroimaging holds great potential for patient stratification and may be less affected by experimental noise than current genetic classifiers. A critical issue for improving selection strategies even further in the future could be to include environmental stress factors, if available. The additional stratification tools, i.e. cognitive and imaging-related phenotypes and environmental stress factors, might help to identify representatives of (sub)groups with verified WM pathophysiology and subsequently to investigate their underlying mechanisms and their impact on cognition in SCZ, e.g. with hiPSC-based cellular modelling Fig. .Fig. 2PrUntil recently, most insights into psychiatric diseases, including SCZ, have been generated from postmortem tissue samples, brain imaging, genetic and pharmacological studies and animal models. Cellular reprogramming methods now provide a new opportunity to model the complex polygenetic conditions in diseases such as SCZ by generating patient-derived hiPSC-based systems.101 and was awarded the Nobel Prize in Physiology or Medicine 2012. Multiple subsequent studies in humans successfully reprogrammed diverse somatic cell types that are more accessible than fibroblasts, such as keratinocytes,102 urine cells103 and (cord) blood samples.108 Peripheral blood mononuclear cells (PMBCs) can be reprogrammed even after cryopreservation.109 Initial reprogramming methods relied on constitutive retroviral and lentiviral expression systems that were limited by insufficient genomic integration and insertional mutagenesis.110 In recent years, integration-free reprogramming methods have been developed on the basis of adenovirus,111 mRNA,112 sendai virus113 and episomal vectors.114 Episomal reprogramming is highly reliable for different somatic cell lines, and the rapid loss of the reprogramming factors from the host genome over short time periods makes this method highly attractive.115 For translational clinical research, it is crucial to choose the best reprogramming method and the most suitable primary cell type, i.e. the cell type that is most easily accessible and can be stored for long periods.116 Because PMBC sampling and cryopreservation are standard procedures and less invasive than skin biopsies, PBMCs represent the best choice for human cell-based studies on psychiatric diseases. Moreover, setting up large PBMC repositories for clinical studies is a routine procedure that might allow post hoc access to patient data, e.g. for validation purposes beyond the ‘subgroup representatives’ selected in first-round reprogramming. Thus, the use of PBMCs (e.g. CD34+ hematopoietic stem cells and erythroid progenitor cells)119 and episomal reprogramming enables hiPSCs to be generated in a safe, efficient and scalable approach.The first successful reprogramming into iPSCs was performed with murine fibroblastsOnce generated, hiPSCs can be differentiated in principle to all specific brain cell types by providing a lineage-specific chemical environment and/or transient overexpression of transcription factors that determine the cell lineage. Protocols have been developed for hiPSC-derived neuronal stem cells (iPSC NSCs), hiPSC-derived glutamatergic and GABAergic neurons (hiPSC neurons), hiPSC astrocytes, hiPSC OPCs and hiPSC OLs (see below). The generation of disease-relevant cell types represents a major step forward in studying aspects of the neuronal and glial contribution to SCZ in 2D or 3D cellular systems without losing the genetic complexity Fig. .120 and hippocampus.121 Specific overexpression of certain transcription factors alone,123 or in combination with small molecules,124 enables more homogeneous and scalable populations of glutamatergic iNeurons to be obtained. Besides excitatory neurons, stem cells can also be differentiated into cortical GABAergic interneurons similar to medium spiny striatal interneurons125 or Parv+ interneurons.127 For more details, we refer readers to comprehensive reviews on the differentiation of iPSC-derived neurons120 and iPSC-derived astrocytes128 or microglia.129The most prominent cell types with significant importance in the modelling of psychiatric diseases are the hiPSC neurons. By using different protocols, glutamatergic excitatory neurons can be generated with similar properties as those of the cortex132 In general, the main disadvantages of such chemical differentiation approaches are the very protracted protocols, which can take up to several months and are expensive, the high variability in the generated OPCs and the limited numbers of differentiated cells produced with the desired properties. Consequently, these approaches are hardly suitable for diagnostic and empirical perturbation purposes. Another strategy to generate OPCs/OLs is the forced overexpression of lineage-determining transcription factors . Rodent fibroblasts have been successfully trans-differentiated in this way.134 Furthermore, several combinations of certain transcription factors can differentiate hiPSCs or predifferentiated hiPSC NSCs into iOPCs and iOLs relatively quickly and efficiently advantages of the different conditions.Thus, several combinations of stage-specific transcription factors seem to be sufficient to differentiate hiPSCs into the OPC/OL lineage by directed overexpression. The studies listed in Table 136 human foetal neurons140 and hiPSC neurons.139 They can be used in co-culture systems to investigate axoglial interactions or, for example, to assess pro-myelinating drugs with potential relevance also for SCZ. After pioneer experiments in mouse pluripotent epiblast stem-cell-derived OPCs,141 studies have also shown that clobetasol, miconazole and pranlukast promote myelination of hiPSC OPCs141 .OLs Fig. .148 Recent studies have focused more on specific neuronal subtypes, such as pyramidal cortical interneurons, cortical interneurons and dentate gyrus (DG) granule neurons; these studies are reviewed in detail elsewhere.149The landmark study by Brennand et al. in 2011 that characterized hiPSC neurons from SCZ patients was performed in a mixed population of glutamatergic, dopaminergic and GABAergic neurons that showed decreased neuronal connectivity, decreased neurites and decreased levels of post-synaptic protein PSD95.150. Additional analysis revealed that potential SCZ-relevant pathways governed FEZ1 expression and post-transcriptional stability150. Lee et al. used hiPSC neurons and hiPSC OPCs from two individuals with a large (289 kb) heterozygous deletion in CNTNAP2 that affected exons 14–15, whereby both the patient with SCZ and the patient’s healthy father, who carried the allele, were heterozygous.151 The research group showed that the expression of exons 14–15 was significantly decreased in hiPSC-derived neurons and OPCs, but not in fibroblasts, whereas the expression of other exons was upregulated, indicating cell-type-specific mechanism.151 Another family-based approach investigated the contribution of two rare missense mutations in CSPG4 (A131T and V901G).152 CSPG4 codes for NG2, a marker of proliferating OPCs, which are frequently termed NG2-glia.154 hiPSC OPCs derived from CSPG4 mutation carriers revealed dysregulated posttranslational processing, different cellular NG2 location, impaired OPC survival and reduced differentiation to mature OLs.152 Transfection of the two risk variants into OPCs generated from healthy non-carrier siblings revealed similar deficits, underlining the contribution of the described mutations to the OPC pathology. By using diffusion tensor imaging (DTI), the investigators observed impairments of white matter integrity in affected mutation carriers but not in unaffected siblings or the general population.152 Interestingly, no pathological signs were detected in carrier-derived hiPSC neurons, underlining the OPC-specific effect of the CSPG4 mutations.152In contrast to the large number of studies on hiPSC-derived neurons, so far only a few studies have investigated the oligodendroglial impact in SCZ-related iPSC models. Chen et al. focused on the known schizophrenia risk gene FEZ1 in murine and human iPSC-derived oligodendroglial cells and showed that FEZ1 knockdown impaired OL developmentThese studies were either prototypical, hypothesis-driven or single-gene interference approaches or they evaluated ‘rare’ variants that are thought to reflect high-penetrance and ‘high-impact’ mutations; thus, the studies are limited in their dissecting mechanisms and restricted to the dysfunction of a single gene or locus. The generation of iPSCs and derived cell systems from patients who are carriers of polygenic risk assemblies could enable a deeper understanding of common molecular roots of SCZ in a given cell type, such as OLs. Moreover, the differentiation protocols applied in the studies mentioned above were solely based on small molecule differentiation protocols, which may be compromised by having a higher level of variability than directed protocols. Nonetheless, direct cell lineage conversion may possibly override subtle developmental deficits, which may be better accessible with chemical protocols (see below).47 SCZ-derived precursor cells displayed impaired glial maturation, altered transcriptomic signatures in vitro and hypomyelination; when grafted into brains of immune-deficient mice, the mice showed psychosis-related behaviours, such as deficits in sensory motor gating.47 This study provides the strongest evidence so far that glial precursor cells have a cell-autonomous effect that is thus a potentially ‘primary’ cause of the disease or at least contributes to it. It seems likely that the majority of SCZ-relevant endophenotypes detected by Windrem et al. were caused by glial dysfunction and related to disturbed myelination and impaired functional connectivity. Nonetheless, more studies are needed to dissect the relative impact of the cell-type-dependent risk in individual patients.In the seminal study by Windrem et al., glial precursor cells, which could mature into both oligodendroglial and astroglial lineage cells, were generated from patients with childhood-onset SCZ and unaffected controls.155 The large heterogeneity of these mixed cell cultures leads to a substantial and hard-to-control level of variability, even when the cells are obtained from the same individual, and decreases the confidence and robustness of the results (i.e. observed SCZ vs. control samples). One very costly and potentially impracticable strategy to tackle this issue is to increase the technical and biological replicates.156 Another, possibly more feasible strategy to increase levels of reliability is to reduce the cellular heterogeneity of hiPSC-based cell systems. This may be possible by the directed generation of cell types via overexpression of defined cell- and stage-specific transcription factors, as described above. These paradigms may bypass certain maturation processes, which might be a disadvantage when studying psychiatric diseases, in which neurodevelopmental aspects are hypothesized to play a role, such as SCZ. As regards SCZ, however, recent analyses of GWAS data did not provide evidence for a strong association between early developmental cell stages and the disease (see above46).To date, hiPSC-based studies in psychiatric disorders have mainly been performed on adherent monolayer cultures and have focused on cell-autonomous molecular and cellular abnormalities. Most protocols applied to generate neuronal cell types also contained cells of mixed temporal and spatial identities and other cell types of glial and non-neural origin.158 To increase reliability, multiple hiPSC clones should be analysed. An alternative strategy could be to initially focus on a pool of clones and to subsequently validate the obtained effects in individual cell clones to substantiate robust findings. In any case, modern array-based genotyping tools should be applied to verify the genomic integrity and genotype beyond karyotyping at the chromosome level.Although great progress has been made with 3D cell-based neurobiological model systems, they still have major limitations. Current protocols for organoids mimic early neurodevelopment but lack mature OLs and do not develop to postnatal maturation stages, including myelination and synapse pruning. However, recent landmark studies have managed to generate myelinating spheroids and brain microphysiological systems and we can expect more elaborated protocols in the future. One of the current challenges is to develop procedures to establish more mature 3D cell systems. For both 2D and 3D cell systems, scalability is an important issue to enable genetic and chemical screenings at high throughput. Another important topic is intra- and inter-patient variability.Even if further progress is made in 2D and 3D cellular systems, they will always be limited regarding construct validity in brain disorders where circuit-level dysfunctions precipitate behavioural and cognitive deficits. We must accept that even patient-specific cellular models, which reflect the genetic background of an individual, only allow us to study certain disease-associated endophenotypes, such as axonal support or multiple aspects of myelination, in isolation. The possibility to generate iOLs and myelinating 3D systems with patient-derived OLs simply will expand the experimental repertoire in psychiatric research.160 Moreover, including longitudinal information into such a clustering will undoubtedly enrich the validity of the clusters in terms of disease progression/trajectory and potentially also treatment response. However, an important limitation of this approach is the high degree of uncertainty regarding the role of the environment in determining the clinical and neurocognitive profile of a patient. Another method of selecting patients is to leverage the current knowledge on the genetic architecture of complex disorders, such as SCZ.78 Given their polygenic nature, one could select those individuals who are carriers of the largest/lowest genetic burden on the basis of their individual PRS and subsequently analyse the characteristics of their cellular systems.78 Such a polygenic score could also be biologically informed on the basis of pathways or cell-type information (cell-type-specific PRS). This approach would be extremely relevant if the hypotheses and cellular systems are based on a specific cell type  and imaging (e.g. DTI) may yield more valid clusters of individuals with an enhanced biological validity, similar to the Research Domain Criteria (RDoC) concept.ype Fig. . Here agImaging techniques might allow patients with impaired white matter tract integrity to be included; these patients might hold the promise of having oligodendroglial pathology, which could be confirmed by combining imaging with cognitive profiling, for example. The subsequent generation of hiPSC cell systems will be very labour- and cost-intensive and represents the bottleneck in the process. This strongly argues for a stringent, at best hypothesis-driven pre-selection of relevant subgroup representatives, which might enable investigators to reveal corresponding molecular mechanisms in SCZ. There is a chance that hiPSC technology might be a future key technology to find new cellular targets by performing genetic and chemical perturbation screenings with relevance for a given subgroup and/or trait, even with very limited numbers of test systems. Ideally, subsequent validation of candidate drugs in clinical trials and approved treatment should be restricted to those patients who fit the initial classifiers used for ‘empirical validation’, such as white matter abnormalities combined with cognitive deficits Fig. .Fig. 3hi161 and might also enable targeted drug development.162 The field of hiPSC technology in neuroscience is rapidly evolving and constantly progressing. We are witnessing a new era of psychiatry research that brings new challenges, new solutions and new possibilities.The advent of hiPSCs combined with the generation of in principle any neuronal or glial cell type holds great potential for ex vivo disease modelling. A wide range of disease modelling iPSC-based systems have been developed, ranging from 2D monocultures to complex 3D myelinating multicellular systems, and we can expect that many more protocols and conditions will be explored in the future. However, the high costs and cellular variability are important limitations and represent crucial challenges, as does the issue of reproducibility. Besides these technical limitations, meaningful patient stratification is another important issue to be addressed in the future. Despite important limitations and challenges, in principle hiPSC-based cellular disease modelling offers a possibility to address cellular phenotypes in patients with SCZ, whereas retaining the genetic background of each individual. This approach, however, requires stringent experimental proof of the stability of the respective genotype throughout the process of reprogramming, cell-line establishment and differentiation. Unfortunately, experiments to prove stability have not yet been implemented in the field. Epigenetic signatures may also introduce an as yet unknown layer of variability. Nonetheless, the decrease in costs for genomic and epigenetic profiling technologies should allow the field to cope with these sources of variability when setting up batteries of iPSC lines that may complement clinical studies. If successful, personalized strategies that include these new technologies might help to address old questions and reveal new molecular pathways that contribute to neuropsychiatric diseases such as SCZ


The Industrial Biotechnology Forum (IBF) on the 13–14.3.2018 was a showcase of international academic and international research activities covering broad areas of expertise encompassing enzyme catalysis, metabolic- and bioprocess engineering as well as downstream bio-separation engineering, synthetic biology, and bio-informatics trends in biotechnology. Not surprisingly the manuscript contributions submitted to the Frontiers IBF special issue covered various current topics on metabolic engineering and biocatalysis, bio-separation technology, and bioprocess engineering.Escherichia coli or Corynebacterium glutamicum using established glucose based fermentation media. However, glucose based fermentations are rather expensive compared to equivalent applications with alternative carbon sources, such as glycerol, which is considered to be a waste stream of biodiesel production processes. In that context, the featured manuscript in this IBF special issue presented by Mindt et al., described the efficient conversion of the sustainable and cost efficient carbon source glycerol into Methylglutamate using a metabolically engineered Pseudomonas putida. This work is remarkable as it expands the microbial chassis that can be applied to directly generate functionalized amino acids. In essence, P. putida is genetically traceable and has the inherent metabolic capacity to convert glycerol in value added products. As such this recombinant production system synergistically expands the available microbial host platforms to enable production of renewable amino acid based commodity chemical intermediates. With a reported product yield of 17.9 g l−1 N-methylglutamate in fed-batch cultivation mode, the reported process has a high potential for consequent technical validation and commercialization. Much like amino acids, aromatic amines are highly sought chemical building blocks in the manufacture of dyes, pesticides, drugs, polymers among other industrial products. To that end, the featured article by Nargesi et al. reports the de-novo engineering of a para-amino-L-phenylalanine biosynthesis pathway utilizing genetic elements of the fungal Ehrlich pathway. The construction of this synthetic biosynthesis route allows recombinant production of the aromatic amines para-amino-phenylethanol (PAPE) and para-amino-phenylacetic acid (4-APA) from glucose in E. coli. Under controlled fed-batch conditions the best recombinant production strains reached yields of 2.5 g l−1 PAPE (11% C mol mol−1 glucose) and 3.4 g l−1 4-APA (17% C mol mol−1 glucose) from glucose respectively. These production levels are the highest obtained for any aromatic amine thus far.In that context, the fermentative production of amino acids and their functionalized derivatives constitutes a major industrial application, as it delivers sustainable building blocks for chemical and pharmaceutical synthesis under economic production constraints. Conventionally, amino acids are either generated in classical, recombinant microbial hosts such as 2 fixation and its subsequent biomass based conversion into value adding industrial products. The high CO2 fixation rate is inherently linked to the enhanced photosynthetic efficiency of microalgae, which is about twice as high as those of equivalent vascular, terrestrial plants. Moreover, microalgae can be cultivated on marginal lands using waste-, brackish-, or salt water. Therefore, algae cultivation does neither compete with agricultural activity nor rely on freshwater resources, that are essential to maintain terrestrial plant-, animal- and human populations. The excellent review by Molazadeh et al. links and reviews bioprocess for algae based CO2 fixation with the algae's ability to remove excess nutrients, such as nitrogen and phosphorous, from agricultural- and municipal wastewater streams. For each of the featured bioprocess options, the article covers details of energy efficiency and performance differences across microalgae species. In its conclusion the article suggests that linking algae biomass growth with wastewater treatment could be the key to resolve economic and ecological barriers that currently hamper industrial deployment of algae based biofuel solutions. While algae cultivation processes are reaching industrial maturity, harvesting and downstream processing of algae biomass from aqueous media remains a scientific and economic challenge. This holds particularly true for biomass separation from highly saline aqueous solutions due to the charge difference between algae biomass and the cultivation medium. In that context, the innovative article by Bracharz et al. describes a new, energy efficient flocculation method. Here a pH shift in combination with the first in class use of a tannin based flocculent, provides a mass efficient algae biomass dewatering process. Moreover, the utilized tannin based flocculent, is derived from a side stream of a tea leave extraction process and is therefore not only cost efficient but also completely biodegradable, which renders this new algae dewatering process highly economical and ecologically efficient.Driven by current climate change concerns, there is an increasing scientific activity in the design- and industrial deployment of microalgae based photoautotrophic bioprocesses, that allow rapid COSchwaminger et al. This review focuses specifically on the rather new and highly specific technology for bio-product purification by the aid of magnetic beads from complex biological matrices. The review  provides an overview of the progress of capture and separation of biomolecules derived from biotechnology and food technology. While several technological challenges, such as development of flexible separation devices and more broadly applicable separation conditions have to be resolved, it is apparent that magnetic separation technologies will increasingly be applied in industrial bioprocesses.The example of algae biomass harvesting demonstrates that efficient bio-separation technologies are at the heart of an economical bioprocess design. The topic of bio-separation is explicitly highlighted in the review of In that respect, the IBF special issue highlights innovations in up- and downstream bioprocess development that will capacitate industry and society alike to establish a globally sustainable bioeconomy in order to reclaim, preserve and protect the natural resources of our planet.TB has orgnized and edited the Frontiers special issue Industrial Biotechnology Forum 2018. Additionally, he wrote the editorial of this special issue.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers.Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined  Among tRTlnKa = ΔH − TΔS). Thus, when the linker of a multivalent construct is completely rigid and allows aptamer-target binding with a perfect fit, binding affinity could be maximally enhanced by entropic optimization. In contrast, small structural mismatches may lead to very weak binding affinity due to a negative impact on enthalpy [et al. connected a 15-mer thrombin binding aptamer with a 29-mer thrombin binding aptamer using a polyethylene glycol (PEG) phosphoramidite linker, and showed that the binding affinity of this bivalent construct was 97-fold higher than those of the individual aptamers [et al. also designed multivalent constructs for mIgM B-cell receptors using PEG phosphoramidite as a linker [Binding affinity is thermodynamically related to the Gibbs free energy  of the connected ligands, resulting in reduced binding affinity  linkers of various lengths  and selected the 5 dTs linker, which had the highest affinity for thrombin . Subsequaffinity , right.KD values of aptamers were calculated by scatchard plot analysis which plots the SPR signal at equilibrium as a function of aptamer concentration. The scatchard plot of bivalent construct without a linker formed a straight line, and this bivalent construct had about 28-fold higher binding affinity (KD = 17 nM) than that of the monovalent aptamer (KD = 4.8 × 102 nM). However, the plots of the bivalent constructs with the poly (dT) linkers did not form straight lines. These data indicate that the bivalent construct without linker would bind to the VEGF-165 with a 1:1 stoichiometry, but the bivalent constructs with poly (dT) linkers would not bind 1:1. The predicted secondary structure of the bivalent construct without a linker indicated the presence of a double stranded DNA (dsDNA) region  region , and theThe probability of finding the extended conformation of a flexible linker is low in solvent , and altet al. developed a bivalent construct with an ideal scaffold using a direct evolution strategy [Ahmad strategy . These iKD of 97 pM had more than 200-fold greater binding affinity than either component aptamer. Hence, with increasing numbers of aptamer pairs, the effects of the multivalent approach to improving binding affinities may be promising.Multivalent interactions may be used to dramatically enhance binding affinities of aptamers to their targets. However, this approach only can be applied to aptamers that bind multivalent proteins or that recognize distinct epitopes of the same targets, and the ensuing aptamer pairs are rare. Recent reports demonstrate selection methods that enable isolation of aptamer pairs with activity against a single target ,71,72. IKD values of aptamers with stable structures were only slight improvements on those of canonical aptamers. Hence, stabilization of aptamer structures may maintain binding properties in various environments, but offers little improvement in binding affinities. Among aptamers comprising natural nucleotides, hydrophobic interactions between aptamers and targets are limited. Thus, oligonucleotide libraries containing unnatural hydrophobic nucleotides have been used to isolate aptamers with hydrophobic moieties and activities against various proteins. Accordingly, introduction of hydrophobic moieties has expanded the repertoire of interactions and structural diversities of aptamers, and has led to substantial improvements in binding affinities. However, further refinements of SELEX with libraries containing modified nucleotides are required, and additional studies of specificities of aptamers containing hydrophobic moieties are warranted. Moreover, because most interactions between proteins are hydrophobic, non-specific binding between hydrophobic modified aptamers and proteins are a concern. Finally, multivalent aptamers form more interactions with target molecules than monomer aptamers, leading to substantial enhancements of binding affinity. Moreover, binding affinities can be tuned by optimizing the scaffolds that connect aptamers of multivalent constructs. Thus, the multivalent approach promises to improve binding affinities of aptamers. However, challenges of this approach include the paucity of aptamer pairs that recognize distinct sites of the same target. Accordingly, selection methods that enable isolation of aptamer pairs have been reported, and have extended the range of potential applications of multivalent aptamers.Because aptamers with desired affinities are not always isolated by SELEX, refinements of selected aptamers are required to improve binding affinity. In our studies, the genetic algorithm- based method ISM enabled identification of optimized aptamer sequences in rugged functional sequence spaces. This approach is a powerful strategy for improving binding affinities of aptamers though sequence optimizations. Moreover, enhancements of binding affinities have been achieved through stabilization of aptamer structures. However,