Axonal injury due to prostatectomy leads to Wallerian degeneration of the cavernous nerve (CN) and erectile dysfunction (ED). Return of potency is dependent on axonal regeneration and reinnervation of the penis. Following CN injury (CNI), RhoA and Rho-associated protein kinase (ROCK) increase in penile endothelial and smooth muscle cells. Previous studies indicate that nerve regeneration is hampered by activation of RhoA/ROCK pathway. We evaluated the role of RhoA/ROCK pathway in CN regulation following CNI using a validated rat model. CNI upregulated gene and protein expression of RhoA/ROCK and caspase-3 mediated apoptosis in the major pelvic ganglion (MPG). ROCK inhibitor (ROCK-I) prevented upregulation of RhoA/ROCK pathway as well as activation of caspase-3 in the MPG. Following CNI, there was decrease in the dimer to monomer ratio of neuronal nitric oxide synthase (nNOS) protein and lowered NOS activity in the MPG, which were prevented by ROCK-I. CNI lowered intracavernous pressure and impaired non-adrenergic non-cholinergic-mediated relaxation in the penis, consistent with ED. ROCK-I maintained the intracavernous pressure and non-adrenergic non-cholinergic-mediated relaxation in the penis following CNI. These results suggest that activation of RhoA/ROCK pathway mediates caspase-3 dependent apoptosis of nitrergic neurons in the MPG following CNI and that ROCK-I can prevent post-prostatectomy ED. Despite nerve-sparing radical prostatectomy for the treatment of clinically localized prostate cancer, there is still a significant amount of men that develop severe erectile dysfunction (ED) post-surgical interventionThe RhoA/Rho-associated protein kinase (ROCK) pathway plays an important NO-independent role in regulating smooth muscle tone in penile tissues and has off target effects on NO signaling in the penis467891111in vitro and in vivo increases axonal regeneration following peripheral nerve injury1718Although increased ROCK activity has been ascribed to cause increased penile vascular tone and ED, its role in regulation of autonomic CN function following injury has not been elucidated. In the central nervous system (CNS), RhoA/ROCK mediates growth inhibitory signals generated by myelin-associated inhibitors via growth factor stimulus of RhoA GTPase activity1314The role of the RhoA/ROCK signaling cascade in the major pelvic ganglia (MPG) and the peripheral autonomic CN following injury is unknown. In this study, we demonstrate that upregulation of the RhoA/ROCK pathway in the MPG leads to direct activation of caspase-3 and selective apoptosis of nitrergic neurons. Using a validated pre-clinical model to study the peripheral effects of CNI11Early temporal increases in RhoA, ROCK1 and ROCK2 mRNA expressions were evident 48 hours to 14 days after CNI and decreased by 30 days post-CNI . The genThe number of TUNEL-positive apoptotic cells was increased in the MPG 7 and 14 days post-CNI . The incElevated gene expressions of RhoA, ROCK1 and ROCK2 14 days following CNI were prevented by ROCK-I in the MPG . FollowiThe production of NO occurs when nNOS is in its dimerized form and when uncoupled can lead to diminished levels of NO and increased oxidative stressFourteen days after CNI, sham, CNI, and CNI rodents treated with Y-27632 were sacrificed. The MPGs were then explanted and cultured in Matrigel. Neurite outgrowth in cultured MPGs was measured after 48 hours of incubation. There was no difference in average neurite length in the sham and CNI MPGs . MPGs cuAs previously shownin vivo erectile responses and ex vivo corporal relaxation results demonstrated that 14 days after bilateral CNI there is a profound decline in nitrergic-nerve mediated erectile responses and corporal vasorelaxation. Identification of alternative pathways contributing to nitrergic axonal degeneration is necessary to advance therapeutic strategies for ED particularly those related to nerve injury and neurological diseases. Thus, we evaluated the activation and expression of RhoA and ROCK in the MPG following CNI in an effort to identify a disease-specific inhibitors of axonal regeneration that contribute to early degeneration of the autonomic innervation of the penis in response to peripheral CNI. The results of the present study demonstrate an early sustained temporal increase in expression of RhoA and ROCK in the MPG after CNI. This increase in gene expression and activation of RhoA and ROCK in the MPG directly correlated with apoptosis in the MPG measured by TUNEL positive cells and caspase 3 gene expression. We also demonstrate that nNOS positive ganglion and gene expression is significantly decreased following bilateral CNI in the MPG. We found that nNOS is uncoupled contributing to the decreased expression and activity of nNOS in the MPG 14 days following bilateral CNI. These data identify RhoA/ROCK as a potential novel signaling pathway activated in response to CNI, which may serve as a regulator of peripheral nitrergic (nNOS) axonal degeneration.In the present study, the There is a desperate need for understanding evolving mechanisms of neuronal degeneration and regenerative pathways in the MPG and CNs after iatrogenic injury. The animal model chosen for this study was a CN crush injury since this type of peripheral nerve injury is designed to mimic the partial nerve damage that occurs during nerve-sparing radical prostatectomy. The model of bilateral CNI causes a time-dependent decline in neurogenic erectile response to CN stimulation and is the model validatedRhoA is a small GTP-binding protein, acting as a molecular switch between the inactive GDP-bound state and the active GTP-bound statein vivo neurogenic erectile responses to CN stimulation following injury have a time-dependent decline in erectile function with the most significant decline at 14 days and improvement in neurogenic-mediated erections up to 60 days post injury25An interesting finding from this study was the rapid decrease in gene expression of nNOS, TH, and ChAT with a temporal increase in expression peaking at 14–21 days with another significant decline in expression in the MPG at 30 and 60 days post-injury. This change in expression follows the temporal trend in expression of the house keeping gene β-tubulin III at the same time points. Previously, we have demonstrated that in vivo and ex vivo nitrergic relaxation of the corpora cavernosa thus providing direct evidence for RhoA and ROCK inhibitory function on nitrergic axons of the MPG.Bilateral CNI reduced nNOS expression in the dorsal nerve of the penis as a result of neurodegeneration following neuropraxia1314Activation of RhoA/ROCK following peripheral nerve injury has been shown to inhibit neurite outgrowth in spinal motor and peripheral sensory neurons293031ex vivo and preserved autonomic innervation to the penis. The mechanism by which this occurs appears to be related to inhibition of caspase-3 dependent apoptosis of ganglia in the MPG. We have previously shown using a whole genomic microarray that pro-apoptotic genes, including caspase-1 and 3, are activated early (48 hours) and 14 days post CNI in the MPG14The intrinsic capability of adult peripheral neurons to regenerate is accompanied by a non-permissive environment consisting of soluble inhibitory molecules, which counteract a regenerative response and promote apoptosis. Recent evidence demonstrates that activation of RhoA and ROCK in peripheral nerves inhibits neurogenesis after injury11ex vivo explanted MPG, we found that daily systemic administration of Y-27632 for 11 days with a three day wash out period to avoid direct vascular effects preserved erectile responses to CN stimulation in vivo and ex vivo corporal myograph responsiveness to activation of NANC nerves that was similar to age-matched sham rats. The CNI group had significantly impaired end organ penile vascular function consistent with a peripheral neuropathy. Systemic ROCK-I prevented both RhoA GTPase activity and total ROCK activity in the MPG and decreased RhoA and ROCK gene expression 14 days after CNI. We chose the 14 day post-crush time point in our peripheral CNI model for all physiologic, molecular, and biochemical analysis in order to evaluate a time when axonal degeneration and early axonal regeneration is occurring with end organ target re-innervationPreviously, in both the CNS and PNS, the use of ROCK-I  enhance axonal regeneration with improvement in motor functionin vivo and ex vivo penile hemodynamics and corporal responsiveness were preserved following daily treatment with the selective ROCK-I, Y-27632, one limitation is that it remains undetermined whether this is a beneficial physiologic effect that can be found after an established injury has occurred to CN and MPG. Moreover, can a delay in treatment with a ROCK-I also promote regeneration of nitrergic axons with reversal of the autonomic neuropathy to the penis? Further studies examining the role of the RhoA/ROCK pathway in the MPG and penile innervation are ongoing in our laboratory to address this question. Also, the upstream activators of RhoA, such as local induction of macrophages and myelin-associated molecules, glycoproteins or chondroitin sulphate proteoglycans (CSPGs) following injury were not addressed in this study and warrant further investigationWhile Besides its pertinence to the field of ED, the study of RhoA/ROCK signaling in axonal degeneration of the CN following crush injury carries additional implications with regard to the field of peripheral axonal regeneration. Knowledge gained in the present study of activated RhoA in the neuroregulation of penile erection provides the first scientific evidence of therapeutic inhibitors of ROCK demonstrating benefit on peripheral nerve regeneration via mechanisms involving inhibition of caspase-3 dependent apoptosis and prevention of nitrergic degeneration in the MPG following peripheral CN-injury.The results of this study indicate that RhoA/ROCK signaling cascade in the MPG promotes apoptosis and nitrergic dysfunction following CNI. Furthermore, systemic administration of the pharmacologic inhibitor of ROCK (Y-27632) prevents caspase-3 dependent apoptosis of nNOS positive axons, nNOS uncoupling and enzymatic activity in the MPG. Inhibition of ROCK promoted neuritogenesis from explanted MPG and preserved nitrergic nerve mediated erections and corporal relaxation in the penis. Therefore, we believe ROCK-I may be an important target to preserve axonal autonomic innervation to the penis following nerve-sparing radical prostatectomy.All experiments were approved by the Johns Hopkins University School of Medicine Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats  weighing 300–325 grams were used and housed in 12 h light/dark lighting cycle with free access to food and water.The first experiment randomly assigned 128 rats to 7 groups to undergo sham or bilateral cavernous nerve injury (CNI) surgery. Subsequent to surgery, MPG tissue was collected for histological analysis, Western blotting, PCR and activity assays after 48 hours, 7, 14, 21, 30 and 60 days .The second experiment used 96 rats to examine the effects of ROCK-I on CNI. Animals were assigned to sham, CNI or CNI plus twice daily intraperitoneal (IP) injections with ROCK inhibitor Y-27632 . ROCK-I was withdrawn 72 hours prior to erectile function testing and tissue collection 14 days post-CNI .Under anesthesia, the prostate was exposed via a midline laparotomy and CN identified1112MPGs from sham, 48 hour, 7, 14, 21, 30, 60 day rats were homogenized and total RNA was extracted and purified using the RNeasy system , quantified and then reverse transcribed MPGs were excised and homogenized in a Tris-HCL (pH 7.5) buffer to evaluate the protein level of ROCK1 and ROCK2 by Western blotIn Situ Apoptosis Detection Kit, Millipore) and counter stained with Hematoxylin . The counting of apoptotic cells was performed by 2 blinded individuals and normalized to the total area of the MPG.The same animals were used for histological analysis and MPG culture with one MPG going to each experimental procedure. MPGs were formalin fixed and immunofluorescence staining was performed as previously describedA separate group of MPGs were homogenized and total ROCK activity was analyzed in MPG lysate with of 0.1 mM adenosine triphosphate by a ROCK activity assay kit using peroxidase coupled anti-phospho-MYPT1 Thr696 monoclonal antibody . RhoA GTPase activity was assayed in MPG lysates by 96-well activity assays  according to manufacturer specifications. Total ROCK and RhoA GTPase activity are displayed as a percent of sham activity, as previously described8NOS activity in MPG tissues was assessed by radiolabeled L-arginine to L-citrulline conversion as previously described 82. Photographs of neurite growth were captured 48 h following implantation using a Nikon TE200 inverted microscope attached to a CCD Camera and digital images were analyzed with Elements software . In each area of growth from the MPG the 5 longest neurites were measuredWhole MPGs were excised and embedded on reduced growth factor Matrigel in serum free media  as described (n = 3/group)Under anesthesia, the left crus was cannulated with a 25 G needle connected to a pressure transducer to measure intracavernous pressure (ICP)2/5% CO2 at 37 °C, stretched 4–5 mN, and equilibrated for an hour−5 M, Sigma) and guanethidine  for 30 mins. Strips were precontracted with phenylephrine (10−5 M) and stimulated at 40 V, 3-ms pulse width at increasing frequencies (1–64 Hz) for 10 sec with 2 min between stimuli. EFS responses were measured as percent relaxation from the pre-contraction to phenylephrine.The penis was cleaned of the urethra, dorsal vessels and nerves, cut in 2 × 10 mm strips and mounted in a muscle strip myograph t-test. P values of less than 0.05 were used as criteria for statistical significance.Data were expressed as mean ± SEM. Differences between multiple groups were compared by analysis of variance (ANOVA) followed by a Tukey’s multiple comparisons test. Two-group analysis was performed by unpaired How to cite this article: Hannan, J. L. et al. Caspase-3 dependent nitrergic neuronal apoptosis following cavernous nerve injury is mediated via RhoA and ROCK activation in major pelvic ganglion. Sci. Rep. 6, 29416; doi: 10.1038/srep29416 (2016).


We have previously shown that granulocyte-colony stimulating factor (G-CSF) treatment alone, or in combination with constraint movement therapy (CIMT) either sequentially or concomitantly, results in significantly improved sensorimotor recovery after photothrombotic stroke in rats in comparison to untreated control animals. CIMT alone did not result in any significant differences compared to the control group . Using a subset of rat brains from this former experiment the present study was designed to evaluate whether dendritic plasticity would parallel improved functional outcomes. Five treatment groups were analyzed (n = 6 each) (i) ischemic control ; (ii) CIMT (CIMT between post-stroke days 2 and 11); (iii) G-CSF (10 μg/kg G-CSF daily between post-stroke days 2 and 11); (iv) combined concurrent group (CIMT plus G-CSF) and (v) combined sequential group . After impregnation of rat brains with a modified Golgi-Cox protocol layer V pyramidal neurons in the peri-infarct cortex as well as the corresponding contralateral cortex were analyzed. Surprisingly, animals with a similar degree of behavioral recovery exhibited quite different patterns of dendritic plasticity in both peri-lesional and contralesional areas. The cause for these patterns is not easily to explain but puts the simple assumption that increased dendritic complexity after stroke necessarily results in increased functional outcome into perspective. Stroke is the leading cause of long-term disability in adulthood . Apart fIn the last years the hematopoietic growth factor granulocyte-colony stimulating factor (G-CSF) has been shown to exert both neuroprotective and neuroregenerative effects in experimental models of ischemic stroke , 6. SeveWe have previously shown that the combined treatment with the hematopoietic growth factor G-CSF and constraint-induced movement therapy did not further enhance neurological outcome compared to treatment with G-CSF alone . In thishttp://www.randomizer.org/). To ensure blinding, behavioral assessment and treatment administration were performed by different investigators. A total of 107 animals has been used in this study in which 7 animals died during surgeries. A subset of brains has been used for the recently published receptor autoradiographic analyses and brain volume quantifications [As recently described , all aniications . Infarctications . At the Photothrombotic ischemia was induced as described in the previously published study . BrieflyForty-eight hours after induction of ischemia, CIMT-treated animals were fitted with one-sleeve plaster casts, as recently described . AnimalsIn all animals, cylinder test and the adhesive tape removal test were performed before ischemia (baseline) as well as on days 1, 12, 21, and 28 after ischemia by an investigator blinded to the experimental groups.For Golgi-Cox analysis, 6 rats per group were used. At the determined endpoint of the experiment rats were deeply anesthetized using a mixture of ketamine hydrochloride (20.38 mg/ml) and xylazine hydrochloride (5.38 mg/ml). Subsequently, mice were transcardially perfused with 0.9% saline followed by PBS. Brains were removed and were then coded with numbers before sectioning to ensure that the experimenter was blinded to treatment. The brains were processed according to current protocols with minor modifications , 23. In Values are presented as mean ± SEM. The normal distribution of the data was verified by Shapiro-Wilk test. Due to the normal distribution all experimental groups were compared pair-wise and significant group effects were confirmed by one-way analysis of variance (ANOVA) and Bonferroni error protection to correct for multiple comparisons. Significant group effects between different groups were confirmed by independent t-test. A p-value <0.05 was considered statistically significant. Analysis was performed using the general statistics module of Analyse-it™ forMicrosoft Excel .As recently published, both motor and somatosensory recovery was significantly increased at 28 days after stroke in the G-CSF treated group and in the two groups with a combination of concomitant or sequential treatment with G-CSF and CIMT compared to untreated ischemic controls. CIMT only did not result in any significant differences compared to the control group .Dendritic plasticity of layer V pyramidal neurons was analyzed in the peri-infarct cortex and the corresponding contralateral cortex in each treatment group  peri-in. HoweverIntersection analysis. Regarding the branching of traced neurons there were no significant changes in the mean number of intersections neither in the peri-infarct cortex nor in the corresponding contralateral cortex when comparing all treatment groups . In the corresponding contralateral cortex the mean number of apical dendrites per branch order was unchanged in the sole treatment groups while for the mean length of these dendrites a heterogeneous pattern of single significant alterations throughout all groups and branch orders was visible (not shown). There were no significant differences between the various experimental groups concerning the mean number of basilar dendrites per branch order in the corresponding contralateral cortex, while 3rd order basilar dendrites of G-CSF treated rats were decreased. Basilar dendrites further showed an increase in the mean length of 4th and 5th order dendrites in the concomitant CIMT/G-CSF group (not shown).As previously shown, G-CSF treatment alone, or in combination with CIMT either sequentially or concomitantly, resulted in significantly improved sensorimotor recovery after photothrombotic stroke in rats in comparison to untreated ischemic control animals, while CIMT alone did not result in any significant differences compared to untreated ischemic controls . This faSurprisingly, a complex and different pattern of dendritic plasticity in the various treatment groups became apparent which can be summarized as follows: (i) a decrease in the mean length of the apical dendrites and an increase of the basilar dendrites in the peri-infarct cortex of the G-CSF group which was associated with a significantly increased number of intersections of higher order; (ii) a significant increase of the mean dendritic length of the apical dendrites in the contralateral cortex of both G-CSF and CIMT treated rats without alterations of the basilar dendrites; (iii) a heterogeneous pattern in the groups with combined treatment.One crucial issue when looking for recovery-associated structural changes after ischemic stroke is the selection of regions to be analyzed. Both in the peri-ischemic as well as in the contralateral homotopic cortex plastic changes have been identified which are thought to represent the structural correlate of spontaneous post-stroke recovery frequently observed in rodents and also in humans , 25–28. In a permanent MCAO model in rats Golgi Cox staining revealed increased perifocal basilar dendrites after 56 days, while at 7 days, number and diameter of basilar dendrites was decreased . The hypUsing in vivo two-photon microscopy, Mostany and Portera-Cailliau  did not In conclusion, animals with a similar degree of behavioral recovery exhibited quite different patterns of dendritic plasticity in both peri-lesional and contralesional cortical areas. A reasonable explanation would be that completely different therapeutic strategies like a pharmacotherapy with the growth factor G-CSF and CIMT which is directed at function as in our previous study are highly likely to trigger different mechanisms eventually resulting in a different distribution of plastic changes. The situation is further complicated by a combination of these strategies either in a concomitant or sequential application. Although we were absolutely aware of this issue, the experimental design of our study was primarily chosen to possibly identify a therapy superior to the others. In a second step we looked whether specific patterns of dendritic plasticity would be associated with improved neurological outcome. It proved that this is only partly the case for the single therapy groups. Another explanation for lack of an association between function and dendritic morphology is the highly underestimated and frequently ignored problem to differentiate between recovery and compensatory movements (for an excellent introduction into this issue see ). IdeallS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Today many subjects with beta thalassaemia major  successfully survive into adult life, due to the remarkable improvement of medical care and better understanding of pathogenesis, clinical manifestations and prevention of endocrine complications.2An emerging endocrine disorder in young adults with β-thal is late-onset male hypogonadism (LOH). LOH is a disorder caused by the inability of the testes to produce the physiologic levels of testosterone and normal number of spermatozoa as a result of a disruption of the hypothalamic- pituitary-gonadal axis . Ten out of our 120 β-thal patients 8.3%) developed this complication and 35% developed hypogonadotropic hypogonadism requiring sex hormone replacement therapy.% developThe causes of male hypogonadism in the general population are multiple. In β-thal hypogonadism is mainly due to iron deposition in the endocrine glands.Iron overload may be the result of economic circumstances (high expense of the chelation therapy), late onset of chelation therapy or poor compliance with treatment.Toxicity starts when the iron load in a particular tissue exceeds the tissue or blood-binding capacity of iron and consequently free non-transferrin iron appears. The ‘free iron’ is a catalyst for the production of oxygen species that peroxidise membrane lipids of cell organelles leading to cell destruction.3The anterior pituitary gland is particularly sensitive to the free radicals produced by oxidative stresses and exposure to these radicals injures the gland. Magnetic resonance imaging (MRI) shows that even a modest amount of iron deposition within the anterior pituitary can interfere with its function. Excess iron deposition in the anterior pituitary leads to degranulation of the adenohypophysis and decreases hormone storage along with hypo-responsiveness to hypothalamic releasing hormones.Abnormalities in the hypothalamic- pituitary growth hormone axis (defective GH secretion in response to stimulation(40%) and GH neuro-secretory dysfunction (up to 20%) and low insulin-like growth factor-1 (IGF-1) synthesis in response to GH).1Abnormalities in the hypothalamic- pituitary- gonadal axis (hypogonadotropic hypogonadism).Combined iron chelation therapy (use of two chelators on the same day), may induce negative iron balance and may reverse hypogonadism and endocrine complications in severe iron overloaded β-thal subjects. Long-term studies have shown that the combined use of deferiprone and deferoxamine (DFO) hastens iron chelation by rapidly reducing liver iron, serum ferritin, and myocardial siderosis. Combined chelation therapy with deferasirox and DFO has also been proved beneficial.1Histologically, a reduced number of cells and moderate siderosis of the parenchymal cells of the anterior pituitary have been found. Virtually very little is known about spermatogenesis in . A summary of the available findings include the followings:A normal sperm count and motility in 45% of fully sexually mature β-thal subjects.3A possible detrimental effect of iron chelation therapy on spermatogenesis. Three out of four patients with serum ferritin levels lower than 500 ng/ml had poor sperm motility.5A higher degree of defective chromatin packaging in β-thal subjects with low sperm concentrations. 5A low seminal plasma concentration of zinc, citric acid and prostate specific antigen. These data suggest impaired prostatic secretion.6An increase of seminal lipo-peroxidation.7An increase of DNA sperm damage and a negative correlation with sperm motility. These findings suggest that iron overload predispose sperm to oxidative injury. 8These findings indicate an increase of oxidative stress in the semen of these patients that could contribute to the impairment of sperm motility.7Blood transfusion is associated with significant acute enhancement of sperm parameters and increased concentrations of serum T, LH, FSH, and IGF-1. These “acute” effects on spermiogenesis are reached by an unknown mechanism and suggest a number of pathways that need further human and/or animal studies.9In addition, abnormal seminal parameters and low serum folic acid levels have been found in subjects with thalassaemia intermedia.10In our experience, β-thal patients develop LOH in their second and third decades of life. It is possible to induce or restore spermatogenesis with exogenous gonadotrophins in some of them.Despite the fact that endocrine complications are very common in multi-transfused thalassaemia patientswww.endothalassemia.org) are to encourage and guide endocrine follow up of multi-transfused patients in developing countries, to promote and support collaborative research in this field, to encourage and guide endocrine follow up of multi-transfused patients, and to educate and train more endocrinologists and other paediatricians/physicians to prevent and improve management of the growth and endocrine complications in these patients.The practical objectives of ICET-A (


Women whose pregnancy was complicated by gestational diabetes have a 7-fold higher risk of developing diabetes, primarily type 2. Early detection can prevent or delay the onset of late complications, for which follow-up screening is important. This study investigated the extent of participation in follow-up screening and the possible consequences of nonattendance in the Region of North Jutland, Denmark.In Danish national registers covering the years 1994–2011 we identified 2171 birthing women whose pregnancy was complicated by first-time gestational diabetes. Control visits to general practitioners and biochemical departments after giving birth were charted. Following national guidelines we defined four intervals for assessment of participation in follow-up screening. Diagnosis of diabetes or treatment with glucose-lowering agents after giving birth were also identified. Participation in follow-up screening and risk of diabetes was calculated. Time to obtaining diagnosis of diabetes or initiating treatment was analysed by Cox regression models. All models were adjusted for age, ethnicity and income.High attendance was found during the first control interval, after which attendance decreased with time after giving birth for both controls at general practitioners and biochemical departments. All differences in proportions were statistically significant. Women attending controls at general practitioners had a significantly higher risk of diabetes diagnosis and treatment after gestational diabetes than women not attending. The results for women attending testing at biochemical departments also showed an increased risk of initiation of treatment. Women attending at least one general practitioners control had a significantly higher risk of early diabetes diagnosis or treatment. Time to initiation of treatment was significantly higher for testing at biochemical departments. Women with high incomes had a significantly lower risk of diabetes diagnosis or initiation of treatment compared to low-income women.Participation in follow-up screening after gestational diabetes is low in the North Denmark Region. Follow-up screening ensures early detection of diabetes and initiation of treatment. Our results emphasize the importance of development of interventions to improve early detection and prevention of diabetes after gestational diabetes. Women affected by gestational diabetes have a 7-fold higher risk of diabetes compared to women with normoglycaemic pregnancies, with the most rapid increase in type 2 diabetes found during the first five years after giving birth, 2. DiabA register-based study of gestational diabetes among inhabitants in The North Denmark Region, from 1 January 1994 to 31 December 2011. The administrative Region encompasses around 580.886 inhabitants, and as The data registers were linked in Statistics Denmark based on the civil registration number. The National Patient Register holds data on all hospital admissions in Denmark. The claFor verification of the gestational diabetes (ICD-10 code: DO244) diagnosis, all collected data regarding other diabetes diagnosis (ICD-10 codes: DE10-14) and claimed prescriptions on glucose-lowering agents (ATC-code: MA10) were divided into three subgroups: prior to pregnancy, during pregnancy and after pregnancy. We thus excluded women who had been diagnosed with any type of diabetes and/or treated with glucose-lowering agents prior to pregnancy. Furthermore, exclusion was performed due to the suspicion of underlying unregistered diabetes prior to pregnancy. Claimed prescriptions or a diagnosis within a year after giving birth is known to be related to gestational diabetes and did not lead to exclusion. This approach has been validated in the literature. Women wThe primary outcome was attendance in follow-up screening at general practitioners or biochemical departments after gestational diabetes in each of the defined time intervals. The outcome was defined by coding of an oral glucose tolerance test or a fasting plasma glucose test. The secondary outcome was the risk of and time to diabetes diagnosis or treatment with glucose-lowering agents among participants and non-participants. Outcomes were predefined.The study applied descriptive statistics (N/%) to illustrate the distribution of age, income and ethnicity. All models were adjusted for these variables. Participation in follow-up screening was analysed using chi-squared tests. Women appearing as missing in the tables were either excluded or censured by death, second birth or end of registration. To determine the association between participation in follow-up screening and diabetes-related outcomes, we applied logistic regression models presented with odds ratios (OR) and 95% confidence intervals (CI). We furthermore examined possible confounding by age, income and ethnicity and interactions between ethnicity and income, and between age and income. Cox’s proportional hazard models were applied to investigate the hazard ratio (HR) of diabetes related outcomes, depending on participation in at least one general practitioner or biochemical department control. All Cox proportional hazard models met the proportional hazard assumption. The SAS statistical software package for Windows, version 9.2, was used .Register based observational studies do not require ethical approval in Denmark. The DanA total of 149.903 women in The North Denmark Region gave birth during the study period, 2238 of whom had a diagnose of first-time gestational diabetes. Diagnosis of diabetes and/or treatment with glucose-lowering agents prior to pregnancy lead to the exclusion of 67 women. The final study population included 2171 women  the highest risk applied for Control 4 for treatment  (Tables The forest plots in Figure Results of the Cox regression analysis showed statistically significant positive associations between attendance to at least one control at either a general practitioner or a biochemical department and time to receiving a diabetes diagnosis or initiation of treatment Table . Women aThis study shows low participation in follow-up screening after gestational diabetes that are recommended in international and national guidelines, 6. The The strength of this study is its basis in the complete and comprehensive data held by the Danish national registers and the negligible risk of loss to follow-up, 26. TheWhile we found high participation rates in the first control after gestational diabetes, other studies have reported rates varying from 14 to 61% for this control. A recent review also finds that screening for diabetes after gestational diabetes generally is low. HoweverIn contrast to other studies examining participation in follow-up screening, 27, 35,Our study has documented the need for preventive health interventions to ensure stringent follow-up screening of women with prior gestational diabetes in order to ensure the early detection of diabetes and initiation of treatment. The general lack of interventions to ensure participation in follow-up screening is described in other studies, 33, 35.In this study of women with prior first-time gestational diabetes we found low participation rates in follow-up screening, except for the first control after which it decreased with time after giving birth. Women who did not attend follow-up screening had a lower risk of diabetes diagnosis or treatment and early detection of diabetes. The higher possibility of early detection of diabetes among women attending follow-up screening reduces their risk of late complications. Contrary to national and international guidelines, 6, the


A 60-year-old man presented to the emergency department complaining of acute onset posterior ankle pain. He reported playing tennis earlier in the afternoon when he suddenly stopped and pivoted, noting a “pop” sensation and pain to the right posterior ankle. The pain was sharp and increased with movement. The patient also experienced difficulty weight bearing and ambulating. There was a palpable defect at the distal end of the expected course of the Achilles tendon and lack of plantar flexion with squeezing the affected calf . Point-oAchilles tendon rupture typically occurs by pushing off the weight-bearing foot with knee extended, sudden dorsiflexion of the ankle, or forceful dorsiflexion of a plantar-flexed foot.7


Drug resistance significantly limits the long-term effectiveness of targeted therapeutics for cancer patients. Recent experimental studies have demonstrated that cancer cell heterogeneity and microenvironment adaptations to targeted therapy play important roles in promoting the rapid acquisition of drug resistance and in increasing cancer metastasis. The systematic development of effective therapeutics to overcome drug resistance mechanisms poses a major challenge. In this study, we used a modeling approach to connect cellular mechanisms underlying cancer drug resistance to population-level patient survival. To predict progression-free survival in cancer patients with metastatic melanoma, we developed a set of stochastic differential equations to describe the dynamics of heterogeneous cell populations while taking into account micro-environment adaptations. Clinical data on survival and circulating tumor cell DNA (ctDNA) concentrations were used to confirm the effectiveness of our model. Moreover, our model predicted distinct patterns of dose-dependent synergy when evaluating a combination of BRAF and MEK inhibitors versus a combination of BRAF and PI3K inhibitors. These predictions were consistent with the findings in previously reported studies. The impact of the drug metabolism rate on patient survival was also discussed. The proposed model might facilitate the quantitative evaluation and optimization of combination therapeutics and cancer clinical trial design. Drug resistance places an often inevitable limit on the long-term effectiveness of targeted therapeutics for cancer patients134567It is widely acknowledged that the inherent heterogeneity9111213in vitro and in vivo experiments, multiple cell types and multiple drug dosages must be considered, in addition to other experimental conditions and challenges in human population studies. As such, these studies are expensive and time consuming. Therefore the systematic development of effective therapeutics to overcome drug-resistance mechanisms has posed a major challenge. Mathematical modeling may potentially serve to bridge molecular/cellular mechanisms of drug resistance and population-level patient survival, and facilitates the quantitative evaluation and optimization of combination therapeutics and cancer clinical trial design.Evaluating cancer therapeutics in the context of tumor heterogeneity and microenvironment adaptation is very complex. In traditional et al.15Many mathematical and computational models have been developed to simulate tumor growth and drug response. For example, the cellular automata model1719202224In this study, we created a multiscale model comprising a set of stochastic differential equations driven by both the Wiener process and Poisson process to describe pharmacokinetics, cellular dynamics, and progression-free survival at the patient level while accounting for microenvironment adaptations . Our modTumors are heterogeneous , resulting in some tumor cells possessing sensitivity to drug therapy and others in the same tumor exhibiting resistance to it. To model growth, transition and dissemination dynamics in drug-sensitive and drug-resistant cancer cells in patients with metastatic disease, we employed the following set of stochastic differential equations (SDEs):82627u represents the mutation rate in drug-sensitive cells as they convert to drug-resistant cells . The third term in equation (1) describes the drug-induced death of drug-sensitive cells. D) via the equation W that is described as N is a unit normal distribution. The third term in Equation (2) is similar. i = 1, 2) represents the diffusion rate. The last terms in equation (1–2) describe the dissemination of existing cancer cells.where λ and describes the count of metastasis within a cancer cell population31λ is the expectation of disseminating cell number within per unit time (Day). In addition, Independently of Therapy-induced drug resistance can intensify tumor metastasis15where Angiogenic growth in tumors is induced by the secretion of angiogenic growth factors . We modeled angiogenesis based on previously established workwhere Pharmacokinetics describe the dynamics of drug absorption, metabolism and elimination by the body37D represents drug concentration in the body. The first term in the above equation models the first-order elimination rate of drugs, and where CS = 0.2, CR = 0.001 and CK = 0.1, simulating the relative cell number ) were easily obtained, since their coefficients satisfies the appropriate growth conditions and are locally Lipschitz continuousThe initial conditions of the equations (1–4) were set to As demonstrated by recent experimental and preclinical studieswhere It was assumed that DIRF secretion and/or degradation in the microenvironment are much faster processes than cellular phenotype switching and shifts in cellular population dynamics. Therefore, using a quasi-steady state assumption we can express the secreted DIRF concentration as follows:where Secreted DIRFs can promote outgrowth, dissemination and metastasis in drug-resistant cells and enhance survival in drug-sensitive cells15α is the regulatory coefficient, and equation (8) describes the dependence of growth rate of sensitive cells on the secreted DIRF concentration, where In this way, we linked the short-term timescale (minutes) associated with intercellular signaling to the long-term timescale (days) necessary for cellular dynamics4243Cancer progression is often clinically evaluated using radiographic imaging. In the below analysis, if a patient’s total tumor cell number or tumor volume exceeded a pre-set threshold, N, which represents number of patients, was set to 100. We calculated the progression-free survival time for each patient in the simulation and then computed overall survival percentages and survival frequencies for the entire patient population under different treatment schedules. In the following text, patient survival refers to progression-free survival unless stated otherwise.where Most of the parameters used to assess cellular dynamics and pharmacokinetics were collected from previous studies263515Currently, in addition to BRAF inhibitors , several other targeted inhibitors, such as MEK inhibitors  and PI3K inhibitors  are in clinical trials for melanoma cancer patients. In this study, we investigated the synergy between BRAF inhibitors in conjunction with each of these other two inhibitor types by incorporating their effects into our model based on their different signaling mechanisms, respectively.MEK is a downstream effector protein of RAF signalingwhere In drug-resistant cells, the PI3-Kinase (PI3K)/AKT pathway is over-activated by DIRFswhere It should be noted that we used dimensionless concentrations of BRAF inhibitor, MEK inhibitor and PI3K inhibitor in the simulation by respectively normalizing them to the Michaelis constants in silico. In We investigated cellular response kinetics following drug treatment For comparison, cellular dynamics without drug treatment are shown in 45We next validated our model using clinical data. In Currently, in addition to BRAF inhibitors , several other targeted inhibitors, including MEK inhibitors  and PI3K inhibitors , are being evaluated in clinical trials for treatment of melanoma patients. In the following study, we investigated whether the co-administration of BRAF-I with either MEK-I or PI3K inhibitor (PI3K-I) produces synergistic effects. To accomplish this, we incorporated the effects of these inhibitors into our model (see details in Model section) based on their specific signaling mechanisms.A Bliss combination index53CI > 0, antagonistic if CI < 0, and additive if CI = 0.where The model predicted that both the BRAF-I & MEK-I combination and the BRAF-I & PI3K-I combination possess dose-dependent synergy but in different patterns . The BRAImportantly, the predicted differences in the synergies of these two combinations are consistent with experimental studies55λ). Since angiogenesis positively supports the sustained growth of the tumor cells and provides an avenue for dissemination and translocation of metastatic cancer cells , therefore the growth/death rates in angiogenic cells as well as metastatic rate of cancer cells were shown to significantly affect cellular dynamics and patient survival.We conducted parameter sensitivity analysis  was used to model the dynamics of heterogeneous cellular populations containing drug-sensitive, drug-resistant, and metastatic cancer cells. An associated random variable characterizing progression-free survival time was subsequently defined. Our approach revealed several interesting features associated with cancer metastasis and progression kinetics. For example, dose-dependent synergy was evident in both BRAF-I and MEK-I co-treatment and BRAF-I and PI3K-I co-treatment. This result suggests that combination therapy with optimized dosages of inhibitors might reduce drug resistance.Our model demonstrated that cancer metastasis and progression occur in bursts A. As suc6061626365We also investigated the impact of heterogeneity in patient drug-metabolism rates on survival percentage. To include the effects of population heterogeneity in our mechanistic models and examine the impact of patient drug metabolism heterogeneity on survival percentage, we considered three patient subclasses with different metabolic rates . As shown in Our model predicted that combined BRAF and MEK inhibitions produced a different pattern of synergy from that created by combined BRAF and PI3K inhibition. As demonstrated inDrug resistance is an obstacle often encountered during oncoprotein-targeted therapy and develops by very complex mechanisms. Tumors frequently display a substantial amount of spatial heterogeneity in both cell population composition and microenvironment factors, such as drug, oxygen and glucose concentrations221In summary, our study utilized a set of SDEs to model the dynamics of targeted therapy-induced drug resistance and metastasis. The model predicted that different patterns of synergy exist for the combination of BRAF-I and MEK-I and for the combination of BRAF-I and PI3K-I. Our study provides insight into the microenvironment-mediated mechanisms underlying drug resistance and delineates the implications associated with optimal dosing and scheduling of combination therapy for melanoma patients with BRAF mutations. It is our hope that this predictive model will facilitate cancer clinical trial design and accelerate the design of effective and robust tumor therapeutics.How to cite this article: Sun, X. et al. Mathematical Modeling of Therapy-induced Cancer Drug Resistance: Connecting Cancer Mechanisms to Population Survival Rates. Sci. Rep.6, 22498; doi: 10.1038/srep22498 (2016).


Event-related ICA (eICA) is a partially data-driven analysis method for event-related fMRI that is particularly suited to analysis of simultaneous EEG-fMRI of patients with epilepsy. EEG-fMRI studies in epileptic patients are typically analyzed using the general linear model (GLM), often with assumption that the onset and offset of neuronal activity match EEG event onset and offset, the neuronal activation is sustained at a constant level throughout the epileptiform event and that associated fMRI signal changes follow the canonical HRF. The eICA method allows for less constrained analyses capable of detecting early, non-canonical responses. A key step of eICA is the initial deconvolution which can be confounded by various sources of structured noise present in the fMRI signal. To help overcome this, we have extend the eICA procedure by utilizing a fully standalone and automated fMRI de-noising procedure to process the fMRI data from an EEG-fMRI acquisition prior to running eICA. Specifically we first apply ICA to the entire fMRI time-series and use a classifier to remove noise-related components. The automated objective de-noiser, “Spatially Organized Component Klassificator” (SOCK) is used; it has previously been shown to distinguish a substantial fraction of noise from true activation, without rejecting the latter, in resting-state fMRI. A second ICA is then performed, this time on the event-related response estimates derived from the denoised data . We hypothesize that SOCK + eICA has the potential to be more sensitive than eICA alone. We test the effectiveness of SOCK by comparing activation obtained in an eICA analysis of EEG-fMRI data with and without the use of SOCK for 14 patients with rolandic epilepsy who exhibited stereotypical IEDs arising from a focus in the rolandic fissure. Event-related functional magnetic resonance imaging (fMRI) is an MRI technique that can be used to detect changes in the Blood Oxygen Level Dependent (BOLD) hemodynamic response to neural activity in response to certain events. The conventional method for detecting event-related responses in fMRI consists of modeling the expected fMRI response to an event by convolving a stimulus presentation time-course with an assumed canonical Haemodynamic Response Function (HRF) and using linear regression to identify voxels with a significant correlation to this expected response , which are pathological patterns of activity generated by the brain of patients with epilepsy between seizures , which allowed for less constrained analyses capable of detecting early, non-canonical responses   using MELODIC  was preformed using the de-noised fMRI data .The above process was performed for both group and individual studies.In the with-SOCK processing stream, ICA is employed to decompose the 4D fMRI time series into a linear combination of spatially independent component maps with an associated time-course .Edge activity: extent of activity in an edge mask.Ventricular activity: extent of activity in a Cerebrospinal fluid (CSF) mask.Temporal Frequency Noise (TFN): the power in temporal frequency beyond 0.08 Hz.SOCK classifies ICs using features likely to indicate motion, physiological noise, or machine or undetermined noise. The algorithm is described in detail elsewhere  into an Unlikely Artifact category.http://www.brain.org.au/software.The SOCK procedure was implemented in MATLAB . Source code of our implementation of the method is available at fsl_regfilt (with the ‘-a’ aggressive filtering option). The de-noised fMRI data along with the original EEG timings are then input to an eICA analysis.After SOCK classification, all ICs classified in the artifact category are discarded from the fMRI data set and a de-noised fMRI data set is assembled from the remaining components. This is done via the FSL function, The eICA method, described in detail elsewhere  to perform the GLM parameter estimation, and the FastICA and ICASSO  using the SPM8 software  and described in detail by Masterton et al. . We summTR = 3 s; TE = 40 ms; FOV = 24 × 24 cm; 128 × 128 matrix; 25 interleaved 4 mm slices with 1 mm gap). In three studies .The patients underwent 30 min of simultaneous EEG and fMRI scanning. fMRI images were acquired in a 3T GE Signa LX scanner  using a BOLD-weighted gradient-recalled echo-planar imaging sequence (Simultaneous EEG was acquired during fMRI scanning using an MR-compatible EEG system (developed in-house) with scalp electrodes positioned in the standard 10–20 locations and filtering to remove the effect of cardioballistic and motion artifacts . Preprocessing included temporal alignment of slices within each volume to the first slice, rigid-body spatial realignment to correct for subject motion, spatial normalization to a symmetric template and spatial smoothing with a Gaussian kernel (FWHM = 8 mm). The symmetric template was created specifically for this patient group using SPM8 software by normalizing each subject's brain to MNI space, averaging these images together , and then smoothing with an 8 mm Gaussian filter . SOCK classified between 27 and 53% of each subject's components as artifact (mean 41%). These ICs were discarded to construct a de-noised fMRI data set for each subject. See Table Fifteen individual analyses were performed for eICA, each with and without the use of SOCK to de-noise fMRI data. This included two analyses for the patient (Subject 5) that had independent left and right-sided CTS, which were analyzed as separate events. The results are summarized in Table eICA estimated an average of 7 (range: 2–11) different components for each analysis. In 11 out of 15 analyses (73%) at least one component was identified showing activity in the vicinity of the ipsilateral rolandic region.After de-noising the fMRI data with SOCK, eICA estimated an average of 6 (range: 1–10) different components for each analysis. In 13 out of 15 analyses (87%) at least one component was identified showing activity in the vicinity of the ipsilateral rolandic region.Figure The associated spatial maps (with and without SOCK) and time courses for all remaining subjects are provided in Figure In two of these analyses (Subjects 8 and 10) activation in the vicinity of the ipsilateral rolandic region was identified when analyzed with eICA after de-noising with SOCK but not when analyzed by eICA alone  estimated 14 different components out of which a single CTS-related component of interest was identified low in the ipsilateral post-central gyrus, extending along its opercular surface into the lateral fissure. A much smaller activation was also seen on the contralateral side  low in the ipsilateral post-central gyrus, extending along its opercular surface into the lateral fissure  and (2) a bilateral component containing de-activation in both the ipsilateral region above and contralaterally  . We would interpret the new finding as distinguishing the activation of the ipsilateral cortex during epileptiform events, and a later bilateral decrease in activity in response to this activation.We have demonstrated a novel application of our ICA classifier, SOCK, to de-noise fMRI prior to an event-related ICA in patients with rolandic epilepsy. The procedure outlined in this paper harnesses the advantage of both techniques: (1) SOCK de-noises fMRI in an objective and automated manner utilizing the entire fMRI time-series. (2) eICA utilizes the EEG information to derive event-related responses which are input into an ICA, thus constraining the final eICA decomposition to a small number of components time-locked to the events of interest. The use of SOCK increased power to detect activity of interest in both individual and group analyses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers . Harnessing the power of these pluripotent stem cells could potentially offer new therapeutic treatment options for a variety of medical conditions. Since the initial derivation of hESC lines in 1998, tremendous headway has been made in better understanding stem cell biology and culture requirements for maintenance of pluripotency. The approval of the first clinical trials of hESC cells for treatment of spinal cord injury and macular degeneration in 2010 marked the beginning of a new era in regenerative medicine. Yet it was clearly recognized that the clinical utility of hESC transplantation was still limited by several challenges. One of the most immediate issues has been the exposure of stem cells to animal pathogens, during hESC derivation and during in vitro propagation. Initial culture protocols used co-culture with inactivated mouse fibroblast feeder (MEF) or human feeder layers with fetal bovine serum or alternatively serum replacement proteins to support stem cell proliferation. Most hESC lines currently in use have been exposed to animal products, thus carrying the risk of xeno-transmitted infections and immune reaction. This mini review provides a historic perspective on human embryonic stem cell culture and the evolution of new culture models. We highlight the challenges and advances being made towards the development of xeno-free culture systems suitable for therapeutic applications. Human embryonic stem cells (hESCs) have emerged as exciting candidates for cell therapies in regenerative medicine due to their capacity to self-renew and differentiate into lineages of all three embryonic germ layers. Studies have demonstrated the ability of hESCs to differentiate into a number of pathologically relevant cell types, including insulin-producing cells , neural Early studies with human ESCs involved direct culture on mouse embryonic fibroblast feeder cells (MEFs) or animal derived extracellular matrices with conditioned medium from MEF feeder cells in medium supplemented with fetal bovine serum (FBS) ,6. This Human pluripotent cells (hPSCs) include not only embryonic stem cells (hESC) but also human induced pluripotent cells (hiPSCs). The latter are derived from adult somatic cells that have been reprogrammed genetically to behave like embryonic stem cells, expressing genes necessary to maintain pluripotency. They represent an additional valuable source of stem cells for therapeutic use as they have the potential to differentiate in to any cell lineage. Although this mini-review concentrates on embryo-derived stem cells many of the described culture systems have also been applied to hiPSCs.The functional definition of embryonic stem cells includes four criteria: 1) Origin from a pluripotent cell population 2) Capable of self-renewal indefinitely in the undifferentiated state 3) Capable of maintaining normal karyotype during growth 4) Clonally derived cells capable of differentiation in to all three embryonic germ layers in vitro or in to teratomas in vivo. In culture, hESC appear as tightly packed colonies with distinct borders. Within the colonies, individual stem cells have a high nucleus: cytoplasm ratio with distinctive nucleoli. Human stem cell identification and characterization in vitro also includes demonstration of a high level of alkaline phosphatase activity and expression of specific embryonic stem cell markers . The pluHuman embryonic stem cells are typically derived from the inner cell mass (ICM) of blastocyst stage embryos. These unique cells can continuously proliferate and also differentiate in to all three embryonic germ layers. The establishment of the first hESC lines  providedThe destruction of embryos for ICM isolation has however raised ethical concerns . An innoEfficiency of human ESC line generation from individual blastomeres has been variable dependent not only on culture conditions to retain “plasticity” or totipotency of the isolated blastomere but also on properties of the isolated blastomere and the embryonic cell stage of isolation. Early efforts to generate ESC lines from morula stage embryos were unfruitful mostly resulting in aggregates of trophoblast-like cells. Blastomere isolation from embryos at earlier cell stages (4 and 8-cells) before cell polarization into outer TE cells and inner non-polarized cells destined to become the ICM were more successful. Initial reports suggested that hESC lines derived from different developmental stages possessed distinct gene expression profiles. More recent data however, demonstrate that hESCs do in fact display a consistent gene profile and preserve a similar pluripotent phenotype, regardless of the source of derivation, ie. ICM-derived hESC and blastomere derived hESC. Slight differences in both the molecular and phenotypic profiles observed between cell lines are most likely attributed to minor differences in derivation and culture protocols ,23.Co-culture of isolated stem cells with mouse embryonic fibroblasts (MEFs) was the first technique used for successful establishment of hESC lines ,14. ThesThe transmission of animal-derived infectious pathogens associated with MEFs remains however a critical concern that precludes the use of current hESC lines for clinical transplantation purposes. MEF cells can contain viral particles capable of infecting humans . Also prHuman feeder layers for hESC culture were therefore explored as a first step towards the ultimate goal of xeno-free culture models. To this end a variety of different human cell types such as human fallopian tube cells , fetal fThese early studies with feeder layers have contributed much to our understanding of the behavior, characteristics and conditions necessary for stem cell renewal in vitro. Basic fibroblast growth factor (bFGF) was determined to be a key growth factor for maintaining hESC pluripotency and shown to be endogenously produced by human feeder cells used for hESC culture ,47,48. DDespite the aforementioned successes, feeder cell dependent hESC culture systems have many limitations that make them unsuitable for future therapeutic applications. Maintenance of feeder layers is very labor intensive with lot-to-lot inconsistencies between feeder populations. This variation can ultimately affect the stem cells as well as their downstream differentiation in to specific cell types for clinical therapy.The focus of current stem cell research has been the design of feeder-free, xeno-free culture systems with chemically defined media formulations. We present some of the most relevant studies in the ensuing sections. Tables One of the first approaches tested for feeder-free growth relied on the use of extracellular matrix proteins in combination with growth factor supplementation to create an in vitro microenvironment that promoted stem cell renewal. The extracellular matrix, Matrigel isolated from mouse Engelbreth-Holm-Swarm teratocarcinoma cells was used by Xu et al.  as a feeFu et al. extracted the extracellular matrix from embryoid bodies (e-ECM) derived from the H9 ESC line . This huThese studies with extracellular matrix have been instrumental in identifying biomolecules involved in stem cell renewal. Inconsistencies in ECM preparations even if of human origin, do however still present a problem. Certainly, use of chemically defined matrices and recombinant proteins based on ECM may be a more reliable approach towards xeno-free culture of therapeutic grade hESC lines. Xu and colleagues analyzed the individual components of Matrigel and determined that not all are equally effective for sustaining undifferentiated growth of ESC lines . LamininLaminin with its role in cell adhesion has been extensively studied. Rodin et al. described a xeno-free system for hESC and hiPSC cultivation using a recombinant form of human laminin-511 with a defined medium, TeSR1 . This seE-cadherin, another cell adhesion molecule that binds to the integrin α6β1 also plays a role in cell:cell binding . Poor suAmongst ECM components, vitronectin is another protein that has emerged as a promising substratum for long term culture of hESC cell serum-free conditions in defined media -85. RecoSynthetic substrata for stem cell culture have also been investigated as alternatives to complex ECM matrices. Synthetic peptide acrylate surfaces (PAS) constructed by conjugating peptides derived from the biologically active region of extracellular matrix proteins can sustain the self-renewal and pluripotency of hESCs for over 10 passages in a xeno-free defined medium X-Vivo 10 . In thisKlim et al.  systematWu et al. recently described a novel synthetic material based on spider silk proteins as a suitable substrate for hESC culture . This arThe cost of peptide synthesis, stability of substratum with attached biological molecules and anticipated difficulties with scale up during manufacture has triggered interest in establishing purely synthetic surfaces for serum-free culture of hESC. This has proved to be particularly challenging. Numerous polymer based synthetic surfaces such as PMEDSAH (poly [2- (methyacryloyoxy) ethyldimethyl-(3-sulfopropyl) ammonium hydroxide]), PMVE-alt-MA ), HIT9 and APMAAm  are often used in culture systems to replace fetal bovine serum but do in fact still contain animal derived serum albumen. Only a few of the media described are completely xeno-free with human serum albumen (HSA) as a protein source in lieu of bovine serum albumen (BSA). However, since HSA is extracted from different pools of donor serum there are batch-to-batch variations. Also HSA manufacturing guidelines only require a purity of ≥96% so most commercial preparations for cell culture contain other undefined plasma proteins and contaminants from the purification process. To technically be classified as a “chemically defined medium”, all constituents of the medium and their concentrations must be known ,104. MedAlthough considerable progress has been made in the design of chemically defined xeno-free media, cell passage, and fold-expansion may be limited by the quality of culture supplements and their stability in culture. Daily replenishing of media is often recommended when using chemically defined media and this can be both costly and labor intensive.Although chemically defined ECM based matrices have helped advance the design of xeno-free hESC culture models, they may still be limited in their ability to physiologically mimic the in vivo stem cell niche. Studies indicate distinct differences in cell signaling and behavior in cells grown in 2-D versus 3-D culture -107. DurNumerous substrates for 3-D culture have been explored and tested. As a biomaterial, hydrogels are perhaps the most versatile and have been used in a variety of engineering applications. Hydrogels are a network of polymer chains either of natural or synthetic origin that are very absorbent and flexible due to high water content, thus resembling natural matrices. Hyaluronic acid (HA) is a glycosaminoglycan (GAG) and a primary component of in vivo extracellular matrices, making it particularly attractive in designing 3-D scaffolding for stem cells. GAG’s are known participators in receptor signaling complexes, cell-cell adhesion, and cell-matrix interactions. HA-hydrogel architecture and rigidity can be easily manipulated. HA encapsulated hESCs were shown to retain pluripotency for 20 days in culture and the need for continuous stem cell passaging was eliminated . The hydOthers have used a hydrogel scaffold composed of chitosan, a deceatylated cationic polysaccharide and alginate . InvestiA plant-derived nanofibrillar cellulose (NFC) has similarly been used to create a flexible 3D environment . This hySynthetic polymers are especially valuable for 3-D scaffolding, offering the advantage of versatility. Microfabrication techniques can be used to generate scaffolds with specific chemical signals immobilized on the matrix. As discussed earlier, combining matrices with tissue-specific bioactive molecules such as cell-adhesion proteins, growth factors and peptides are particularly promising. Derivatives of polyethylene glycol (PEG) combined with tissue-specific bioactive molecules such as cell-adhesion proteins, growth factors and peptides are particularly promising. The presentation of physiologically relevant signals allows the hESC microenvironment to be tailored towards self-renewal or differentiation as needed. PEG hydrogel constructs cross-linked with matrix metalloproteinase (MMP)-sensitive peptides and coupled with integrin-specific adhesion ligands are able to sustain the pluripotency of mouse and human stem cells ,119. TheSuspension culture of hESCs on microcarriers (MC) has also been used to create a pseudo 3D microenvironment that can be easily manipulated to direct cells towards undifferentiated proliferation or directed cell differentiation ,120-127.Suspension culture of hESCs without the use of coatings or microcarriers is also possible -131. AggConsiderations in selection of optimal matrices for stem cell culture should also include ease of stem cell release for passaging, stability over time and compatibility with sterilization techniques. The latter is often overlooked but is especially critical if substrates containing biological components like growth factors and peptides are to be used. Another consideration is the cost of using exclusively xeno-free synthetic media, recombinant growth factors and other additives. Culture design must also allow for large-scale propagation to meet the stem cell dosages needed for patient treatment cycles. The ability to effectively direct terminal differentiation of stem cells under these same xeno-free conditions is also of paramount importance in developing future therapeutic applications.Currently, the FDA requires all hESC (and hiPSC) lines intended for use in clinical trials to be developed in compliance with current Good Manufacturing Practices (cGMP) and in accordance with the Guidelines for the Clinical Transplantation of Stem Cells . ManufacFDA approval of the first clinical trials of hESCs for treatment of spinal cord injury and macular degeneration in 2009 marked the beginning of a new era in regenerative medicine -141. GerAdvance Cell Technology  has two on-going clinical trials. The first is for Stargardt’s Macular Dystrophy, which results in blindness from degeneration of the retinal epithelium and loss of photoreceptor cells. The second is for age-related dry macular degeneration. Retinal pigment epithelial cells (RPE) derived from hESC were used to restore the degenerative retinal epithelial monolayer. Scwartz et al. published the first report on transplantation of hESC-derived RPE in to a human patient in 2012 . A dosagStem cell therapy for treatment of Type I Diabetes is the newest arena for therapeutic use of hESC cells. Loss of insulin secreting cells in the pancreas is the cause of the disease. In August 2014, a diabetes clinical trial with hESC derived islet cells was approved . The comMinimizing immune rejection and thus the longevity of transplanted cells is also a priority for successful use of hESC based therapies. Besides immune response from any animal derived products, there is the risk of transplantation rejection due to HLA or ABO blood group antigen mismatch. Undifferentiated hESC’s have been shown to express low levels of MHC class I molecules, which are up-regulated during differentiation . The issDeveloping effective cell based regenerative therapies remain a challenge. Xeno-free culture models using extracellular matrices, biosynthetic surfaces and chemically defined media to create therapeutic grade hESC represent an important stepping stone in expanding such therapies. More data is still needed on the continued functionality of hESC cells after long term culture and continued passage in these new culture models. In depth analysis of cellular level mechanisms involved in continuous self-renewal as well as terminal differentiation will ultimately be necessary to truly harness the power of these pluripotent cells.


While ethnic diversity is increasing in many Western countries, access to youth mental health care is generally lower among ethnic minority youth compared to majority youth. It is unlikely that this is explained by a lower prevalence of psychiatric disorders in minority children. Effective screening methods to detect psychiatric disorders in ethnic minority youth are important to offer timely interventions.School-based screening was carried out at primary and secondary schools in the Netherlands with the Strengths and Difficulties Questionnaire (SDQ) self report and teacher report. Additionally, internalizing and psychotic symptoms were assessed with the depressive, somatic and anxiety symptoms scales of the Social and Health Assessment (SAHA) and items derived from the Kiddie-Schedule for Affective Disorders and Schizophrenia (K-SADS). Of 361 Moroccan-Dutch youths (ages 9 to 16 years) with complete screening data, 152 children were diagnostically assessed for psychiatric disorders using the K-SADS. The ability to screen for any psychiatric disorder, and specific externalizing or internalizing disorders was estimated for the SDQ, as well as for the SAHA and K-SADS scales.Twenty cases with a psychiatric disorder were identified (13.2 %), thirteen of which with externalizing (8.6 %) and seven with internalizing (4.6 %) diagnoses. The SDQ predicted psychiatric disorders in Moroccan-Dutch youth with a good degree of accuracy, especially when the self report and teacher report were combined . The SAHA scales improved identification of internalizing disorders. Psychotic experiences significantly predicted psychiatric disorders, but did not have additional discriminatory power as compared to screening instruments measuring non-psychotic psychiatric symptoms.School-based screening for psychiatric disorders is effective in Moroccan-Dutch youth. We suggest routine screening with the SDQ self report and teacher report at schools, supplemented by the SAHA measuring internalizing symptoms, and offering accessible non-stigmatizing interventions at school to children scoring high on screening questionnaires. Further research should estimate (subgroup-specific) norms and optimal cut-offs points in larger groups for use in school-based screening methods. While ethnic diversity is increasing in many Western countries, access to youth mental health care is generally lower among ethnic minority youth compared to majority youth , 2. It iSchools may play an important role in the early detection of psychiatric disorders outside the mental health care system. If school-based screening for psychiatric disorders is effective among ethnic minority youth in Western societies, it might provide a pathway to care for ethnic minority youth and an opportunity to bridge the treatment gap observed in this group.Since most screening instruments have been developed for Western populations and cross-cultural biases are likely to influence psychometric properties , it is nThe Strengths and Difficulties Questionnaire (SDQ)  is a queStudies report cultural variations in the presentation or symptom expression of internalizing disorders . TherefoThe present study was carried out among Moroccan-Dutch youth. In the Netherlands, Moroccan-Dutch youth is the largest ethnic minority population in its age group, has an increased risk to develop childhood psychiatric problems  or psychThe aim of this study was to examine if school-based screening for psychiatric disorders, using children and teachers as informants, is effective in Moroccan-Dutch youth. We examined the ability of the SDQ to predict any psychiatric disorder, and specific externalizing or internalizing disorders among Moroccan-Dutch youth. Since internalizing disorders are best detected by self-report measures  and exteThe study had two phases, a screening part and a diagnostic part.In the first phase of the study, schools with various educational levels in districts with small and large Moroccan-Dutch populations (range 1.9-9.2 %) were approached, in order to obtain a large sample of Moroccan-Dutch youth with various socio-economic backgrounds. Eight primary schools and ten secondary schools (78.2 %) participated. Children in years six to eight of primary schools (9–12 year olds) and years one to three of secondary schools (12–15 year olds) were included. The overall participation rate was 85.7 %. The total sample consisted of 1563 participants.According to the ethnic classification of Statistics Netherlands, children were categorized as Moroccan-Dutch when they and one or both parents (first-generation migrants) or when one or both parents (second-generation migrants) were born in Morocco (n = 407).Teachers filled out a questionnaire on 88.7 % of the Moroccan-Dutch children and adolescents .2 = 0.019; df = 1; p = 0.89) or age groups . The sampling procedure and response are presented in Fig. In the second phase of the study, a high-risk and a low-risk subgroup of the total Moroccan-Dutch screening sample were selected for in-depth psychiatric diagnostic assessment. Only youth with complete data (self report and teacher report) were eligible n = 361). Cut-offs were calculated for children  and adolescents  separately. Cut-offs for high-risk and low-risk subgroup selection, were based on scores on nine (sub)scales measuring psychiatric problems: subscales emotional symptoms, conduct problems and hyperactivity of the SDQ self report, subscales conduct problems and hyperactivity of the SDQ teacher report , subscal. Cut-offThe ethics committee of the VU Medical Centre approved the study. In the screening phase (2009–2010), a letter of introduction and a description of the study were sent to children and parents at their home address in separate envelopes. Parents or primary caregivers additionally received a passive informed consent form, which they could sign and return when they did not want their child to participate. Children had the option to decline at the time the survey was administered. They completed the web-based survey during a regular school day. A trained research assistant introduced the study and at least two research assistants were available in the classroom to answer the children’s questions during administration. Teachers were not involved in the actual administration. For every child, the teacher filled out a paper version of the SDQ teacher report (see section Measurements). All instruments were administered in Dutch.In the diagnostic phase (2010–2011), the selected Moroccan-Dutch youths and their parents received a letter in Dutch and Moroccan Arabic introducing the study. Parents or primary caregivers were asked to inform the researchers if they refused participation. The remaining families were visited at home in a face-to-face approach. We worked exclusively with female interviewers, because for some Moroccan-Dutch families it is more accepted to welcome unknown females than males into their homes. Additionally, each team consisted of at least one Moroccan-Dutch research assistant to have the option to inform families about the study in Dutch, Moroccan Arabic or a Berber (Tamazight) language. Written informed consent was obtained from all parents and children. Children were interviewed on psychiatric symptoms and impairment by medical doctors trained in transcultural psychiatry using the K-SADS (see section Measurements) during a separate appointment at school or at home. The average time between the screening and diagnostic phase was 13 months.Children filled out questions on demographic characteristics, such as their gender, age, and child’s and parents’ country of birth. A measure of the socioeconomic status of the neighbourhood was obtained from the Netherlands Institute for Social Research . Readingnot true) to 2 (certainly true). A total difficulties score is generated by summing the scores on four subscales: emotional symptoms, conduct problems, hyperactivity and peer problems (range 0–40). In order to create an aggregated measure of the self report and teacher report, we added the self-report and teacher-report total difficulties scores. Cronbach’s alphas in the screening sample (n = 361) were good for the self report (α = 0.76), the teacher report (α = 0.87), as well as for the aggregated self report and teacher report (α = 0.87) total difficulties scores.Children and teachers completed the self-report and teacher-report versions of the SDQ , consistComparisons of SDQ scores between Moroccan-Dutch youth and other ethnic groups are reported elsewhere .depressive symptoms scale consists of 15 items, with 11 negative  and four positive (e.g. ‘I enjoyed doing things’) statements. The somatic symptoms scale consists of 12 items representing somatic symptoms commonly reported by children and adolescents . Children reported on the presence of depressive symptoms and somatic symptoms during the past month on a three-point scale . The anxiety symptoms scale consists of 13 items . Children reported on the presence of anxiety symptoms on a three-point scale . By summing the scores of the depressive, somatic and anxiety scales a total internalizing symptoms scale was computed. The Cronbach’s alpha of the total internalizing symptoms score in the screening sample (n = 361) was excellent (α = 0.90).Children additionally completed selected scales of the SAHA . The SAHpsychotic experiences. These items had high resemblance to items that were predictive of adult schizophreniform disorder [psychotic experiences score. The Cronbach’s alpha of the psychotic experiences score in the screening sample (n = 361) was good (α = 0.76).Eight items from the K-SADS , adapteddisorder . Six iteDetails on the prevalence of psychotic experiences among the Moroccan-Dutch screening sample compared to other ethnic groups have been reported elsewhere .Children were interviewed using the K-SADS , a semi-Analyses were performed using the Statistical Package for Social Sciences (SPSS), version 20.0. First, demographic characteristics, SDQ, SAHA and K-SADS psychotic experiences scores and numbers of psychiatric disorders in the screening and diagnostic sample were described. There were no significant differences in SDQ, SAHA and K-SADS psychotic experiences scores for migrant status or reading skills. Second, logistic regression analyses were performed to assess the ability of (sub)scales of the used screening instruments to predict a diagnosis of any, externalizing or internalizing psychiatric disorder. Third, Receiver-Operating Characteristic (ROC) analyses were run for the SDQ total difficulties , SAHA total internalizing symptoms and K-SADS psychotic experiences scores assessing the diagnostic performance to predict any psychiatric disorder, an externalizing disorder and an internalizing disorder. For all ROC curves the Area Under the Curve (AUC), the optimal cut-off point, based on the cut-off point extending the highest towards the upper left corner, sensitivity and specificity were determined. Finally, the prediction of the optimal cut-off point of the screening instrument with the highest AUC for any psychiatric disorder was shown in a cross-tabulation, with a range of test values. The added value of the SAHA and K-SADS psychotic experiences scales were calculated as well.Demographic characteristics and SDQ, SAHA and K-SADS psychotic experiences scores of the Moroccan-Dutch screening and diagnostic sample are presented in Table The prevalence of psychiatric disorders in the Moroccan-Dutch diagnostic sample was 13.2 % , additional cases of internalizing disorders were found. The SAHA did not predict externalizing disorders, which is interesting because it confirms that the measurement of internalizing symptoms and externalizing symptoms in the screening phase captured different constructs.Considering the lower discriminatory powers of the psychotic experiences scale and the absent added value to the SDQ self report and teacher report in detecting psychiatric disorders, the additional assessment of psychotic symptoms is not needed for screening purposes. Interestingly though, psychotic experiences were significantly related to self- and teacher-reported psychiatric problems , and extIdentification of internalizing psychiatric disorders in general screening and diagnostic procedures in ethnic minorities is hampered by cultural variations in presentation or symptom expression . In our School-based screening is a practical way to gather multi-informant data of large groups of children, especially when using a web-based survey. A next step is to investigate how to connect children from school-based screening to mental health care. An option is to offer accessible, non-stigmatizing interventions at school to children scoring high on screening questionnaires. If psychiatric problems turn out to be more severe, children should be referred to specialized mental health care centres for further diagnostic assessment and treatment. Such stepped care methods ensure that children detected by screening methods also receive timely intervention. For ethnic minority youth, school-based screening in combination with stepped care interventions may provide an alternative pathway to care and an opportunity to bridge the treatment gap observed in this group.The findings of this study are subject to several limitations. First, although studying the performance of screening instruments in one separate ethnic minority group is preferable to overcome possible cross-cultural biases, it precludes generalization of results to other ethnic groups. Second, only Moroccan-Dutch youth scoring very high (above two standard deviations above the mean) or average/low (beneath one standard deviation above the mean) on selected subscales measuring psychiatric problems were included in the diagnostic phase of the study. These cut-off points were chosen to form a group of Moroccan-Dutch youth exhibiting high numbers of internalizing, externalizing or psychotic psychiatric symptoms and to create a contrasting comparison group of Moroccan-Dutch youth with few symptoms on any group of psychiatric symptoms. Since Moroccan-Dutch youths with medium scores were excluded and in these cases it is usually more difficult to make a diagnosis, results on the performance of the screening instruments might be somewhat inflated. Moreover, we did not use the algorithm, based on scores reported by parents, teachers and children, developed for the SDQ for the selection or prediction methods . This waStrengths of this study were the use of a web-based survey, a high participation rate and multi-informant data on psychiatric problems in a community sample at school in the screening phase of the study, and in-depth diagnostic assessment with a semi-structured psychiatric interview in a subgroup of Moroccan-Dutch youth in the diagnostic phase of the study.School-based screening for psychiatric disorders is effective in Moroccan-Dutch youth. We suggest routine screening with the SDQ self report and teacher report at schools, with additional administration of the SAHA measuring internalizing symptoms to enhance identification of internalizing disorders. To make the connection with actual treatment, we suggest offering accessible non-stigmatizing interventions at school to children scoring high on screening questionnaires and referring severe cases to specialized mental health care centres. Further research should estimate (subgroup-specific) norms and optimal cut-off points in larger groups for use in school-based screening methods. School-based screening in combination with timely interventions may reduce the treatment gap observed in Moroccan-Dutch youth in the Netherlands. Other countries with similar underrepresentation of ethnic minority youth in mental health care could possibly benefit in the same way.