Hypertrophic cardiomyopathy (HCM) is characterized by substantial genetic and phenotypic heterogeneity, leading to considerable diversity in clinical course including the most common cause of sudden death in young people and a determinant of heart failure symptoms in patients of any age. Traditionally, two-dimensional echocardiography has been the most reliable method for establishing a clinical diagnosis of HCM. However, cardiovascular magnetic resonance (CMR), with its high spatial resolution and tomographic imaging capability, has emerged as a technique particularly well suited to characterize the diverse phenotypic expression of this complex disease. For example, CMR is often superior to echocardiography for HCM diagnosis, by identifying areas of segmental hypertrophy  not reliably visualized by echocardiography (or underestimated in terms of extent). High-risk HCM patient subgroups identified with CMR include those with thin-walled scarred LV apical aneurysms (which prior to CMR imaging in HCM remained largely undetected), end-stage systolic dysfunction, and massive LV hypertrophy. CMR observations also suggest that the cardiomyopathic process in HCM is more diffuse than previously regarded, extending beyond the LV myocardium to include thickening of the right ventricular wall as well as substantial morphologic diversity with regard to papillary muscles and mitral valve. These findings have implications for management strategies in patients undergoing invasive septal reduction therapy. Among HCM family members, CMR has identified unique phenotypic markers of affected genetic status in the absence of LV hypertrophy including: myocardial crypts, elongated mitral valve leaflets and late gadolinium enhancement.The unique capability of contrast-enhanced CMR with late gadolinium enhancement to identify myocardial fibrosis has raised the expectation that this may represent a novel marker, which may enhance risk stratification. At this time, late gadolinium enhancement appears to be an important determinant of adverse LV remodeling associated with systolic dysfunction. However, the predictive significance of LGE for sudden death is incompletely resolved and ultimately future large prospective studies may provide greater insights into this issue. These observations underscore an important role for CMR in the contemporary assessment of patients with HCM, providing important information impacting diagnosis and clinical management strategies. Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiomyopathy  caused by mutations in genes encoding proteins of the cardiac sarcomere ,6. HCM iGenetics. HCM is caused by vast genetic heterogeneity with > 1,400 mutations in 13 or more genes encoding contractile proteins of the cardiac sarcomere (or in sarcomere-associated proteins) with cardiac β-myosin heavy chain (MYH7) and cardiac myosin binding protein C (MYBPC3), lysosome associated membrane protein (LAMP2) and the recessive disorder Fabry disease caused by mutations in the gene α-galactosidase (GLA) ) ,21,23. AFurthermore, these observations support an expanded role for CMR in earlier diagnosis of relatives within HCM families ,58. For Although sarcomeric HCM accounts for the majority of unexplained left ventricular hypertrophy seen in adults, a number of other non-sarcomeric diseases can produce increased wall thickness of the myocardium as part of their phenotypic expression. Infiltrative cardiomyopathies such as cardiac amyloidosis, glyocogen/lysosomal storage diseases including Fabry's, Danons, and AMP kinase are considered to be the most common non-sarcomeric diseases in which cardiac phenotypic expression can mimic that of HCM  . Fabry'sAn accurate diagnosis early in clinical presentation is critical, as treatment strategies and prognosis may differ for these diseases compared to HCM . CMR mayFurthermore, due to its super spatial resolution in imaging the distal LV myocardium, CMR may also help clarify  diagnosis by demonstrating the presence of prominent trabeculations  consistent with a diagnosis of LV noncompaction in patients initially diagnosed with apical HCM . In thisDifferentiating HCM from wall thickening due to hypertension has historically represented a clinical challenge. Invariably, exposure to long-standing systemic hypertension will result in nearly identical wall thickening in the septum and LV lateral wall . In addition, hypertensive cardiomyopathy is very rarely associated with resting LV outflow tract obstruction due to typical systolic anterior motion (SAM) with septal contact. Likewise, LV wall thickening in HCM is almost always asymmetric with resting outflow obstruction present in over one-third of patients.In addition, a subset of HCM patients demonstrate non-contiguous patterns of LV wall thickening . This moCMR can also be particularly helpful in detecting changes in serial measurements of LV wall thickness after treatment with antihypertensives, in which regression of hypertrophy would favor a diagnosis of hypertensive cardiomyopathy. In addition, when the distinction between these two disease entities still remains otherwise ambiguous in an individual patient, detection of HCM in family members who were previously undiagnosed or identification of a sarcomere mutation with genetic testing would provide additional evidence strongly favoring a clinical diagnosis of HCM.In addition, it may also be difficult to differentiate HCM from situations in which increased LV wall thickness results as a secondary response to intense athletic training. CMR can have a role in differentiating these conditions by identifying focal, limited hypertrophy not well visualized by echocardiography . CMR is Cine CMR can accurately identify the mechanism of subaortic obstruction in HCM with SAM-septal contact in both long-axis and basal short-axis images. Subaortic obstruction will result in high velocity blood flow in the outflow track area which can often be visualized as a systolic signal void jet  in the region of SAM-septal contact . In addiIn HCM patients with outflow tract obstruction, phase velocity flow-mapping sequences can be performed in order to determine the peak velocity of blood flow through the outflow tract as well as to quantify the amount of mitral regurgitation. However, only a small number of studies have assessed the accuracy of CMR-derived LV outflow tract velocities compared to continuous-wave Doppler derived pressure gradients . TherefoHCM patients with LV outflow obstruction gradient ≥ 50 mmHg at rest or with provocation, who have advanced heart failure refractory to medical therapy, are candidates for invasive septal reduction therapy to relieve obstruction and improve limiting symptoms ,70. SurgCMR has a role in pre-operative surgical myectomy planning in those HCM patients who are considered candidates for the operation by characterizing a number of important morphologic abnormalities related to outflow tract anatomy as well as the mitral valve and submitral apparatus ,53-55. FCMR can identify additional morphologic abnormalities of the mitral valve and papillary muscles, which are important contributors to the mechanism responsible for subaortic gradients. Specifically, in those patients with substantially elongated mitral valve leaflets Figure  the mitrIn addition, a number of other structural abnormalities of the submitral apparatus notable for proper surgical myectomy planning can be routinely identified by CMR. Apically displaced accessory anterolateral Figure  or a douAlcohol septal ablation (ASA) is an alternative invasive septal reduction therapy in which 1-2 cc of alcohol are injected into an anatomically appropriate septal perforator artery supplying the basal septum  creating a myocardial infarction resulting (ultimately) in septal thinning, widening of the LV outflow area and reduction of obstruction . For HCMNoninvasive imaging of LV wall thickness has proven to have a role in risk stratification with massive LV hypertrophy of ≥ 30 mm demonstrated by 2-D echocardiography anywhere in the LV chamber identifies those HCM patients at highest risk and potentially deserving of ICD therapy for primary prevention of sudden death ,41,78. IDue to the variable distribution of LV hypertrophy in regions of the LV chamber remote from maximal wall thickness, CMR-derived LV mass provides the most accurate assessment of the overall extent of LV hypertrophy in this disease. As a result, LV mass may represent a marker for adverse risk and would therefore seem to hold promise for aiding in risk stratification . Howeverin vivo detection of myocardial fibrosis as a prognostic marker in HCM [Recently, considerable interest has emerged in using non-invasive r in HCM ,28,30,32r in HCM -39.The precise pathophysiologic mechanism responsible for LGE in HCM currently remains uncertain. Nevertheless, observations derived from contemporary imaging and histologic studies provide support for the principle that LGE may derive from a pathophysiologic cascade in which repetitive bouts of microvascular ischemia result from structurally abnormal intramural coronary arteries (with impaired vasodilatory capacity) responsible, over a period of time, for myocardial ischemia-mediated myocyte death, ultimately triggering repair in the form of replacement fibrosis .Indeed, there are a number of avenues of support for the principle that LGE probably constitutes (or largely represents) areas of myocardial replacement fibrosis. Studies in HCM patients with stress-CMR and PET have demonstrated myocardial blood flow to be substantially reduced in LV segments occupied by LGE and severely blunted in areas situated adjacent to LGE ,81. In vVirtually any pattern, distribution and location of LGE can be observed in HCM, although LGE never corresponds to a coronary vascular distribution Figure 37,87,88,8887,88.Transmural extent of LGE in not uncommon, occurring in one-half of HCM patients . A signiOne of the strongest and most consistent observations derived from a number of contemporary cross-sectional studies is the inverse relationship between ejection fraction and LGE in HCM. LGE extent is greatest in HCM patients with EF < 50%.  while those with hyperdynamic LV systolic function have comparatively minimal LGE ,87,88. HAt present, the precise impact of LGE on the development of heart failure symptoms is unresolved. A few studies have reported a weak but significant relationship between LGE and progressive heart failure symptoms/death in HCM  ,38. HoweDespite considerable advances, risk stratification in HCM remains incomplete as some at-risk patients are not identified by the conventional risk factors suggesting a need to identify novel markers of susceptibility to sudden death risk ,26,91,92Since nonsustained ventricular tachycardia is an independent risk factor for sudden death in this disease , the relTherefore, available data do not strongly support LGE as a primary independent risk factor for sudden death in HCM . As a reThe clinical significance of LGE is currently one of the most important and debated clinical questions in HCM ,99. Resoextent of LGE or when LGE is combined with the other currently established prognostic markers into a composite risk score for sudden death.Another point in establishing clinical relevance for contrast-enhanced CMR is the relationship of presence vs. extent of LGE with respect to outcome. The majority of follow-up contrast-enhanced CMR studies have reported only an association between the presence of LGE and sudden death ,37,39. HOver the last several years, it has become increasingly common in the HCM literature and clinical arena to equate areas of LGE directly to myocardial fibrosis ,95,100. However, a number of CMR observations in HCM would suggest that, in fact, not all LGE may represent myocardial fibrosis in this disease. First, LGE in HCM is more commonly found in those LV segments which are the thickest with normal regional systolic function ,87-89. TTherefore, based upon the totality of these observations it would be reasonable to consider that some LGE in HCM may represent histopathology which is not a result of myocardial scarring from replacement fibrosis. For instance, gadolinium may deposit within the diffuse interstitial collagen (matrix) compartment between normally aligned myofibrils or areas of perivascular fibrosis. Matrix collagen constitutes a greatly expanded extracellular tissue volume partially responsible for increased LV wall thickness, represents a primary morphologic feature of HCM, and provides an opportunity for gadolinium accumulation that may not reflect true replacement fibrosis. In addition, disorganized myocyte architecture will result in expanded areas of myocardial matrix where gadolinium could aggregate. Consistent with this point is the fact that one of the most common locations of focal LGE in HCM is at the insertion of RV wall into anterior or posterior ventricular septum ,39,87,88With current CMR technology it is not possible to reliably distinguish interstitial (matrix) from replacement fibrosis, or an expanded matrix compartment created by myocyte disarray . This maNevertheless, there has been interest in using CMR to differentiate myocardial substrates of different histologic composition. This principle has been addressed predominantly in patients with atherosclerotic coronary artery disease and prior myocardial infarction, in which areas of intermediate LGE signal intensity  correlate histologically to regions of 'tissue heterogeneity' (mixture of isolated myocytes and fibrosis), while regions of higher signal intensity LGE correlate with core infarct zones comprised of only replacement scar. IdentifIn HCM, areas of lesser signal intensity LGE are also common but have largely been ignored in prior analysis of hyperenhancement in this disease and therefore their potential for generating life-threatening ventricular arrhythmias largely ignored -39,89,93Over the last several years, numerous techniques have been proposed to assess extent of LGE in HCM reflecting the uncertainty which persists regarding the most appropriate strategy for assessing hyperenhancement in this disease -112. TheThese thresholding algorithms has been most extensively studied in patients with ischemic cardiomyopathy, where grayscale techniques using 2 SD above the mean of normal myocardial SI or FWHM have been shown to correlate well with the spatial extent of the infarct ,113. In ex vivo histopathology in an HCM patient with preserved EF , it seems most reasonable at this time to promote the use of either of these two methods for quantifying LGE in HCM.However, HCM represents a distinctly different disease entity in which abnormal myocardial substrate  involve large proportions of LV myocardium ,114-116.Furthermore, there are a number of technical issues to consider as potential limitations with respect to LGE images. The quality of contrast-enhanced images can be affected by the selection of poor inversion times and motion artifact, making it difficult to reliably determine in such cases whether LGE represents abnormal histopathology versus "background noise" . In addiAt present, the totality of these data suggests a note of caution is appropriate in interpreting the significance of LGE from a histologic perspective in patients with HCM. It is reasonable to conclude that in end-stage HCM patients the majority of LGE represents myocardial replacement fibrosis responsible for the adverse LV remodeling evident. It remains less clear to what extent LGE equates to scar formation among the more typical HCM patients with preserved EF, who comprise the vast majority of patients with this disease. Nevertheless, even if all hyperenhancement does not equate with fibrosis, LGE may still prove to be an important clinical marker for prognosis in this disease. In addition, at this early juncture, there still remains a great need for standardization of LGE quantification methods and sequence acquisition, given that the substantial amount of variability evident among these techniques represents a potential limitation in applying CMR to routine clinical decision-making.Over the last decade, the unique imaging strengths of CMR have led to improved diagnostic capabilities and expanded our understanding and appreciation of the diverse phenotypic expression of this complex genetic heart disease. In addition, CMR has an emerging role in the assessment of risk in patients with HCM, as substantially increased LV mass and late gadolinium enhancement have been associated with increased likelihood of future adverse cardiovascular events. Taken together, these observations underscore an important and growing role for CMR in the contemporary evaluation of HCM patients and support the need for future longitudinal studies to clarify whether CMR-derived variables will be independent predictors of sudden death and disease progression.CMR: cardiovascular magnetic resonance; EF: ejection fraction; HCM: hypertrophic cardiomyopathy; ICD: implantable cardioverter defibrillator; LGE: late gadolinium enhancement; LV: left ventricle; RV: right ventricle; SAM: systolic anterior motionConsultant PGx HealthI have read and approved the manuscriptDr. Martin Maron is an assistant professor of medicine and Director of the Hypertrophic Cardiomyopathy Center and Co-Director of Advanced Cardiac Imaging at Tufts Medical Center. His research interests involve the role of cardiovascular magnetic resonance (CMR) in the evaluation and management of patients with HCM as well as investigating novel drug therapy to modify the HCM phenotype. Dr. Maron also collaborates with members of the Molecular Cardiology Research Institute (MCRI) at Tufts on several projects aimed at better characterizing the molecular pathways which contribute to disease expression in HCM. Several of these on-going projects include evaluating the relationship of novel polymorphisms with regard to the expression of hypertrophy and characterizing signaling pathways which promote the upregulation of cardiac fibroblasts and their promotion of adverse remodeling in HCM.


Snakebite is classified by the WHO as a neglected tropical disease. Envenoming is a significant public health problem in tropical and subtropical regions. Neurotoxicity is a key feature of some envenomings, and there are many unanswered questions regarding this manifestation. Acute neuromuscular weakness with respiratory involvement is the most clinically important neurotoxic effect. Data is limited on the many other acute neurotoxic manifestations, and especially delayed neurotoxicity. Symptom evolution and recovery, patterns of weakness, respiratory involvement, and response to antivenom and acetyl cholinesterase inhibitors are variable, and seem to depend on the snake species, type of neurotoxicity, and geographical variations. Recent data have challenged the traditional concepts of neurotoxicity in snake envenoming, and highlight the rich diversity of snake neurotoxins. A uniform system of classification of the pattern of neuromuscular weakness and models for predicting type of toxicity and development of respiratory weakness are still lacking, and would greatly aid clinical decision making and future research. This review attempts to update the reader on the current state of knowledge regarding this important issue. Snakebite is a neglected tropical disease of global importance Snakebite-related mortality is highest in resource-poor countries, and is directly related to socioeconomic indicators of poverty Bungarus spp.) Naja spp.) Oxyuranus spp.) Micrurus spp.) Acanthophis spp.) Notechis spp.) Crotalus spp.) Daboia russelii) in Sri Lanka and South India Vipera aspis) Vipera berus) Vipera ammodytes) Neurotoxicity is a well-known feature of envenoming due to elapids (family Elapidae) such as kraits , and by the non-neurotoxic neurological manifestations of envenoming such as those due to coagulopathy. Comparing findings from different studies is difficult, as there is a lack of uniformity in description or grading of neuromuscular weakness, or in assessment of response to treatment. Interpretation of neurophysiological findings is also difficult as different methodologies have been used between studies.The effects of a bite from one snake species can also vary, as venom constituents in one species may vary seasonally, geographically, as well as ontogenetically, and some venoms contain a number of different neurotoxins.Accurate case definition is the key to meaningful interpretation of available data and comparison between studies. However, this is hampered by the difficulties in identifying envenoming snakes, which have been previously highlighted We searched PubMed with varying combinations of the search terms “snake bite,” “snake envenoming,” “snake venom,” “neurotoxicity,” “neurology,” and “neurological manifestations.” We acquired only English-language articles. We also obtained articles on “neuromuscular junction” and “neuromuscular block.” Additional related articles were obtained from citation tracking of retrieved articles and tracking of “related citations” in PubMed. Altogether, 624 titles and abstracts were screened, and 287 full articles were retrieved and read by a single author (UKR) for data acquisition. The reference list was further modified following reviewers' comments.The peripheral neuromuscular weakness after snakebite results from defective neuromuscular junction (NMJ) transmission. It is pertinent to briefly review the current knowledge on NMJ transmission and neuromuscular block, to better understand the effects of snake venom at the NMJ At the pre-synaptic level, the motor nerve axon terminal is responsible for the synthesis, packaging, transport, and release of the neurotransmitter acetylcholine (ACh). Release of ACh in response to an incoming nerve action potential is triggered by the opening of voltage-gated calcium channels and the influx of calcium ions. Increased intracellular calcium concentration triggers a cascade of events that leads to the formation of a fusion complex made up of SNARE  proteins, which enables fusion of ACh vesicles to the nerve terminal membrane and ACh release ACh released from the nerve terminal diffuses rapidly across the synaptic cleft. Degradation of ACh at the synaptic cleft by acetyl cholinesterase (AChE) is necessary for the termination of its action.At the post-synaptic level, ACh binds to muscle nAChRs (adult or mature type—α1β1εδ) on the post-synaptic membrane. nAChRs are ligand-gated ion channels, and their activation by ACh leads to an influx of sodium and calcium cations, accompanied by efflux of potassium ions through potassium channels, and produces an end-plate potential. If adequate ACh is released, this end-plate potential is propagated by the opening of sodium channels along the perijunctional zone and muscle membrane and initiates calcium release and muscle contraction Neuromuscular block at the post-synaptic level is classified into non-depolarising and depolarising types. Depolarising neuromuscular blocking agents (NMBAs) (such as suxamethonium) bind irreversibly to the post-synaptic muscle nAChRs, and produce a non-competitive block, which is not reversed by acetyl cholinesterase inhibitor drugs (AChEIs). Depolarising NMBAs initially produce excessive depolarisation Traditionally it has been considered that snake venom toxins cause two types of neuromuscular blockade, pre-synaptic and post-synaptic; but this view may be oversimplistic and needs to be reviewed in view of the recent insights into neuromuscular transmission and descriptions of different patterns of neurotoxicity. Much of the current understanding of neurotoxicity has come from animal studies using purified individual toxins.2 toxins, PLA2s) bind to the motor nerve terminals, leading to depletion of synaptic ACh vesicles, impaired release of ACh, and later, degeneration of the motor nerve terminal The pre-synaptically active neurotoxins (beta-neurotoxins—mostly neurotoxic phospholipase ABungarus spp.) which predominantly has potent PLA2 enzymatic activity. Dixon and Harris (1999) first highlighted the significance of denervation in producing the treatment-resistant paralysis in krait bite Oxyuranus spp.) and notexin  had effects similar to beta-bungarotoxin Pre-synaptic toxins are best illustrated by beta-bungarotoxin (b-BuTX) of kraits  bind to the post-synaptic muscle nAChRs. Alpha-neurotoxins belong to the group of “three-finger toxins” (3FTXs) characterized by a shared toxin structure resembling three outstretched fingers of a hand Lee et al. (1977) showed that alpha-bungarotoxin (a-BuTX) produced a pure post-synaptic, non-depolarising, but almost irreversible neuromuscular blockade Bungarus candidus), is a non-conventional 3FTX with structural similarities to alpha-bungarotoxin The recent insights into NMJ transmission have enabled better and more comprehensive characterization of the more recently described toxins. Candoxin, a novel toxin isolated from the venom of the Malayan or blue krait  and the black mamba (D. polylepis), enhance ACh release from the nerve terminals by inhibiting potassium channels and produce a neuromuscular block similar to depolarising block D. angusticeps acts as an AChE inhibitor, thus increasing the availability of ACh at the NMJ. They have been named fasciculins due to their effect of producing generalized, long-lasting fasciculations Some snake venom toxins interfere with NMJ transmission through various other mechanisms. Some pre-synaptic toxins, such as the dendrotoxins from venoms of the Eastern green mamba (Naja atra) venom  Snake venoms do not contain a homogenous single toxin, but are complex cocktails of enzymes, polypeptides, non-enzymatic proteins, nucleotides, and other substances, many of which may have different neurotoxic properties Daboia russelii) Bungarus spp.) consists of several different types of neurotoxins. In addition to the alpha-bungarotoxin (post-synaptic block) and beta-bungarotoxin (pre-synaptic block) already described, it also contains kappa-bungarotoxin which binds to the neuronal nAChR at the post-synaptic level in central cholinergic synapses in autonomic ganglia Characterization of new toxins continues to add to the rich diversity of snake venom, and many types of venom are now known to contain both pre- and post-synaptically active toxins. For example, a post-synaptic toxin (DNTx-I—Daboia Neurotoxin 1) has been isolated from the venom of Russell's viper , which feeds mainly on rodents and reptiles, preferentially binds to murine nAChRs rather than to those of chick origin Boiga irregularis) shows taxa-specific lethal toxicity to birds and lizards, but not toward mice. In vitro studies showed that it produced potent post-synaptic toxicity similar to alpha-bungarotoxin at avian NMJs, but not in mammalian NMJs Tropidolaemus wagleri) binds more tightly to mouse nAChRs than to those from rats or humans For example, candoxin from the Malayan or blue krait (Tropidolaemus wagleri) and azemiopsin from Fea's viper (Azemiops feae) have shown specificity toward the ontogenetic state of the nAChR, with higher binding affinity to the adult (or mature) form than the foetal form Furthermore, some in vitro studies of toxins have been done on nAChRs of αβγδ type, which is the foetal type of nAChR, in contrast to the adult (or mature) type (αβεδ) of nAChR normally expressed in the NMJ Reported prevalence rates of neuromuscular weakness vary between series, and with different snake species and different study settings. As seen from above, each toxin can have a multitude of effects, venom of one snake species is a mix of multiple toxins, and venom composition varies between species of same genus. In addition, intraspecies variations in venom compositions are well known. It is therefore not surprising that wide variation is seen in the neurotoxic effects.Possible methodological differences and lack of uniformity in description contribute to the large variation between studies, and make interpretation of data from different studies difficult. Ptosis is reported in between 70–93% of patients in most series, and extraocular muscle weakness in 68–82% The envenoming snake species is highly likely to influence the clinical presentation and outcome, but many studies have considered together bites from different snake species Many patients with neurotoxicity develop ptosis and extraocular muscle weakness, but only a few will develop respiratory muscle weakness. Factors that determine development of respiratory muscle weakness in some patients are not clear. The traditionally held view that it is related to the dose of venom and the severity of envenoming, perhaps modified by antivenom therapy, has not been adequately addressed. The possibility that distinct patterns of neuromuscular weakness exist in snake envenomation has not been studied. A parallel may be drawn with myasthenia gravis where two forms of weakness, ocular and generalized, are well known. Extraocular muscles are developmentally, histologically, ultrastructurally, immunologically, metabolically, and functionally different to other skeletal muscle groups Ptosis and extraocular weakness are commonly reported in Sri Lankan Russell's viper envenoming It is known that different toxins have different affinities to the two isoforms of muscle nAChRs Bungarus multicinctus) in Vietnam for whom antivenom was not available, 87% needed mechanical ventilation for a mean of 8 days, the mean duration of the ICU stay was 12 days, and hospital mortality was 7% The natural history of neurotoxic envenomation is likely to vary with the degree of envenoming and snake species, and between patients. There are little data on the natural course, as it can be affected by treatment. In a rare case series of 60 patients with envenoming by the many-banded krait (Naja philippinensis) produces more neurotoxicity and less local swelling Daboia russelii) in Sri Lanka and South India, in contrast to reports of bites by Russell's viper from other countries There is a clear variation in the propensity of similar species of snakes to produce different patterns of neuromuscular weakness in different geographical locations. For example, the Philippine cobra (Daboia russelii) and South India (D. russelii) was found to be different from that found in Pakistan (D. russelii), Thailand (D. siamensis), and Taiwan (D. siamensis) Daboia russelii) in India Crotalus scutulatus) in the United States Vipera aspis) in France Notechis scutatus) in Australia These geographical differences may be due, at least in part, to interspecies and intraspecies differences in venom compositions. The venom composition in Russell's viper in Sri Lanka  envenoming, antivenom was not found to be effective B. multicinctus) Naja nigricollis toxin-alpha were able to reverse toxin binding to the AChRs and the resultant neuromuscular paralysis There are many unresolved questions regarding the use of antivenom in snake envenomation–related neurotoxicity, and available reports show conflicting results. Differences in study methodology  are likely to contribute significantly to the reported variations in response to treatment. In addition, such variations may well be related to the differences between pre-synaptic and post-synaptic types of toxin in snake venom, and also to the specificity of antivenom to the envenoming snake species. How much of the reported recovery may be attributable to spontaneous recovery is not clear, and there are a number of reports of recovery from neuromuscular paralysis without antivenom Neuromuscular weakness, especially due to non-depolarising post-synaptic blockade, has similarities to myasthenia in pathophysiology, and it is theoretically plausible that AChEIs are effective in this type of neurotoxic envenoming. However, the evidence for benefit of AChEI is conflicting. This may well be due to the confounding effects of any natural recovery, co-administered antivenom, and different types of envenomation by different species.Naja philippinensis) envenoming There are several reports of benefit from AChEIs It is likely that a good response to AChEIs is seen only in the competitive, reversible type of post-synaptic toxicity Several other interesting acute neurological features have been reported after snake envenomation, which are likely to be direct neurotoxic effects. The mechanisms of many of these acute manifestations are not clear, and there has been no systematic study of these in a large series.Crotalus spp.) envenoming, providing further evidence of variation in neurotoxicity with species and geographical differences Crotalus spp.) venom has been shown to act on voltage-gated sodium and potassium channels Crotalus spp.). Inhibition of pre-synaptic voltage-gated potassium channels is seen in neuromyotonia, which is an autoimmune disorder presenting with continuous fasciculations. It would be interesting to see whether a similar mechanism exists in myokymia due to rattlesnake envenoming Myokymia has been reported mainly from the United States following rattlesnake  Micrurus fulvius) Autonomic involvement, especially parasympathetic denervation effects, are reported in several case series and case reports, and almost all these reports are following krait bites Bungarus caeruleus), 38 patients had delayed neurological deficits. Fourteen of them had nerve conduction defects that lasted for 2 weeks to 6 months before complete recovery Vipera aspis. They have demonstrated a potential immunological basis for the syndrome, with cross-reactivity shown between glycosidic epitopes of venom proteins and neuronal GM2 ganglioside, without evidence of direct neurotoxicity of the venom There are several reports of delayed neurological manifestations after snake envenomation. Some are reports of persistence of neurological deficits which first developed during the acute stage. Distinction from critical illness neuropathy and myopathy may be difficult when symptoms are first noticed soon after recovery from the acute phase, especially with a background of ventilation, ICU care, or sepsis There are few robust studies of long-term neurological effects. In the first detailed clinical and neurophysiological study of long-term neurological deficits, Bell et al. studied 26 asymptomatic survivors who had evidence of neurotoxicity during acute envenomation one year earlier The factors responsible for the causation of long-term neurological effects need further study. Persistent axonal damage due to neurotoxins, and delayed immune-mediated reactions to toxins or antivenom are possible explanations. There is also some experimental evidence for delayed neuropathic effects. In their report of beta-bungarotoxin–induced toxicity in rats, Prasarnpun et al. observed loss of myelinated axons at 6 months after inoculation Although the clinical manifestations of acute neuromuscular weakness with respiratory involvement are well recognised, it is surprising how many questions remain unanswered regarding neurotoxicity. This lack of clarity may at least partly be explained by the emerging evidence that has led to an increased understanding of neuromuscular transmission. This suggests that previously held traditional models of two different types of neurotoxicity (pre-synaptic or post-synaptic) are inadequate to explain all of the differences seen in symptom evolution and recovery, patterns of weakness, respiratory involvement, and responses to antivenom or AChEI therapy. For example, it is becoming clear that many of the post-synaptic toxins produce nearly irreversible binding, and long-lasting effects. The importance of the reversibility of post-synaptic toxicity, and the potential for blockage of pre-synaptic nAChRs by “post-synaptic” toxins after envenoming have not been addressed in adequate detail. This variability in toxicity may partly explain the differences in the pattern of envenomation by different species in different geographical regions, and it is highly likely that the presence of a number of different toxins in one venom also contributes. Detailed analysis of venoms from different snake species from different regions may help further elucidate these.In addition to neuromuscular failure, several other interesting acute and delayed neurological manifestations have been described after snake envenomation, and there is very little understanding of their pathophysiological basis. These are further pointers to the diversity of the types of neurotoxicity produced by different snake species. There is no agreed time cut-off for classifying neurological manifestations into “acute” and “delayed/late.” There is a clear need for a uniform classification of delayed neurological manifestations. We propose that changes be classified as acute , delayed (onset within 2–8 weeks), and late (onset after 8 weeks of envenoming).Improved case definitions are the key to a better understanding of neurotoxicity from different snakes. This can only be achieved by either the identification of dead snakes or the use of laboratory or near-patient detection of venom antigen. Further development of such techniques for developing countries where snakebites are common is vital to allow accurate and meaningful clinical descriptions of neurotoxicity.Given the high morbidity and mortality, better treatment options are clearly needed in neurotoxic envenoming. There are several exciting reports of the use of plant extracts in the treatment of neurotoxicity Given the lack of clarity over mechanisms of neurotoxicity, the lack of consensus on the value of antivenom or AChEI therapy in snake envenoming is not surprising. Conflicting reports of their efficacy are likely to reflect different mechanisms of neurotoxicity produced by different snake species, and potentially, variations in antivenom efficacy and time of administration. Models to predict type of toxicity, and a better understanding of the type of toxicity produced by different species, would perhaps enable better use of these treatment strategies. More data are needed on their efficacy, and may be obtained only from clinical trials in envenomation by different snake species. Electrophysiological studies may also be valuable in helping us to understand the complex processes in human neurological envenoming.Snake neurotoxins have contributed significantly to our understanding of neuromuscular transmission and receptor function, and recent studies have highlighted many of their other properties, e.g., the ability to enter actively proliferating cells, nuclear localization, preferential binding in specific cell division phases, inhibition of apoptosis, anti-inflammatory and analgesic actions, and antimicrobial effects Snake venoms are complex mixtures of different toxins, and each neurotoxin has diverse neurotoxic effects.There is considerable geographical, interspecies, intraspecies, as well as possibly ontogenetic variation in neurotoxicity with snake envenoming.Accurate identification of envenoming snakes and uniform case definitions are needed to improve comparability of different reports of neurotoxic envenoming.There are many interesting acute and delayed neurotoxic manifestations other than neuromuscular weakness, and these may reveal valuable information that may lead to a better understanding of other neurological diseases.The evidence for antivenom and AChEIs in treatment of neurotoxic envenoming is not strong, and large randomized trials are urgently needed.Prasarnpun S, Walsh J, Awad SS, Harris JB (2005) Envenoming bites by kraits: the biological basis of treatment-resistant neuromuscular paralysis. Brain 128: 2987–2996.Lee C, Chen D, Katz RL (1977) Characteristics of nondepolarizing neuromuscular block: (I) post-junctional block by alpha-bungarotoxin. Can Anaesth Soc J 24: 212–219.Bungarus caeruleus) bite in Anuradhapura, Sri Lanka: a prospective clinical study, 1996–98. Postgrad Med J 78: 276–280.Kularatne SA (2002) Common krait (Oxyuranus scutellatus canni): paralysis, hemostatic and electrocardiographic abnormalities, and effects of antivenom. Am J Trop Med Hyg 52: 525–531.Lalloo DG, Trevett AJ, Korinhona A, Nwokolo N, Laurenson IF, et al. (1995) Snake bites by the Papuan taipan (Naja naja philippinensis). A placebo-controlled study. N Engl J Med 315: 1444–1448.Watt G, Theakston RD, Hayes CG, Yambao ML, Sangalang R, et al. (1986) Positive response to edrophonium in patients with neurotoxic envenoming by cobras (


In the last decade, both regenerative medicine and nanotechnology have been broadly developed leading important advances in biomedical research as well as in clinical practice. The manipulation on the molecular level and the use of several functionalized nanoscaled materials has application in various fields of regenerative medicine including tissue engineering, cell therapy, diagnosis and drug and gene delivery. The themes covered in this review include nanoparticle systems for tracking transplanted stem cells, self-assembling peptides, nanoparticles for gene delivery into stem cells and biomimetic scaffolds useful for 2D and 3D tissue cell cultures, transplantation and clinical application. Regenerative medicine is a broad interdisciplinary science that attempts to restore lost, damaged, or aging cells and tissues to a state as close as possible to its native architecture and function. Therapeutic approaches in regenerative medicine may be divided into: (i) cell-based; (ii) biomaterials-based or (iii) combined tissue engineering strategies. Over the past twenty years, there have been tremendous advances in the fields of stem cell biology, nanotechnology and bioengineering, the intersection of which offers increasing potential to achieve the goal of truly regenerative therapies for myriad pathologies.Stem cells are defined by their ability to retain their stem cell capacity through controlled proliferation  while also dividing to produce differentiated daughter cells, which lose some degree of “stemness” and proceed to form mature cells of all lineages in the body . These iNanotechnology is defined by the size of a material  or manipulation on the molecular level, and it involves a broad range of nanoscaled materials used in various fields of regenerative medicine, including tissue engineering, cell therapy, diagnosis and drug and gene delivery. Moreover, the growing interest and expansion in nanomedicine during the last years has led to new perspectives and advances in biomedical research as well as in clinical practice. To date, nearly 30 nanotechnology-based products have been approved for clinical use, focused mainly on liposomal formulations and stealth polymer-drug conjugates. The application of nanotechnology tools to the development of structures at the molecular level enables the improvement of the interactions between material surfaces and biological entities. Thus, nanomedicine provide the possibility to produce surfaces, structures and materials that can mimic the natural environment of cells, to promote certain functions, such as cell adhesion, cell mobility and cell differentiation [Since Langer and Vacanti in 1993  proposedNatural or synthetic materials are used to make scaffolds and depending on the final purpose, barriers (membrane or tubes), gels or 3D matrices are developed to mimic the extracellular environment of a target tissue or organ. Natural materials are derived from human or animal (xenogeneic) sources and are composed of extracellular components . They inAnother promising strategy for tissue regeneration is the use of nanomaterials as cell delivery vehicles. The most commonly used nanomaterials are peptide amphiphiles, self-assembled peptides, carbon nanotubes (CNTs), electrospun fibers and layer-by-layer structures . The devControlled release of biomolecules is crucial in the support and enhancement of tissue growth in tissue engineering applications. Nanotechnology approaches in delivery systems can enhance the success of specific therapeutic agents, such as growth factors, proteins, peptides, DNA, RNAi and small drugs, which are of vital importance for tissue regeneration .Further progress in cell therapy leading to clinical trials requires the crucial use of non-invasive techniques for monitoring the efficacy of cell therapy and graft survival in the host organism. For pre-clinical and clinical trials, it will be important to track SCs noninvasively in order to evaluate their therapeutic effect and grafting location to rule out potentially dangerous side effects. Moreover, nanoparticle (NP)-labeled SCs/progenitor cells might contribute to our understanding of the cell migration processes in numerous diseases, such as neurologic diseases, myocardial infarction and cancer . In addiThe aim of this review is to highlight some of the most recent advances in the exciting fields of nanotechnology and regenerative medicine. We will focus in the functionalization of nanostructured biomaterials with potential applications in regenerating different tissues. Therefore, the themes covered in this article include: (i) nano- or micro-fabricated scaffolds with different biomolecules (depending on the targeted cells) and nano-modified surfaces; (ii) stem cell encapsulation with nano-fabrication techniques; (iii) NP systems for tracking transplanted SCs using several imaging modalities; and (iv) NPs for gene and drug delivery targeting SCs. We also emphasize some of the ways they are being used in translational and clinical practice.in vivo condition the cells are located in three-dimensional (3D) microenvironments, where they are surrounded by other cells and by the extracellular matrix (ECM), whose components, such as collagen, elastin, and laminin, are organized in nanostructures  with specific bioactive motifs that regulate the cell homeostasis. It is therefore essential to develop scaffolds that create synthetic microenvironments providing 3D support, so as to control and direct the cellular behavior and to promote specific cell interactions [A main strategy of regenerative medicine is the construction of a biocompatible scaffold that, in combination with living cells and/or bioactive molecules, replaces, regenerates or repairs damaged cells or tissue. Numerous studies, using nanostructured materials, have demonstrated the validity of this approach for a variety of cell types and the application in regenerating different tissues  by enhancing the biological properties of the cells such as cell adhesion, cell mobility and cell differentiation ,39–42. Iractions ,44. The ractions –47. In tractions ,49. Moreractions . The devThe ideal situation for hard tissue regeneration is to utilize “bioactive” materials that stimulate active tissue regeneration. Substrate rigidity or flexibility has been shown to regulate cell behaviour and is a critical determinant of tissue function . Stifferin vitro culture.The bone tissue is a mineralized organic matrix mostly formed from collagenous fibres and calcium phosphate in the form of hydroxyapatite (HA), with embedded osteoblasts, osteocytes and osteoclasts as cell components. For bone reconstruction, nanoscaffolds have been provided with suitable biophysical properties, such as hardness and porosity, as well as support for cell growth and differentiation. In the past several years, various nanofibrous matrices have been created. Most of the nanoscaffolds produced emulate the structural, compositional and biological characteristics of natural bone. They are based primarily on nano-HA, collagen, electro-spun silk, anodized titanium and nano-structured titanium surfaces –55. NanoOn the other hand, SCs are an attractive option because their large proliferative capacity and their ability to differentiate into multiple cell types . Numerouet al. showed that hMSCs on aligned CNTs networks exhibited enhanced proliferation and osteogenic differentiation compared to those on randomly oriented CNT networks due to mechanotransduction pathways triggered by high cytoskeletal tension in the aligned hMSCs [et al. have suggested that the rapid osteogenic differentiation could be due to the ability of graphene to act as a platform for the accumulation and interactions of osteogenic inducers included in the conditioned medium such as dexamethasone and β-glycerolphosphate [Other promising materials for bioengineering applications are carbon nanotubes (CNTs). CNTs are conductive and have nanostructured dimensions that mimic the 3D structure of proteins found in ECM. Large CNTs lead to a dramatic stem cell elongation, inducing cytoskeletal stress and selective differentiation into osteoblast-like cells . Furthered hMSCs . In addied hMSCs ,68. Engied hMSCs , and miged hMSCs . Lee et hosphate . Specifihosphate .et al. seeded HA and nano-HA coated and uncoated electrospun PLLA fibres with human cord blood derived SCs and implanted the scaffolds subcutaneously into mice [In two recent studies Seyedjafari nto mice ,73. Aftento mice . Additionto mice ,73.et al. examined the effects of the nanofibrous architecture in both two-dimensional (2-D) PLLA thin matrices and 3D PLA scaffolds to assess their effect on the osteogenic differentiation of human ESCs (hESCs) in vitro compared to more traditional solid films and solid-walled (SW) scaffolds. After 6 weeks of 3D culture, the hESCs on the NF scaffolds expressed significantly more osteocalcin mRNA compared to these on the SW scaffolds. The data indicates that the NF architecture enhances the osteogenic differentiation of the hESCs compared to more traditional scaffolding design [in vitro showed highly mineralized tissues developed with specific bone marker genes expressed. These results indicate the promise of hESCs-MSCs for bone regeneration [Nanofibrous (NF) matrices have also been shown to enhance the expression of osteogenic genes and proteins and calcium staining of pluripotent embryonic stem cells (ESCs) . Recentlg design . In addineration .Reprogramming somatic cells into an ESC-like state, induced pluripotent stem (iPS) cells, has emerged as a promising new venue for customized cell therapies that avoided the controversy surrounding human ESCs. A recent study demonstrated the ability of murine iPS-derived osteoblasts seeded in a gelfoam matrix followed by subcutaneous implantation, in syngenic imprinting control region mice, to express matrices characteristic of bone . MoreoveCartilage is an avascular tissue composed of chondrocytes entrapped in an ECM rich in proteoglycans and collagens. Chondral defects suppose a challenging clinical problem as the proportion of elderly people in the population increase. Cartilage injuries lead to joint pain and loss of function with limited capacity for self-repair . Innate Tissue engineering strategies have long been used for cartilage regeneration and have been based mostly on matrix seeded with either chondrocytes or MSCs . Biomatein vivo. The capacity of cartilage regeneration of BM-derived hMSCs on PVA/PCL nanofibre scaffolds has been assayed after implantation into rabbit full-thickness cartilage. To improve the surface hydrophilicity, the biocompatible water-soluble synthetic biodegradable polymer PVA was selected to be electrospun concurrently with PCL via hybrid electrospinning. Results showed that PVA/PCL scaffolds supported the proliferation and chondrogenic differentiation of MSCs in vitro and when cell-seeded PVA/PCL scaffolds were implanted the animals treated showed improved healing of defects compared with untreated control and those which received cell-free scaffolds [in vitro and in vivo [in vivo study was conducted in a swine model, and showed the applicability of biodegradable PCL NF scaffold, in this case, seeded with allogeneic chondrocytes or xenogeneic hMSCs. Thus, NF scaffolds effectively deliver therapeutic cells to cartilage lesions and support chondrogenic activity during the cartilage regeneration in vivo. The xenogeneic hMSCs-based treatment proved to repair cartilage defects and restore biomechanical functions of cartilage, suggesting the potential of MSCs xeno- or allo-grafting for cartilage tissue engineering [The applicability of stem cell-seeded on NF scaffolds has also been evaluated caffolds . Combina in vivo . Anotherineering .l-lysine) microcapsules harbouring rat islet cells, that naturally secreted insulin, for the treatment of diabetes [Cells in native tissue are embedded within a complex 3D microenvironment consisting of soluble molecules (cytokines and growth factors) and non-soluble factors (mainly ECM). The microenvironment not only provides structural integrity, but also controls numerous signal transduction processes that direct cell survival, cell cycle progression, and the expression of different phenotypes . Cell endiabetes . This waet al., where the measurement of the surface electrical charge, by means of zeta potential, was found to predict the interfacial reactions between the biomaterial and the surrounding tissue [The cellular recognition of biomaterials and the progression of subsequent cellular events are essentially based on integrin-mediated interactions, but depend in particular on the chemical and physical characteristics of biomaterials. The micro-fabricated biomaterials offer several advantages: (i) the individual parts of living cells exist in nano- or micro-scale lengths, and thus micro- or nano-fabrication techniques enable the recapitulation of their features; (ii) the micro-fabricated substrate can present simultaneously multiple adhesive or morphogenic signals in a small dimension simultaneously; and (iii) multiple parameters governing cell-biomaterial interactions could be analyzed separately with a small amount of analytes . Microcag tissue . Mechaniet al. revealed that cellular reaction to the smaller capsules was much lower than that to the larger capsules [i.e., aqueous medium, no reactive species, no organic solvent). It is important to choose a reproducible method in which different parameter can be tightly controlled, for instance, permeability, size, and surface area. So far, different methods have been used to prepare cell-loaded microcapsules, including extrusion , emulsion/thermal gelation (agarose as core polymer), and microfluidic flow focusing approach [Another main concern is the immunoreactions against the capsule, which probability will trigger the immune system of the body. So encapsulated cells should be immunoprotected not only from immune cells but also from antibodies and cytokines . An intecapsules . Thus, rcapsules ,96. Otheapproach ,97,98.in vivo stability of electrostatic and hydrogen bonds maintaining the structure of microcapsules is limited, and the mechanical resistance of capsules is still a concern. Liquid-core/shell microcapsules where cells are embedded in liquid seem to allow better cell growth and protein production, because diffusion of gases and nutrients is higher in liquid than in gel, although their mechanical resistance is further diminished compared to matrix-core capsules. In this respect, semi-liquid cores and cells-core/shell where cells are directly surrounded by a coating layer appear to be good options. However, several questions pertaining to mechanical resistance (related to strength of the cell aggregates), the consequences of cell growth and death on the integrity of the immunoisolation barrier, and finally, long-term stability and performance, need to be addressed [The first assays of encapsulation were made using matrix-core/shell microcapsules. Cells were embedded in hydrogel matrix, often surface-modified by one or more external layers of different materials to improve permeability and stability properties, preventing cell release from the beads and ensuring biocompatibility . Despiteddressed ,97. Figuet al. evaluated benefits of microencapsulation in alginate when performing cell cultures. This work established that microencapsulation technology may be a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications [Alginate is the most common encapsulation material due to its intrinsic properties including biocompatibility, biosafety and permeability. The production of alginate cell microcapsules can be performed under safe and physiological conditions  and using good manufacturing practice (GMP) guidelines, a fact which potentiates the use of this technology in cell-based therapies –101. Recications . The potAt the present, many studies focus on the encapsulation of SCs as a starting point for regenerative medicine, engineering tissue and gene therapy . Therefoin vivo without the modification of the host genome [In summary, cell encapsulation allows continuous delivery of products from the cells for a longer period of time and, also, the transplantation of non-human cells, which could be considered as an alternative to the limited supply of autologous tissue. In addition, genetically modified cells can be immobilized to express any desired protein t genome ,129 and t genome ,130. Nevt genome ,132.versus-host disease and blood disorders is widely recognized. In addition these cells are useful tools for modelling a broad range of diseases, both in vivo animal and in vitro. Among the intrinsic characteristic of SCs, their immunomodulatory properties and their low immunogenicity are essential features to be ideal candidates for cell therapy treatments. Adult MSCs have been shown to generate a local immunosuppressive microenvironment via cytokines activity and could produce key factors to inhibit both fibrotic and apoptotic phenomena [The therapeutic potential of SCs in illnesses such as myocardial infarction, stroke, regeneration of bone and cartilage defects, spinal cord injury, graft-henomena .ex vivo to distinguish the implanted cells from the host tissue cells and follow their survival, migration, differentiation and regenerative impact in living subjects [Assuming that SCs may not trigger the immune response it is still important to consider how these cells can be guided to specific locations once they are transplanted into the patient. To monitor and evaluate the engraftment in the host, cells are labelled subjects .In vivo imaging requires that a contrast agent associated with the transplanted cells exert an “effect size” sufficient for detection by the imaging hardware. Fluorescence labelling techniques are not sensitive enough to track in vivo injected cells because this approach requires invasive methods in order to address location of labelled cells [in vitro studies, microscopic optical imaging techniques include light and fluorescence microscopy, confocal microscopy, and two photon microscopy.In these terms NP technology could help to track and localize transplanted cells. Nanocapsules are made with unique optical and/or magnetic properties to allow a non-invasive, accurate and real-time cell tracking . Ideallyed cells . For in in vivo. X-ray, ultrasound and computed tomography (CT) have the disadvantage to require high concentrations of high density, high atomic number materials such as iodine, gadolinium, or metals, in order to achieve good contrast for image acquisition. Multimodality imaging techniques such as CT/positron emission tomography (PET), CT/single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI)/PET combine different “anatomical” and “functional” methods. In this regard, MRI provides a very safe method for human in vivo imaging using a powerful magnetic field to align the nuclear magnetization of hydrogen atoms, which are responsible for the majority of MRI signals [Conventional technologies and new approaches in the clinical radiology field have been used to track injected cells  signals ,138. How® and Endorem® or as ultrasmall NPs . Coating SPIO NPs with a protective layer of dextran or other polysaccharide helps to prevent their aggregation, induces the efficient internalization of the contrast agent into the cell and minimizes any deleterious effects on cellular function [Combination of NPs and MRI are currently used for biomedical applications, however, contrasts agents used are mostly iron particles which accumulate in the liver. To improve imaging probes sensitivity and decrease particle toxicity, new strategies to coat the iron particles have been developed, consisting of nonstoichiometric microcrystalline magnetite cores, which are coated with dextrans (in ferumoxide) or siloxanes (in ferumoxsil). These SPIO are now used as MRI contrast probes for studying the fate of transplanted cells . Commercfunction .In summary, these novel contrast agents present the advantages of suitable magnetic saturation, superparamagnetic properties, and also their colloidal stability and biocompatibility ,142. In in vitro cell cultivation, and accumulate in the endosomes. Depending on the size of them, iron oxide NPs can be internalized by several phenomena, including phagocytosis for SPIO and pinocytosis for ultrasmall superparamagnetic iron oxides particles (USPIO) and magnetodendrimers micron sized iron oxide particles (MPIOs) (L-lysine or lipofectamine), electroporation [Iron oxide NPs are sequestered into SCs through endocytosis during  (MPIOs) . This prporation , specifiporation , magnetoporation , or tranporation –148. AccDifferent studies using animal models have been carried out to evaluate the use of SPIO as a biomedical tool. SPIO labelling technique has been successfully used to study cell function and integration after transplantation in the cerebral nervous system (CNS), heart, liver, and kidney as well as in pancreatic islets of animal models.in vivo tracking conditions, SPIO NPs have been used to establish how intravenous delivery of human adipose stem cells (h-ADSC) can home to the radiofrequency ablated canine myocardial lesion and express a cardiomyocyte-like phenotype [Besides henotype . In diabhenotype . In a sahenotype .in vivo the functional response of glial-committed neural precursor cells (NPCs) labelled with SPIO. Results showed that labelled cells migrated over comparable distances and differentiated into glial lineages in the same way as unlabeled cells [in vivo studies have monitored the progression of neural SC therapy using NPs for up to 6 weeks [Cell therapies developed to cure neurological disorders such as multiple sclerosis (MS) have used SPI-labelled cells for high resolution monitoring of the bio-distribution of cells after transplantation into the CNS. Experimental autoimmune encephalomyelitis (EAE) mice were used to examine ed cells . NPCs poed cells ,154 and ed cells ,156. Oth 6 weeks . A recen 6 weeks .Although NPs have important applications for tracking SCs, however, there are potential scientific issues as well as safety issues that must be considered before clinical trials. In fact, clinical trials are hampered by basic research; nevertheless, few clinical trials using NPs are recruiting people at the moment.in vivo cell tracking. QDs are able to maintain fluorescent intensity in cell culture for a prolonged time, up to a few hours using confocal microscopy [Quantum dots (QDs) are another class of nanomaterials with unique optical features, typically in the size range of 2–10 nm. They are inorganic fluorescent semiconductor NPs coated with an outer shell of a different material. First studies demonstrated the use of QDs as luminescent cell markers ,160. Curcroscopy . In factcroscopy . Due to croscopy ,160.in vivo after transplantation is essential for determining the efficacy of SC therapy. In a recent study, QDs were used to label ASCs, in combination with heparin, and analyzed their behaviour and organ-specific accumulation after having been transplanted in a mouse model of acute liver failure [The major potential applications of QDs in regenerative medicine are their use for labelling and tracking of implanted SCs. Monitoring the cells  failure .et al. showed that passive loading of QDs resulted in uniform diffused cytoplasmic labelling of a population of hMSCs and maintained similar proliferative and differentiation capacities compared to unloaded hMSCs. Then, passive loading of QDs in hMSCs is an effective method for uptake and did not significantly affect the features of the cells [QDs could integrate into SCs through passive loading, receptor-mediated endocytosis or transfection. Rosen he cells .in vivo it is necessary both to increase cellular uptake of QDs and to promote endosomal escape into the cytosol. In this matter, specific peptides such as cholera toxin [Progress in SC research has prioritized the refinement of cell-labelling techniques. Therefore, for effective labelling of MSCs and long observation by QDs ra toxin , TAT-pepra toxin , octa-arra toxin  or the pra toxin  have beera toxin ,169, whiet al. conducted an assay directing QDs labelling of mammalian stem and progenitor cells in vivo to study the developing mammalian central nervous system. Ultrasound guided in vivo delivery techniques were developed to specifically and efficiently label NSCs and progenitor cells (NSPCs) with QDs. QDs were found in the cell types generated by NSPCs at substantial distances away from the initial site of injection, suggesting that QD loading of NSPCs in vivo did not inhibit their migration or differentiation during the developmental cycle. Therefore, QDs labelling may be particularly useful for embryonic studies about the formation of nascent primordial layers [Different studies have been carried in order to analyze the effect of QDs on SCs properties; Slotkin l layers .in vitro or in vivo due to their inherent long-term fluorescence intensity [QDs are also attractive nanomaterials for monitoring stem cell survival, location, and differentiation either ntensity –167. Stentensity –173.in vivo trafficking of therapeutic cells. However, further studies are necessary to demonstrate the outcomes of QDs on long-term effects and their degradation products on SCs. In fact, the release of reactive oxygen species during the degradation of QDs contributes to its cytotoxicity [in vivo degradation.In conclusion, QDs based cell labelling can be used as a safe and effective means to facilitate toxicity ,175. AltAfter decades of fine-tuning many biological materials can now be designed and fabricated at the molecular level for particular uses. Because of their chemical complexity and structural sophistication, biological materials of natural origin have been more difficult to design and fabricate at the molecular level. However, over the last two decades the efforts of chemical scientists, chemical engineers, materials scientists and medical doctors have borne fruit and now a wide range of biomaterials for medical applications exist.Peptide nanostructures containing bioactive signals that combine bioactivity for multiple targets with biocompatibility, improve the possibility to deliver proteins, nucleic acids, drugs and cells. Their chemical design versatility leads to a variety of possible secondary, tertiary and quaternary structures through folding and hydrogen bonding. In particular, β-sheet forming peptides have demonstrated the extraordinary ability to use intermolecular hydrogen bonding for the assembly of one-dimensional nanostructures. Furthermore, the design of self-assembly peptides for targeted functions can also include their modification with other biomolecular units such as sugars, lipid components and nucleic acid monomers. These oligopeptides, whilst designed for supramolecular self-assembly, could also serve to functionally mimic large proteins. For these reasons, self-assembling, nonimmunogenic peptides project as promising new therapeutics for human disease –178.The first self-assembling peptide discovered was the EAK16, also named zuotin protein for its ability to bind to left-handed Z-DNA, which contains ubiquitous right-handed B-DNA and other DNA structures . This peSince EAK16 was discovered, many peptides and combinations are being studied. These peptide amphiphiles (PAs) not only have been used as scaffolds that provide structural support and bioactive signals, but these materials have also been moulded to allow for the study of the effects of matrix geometry on the behaviour of cells.Some examples of different types of peptides that are being investigated and their possible application in regenerative medicine are:in vitro within networks of IKVAV PA quickly undergo selective and rapid differentiation into neurons with the formation of astrocytes being largely suppressed [in vitro results suggested that the IKVAV PA may be a useful material in the treatment of spinal cord injury, where the formation of a glial scar, comprised primarily of astrocytes, prevents axonal regeneration after injury [The Ile-Lys-Val-Ala-Val (IKVAV) peptide sequence, derived from laminin, has been incorporated into PAs for applications in neural regeneration to enhance neural attachment, migration and neurite outgrowth. Neural progenitor cells cultured ppressed . Controlr injury . Mice trr injury . This war injury .An interesting PAs with angiogenesis properties is the heparin-binding peptide amphiphile (HBPA), which was designed with a Cardin-Weintraub heparin-binding domain to specifically bind heparan sulphate-like gylcosaminoglycans (HSGAG). This glycosaminoglycan displayed charges on the HBPA molecules, triggering PA self-assembly into nanofibres that presented heparin on their surface. Moreover, they were able to capture many potent signalling proteins through their heparin-binding domains, including fibroblast growth factor 2 (FGF-2), bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF). This material was biodegraded and quickly remodeled into a well vascularised connective tissue without the addition of any exogenous growth factors –185.Since nitric oxide has long been recognized as a possible solution to prevent complications of neointimal hyperplasia during angioplasty treatment in patients with atherosclerosis, PAs presenting heparin were mixed with diazeniumdiolate nitric oxide donors to prepare nitric oxide releasing nanofibre gels . When apin vitro, whilst cells did not attach to the non-functionalized NiTi [PA nanofibres were explored as a means to functionalize the metal implants to enhance bioactivity and prompt tissue growth around the implant to assist in long-term implant fixation. A nickel-titanium (NiTi) alloy that is frequently used for stents, bone plates, and artificial joints was modified through covalent attachment of PA nanofibres using standard silanization and cross-linking chemistry ,188. Modzed NiTi .in vitro, these cells showed an enhancement in proliferation and increased their expression of amelogenin and ameloblastin, two proteins secreted by ameloblasts during enamel formation [in vitro scaffold for dental SCs, where SCs from human exfoliated deciduous teeth proliferate and secrete a soft collagen matrix when encapsulated within the PA, whilst dental pulp SCs differentiate into an osteoblast-like phenotype and deposit mineral when encapsulated within the gel [Branched RGDS-presenting PA nanofibres have been also used as scaffolds for ameloblast-like cells and primary enamel organ epithelial cells that initiate the process of enamel formation. When treated with branched RGDS PA nanofibres ormation . PAs hav the gel .de novo hydroxyapatite when incubated in mineralizing solutions [The β-sheet peptide nanostructures have been also evaluated for the treatment of enamel decay, resulting in significant gains of net mineral within the lesions over the 5-day study. The peptide gels also nucleated the formation of olutions . The samolutions .in vitro have found that these β-hairpin hydrogels can support survival, adhesion, and migration of fibroblasts, and can be used to encapsulate MSCs and hepatocytes. These gels have also been found to have inherent antimicrobial properties; showing selective toxicity to bacterial cells compared with mammalian cells [Another peptide design that captures the self-assembling potential afforded by the β-sheet was prepared from monomers of alternating hydrophilic and hydrophobic residues, lysine and valine, respectively, flanking an intermittent tetrapeptide designed to mimic a Type II b-turn, termed a β-hairpin peptide. These peptides are designed to be hydrated in pure water, adopting a random coil conformation. Studies an cells ,193,194.The ionic self-complimentary peptides based on β-sheet-rich proteins from nature, prepared from sequences of alternating hydrophobic and hydrophilic residues, have the ability to support cell attachment to promote the survival, proliferation, differentiation and neurite growth for neural cells. Moreover, they were capable to promote differentiation of liver progenitor cells into hepatocyte spheroids and serve as scaffolds for human endothelial cells, as well as for chondrocytes and for osteogenic differentiation of hESCs ,195–197.N-terminal end of di- and tri-peptides, demonstrating the formation of very stable, highly aunable hydrogels. A number of these sheets twist together to form nanotubes. These materials can also support chondrocyte survival and proliferation in both 2D and 3D [Self-assembling peptides can also use conjugated aromatic groups such as carbobenzyloxy, naphthalene, or fluorenylmethyloxycarbonyl on the D and 3D ,199.Furthermore these systems can be used as templates for nanofabrication and biomineralization of synthetic replacements in biological tissues, with specific medical applications including diagnostic technologies. Indeed, functionalized peptide nanostructures are part of the immediate future with high relevance in regenerative medicine and drug delivery.When a drug is introduced in the human body using traditional administration methods, a cascade of biotransformations occurs as result of its interaction with the biological environment. These processes are part of the drug metabolism. Drug metabolism is very complex and comprises oxidation, reduction, hydrolysis and conjugation reactions leading to final excretion from the body. Depending on the anatomical route, administered drugs are passing, upon absorption, through several tissues and organs  before reaching the systemic circulation. In those organs, drugs may be subjected to chemical or enzymatic degradation. As a result, a higher dose of drug is necessary to ensure relevant therapeutic levels. Gene delivery methods using viral vectors, such as retroviruses, adenoviruses, and adeno-associated viruses, are extensively used and show superior transfection efficiency ,201. HowIn recent years, SC nanotechnology has emerged as a new exciting field since it overcomes some of the major limitations of conventional delivery system . Novel nDrug delivery systems into stems cells are associated above all with the field of the cancer stem cells (CSCs). Carcinogenesis in humans is a multistage process, and the two major stages have been designated initiation and promotion. Although the biochemical basis for initiation and promotion remains to be established, recent research has provided important insights into potentially significant biologic mechanisms. Today is known that the initiation of carcinogenesis may result from cellular immortalization and the development of defects in the integrated control of stem cell proliferation and differentiation and that the promotion of carcinogenesis may result when such initiated SCs develop aberrant auto-regulatory growth-control properties. Understanding the role of CSCs during carcinogenesis, from tumour initiation to metastasis formation, has become a major focus in stem cell biology and in cancer research . Nano-scet al. designed a tailor-made dual pH-sensitive polymer-doxorubicin conjugate nanoparticulate system to overcome the challenges. The NP is capable of reversing its surface charge from negative to positive at tumour extracellular pH (±6.8) to facilitate cell internalization. Subsequently, the significantly increased acidity in subcellular compartments such as the endosome (±5.0) further promotes doxorubicin release from the endocytosed drug carriers. This dual pH-sensitive NP has shown enhanced cytotoxicity in drug-resistant cancer SCs, indicating its great potential for cancer therapy [In the past decade, NP-based drug delivery systems have shown exciting efficacy for cancer treatments due to their improved pharmacokinetics and biodistribution profiles via the enhanced permeability and retention (EPR) effect. Efficient delivery of therapeutics into tumour SCs to increase the intracellular drug concentration is a major challenge for cancer therapy due to drug resistance and inefficient cellular uptake. In this context, Du  therapy .et al. proved that these cells could be used as NP delivery vehicles [In addition, the combination of SCs and drug-loaded NPs for therapeutic applications in glioma therapy is a promising strategy, as NP could protect therapeutic agent and could allow its sustained release. The prognosis of patients with malignant glioma remains extremely poor, despite surgery and improvements in radio- and chemotherapies. However, new paradigms allowing tumour specific targeting and extensive intratumoral distribution must be developed to efficiently deliver NPs. Taking advantage into the fact that MSCs have a natural tropism toward brain tumours, Roger vehicles . Two typvehicles ,214.In vivo experiments proved that the burdened MSCs could track down the U251 glioma tumour cells more efficiently and deliver doxorubicin with wider distribution and longer retention lifetime in tumour tissues compared with free doxorubicin and silica nanorattle-encapsulated doxorubicin. The increased and prolonged doxorubicin intratumoral distribution further contributed to significantly enhanced tumour-cell apoptosis. This strategy has potential to be developed as a robust method for targeted tumour therapy with high efficiency and low systematic toxicity [Low targeting efficiency is one of the biggest limitations for NPs drug delivery system-based cancer therapy. In another study MSCs were used as targeting vehicle and a silica nanorattle was employed as drug carrier ,216. Thetoxicity .in vivo therapeutic transfer of genes to the host’s cells. These genes affect the host tissue in situ and induce tissue formation [Tissue engineering can generally be divided into three main methodological areas of interest: direct gene delivery, cell therapy without involving genetic modification, and genetically modified cell-mediated therapy. The direct approach, gene delivery, focuses on an ormation  and regeormation . Cell thormation ,221. Genormation . Gene-moormation , resistaormation , and dirormation . Thus, nReprogramming human somatic cells to induce pluripotent stem cells (iPSCs) by defined transcription factors   has revoin vivo distribution. In this context, recently Lee and collaborators [et al. [Although iPSCs offer great possibilities, there is a crescent concern regarding the procedures used to induce the pluripotentiality. The main techniques imply the use of viral vectors, such as retrovirus and lentivirus, which integrate the reprogramming factors into the host genomes and may increase the risk of tumour formation. Several non-integration methods have been reported to overcome the safety concern associated with the generation of iPSCs, such as transient expression of the reprogramming factors using adenovirus vectors or plasmids, and direct delivery of reprogramming proteins. Although these transient expression methods could avoid genomic alteration of iPSCs, they are inefficient . The appborators  generate [et al.  successfThe ability to deliver biomolecules via an intracellular route, including growth factors, genes, proteins, and small chemicals, presents an excellent tool to direct SCs differentiation into specific cell types as bone, cartilage, muscle, adipose and cardiac. Some of these biomolecules/chemicals (i) have poor solubility, (ii) can be quickly cleaved by cellular enzymes, and (iii) have side effects when administered systemically. Biodegradable and biocompatible NPs able to target SCs and release the payload in their cytoplasm, with consequent activation of signalling cascades, will be of great interest.in vivo, they are difficult to administer to sites of damaged tissue at therapeutic concentrations and for sustained periods of time. Thus, the way these molecules should be delivered to the injury site plays a crucial role for their success as therapeutic agents. To overcome the limitations of traditional methods of administration, several technologies using biomaterials have been explored to achieve a better control over the growth factor release. In this context, recently was reported a new approach for the delivery of vascular growth factors into hESCs, by incorporating growth factor-release particles in human embryoid bodies (EBs) [Growth factors are naturally occurring proteins capable of stimulating cellular proliferation, migration and/or differentiation into a specialized phenotype. Because they are involved in the regulation of several cellular functions, they can enhance the healing and regeneration processes of diverse tissues . Nowadayes (EBs) . The incet al. [in vitro culture systems. Recently, in the field of adipogenesis a systematic study in rat mesenchymal stem cells (RMSC) demonstrated that the biocompatible silica NP-insulin (SiNP) conjugates induce in vitro adipogenic differentiation [et al. [in vitro as well of in vivo transplanted cells, using the adipogenic transcription factors C/EBP-α and C/EBP-β complexed with PEI coupled with biodegradable PLGA nanospheres. The expression of specific adipogenic genes and proteins in hMSCs was significantly elevated compared to the controls.Moreover, use of NPs, as delivery system, could be the best choice for bone regeneration, as there is no need for long-term overexpression of bone morphogenetic proteins (BMP) to induce valid bone formation. Transient overexpression of protein for a few weeks is sufficient to initiate the regeneration process. Indeed, many studies have shown the potential for MSCs that transiently overexpress BMP to induce bone formation on nanofibres  and chitet al.  employedntiation . Moreove [et al.  achievedin vitro or in vivo and may chemically degrade, making it difficult to relate systematically nanoparticle toxicity to such a diverse set of materials [in vitro and in vivo mechanisms of toxicity depend on the type of NPs [The use of NPs in biomedical research implies a detailed consideration of their toxicity . Thus, aaterials . Severale of NPs ,242,243.e of NPs ,245, mESe of NPs , and NSCe of NPs  showed ie of NPs . In concSignificant advances achieved in both regenerative medicine and nanomedicine offer the promise of tissue and organ specific regenerative treatments. The development of functionalized bioactive scaffolds and NPs that promote cell proliferation, migration and differentiation has opened expectations for clinical application. However, some questions about safety and effectiveness of SC-based therapy and nanomaterials need to be addressed for widespread diagnostic and therapeutic use. A deep understanding of biological mechanisms responsible of regenerative processes based on nanomaterials-SC cell therapy is needed to obtain valuable information concerning cell behaviour, efficacy and undesirable effects of nanomaterials. Functionalization at molecular level of bioactive and biodegradable nanoscale filaments that mimic ECM must be explored in animal models before translation to clinic. After that, we believe clinical trials with current and novel nanomaterials will revolutionize application of regenerative medicine in patients.


KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defined subgroup. We performed a prior free analysis of CRC heterogeneity on 1113 CRC gene expression profiles and confronted our findings to established molecular determinants and clinical, histopathological and survival data. Unsupervised clustering based on gene modules allowed us to distinguish at least five different gene expression CRC subtypes, which we call surface crypt-like, lower crypt-like, CIMP-H-like, mesenchymal and mixed. A gene set enrichment analysis combined with literature search of gene module members identified distinct biological motifs in different subtypes. The subtypes, which were not derived based on outcome, nonetheless showed differences in prognosis. Known gene copy number variations and mutations in key cancer-associated genes differed between subtypes, but the subtypes provided molecular information beyond that contained in these variables. Morphological features significantly differed between subtypes. The objective existence of the subtypes and their clinical and molecular characteristics were validated in an independent set of 720 CRC expression profiles. Our subtypes provide a novel perspective on the heterogeneity of CRC. The proposed subtypes should be further explored retrospectively on existing clinical trial datasets and, when sufficiently robust, be prospectively assessed for clinical relevance in terms of prognosis and treatment response predictive capacity. Original microarray data were uploaded to the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under Accession Nos E-MTAB-990 and E-MTAB-1026.The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI,  BRAF or KRAS genes .We have built two non-overlapping data collections: a discovery collection, comprising four publicly available (425 samples) and two previously unpublished datasets  with known stage status, and a validation collection of eight publicly available datasets (720 CRC samples) . Observations derived from the analysis of 64 normal samples were further validated on five publicly available datasets, with both carcinoma and normal samples available in one batch . Copy number data was available for 154 of the PETACC3, as in http://www.ebi.ac.uk/arrayexpress/), under Accession Nos E-MTAB-990 and E-MTAB-1026, respectively. Gene expression data were processed by standard tools to obtain normalized, probeset-level expression data. For each EntrezID in the datasets, the probeset with the highest variability was selected as representative and the number of EntrezIDs entering the analysis was reduced to 3025 by applying non-specific filtering. For PETACC3 and normal colon samples, patients signed an informed consent form in which the use of tissue specimens was included, and all marker study proposals were subjected to the approval of the trial steering committee.The discovery collection contained the previously unpublished 688 CRC formalin-fixed, paraffin-embedded (FFPE) samples of PETACC3 gene modules (groups of genes with correlated expression), from which non-robust modules  and a gender-related module were discarded. Each expression profile was then reduced to a vector of meta-genes by taking the median of the values of genes in each gene module. The meta-genes were then further grouped into clusters using hierarchical clustering.For model development  only the 1113 CRC samples of the discovery set were used, no sample in the validation collection being used for any model tuning. Hierarchical clustering  and dynamic cut tree core samples—those samples from the discovery collection that were assigned to clusters by hierarchical clustering, using a consensus distance subtypes. The rest of the samples from the discovery collection, not assigned to subtypes by this procedure, were called non-core samples. This approach allowed the reduction of noise in subtype-defining samples, and thus a higher consistency of the resulting subtypes defining the ground truth for downstream analyses. The stability of the obtained clusters was assessed under different perturbations of the processing pipeline (different parameters and clustering methods) to ensure that the results were not simple artefacts . A multiclass linear discriminant (LDA) The subtypes were defined in terms of t statistic In order to validate the existence of subtypes (and their independence on data selection) and the modelling choices in subtype discovery, we applied the same subtyping procedure (including parameters) to the validation collection. The clusters identified in the validation collection were put in correspondence with the subtypes in the training set by LDA predictions and correlations of subtype-specific moderated p values were adjusted for multiple hypothesis testing, using the Benjamini–Hochberg procedure. Significance level was set at 0.1. Pathway analysis for each set of gene modules was carried out using the Database for Annotation, Visualization and Integrated Discovery (DAVID) If not specified differently, all the reported The identified subtypes were characterized histologically in terms of six different architectural patterns: complex tubular; solid/trabecular; mucinous; papillary; desmoplastic; and serrated A, which We identified 54 gene modules, reproducible across all datasets in the discovery collection, comprising 658 genes from an initial list of 3025 identified as the most variable. The assignment of genes to gene modules and gene module clusters is listed in Table S1 ; meta-gene expression profiles for the discovery set are shown in t statistic . All five major subtypes reappeared in the validation set, confirming the robustness of our approach. Figure S2  presents gene expression profiles of both discovery and validation sets. Two notable differences were observed: (i) subtype B in the validation set was split into two subgroups , as observed in the discovery set too, but only at lower pruning height; (ii) another cluster passed the minimal size criteria, corresponding to the small subtype (F) which, in the discovery set, was not considered for further characterization because of small sample size. Validation of other subtype characteristics (to the extent of available information) is described in each of the respective sections.In the validation set of 720 CRC samples we identified a set of subtypes comprising 602 samples  and associated them with our discovery subtypes using the subtype classifier  and correlations of subtype-specific patterns based on moderated EREG gene  or subtype B , confirming subtype A as the most normal-like. The 80 carcinoma samples were distributed over all subtypes .p values of pairwise differences between subtypes are shown in We assessed whether subtypes differ in survival, as a general read-out of biological significance, and then tested the association of each meta-gene with prognosis, using the complete discovery set of 1113 patients  showed that subtype C is enriched for MSI tumours and β-catenin nuclear immunoreactivity at the invasion front . Subtypes B and E showed the highest percentages, while subtypes A and D showed significantly lower percentages of the β-catenin-positive nuclei. Subtype C exhibited almost no β-catenin nuclear immunoreactivity at the IF. We analysed CSC signatures derived from low colon crypt compartment cells that had been identified either by a Wnt reporter construct TOP GFP or by high surface expression of EphB2. Subtypes D and E expressed both TOP GFP and EphB2-derived CSC signatures, while subtype B mainly expressed only the TOP GFP signature . Subtype D was not significantly enriched for any of the tested variables except for the BRAF mutated signature and possibly represents a mixture of tumours that have the EMT/stroma signature in common. KRAS mutants occurred in all subtypes , supporting the emerging notion that KRAS-mutated CRC are substantially heterogeneous KRAS varying per specific mutation and the molecular background of the tumour in which it occurs BRAF mutant signature we identified earlier An important goal of this study was to assess how our molecular subtypes complement known clinical variables and molecular markers. We found that MSI, subtypes , but notTo study whether or not our molecular subtypes are associated with histological patterns, we examined haematoxylin and eosin (H&E)-stained paraffin sections of a randomly selected subset of each subtype . In attempting to match histological morphotypes to molecular subtypes, architectural patterns were used, as illustrated in Our approach, using gene modules on a large panel of samples, allowed us to identify five main CRC gene expression subtypes Table . It is rThe combination of gene expression, clinical, mutational, survival and morphological data contributes new insight into the heterogeneity of CRC. While the validation confirmed the robustness of our findings across different platforms (ALMAC versus Affymetrix), sample preparation methods (FFPE versus fresh-frozen) and dataset collections, larger datasets are necessary to assess and characterize the relevance of lower frequency subtypes . Our data indicate that several major biological processes are key determinants of a complex subtype structure of CRC. Therefore our subtypes defined by gene expression do not substitute but complement groups defined by current clinicopathological variables and molecular markers. Notably, morphological subclassification of CRC has clearly reached its limits, given the often striking intratumour heterogeneity, which made us use a (primary and secondary) architectural pattern approach rather than the canonized histological subtypes (WHO). Profiling of microdissected patterns within a single tumour might reveal molecular mechanisms responsible for these morphotypes. This additional heterogeneity within the subtypes may reflect tumour polyclonality, similar to breast cancer A striking association was found between the stromal subtype D and the EMT signature. The previously discovered EMT β-catenin-positive nuclei than subtype B, with its Wnt-associated gene expression and better survival.EMT, however important, only partly explains CRC heterogeneity, as even subtypes with similar expression of EMT-associated genes (A–C or D–E) differ in survival, mutational, clinical and gene expression characteristics. Additional biological components, such as differentiation, immune response, proliferation, chromosome 20q or cluster of genes deregulated in CRCs, are important co-determinants that underpin a need for further subdivision of CRCs. The findings from the analysis of CSC and WNT signatures support the recently suggested hypothesis that the colon stem cell signature under the condition of silenced canonical WNT targets is associated with higher risk of recurrence (subtype D) BRAF share distinct gene expression patterns. Subtype C was enriched for both MSI and BRAF mutants and had one of the best outcomes for RFS, but the worse outcome in SAR, in concordance with previously reported results BRAF wild-type patients. Our data suggest that subtype C represents tumours with a common biology and a gene expression pattern that might best characterize a group of tumours resistant to chemotherapy, once metastatic. In this sense, our work not only agrees with the current known markers (BRAF mutation status and MSI) but clearly adds new insight, putting together these previously unrelated clusters into one biologically meaningful group. This observation is in line with recently published work MSI tumours represent a subclass in most unsupervised analyses and can be recognized at the gene expression level in vitro functional screens. High-risk subtypes D and C might contribute to therapeutic decision making in either adjuvant or metastatic settings. Retrospective analysis of clinical trial series may identify drug sensitivity associated with particular subtypes, and might open new treatment optimization strategies to be tested in clinical trials with stratified cohorts, similar to the I-SPY2 trial for breast cancer Our observations show that gene expression profiling contributes substantially to our insight into CRC heterogeneity in confirming and complementing data from sequencing, CNV and promoter methylation analysis. Our subtypes can be further functionally interrogated for driving oncogenes/events by In conclusion, our unsupervised approach using gene modules resulted in the identification of distinct molecularly defined CRC subtypes, which adds a new layer of complexity to CRC heterogeneity and opens new opportunities for understanding the disease. The challenge is now to assimilate conventional and these new molecular approaches into a comprehensive consensus classification, which might then be used in further clinical studies for patient stratification and experimental studies to further elucidate mechanisms involved in the development and progression of CRC.