This is a case report of a 56-year-old male with hypertension who presented with urinary retention and bowel incontinence. CT and MRI of the abdomen and pelvis showed a large complex cystic and solid enhancing mass in the right presacral space. Pathology biopsy result showed malignant peripheral nerve sheath tumor (MPNST) with extensive necrosis. The diagnosis of MPNST is extremely difficult due to the lack of (1) conclusive immunohistochemistry or unique chromosomal anomaly, (2) universal distinctive histopathology, and (3) clinical criteria. The clinical, radiologic, and histologic presentation of MPNST isimportant in its diagnosis. A rare case of MPNST that produced urinary retention and bowel incontinence is presented that may aid clinicians in the diagnosis of this rare clinical entity. Motor weakness, central enhancement, and immunohistochemistry may assist in the diagnosis of MPNST and differentiation between benign peripheral nerve sheath tumor (BPNST) and MPNST.  This is a 56-year-old male with history of hypertension and neurofibromatosis who presented with sudden onset of urinary retention and bowel incontinence.MRI of the lumbar spine showed a large mass extending anteriorly from the right S2 nerve root and in the posterior right lower back musculature at the level of L4-L5 and mildly enlarged left L2 ganglion and multiple skin neurofibromas. CT of the abdomen and pelvis showed a large complex pelvic mass measuring greater than 10 cm displaying mass effect on the posterior surface of the rectum arising from the right S2 neuroforamen. MRI of the abdomen and pelvis showed 9.6 × 9.8 cm complex cystic and solid enhancing mass in the right presacral space, extending from the right S2 neural foramen, displaces the rectum anteriorly and to the left Figures .Pathology biopsy result showed malignant peripheral nerve sheath tumor (MPNST) with extensive necrosis.MPNST is a term that replaces malignant schwannoma, malignant neurofibroma, neurosarcoma, and neurofibrosarcoma. MPNSTs which account for 3–10% of all soft tissue sarcomas are rare and arise in proximity to large peripheral nerves. The most common location of these tumors is the trunk, extremities, or head and neck, being extremely rare in abdomen. The majority of MPNSTs are high grade sarcomas with high probability of recurrence and metastases. About half occur in patients with neurofibromatosis type 1 which have a poorer prognosis than MPNST in patients without neurofibromatosis type 1. Approximately two-thirds of MPNSTs result from malignant transformation of tumor associated with neurofibromatosis type 1. The overall lifetime risk of developing MPNST in neurofibromatosis type 1 patients is 8–13%. Neurofibromatosis type 1 is autosomal dominant disorder with highly variable phenotypic expression and natural history. The involvement of the abdomen and pelvis by neurofibromatosis type 1 is rare. Neurofibromatosis type 1 associated MPNST occurs in young adults (20–35 years of age) whereas sporadic MPNST occurs in middle age and older adults (peak: 5th and 6th decades). Pelvic peripheral nerve sheath tumors are very uncommon. Differential between benign peripheral nerve sheath tumor (BPNST) and MPNST is difficult because the former may be large and have marked atypical nuclei. The age range and size of BPNST and MPNST are 10–76 years  and 4–20 cm (median size 9 cm) and 16–61 years  and 9–17 cm , respectively. MPNSTs occurring in central locations such as the paraspinal region of the retroperitoneum have lower 5-year survival rates, higher recurrence rates, and higher frequency of metastasis compared to tumors in other parts of the body. MPNST may occur as solitary or multiple enlarged masses associated with major nerve trunks such as the brachial plexus, sacral plexus, and sciatic nerve and may be asymptomatic or present with various sensory and motor symptoms, including projected pain or compressing and infiltrating surrounding tissues and structures. About 10% of MPNSTs occur due to therapeutic or occupational radiation with a latent period of greater than 15 years [BPNST is frequently asymptomatic whereas MPNST produces pain or neurological deficit. Pain at rest, tumor size, and duration of symptoms are of little value in differentiation between BPNST and MPNST. Faint motor weakness may be present in BPNST whereas severe motor weakness is seen exclusively in MPNST. MPNST may occur as solitary or multiple enlarged masses associated with major nerve trunks such as the brachial plexus, sacral plexus, and sciatic nerve and may be asymptomatic or present with various sensory and motor symptoms, including projected pain or compressing and infiltrating surrounding tissues and structures. The involvement of the abdomen and pelvis by neurofibromatosis type 1 is rare presenting with dysfunction and obstruction of genitourinary and gastrointestinal tracts. Patients with BPNST present with pain at rest (79%), sensory disturbance (53%), and faint-to-mild motor weakness (32%). The duration of the symptoms is 2–120 months . All patients with MPNST have pain at rest (100%), some patients have sensory disturbance (73%), and other patients have faint-to-severe motor weakness 64%). Symptom duration was 3–96 months  [%. SymptoThe correct histological diagnosis of MPNST is made in about 17–41% of new patients, whereas about 52% were diagnosed as having another type of sarcoma, about 4% were classified as suspicious, and 4% represented false negatives due primarily to profound cytological overlap with other sarcomas. The diagnosis of MPNST is extremely difficult due to the lack of (1) conclusive immunohistochemistry or unique chromosomal anomaly, (2) universal distinctive histopathology, and (3) clinical criteria. The clinical, radiologic, and histologic presentation of MPNST is important in its diagnosis [In the present case report, MPNST has the following: (1) MRI signal characteristics: high signal intensity: STIR, T2, and STIR > T2; intermediate signal intensity: PD FS, T1 FS, and PD FS > T1 FS; low signal intensity: T1, isointense to muscle; heterogeneous enhancement, whereas skin neurofibromas have high signal intensity: STIR, T2, and STIR > T2; intermediate signal intensity: T1; uniform enhancement; and (2) CT with contrast attenuation characteristics: heterogeneous attenuation; small lesions: attenuation lower than muscle; skin neurofibromas: attenuation same as muscle, with no significant enhancement. MRI is better than CT at detecting small lesions. CT did not show the smallest lesions but it is adequate for diagnosis if the patient cannot have MRI. The most sensitive MRI sequence for detection of MPNST is STIR. Heterogeneous hypointense signal on T1WI and heterogeneous hyperintense signal on T2WI are due to high water content of myxoid matrix or cystic or necrotic degeneration. Central areas of low signal intensity may be seen due to fibrosis. Post-IV contrast CT may show no contrast enhancement due to areas of necrosis, hemorrhage, or cystic degeneration. About 40–50% of BPNSTs may have central enhancement on CT corresponding to central zone of tightly packed cellular components (Antoni A) surrounded by hypocellular myxoid material (Antoni B) corresponding to target sign on MRI T-2 weighted images . The central enhancement and target sign are rarely seen in MPNST. In large tumor, there is central necrosis or degeneration, with peripheral tumor enhancement. The borders in MPNST are frequently irregular and infiltrative borders may invade adjacent structures or destroy adjacent bones. CT and MRI cannot accurately differentiate between MPNST and BPNST because tumor heterogeneity, irregular or infiltrative borders, and bone erosion may be seen in both MPNST and BPNST .MPNST is high grade tumor with high mitotic rate. However, histological diagnosis of MPNST is very difficult due to the lack of 1) histological and immunohistochemical markers specific for malignant peripheral nerve sheath tumor and (2) standardized diagnostic criteria. However, S-100 protein is seen in 50–90%, Leu-7 in 50%, and myelin basic protein in 40% and Ki-67 indices are seen in 5–65% of MPNSTs. Coinactivation of TP53 and Rb pathways is seen in 75% of MPNSTs [ histologThe most common metastatic site of MPNST is the lung, followed by the bone, pleura, retroperitoneum, and subarachnoid space of the spine . The patSurvival of MPNST is associated with complete tumor resection. The overall 5-year survival rate is about 44–50% which is affected by the patient's age, size of tumor, location of tumor, and margins affecting survival. Tumor size is the most reliable independent prognostic factor, with larger tumors having worse prognosis. Tumors occurring in central locations such as the paraspinal region of the retroperitoneum have lower 5-year survival rates, higher recurrence rates, and higher frequency of metastasis compared to tumors in other parts of the body. Longer survival is associated with early diagnosis (due to improved imaging), aggressive treatment with complete surgical resection and neoadjuvant therapy, small tumor size (<5 cm), and presence of low grade components. Negative staining for S-100 is associated with better prognosis for completely resected tumors .A rare case of MPNST that produced urinary retention and bowel incontinence is presented that may aid clinicians in the diagnosis of this rare clinical entity. Motor weakness, central enhancement, and immunohistochemistry may assist in the diagnosis of MPNST and differentiation between BPNST and MPNST.


Diphencyprone (DPCP) is a hapten that causes delayed-type hypersensitivity (DTH) reactions in human skin, and is used as a topical therapeutic for alopecia areata, warts, and cutaneous melanoma metastases.  We examined peak DTH reactions induced by DPCP  by comprehensive gene expression and histological analysis.  To better understand how these DTH reactions naturally resolve, we compared our DPCP biopsies to those from patients with psoriasis vulgaris, a chronic inflammatory disease that does not resolve.  By both microarray and qRT-PCR, we found that psoriasis lesional skin has significantly lower expression of many negative immune regulators compared to peak DPCP reactions.  These regulators include: interleukin-10, cytotoxic T lymphocyte-associated 4 (CTLA4), programmed cell death 1 (PD1), programmed cell death 1 ligand 1 (PDL1), programmed cell death 1 ligand 2 (PDL2), and indoleamine 2,3-dioxygenase (IDO1).  Their decreased expression was confirmed at the protein level by immunohistochemistry.  To more completely determine the balance of positive vs. negative immune regulators in both DPCP reactions and psoriasis, we developed one comprehensive gene list for positive regulatory (inflammatory) genes, and another for negative regulatory (immunosuppressive) genes, through Gene Ontology terms and literature review.  With this approach, we found that DPCP reactions have a higher ratio of negative to positive regulatory genes (both in terms of quantity and expression levels) than psoriasis lesional skin.  These data suggest that the disease chronicity that distinguishes psoriasis from transient DTH reactions may be related to absence of negative immune regulatory pathways, and induction of these is therefore of therapeutic interest.  Further study of these negative regulatory mechanisms that are present in DPCP reactions, but not in psoriasis, could reveal novel players in the pathogenesis of chronic inflammation.  The DPCP system used here thus provides a tractable model for primary discovery of pathways potentially involved in immune regulation in peripheral tissues. The mechanisms by which DPCP decreases pathogenic immunity for the promotion of hair growth in alopecia areata are incompletely understood. DPCP has been shown to alter the cytokine profile in treated alopecic scalp, in particular increasing interleukin (IL)-2 and IL-10 expression4. This increased IL-10 expression has been hypothesized to inhibit the lesional T cells of alopecia areata, but a comprehensive evaluation of other negative immune regulators induced by DPCP is lacking. We have previously shown that human skin responses to DPCP evolve from an inflammatory/effector peak at 3 days post-challenge to a more regulated immune response, with diminished markers of T cell activation, at 14 days. This study included comprehensive gene expression profiling, by microarray and qRT-PCR approaches, of biopsies from DPCP-challenged healthy volunteer skin at 3 days (peak reaction), 14 days (actively resolving reaction), and 120 days  compared to placebo-treated skin5. We have also previously performed similar transcriptomic profiling of psoriasis vulgaris lesional vs. non-lesional skin. This resulted in a meta-analysis derived transcriptome (MAD3) which combined the results of 3 individual microarray experiments, in an effort to address the variability in differentially expressed genes observed between experiments6. In this study, we expand our previous characterizations of transient DTH reactions and chronic psoriasis biopsies by directly comparing them to each other, particularly in relation to positive and negative immune regulation.Diphencyprone (DPCP) is a hapten that induces delayed-type hypersensitivity (DTH) reactions in human skin, and is used therapeutically for alopecia areatahttps://clinicaltrials.gov/ct2/show/NCT01452594). For each volunteer, biopsies were taken of placebo-treated skin as well as DPCP reactions 3, 14, and 120 days after challenge, as previously described5.For diphencyprone (DPCP) reaction microarray, qRT-PCR, and immunohistochemistry studies, skin biopsies were obtained from 11 volunteers under a protocol approved by The Rockefeller University’s Institutional Review Board (IRB Number JKR-0742). Written, informed consent was obtained from all subjects and the study adhered to the Declaration of Helsinki principles. This trial is registered at clinicaltrials.gov under NCT01452594  transcriptome described inTotal RNA was extracted using the miRNeasy Mini Kit  according to the manufacturer’s protocol with on-column DNase digestion. The amount of RNA was assessed by NanoDrop 1000 spectrophotometer . The quality of extracted RNA was examined using Agilent Bioanalyzer 2100 . RNA was hybridized to HGU133 Plus 2.0 chips  to measure relative gene expression.Statistical analysis. Microarray data were analyzed using R/Bioconductor packages (http://www.r-project.org). The Harshlight package7 was used to scan Affymetrix chips for spatial artifacts. Expression values were normalized using the GeneChip Robust Multi-array Average (GCRMA) algorithm. Genes with low variation and low expression in most samples were filtered out prior to the analysis. Batch effect due to hybridization date was adjusted using ComBat8. To identify differentially expressed genes, we fitted by REML (Restricted Maximum Likelihood) a linear mixed effect model with treatment (placebo/DPCP) and day (3/14) as fixed effects and a random intercept for each patient. Hypotheses of interest were tested using contrasts in R’slimma package framework. Thep-values resultant from the moderated paired Student’st-tests were adjusted for multiple hypotheses using the Benjamini-Hochberg procedure, which controls for the false discovery rate. The DPCP data discussed in this publication have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus . Psoriasis data were derived from6.Pre-amplification quantitative RT-PCR technique was used for measuring various genes in total RNA extracted from skin biopsy samples according to the company’s instructions. Briefly, 5 ng of total RNA was subjected to first-strand cDNA synthesis using High Capacity cDNA Reverse Transcription kits . The resulting cDNA was subjected to 14 cycles of pre-amplification using TaqMan PreAmp Master Mix Kit (Applied Biosystems) with desired pooled assay mix. The Gene Amp PCR System 9700 (Applied Biosystems) was used for the pre-amplification reaction with the following thermal cycler conditions: 10 min at 95°C and 14 cycles of 15 seconds at 95°C followed by 4 min at 60°C. 12.5 μl of pre-amplified cDNA was then used for quantitative RT-PCR reaction using TaqMan Gene Expression Master Mix (Applied Biosystems). The 7900HT Fast Real-Time PCR System was used for PCR reactions, and the thermal cycler conditions were as follows: 2 minutes at 50°C, 5 minutes at 95°C, and 40 cycles of 15 seconds at 95°C followed by 60 seconds at 60°C. Data were analyzed by the Applied Biosystems PRISM 7700 software  and normalized to human acidic ribosomal protein (hARP) housekeeping gene.All assays were from Applied Biosystems and inventoried assays used in this study were as follows: IL10 (Hs00961622_m1), CTLA4 (Hs03044418_m1), PDCD1 (PD1) (Hs01550088_m1), CD274 (PDL1) (Hs01125301_m1), PDCD1LG2 (PDL2) (Hs01057777_m1), IDO1 (Hs00984148_m1), and LAG3 (Hs00158563_m1). For RPLP0/hARP, a custom primer/probe set was used .Frozen sections of skin biopsies were dried at room temperature and then fixed for 2 minutes in acetone. Next, the samples were blocked with 10% normal serum of the species in which the secondary antibody was made and then the samples were incubated overnight at 4°C with the appropriate primary antibody. Biotin-labeled secondary antibodies  were amplified with avidin-biotin complex (Vector Laboratories) and developed with chromogen 3-amino-9-ethylcarbazole  to produce a red color indicative of positive staining.Primary antibodies used in this study are as follows : IL10 , CD95 (FAS) , LAG3 , PD1 , PDL1 , PDL2 , IDO1 , and CTLA4 .9. To globally assess the balance of positive vs. negative immune regulators in both DPCP reactions and psoriasis using our microarray data, we developed one comprehensive gene list for positive regulatory or inflammatory genes and another gene list for negative regulatory or immunosuppressive genes , cytotoxic T lymphocyte-associated 4 (CTLA4), indoleamine 2,3-dioxygenase (IDO1), programmed cell death 1 (PD1), programmed cell death 1 ligand 1 (PDL1), programmed cell death 1 ligand 2 (PDL2), and IL-10 . We conf10. In addition, these negative immune regulators are likely involved in the therapeutic applications of DPCP, particularly alopecia areata where IL-10 has already been implicated4. Further study of these regulatory mechanisms that are present in DPCP reactions, but not in psoriasis, could reveal novel factors in the pathogenesis of chronic inflammation. The DPCP system used here provides a tractable model for primary discovery of pathways potentially involved in immune regulation in peripheral tissues.These data suggest that disease chronicity in psoriasis could be related to absence of several negative immune regulatory pathways, with the implication that strategies to obtain stable clearance/restore tolerance in skin lesions may need to focus on increasing these negative pathways. These negative immune mechanisms may be of more general importance for maintaining skin homeostasis as non-inflammatory in the presence of a large population of effector memory T cells that normally reside in skin The paper under review provides interesting research data with immunopathogenic implications for both delayed-type hypersensitivity (DTH) reactions and psoriasis. Using both microarray and quantitative RT-PCR the authors found that peak DTH reactions induced by diphencyprone (DPCP) 3 days post-challenge has significantly higher expression of negative immune regulators than psoriasis lesional skin. These regulators include interleukin-10 (IL-10), cytotoxic T lymphocyte-associated 4 (CTLA4), programmed cell death 1 (PD1), programmed cell death 1 ligand 1 (PDL1), programmed cell death 1 ligand 2 (PDL2), and indoleamine 2,3-dioxygenase (IDO1), and their increased expression was confirmed at protein level by immunohistochemistry. According to the authors, these findings might explain at least in part the disease chronicity in psoriasis and might provide new therapeutic avenues.et al., 2014Gulati) detailed microarray and quantitative RT-PCR data on the time course of evolving lesions of psoriasis is lacking. Koebner phenomenon, guttate psoriasis and rebounds following clobetasol propionate suppression of plaque psoriasis might provide interesting models in this respect. The authors discuss the potential role of IL-10 regulation in DPCP immunotherapy of alopecia areata, and it might be interesting in this context to recall “Renbök phenomenon” or inverse Koebner phenomenon, namely the observation of normal hair growth in psoriatic plaques in patients with co-existing psoriasis and alopecia areata .Of course, further studies are required to extricate the dynamics of immune activation in DTH vs psoriasis lesions; even data on DPCP reactions at 14 days can be found in another paper by the same authors , even though IL-1 β activation of dendritic epidermal T cells and production of IL-17 seems to be important in experimental contact hypersensitivity . On the other hand, nickel-sensitized psoriasis patients develop a delayed but otherwise typical allergic contact dermatitis (ACD) even in close proximity to pre-existing psoriasis plaques, with a predominantly Th17 response in psoriasis and a stronger Th2 and cytotoxic immune response in ACD . Th9 cells are skin homing or skin resident cells which have been identified both in psoriasis and ACD, but IL-9 can have pro-inflammatory or regulatory activities depending on context . The innate immune responses to damage associated molecular patterns and reactive oxygen species are of foremost importance for the elicitation of ACDin vivo .Pathogenic differences between DTH and psoriasis are not limited to the expression of genes involved in negative immune regulation, as illustrated by the lack of effect of blocking Th1/Th17 pathways with ustekinumab on the elicitation of ACD by patch testing  and duration of exposure. Nickel and other metal ions are able to elicitate ACD through the activation of toll-like receptor 4 (TLR4) , direct activation of the NLRP·inflammasome with production of IL-1β  and other mechanisms .Furthermore, the gene expression signatures and pathomechanisms of DTH reactions may differ according to the antigen  and membrane proteins which are greatly interdependent and subject to cross-regulation, and several cell types, including Foxp3+ regulatory T cells, monocytes and B cells can be involved in down-regulation of inflammatory and autoimmune processes. Some of these cell types are characterized by their plasticity, so their phenotype might be subject to therapeutic modulation.In conclusion, the paper by Gulati and coworkers represents a seminal work in the study of the comparative dynamics of immune activation and downregulation in several skin disorders, with potential therapeutic implications.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this paper, Gulati and colleagues have tested the hypothesis that psoriatic skin lesions have significantly lower expression of negative immune regulators compared to peak DTH reactions from diphencyprone (DPCP). This study was done using two previously generated data sets described in previous publications from this group: 1) a study in 11 healthy human volunteers of recall responses to DPCP at 3-day (peak) and 14-day (resolution) using immunohistochemical techniques to visualize the infiltrating immune cells and gene expression (Affymetrix HGU133 Plus 2.0 Arrays) to perform a molecular profiling of the day 3 and day 14 DPCP responses, and in 6 cases 4-8 months later (reference 5); and 2) a meta-analysis derived (MAD) transcriptome of psoriatic skin from 3 different studies using Affymetrix HGU133 Plus 2.0 arrays in which microarray raw data were available through public databases (reference 6), termed the MAD-3 transcriptome.The results show an imbalance in the expression of negative regulatory genes in DPCP day-3 peak reactions compared to psoriatic skin lesions, favoring the expression of more negative immune regulators in the former vs. the latter group (52 genes vs 7 genes). They generated odds ratios for the positive and negative immune regulators from a list of negative regulatory and positive regulatory genes derived from a GO search. The odds ratio for negative immune regulators was significantly higher in the DPCP day-3 (peak) group than the psoriatic skin group, while the odds ratios for the positive immune regulators were not statistically significantly different between the two groups. The enrichment for negative immune regulators held true also at day-14 (resolving) and day 120 (late). These findings were confirmed by qRT-PCR of several key genes and by immunohistochemistry.This study is technically sound and the paper clearly written, with acceptable figures and tables. The title and the abstract are appropriate for the research described in the article. The methods are described in sufficient detail for the experiments to be replicated, although it would be useful to provide more detail about the methods underlying the calculations of the odds ratios. The interpretation of the results are reasonable based on the data and the conclusions provide a novel conceptual framework for understanding the fundamental mechanisms of skin homeostasis in psoriasis. I had one question about the results and their interpretation. In the study from reference 5, the results suggested different patterns of immune responses after DPCP challenge, referred to as subgroups A and B, where subgroup B had higher levels of negative immune regulators at day-3 than subgroup A. Interestingly, subgroup B fit the typical kinetics of a DTH response in which T cell and DC infiltrates peaked at day-3 and were diminished at day-14; whereas, subgroup A showed an unexpected increase in the numbers of T cells and DC’s at day-14 compared to day-3. It might be useful to know if the higher expression of negative immune regulators in DPCP skin compared with psoriatic skin was driven by the higher levels of these genes in subgroup B, or if it was generally true across both subgroups.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In this welcome addition to the field of psoriasis transcriptomics, Gulati and colleagues sought to test whether expression of genes involved in immune regulation in transient condition, diphencyprone (DPCP) induced delayed-type hypersensitivity (DTH), was significantly different from expression of genes involved in immune regulation of a chronic T cell-mediated disease, psoriasis vulgaris. Employing microarray and qRT-PCR based approaches to profile the transcriptomes from biopsied samples, the authors found that negative immune regulatory genes were significantly overexpressed in the DPCP vs. healthy controls comparison, while such a pattern of overexpression was not observed in the psoriasis vs healthy controls comparison. This lack of overexpression of key negative regulatory genes was found in both the microarray and the qRT-PCR analyses. The authors conclude that the relative dearth of negative immune regulatory genes in psoriasis may be associated with the persistent inflammation found in psoriasis and that strategies to restore negative regulators may have therapeutic implications.This paper is technically sound, with well accepted protocol employed in all wet experiments. The statistical analysis was also performed with well accepted and sufficiently rigorous methodology. The evidence supports the authors’ central claims. P-values for differential expression analysis were adjusted for multiple hypotheses by the Benjamini-Hochberg method to control for the FDR.The authors note that “genes with low variation and low expression in most samples were filtered out prior to the analysis”. We think that the filtering threshold for variance and expression should be reported as well.Odds ratios are reported in the results but it is not clear how they were estimated in the methods. Furthermore, interpretation of the odds ratios is unclear as well.In Table 1, we have questions regarding the FCH and the p and FDR values. Is the FCH the absolute FC? Is it log2? We think that the labeling should be clearer. What do p and FDR values of “0E+00” indicate? Is it for p and FDR values that are high or low?Could the authors’ results be confounded by the fact that psoriatic skin has a larger proportion of keratinocytes relative to DTH skin? If the negative regulators were primarily expressed in non-keratinocyte cell types, then biopsy of psoriatic skin compared to DTH skin might show a lower proportion of negative regulators based simply on cell proportions rather than intrinsic immunologic differences.et al., 2014Ferraro). Is the transcriptional profile of the observed DTH reaction consistent with the effects of a T regulatory cell effect, for example as described inet al. (2011)Rosenblum?We feel that the biology of psoriasis and DTH (or contact dermatitis) can be expanded upon in the discussion section. For instance, a number of the genes listed in Table 1 are known T regulatory cell signature genes  in response to an obligatory hapten antigen, diphencyprone (DPCP), and psoriasis vulgaris, a chronic T-cell mediated inflammatory skin disease. Both diseases are particularly suited for this approach. DPCP-specific DTH reactions are immune mediated, resolve naturally and have well-defined self-limited kinetics. This allows for the accurate definition of time points for the analysis of inflammatory peak reactions and resolving inflammatory activity. Psoriasis instead constitutes a persistent T-cell mediated skin inflammation.The approach to obtain insights into the mechanisms perpetuating inflammation consists of the analysis and comparison of gene expression profiles in lesional and non-lesional skin probes of both diseases. Regarding psoriasis the authors refer to meta-analysis derived transcriptome data that cover the gene expression profiles from 193 lesional and non-lesional biopsy pairs as recently published. For DPCP-treated patients 11 lesional/non-lesional sample pairs were included for different time points during inflammation. Like the psoriasis transcriptome analyses the data acquisition in DCPC samples has been peer peer-reviewed in a high ranking scientific journal.To achieve meaningful results regarding the posed question, the authors define two groups of genes for comparison. One comprehensive gene list includes positive regulatory or inflammatory genes that enhance inflammation, the other genes particularly related to negative regulatory or immunosuppressive functions. The composition of the gene lists is based on scientifically verified Gene ontology terms. In addition to the microarray data, for select genes expression levels are verified by quantitative RT-PCR and by immunohistochemistry analyses of protein expression in skin samples.vs. negative regulatory transcripts in psoriasis compared to DTH reactions, which is maintained throughout DTH healing.As a result the authors report, that at the time of the peak inflammatory response DPCP-induced DTH reactions contain significantly more genes related to suppression of inflammation than psoriasis lesions. This results in an altered balance between positiveThe authors conclude that in psoriasis, but also beyond this disease the lack of negative immune regulatory genes may be related to inflammatory disease chronicity. They furthermore propose that DPCP reactions may serve to examine regulatory immune pathways.The study addresses a highly relevant question. The scientific approach is novel, original and represents a truly innovative solution to obtain novel insights into immune regulation. The comparison of psoriasis and DCPC reactions is reasonable because in both of them inflammatory reactions result from T-cell driven mechanisms. The methods are sound and appropriate for the analysis. The underlying data are extensive and due to the large sample size create a reliable and credible basis for the study. Accordingly, results and conclusions are well supported. The abstract provides an adequate summary of the study. The results are clearly presented. The discussion is short, draws a clear and valid conclusion and avoids unnecessary speculations. Indeed, the insights from the study provide a novel approach to understand the chronicity of inflammation.  My personal assessment is that of an intriguing approach, which by itself is revealing and at the same time opens novel access paths into the investigation of chronic immune-mediated inflammation.I support the publication of this article in the present form without any reservation. There are no changes necessary.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis bothin vitroandin vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases’ seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease.Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by  Osteosarcoma (OS) is an aggressive malignant tumor arising from primitive transformed cells that exhibit osteoblastic differentiation and produce malignant osteoid tissue13in vivo and in vitro and its expression is absent or reduced in the majority of OS casesWWOX gene by genome-wide array CGH analysisWWOX mRNA by real-time PCRWwox null mice die prematurely at age of 3–4 weeks with metabolic and neurological disorders151617Wwox mutant mice prior to their death revealed that WWOX possesses tumor suppressor functions151719Wwox results in lesions resembling OS in juvenile mice1215Wwox-heterozygous mice develop spontaneous and chemically-induced tumors1519Inactivation of tumor suppressors is a hallmark of cancer8910in vitro and metastasis in vivo through inhibition of key metastatic genes implicated in OS.The current therapeutic strategies have a limited efficacy in the treatment of metastatic disease, and the metastatic relapse or recurrent conditions have remained unchanged over the last three decadesHOS cell line was obtained from the American Type Culture Collection. LM7 cell line was a gift of Dr. Eugenie Kleinerman . Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum.To generate a lentiviral WWOX vector, human WWOX cDNA was cloned in lentiviral vector containing neomycin as the selection marker using the Gateway cloning system . Cells were transduced with Lenti-WWOX or Lenti-EV at appropriate multiplicities of infection. Cells were next labeled with green fluorescent protein (GFP) using Lenti-GFP particles and assessed by immunoblotting using anti-WWOX polyclonal antibody and visualization of GFP. Cells were put in selection with 2 μg/mg of neomycin.Cells were grown to confluence in a 6-well plate, and then scratched using a 200 μl sterile pipette tip. Images of wounded monolayers were photographed under an inverted microscope at the time of wounding (0 h), 9 and 24 hrs later. Quantification of the % of wound closure in WWOX-manipulated cells was done using the software ImageJ in three spots of the wound on each triplicate. Results were expressed as mean ± STDV.5), suspended in DMEM containing 0.1% bovine serum albumin, were added to the upper chamber. Conditioned medium of NIH3T3 fibroblasts was applied as a chemoattractant and placed in the lower compartment of the Boyden chamber. Assays were carried out at 37 °C in 5% CO2. Over 90% of the cells attached to the filter after incubation for 7 h. At the end of the incubation, the cells on the upper surface of the filter were removed by wiping with a cotton swab. Cells that passed trough the filter into the bottom side of the membrane were fixed with methanol and stained with Giemsa. Each assay was done in triplicate and ten representative fields in each well were quantified to determine the number of invasive cells under a light microscope.Blind well chemotaxis chambers with 13-mm-diameter filters were used for this assay. Polyvinylpyrrolidone-free polycarbonate filters, 8-mm pore size , were coated with basement membrane Matrigel (25 μg per filter). Briefly, the Matrigel was diluted to the desired final concentration with cold distilled water, applied to the filters, and dried under a hood. HOS or LM7 cells , and injected intravenously (i.v.) into the lateral tail vein of mice at a density of 1 × 107 cells/ml. 1 × 106 cells/0.1 ml serum-free medium were injected into the right proximal tibia. Briefly, the animals were anesthetized with ketamine (80 mg/kg) and xylazine (7 mg/kg). The knee of the NOD SCID mouse was flexed beyond 90o and a 100 μl of the cell suspension was injected into the proximal tibia using a 25-gauge needle. Five animals were injected with each OS-manipulated cell line. Animals were monitored daily, and tumor sizes were measured every week. At the end point animals were sacrificed, tumors were weighed, and tumor volumes were calculated as previously describedFor intratibial injections, exponentially growing GFP-tagged OS cells were harvested, counted, and resuspended in PBS to a final concentration of 10RNA was isolated from cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was synthesized with oligo (dT) primers using the super-script first strand synthesis kit (Invitrogen) according to the manufacturer’s protocol. RealTime PCR was run on Applied Biosystem 7900HT machine using Sybr Green probe ) at standard conditions. The primers used for qRT-PCR used are summarized in the in vitro and in vivo experiments were expressed as mean ± SD or SE. Fisher’s exact test and Student’s t test were used to compare values of test and control samples. P < 0.05 indicated significant difference.Results of in vitro. Overexpression of WWOX was validated by immunoblotting using anti-WWOX antibody  and followed formation of primary tumors and examined for lung metastasis, the predominate site of OS metastasis. By three weeks of IT injections, HOS EV cells formed large tumors that mice had to be removed from the experiment    To compactively) . These dWe next determined whether WWOX impact on invasion and metastasis was also attributable to effects on later steps of the invasion-metastasis cascade, independent of its influence on local invasion (IT model). Thus we injected WWOX-expressing HOS or LM-7 cells directly on the circulation of mice, thereby circumventing the initial steps of local invasion and intravasation. To this end, GFP-fluorescent OS cells were injected intravenously (IV) and formation of GFP+ metastatic lung nodules was assessed. Two months after tail-vain injection, WWOX-expressing HOS cells generated fewer lung metastases than did controls . In factOur results so far indicate that WWOX restrains OS metastasis. To shed light on the mechanism by which WWOX could exert its anti-metastatic activity, we perform real-time PCR analysis on known metastatic genes implicated in OS progression. To this end, total RNA was isolated from manipulated HOS and LM-7 cells. Our results indicate that the expression of ezrin, integrins alpha 4 and 5, MMP13 and VEGF were reduced upon WWOX expression in HOS and LM7 cells .WWOX overexpression is accompanied by the downregulation of genes that are involved in tumor cell migration and invasion rendering them less metastatic and less aggressive.Since it has been shown that RUNX2 regulates the expression of genes involved in cell motility and adhesion in OS12in vitro and in vivo, likely through regulation of RUNX2 levels and activity.Several studies have demonstrated that WWOX inactivation is associated with a more aggressive tumor behavior in different cancers10121314The data presented here shed light on pleiotropic functions of WWOX in suppressing tumoriogenesis. Ectopic expression of WWOX has been shown constantly to suppress primary tumor growth of different cancer types not only in OSWwox-deficient mice32343538et al. have recently analyzed the genomic function of RUNX2 in OS cells to gain insight into molecular pathways that are perturbed in bone cancer. Their results showed that RUNX2 controls pathways that are broadly related to cell adhesion and motility and renders OS cells more aggressive384142Another finding that emerge from our study is that WWOX suppresses RUNX2 transactivation function of target genes implicated in migration and adhesion in OS cells. RUNX2 is the principle regulator of osteoblast differentiation and has been shown to be upregulated in OS122732et al. suggested that loss of WWOX contributes to the peritoneal dissemination of human ovarian cancer cells via modulating the interaction between tumor cells and the extracellular matrixEmergence evidence is associating WWOX function with metabolism and genome stability. Depending on timing of WWOX loss and context of what other tumor suppressors or oncogenes are modulated, the functional outcome of WWOX is shaped. For example, WWOX could be lost during early stage of carcinogenesis impairing DNA damage response and hence promoting genome instability and cancer evolutionWWOX gene in human patients are associated with epileptic encephalopathies and early death, in most patients, precluding adult tumor analysis184950WWOX locus (CNV-67048), which is associated with reduced WWOX expression, has been recently correlated with predisposition to lung cancer and gliomas52535556Another emerging function of WWOX is maintaining homeostasis of the nervous system. Modeling loss of WWOX in animal models is associated with epilepsy and ataxia18In conclusion, our data suggest that WWOX expression, which is commonly lost or reduced in a large portion of human OS, could suppress OS pulmonary metastasis likely through regulation of RUNX2 function.How to cite this article: Del Mare, S. and Aqeilan, R. I. Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function. Sci. Rep.5, 12959; doi: 10.1038/srep12959 (2015).


These findings show that tricaine used at standard concentrations in zebrafish larvae does not paralyse muscle, thereby diminishing concern that a direct action on muscle could mask a lack of general anaesthesia.Movements in animals arise through concerted action of neurons and skeletal muscle. General anaesthetics prevent movement and cause loss of consciousness by blocking neural function. Anaesthetics of the amino amide-class are thought to act by blockade of voltage-gated sodium channels. In fish, the commonly used anaesthetic tricaine methanesulphonate, also known as 3-aminobenzoic acid ethyl ester, metacaine or MS-222, causes loss of consciousness. However, its role in blocking action potentials in distinct excitable cells is unclear, raising the possibility that tricaine could act as a neuromuscular blocking agent directly causing paralysis. Here we use evoked electrical stimulation to show that tricaine efficiently blocks neural action potentials, but does not prevent directly evoked muscle contraction. Nifedipine-sensitive L-type Ca Increased cytoplasmic Ca++ concentration promotes actomyosin contractility In the 18+, Ca++ and K+ channels + (Nav) channels initiate the action potential, rapidly activate and inactivate in a few milliseconds and have been widely studied ++ (Cav) channels can make a significant contribution to the action potential V1.1 channels play an essential role in calcium release in skeletal muscle through their role as components of the DHPR, which provides a voltage-sensitive mechanical link that triggers release of Ca++ from the sarcoplasmic reticulum Excitability arises from the sequential opening and closing of voltage-gated NaDanio rerio offer many advantages for drug screening and investigating the relationship between ion channel expression and nerve and muscle function in vivowww.accessdata.fda.gov/scripts/animaldrugsatfda/). Previous reports have shown a suppressive effect of tricaine on zebrafish peripheral and central nervous system The larvae of the zebrafish v channels e.g. scn) gene family consists of four sets of duplicated alpha subunit genes. scn1a and scn1Lab form the Nav1.1 channels expressed in cardiac muscle scn4aa and scn4ab contribute to Nav1.4 channels and are expressed in skeletal muscle scn12aa and scn12ab (putatively forming Nav1.5 channels) is transient in zebrafish muscle Scn5a is expressed in neonatal skeletal muscle and limbic system and in adult muscle after denervation scn8aa and scn8ab (Nav1.6 channels) are expressed in brain eye, spinal cord, retina, and peripheral nervous system v channels targeted by tricaine are unclear.Local anaesthetics of the amino amide class, to which tricaine belongs, are known to inhibit certain mammalian Nav channels is unknown, but such effects have been reported in other species v channels have similarities to Nav channels, in that their alpha subunits have clear structural and functional similarities cacna1sa (CaV1.1)cacna1a is expressed in brain, neuronal cells and heart Whether amino amide anaesthetics also inhibit zebrafish Ca++ channel antagonist. Our findings show that, at the normal concentrations used for anaesthesia, tricaine does not prevent muscle contraction or interfere with excitation-contraction coupling.We set out to investigate the tissue target(s) of tricaine in the zebrafish, particularly in muscle. We report the impact of tricaine on zebrafish skeletal muscle at early developmental stages. We also studied the impact of nifedipine, an L-type Casb13chrnd  mutants Danio rerio, either Tübingen wild type or Fish larvae were subjected electrical stimulation at room temperature using a custom-built electric stimulation set-up (ESS) . To miniFor wash-out, fish were later transferred to a dish containing fresh fish water and were left there for over 30 minutes. Care was taken to exchange the fish water five times to remove residual tricaine.Observations were made using a dissecting microscope . All videos were recorded using Olympus (DP70) camera with DP Controller software at a frame rate of 60 frames per second in total time of 25 seconds.d” was measured at the level of the last somite by superimposing images from video. Two images, before and after the onset of stimulation, were overlaid using Adobe Photoshop by false-colouring one image red and converting it to transparent mode.Fish tail displacement “Fish were bred and experiments performed under licences from the UK Home Office and conform to all institutional, national and EU guidelines.fixe mutant larvae, which lack functional acetylcholine receptors The performance of ESS was tested at a range of voltage and pulse train parameters to define a stimulation regime that was not toxic to the fish yet triggered motility. The response of fish to the external stimulus was immediate oscillatory swimming movement with the appearance of an escape response . Escape fixe sibling larvae at the concentration widely used for anaesthetizing zebrafish fixe mutants . These results suggest that nifedipine-sensitive L-type Cav channels are functional in neurons of the motor pathway, but do not significantly contribute within the muscle itself.Why is muscle resistant to doses of tricaine that act as a general anaesthetic? One hypothesis is that the voltage-gated channels in skeletal muscle are distinct from those inhibited by tricaine in the nervous system. As Nav channels. Lastly, nifedipine-sensistive L-type voltage-gated calcium channels also act in the nervous system but appear to have little direct action within zebrafish muscle to promote contraction.There are four major results from the current study. First, a simple and custom built electric stimulation set-up shows that larval zebrafish muscle contraction can be directly evoked using external electric stimuli. Second, that tricaine anaesthesia at normal doses prevents movement by effectively inhibiting neural but not skeletal muscle action potentials. Thirdly, tricaine at higher concentrations also blocks muscle action potentials, probably via effects on NaMovements in skeletal muscle can be elicited either by neurons  or by external electrical stimulation , in which muscle plasma membrane depolarization caused by the applied voltage triggers an action potential. In the current study, we show that EES can initiate contraction in mutants that lack the acetylcholine receptor delta subunits and therefore cannot receive natural input from motorneurons v channels and thus block the conduction of action potentials in excitable cells v channels, blocking Na+ movement through the channels, but can also affect other channels Amino amide anaesthetics are known to block Nav channel alpha subunit genes, only scn4aa and scn4ab are expressed in zebrafish skeletal muscle around 3 dpf v1.4) may be less sensitive to tricaine than those made with classical neural homologues, such as scn1a (Nav1.1) and scn8a (Nav1.6) subunits. The Nav1.5 channel alpha subunit gene (scn12ab in zebrafish) appears to be expressed transiently in early developing slow muscle fibres, but may become restricted to cardiac muscle later v1.5 channel is up-regulated in skeletal muscle on denervation fixe mutants reflects up-regulation of similar channels sensitive to tricaine in inactive somitic muscle. Indeed, mammalian Nav1.5 appears several fold more sensitive than Nav1.4 to lidocaine v channel expressed at early stages , fixe mutants (sb13/sb13chrnd) or fixe sibling (sb13/+ or chrnd+/+chrnd) fish in response to EES. Fish were treated with the water control or tricaine at standard or fivefold dose, as indicated before each clip. A pulse train of 0.1 s duration was applied every 5 seconds. Movie is sped up about 2.5-fold for ease of viewing. Note increased movement in response to the first pulse, possibly reflecting the need for channel activation for tricaine to bind to Nav channels. Heartbeat shows all fish are alive. In ‘fixe + 5X tricaine' clip, heartbeat shows fish are alive but fail to respond to stimulation.(MP4)Click here for additional data file.


Objective: It has been found that asphyxia influences proliferation and differentiation of brain neural stem cells in newborn animal models, and that peripheral blood stem cells play an important role in repairing brain damage. But it has not been reported yet whether asphyxia influences peripheral blood stem cells differentiating into neural cells, and whether with the progress of the disease there is a change of peripheral blood stem cells differentiating into neural cells in newborns with hypoxic ischemic encephalopathy (HIE).Methods: Fifty term HIE infants were enrolled in research from March, 2007 to March, 2010. There were 10 cases of the severe HIE patients with good improvement, the severe HIE patients with poor improvement, the moderate HIE patients, the mild HIE patients and the controls, respectively. The peripheral mononuclear cells collected within 24 hours and on 7th day after birth were cultured in vitro for 10 days to differentiate into neural cells. The induced nestin positive cells were identified with Immunohistochemistry and counted.Findings: Within 24 hours after birth, there were no difference of induced nestin positive cells among the severe HIE patients with good improvement (68.99±7.85), the severe HIE patients with poor improvement (71.43±6.88), the moderate HIE patients (73.34±6.46), the mild HIE patients (70.46±6.66) and the controls . In the severe HIE patients with obvious improvement, the induced nestin positive cells from 7th day peripheral blood mononuclear cells (94.50±15.57) increased markedly compared with that within 24 hours , and were higher than the induced nestin positive cells from 7th day peripheral blood mononuclear cells in the severe HIE patients with no obvious improvement .Conclusion: The ability of peripheral mononuclear cells differentiating into neural cells in term infants with good improvement suffering from severe HIE was enhanced, which may suggest possible relationship between the brain repair and the peripheral stem cells. It has been found that asphyxia influences proliferation and differentiation of brain neural stem cells in newborn animal models[2-4], and that peripheral blood stem cells play an important role in repairing brain damage[5]. But It has not been reported yet whether asphyxia influences peripheral blood stem cells differentiating into neural cells, and whether with the progress of the disease there is a change of peripheral blood stem cells differentiating into neural cells in newborns with HIE. This research will focus on these questions.Neonatal hypoxic ischemic encephalopathy (HIE) is one of the most common causes of death and long-term neurological impairment in full-term neonates worldwide. Up to now, there is no specific effective treatment for the severe HIE. The potential use of stem cells to reduce brain damage is a possibilityFifty term newborn infants from the pediatric department of Shanghai Tongji hospital affiliated to Tongji University, Shanghai, China, were enrolled into the study from March 2007 to March 2010. 72 hours after birth, the severity of HIE was evaluated according to The Sarnat Grading Scale of HIE, and 20 serious cases, 10 moderate cases, and 10 mild cases were identified. All the patients met the HIE diagnostic criteria. 10 severe cases improved obviously 14 days after birth, which were defined as no consciousness, no seizure, no abnormal muscle tone, no brain stem symptom, whereas 10 severe cases poorly improved, which were defined as having one or more of the above manifestations 14 days after birth or died. 2 cases died within 1 week, and 6 cases died after 1 week. 10 newborn infants with wet lung acted as controls, presenting with breath difficulty, but oxygen saturation was normal. The study protocol was reviewed and approved by the local institutional ethics committee and written informed consent was obtained from all guardians prior to the studyBlood sample collection: th day after birth if the patients were still alive. The mononuclear cells were separated and collected with Ficoll separating medium (GIBCO ) 1500r/min×5min, re-suspended in proliferation culture medium (see below), which were applied in the following process. 5 ml peripheral blood was collected from all newborn infants within 24 hours after birth. Again 5 ml blood was collected from severe HIE patients on 7The proliferation culture of peripheral mononuclear cells (PMCs)[6]:7 cells/ml with volume of 2 ml per well, cultured for 3 days in a humidiﬁed atmosphere .Proliferation culture medium was prepared, which consisted of DMEM culture medium (GIBCO), 10% fetal bovine serum , penicillin  100U/ml, streptomycin  100µg/ml. PMCs were plated into 6 well culture-plates at the density of 1×10The differentiation culture of peripheral mononuclear cells (PMC)[6]:2), and half volume of culture medium in each well was renewed with the same medium with the original medium in the well (Proliferation culture medium or differentiation culture medium) every 3 days. The cell shape and growth were daily observed under inverted phase contrast microscope. On the 10th day of differentiation culture, the immunohistochemistry was carried out to identify the differentiated cells.After proliferation culture of peripheral mononuclear cells for 3 days, the culture medium was eliminated, and sterilized PBS was added, then PMCs were gently repeatedly beaten by pipette so that the unattached cells could be eliminated. 2 wells were again continually added with proliferation culture medium as negative controls, and the other 4 wells were added with changed culture medium, namely differentiation culture medium, which consisted of DMEM/F12 (GIBCO), 10% fetal bovine serum (GIBCO), penicillin  100U/ml, streptomycin  100µg/ml, all trans retinoid acid (Sigma Co) 0.5 µM, nerve growth factor (Sigma Co) 100ng/ml, insulin (Bo-Yun Biotech) 25 µg/ml, transferring (Bo-Yun Biotech) 100µg/ml, heparin  2 µg/ml, progesterone  60 nM. PMCs were cultured in a humidiﬁed atmosphere  from immunohistochemistry kit was dropped into culture wells (Hangzhou Baitong Biotechnology Co Ltd), incubated and protected from light for 45 min to block antigens. 5μg/ml anti-Nestin antibody (R&D systems) was added, incubated and protected from light at 4℃ overnight. Each cell was rinsed and washed with PBS for 5 min×5 times, and agent B (biotin labeled second antibody) from immunohistochemistry kit was added, incubated and protected from light at room temperature for 60 min, rinsed and washed with PBS for 5min×5 times. Agent C (horseradish peroxydase complex) was added and incubated at room temperature for 15 min, washed with PBS 5 for 5 min×5 times. DAB was added into each well to react for 3 min, and then immediately washed with double distilled water thoroughly. Immunohistochemistry positive cells in each well further identified by cell shape, which had at least two long cell processes, were counted under microscope, and the average number of positive cells in 4 wells (added with differentiation culture medium) was taken to be the positive cells number.Statistical methods:th day was analyzed by t test.SPSS13.0 for windows was adopted to analyze the data. The difference of Neuron marker nestin positive cells, time after birth, gestational weeks, birth weight among HIE newborns with different severity within 24 hr was analyzed by ANOVA, The difference of sex and delivery mode among HIE newborns with different severity was analyzed by chi-square test. The difference of Neuron marker nestin positive cells in the severe HIE newborns between different improvement groups on 7The patients’ clinical data was as follows . There wth day. The number of cells with neuron marker nestin had no difference among the mild HIE group, the moderate HIE group, the severe HIE with good improvement group, the severe HIE with poor improvement group and the controls . The numThe number of cells with neuron marker nestin in the severe HIE with poor improvement on 7th day was not different from that within 24 hours after birth .[1], and there are also investigations revealing that peripheral blood stem cells have close relation to the damage repair of HIE brainHIE is one of the main causes of death and long- term neurological impairment in full-term neonates. There is no specific effective treatment for the severe HIE. Many investigations have identified endogenous neural stem cells were mobilized when HIE took place, namely theneural stem cells in brain take part in HIE repair[12] and mesenchymal stem cells[13] with neuron differentiation potential. Umbilical blood is a part of the neonatal peripheral blood, so it is likely that the neonatal peripheral blood cells possess similar character of the umbilical blood cells, namely consist of stem cells with neuron differentiation potential. Umbilical mononuclear cells consist of multipotent stem cells[14] and CD133+ stem cells in umbilical blood[15] can differentiate into cells with neural markers. It needs to be further confirmed which PMCs gradients in HIE newborn patients can differentiate into neuron cells. Our data confirmed that PMCs from newborns and newborns with HIE could differentiate into cells with neural marker namely possessing neuron cells differentiation potential. Human peripheral CD14+ monocytes. We could not find the influence of asphyxia on the ability of neonatal PMCs differentiating into neural cells, which may suggest the different influence of asphyxia on central neural stem cells and peripheral blood stem cells with neuron differentiation potential.  The influence of asphyxia on neonatal peripheral blood stem cells differentiating into neuron cells has not yet been reported by now. Ischemia and anoxia have definite influence on neural stem cells in brain[18-27]. The different differentiation ability of PMCs with the progress of HIE has close relationship with the prognosis. If the differentiation ability of PMCs can be identified as early as possible by improving the culture methods to shorten the culture time, it may be valuable to judge the prognosis.  We found the ability of PMCs from severe HIE with good improvement on 7th day after birth to neural cells obviously increase, which may suggest these patients predominate at promoting neural cells differentiation, and may also suggest peripheral blood stem cells play a possible role in the HIE repair. The mechanism of peripheral blood stem cells taking part in central neural cells repair is unclear, which may relate to directly entering central nervous system to differentiate into neural cells to replace the injured brain tissues or to produce neurotrophin to promote the proliferation and differentiaton of brain internal neural stem cells The ability of peripheral mononuclear cells differentiating into neural cells in term infants with good improvement suffering from severe HIE was enhanced, which may suggest possible relationship between the brain repair and the peripheral stem cells. The mechanism of PMCs taking part in the protection of central nervous system injury suffered from asphyxia need to be further investigated.The ability of peripheral mononuclear cells differentiating into neural cells in term infants with good improvement suffering from severe hypoxic ischemic encephalopathy was enhanced, which may suggest possible relationship between the brain repair and the peripheral stem cells.


Glycine max (L.) Merr.] genotypes was grown in two years and total leaf carotenoid content was determined using three different methods. The first method was based on extraction and spectrophotometric determination of carotenoid content (eCaro) in leaf tissue, whereas the other two methods were derived from high-throughput canopy spectral reflectance measurements using wavelet transformed reflectance spectra (tCaro) and a spectral reflectance index (iCaro). An association mapping approach was employed using 31,253 single nucleotide polymorphisms (SNPs) to identify SNPs associated with total carotenoid content using a mixed linear model based on data from two growing seasons. A total of 28 SNPs showed a significant association with total carotenoid content in at least one of the three approaches. These 28 SNPs likely tagged 14 putative loci for carotenoid content. Six putative loci were identified using eCaro, five loci with tCaro, and nine loci with iCaro. Three of these putative loci were detected by all three carotenoid determination methods. All but four putative loci were located near a known carotenoid-related gene. These results showed that carotenoid markers can be identified in soybean using extract-based as well as by high-throughput canopy spectral reflectance-based approaches, demonstrating the utility of field-based canopy spectral reflectance phenotypes for association mapping.Carotenoids are organic pigments that are produced predominantly by photosynthetic organisms and provide antioxidant activity to a wide variety of plants, animals, bacteria, and fungi. The carotenoid biosynthetic pathway is highly conserved in plants and occurs mostly in chromoplasts and chloroplasts. Leaf carotenoids play important photoprotective roles and targeted selection for leaf carotenoids may offer avenues to improve abiotic stress tolerance. A collection of 332 soybean [ Carotenoids are organic pigments that are produced predominantly by photosynthetic organisms and comprise the red, yellow and orange colors of flowers, fruits and other plant organs . In addiIn plants, carotenoids play important roles in photosynthesis as accessory pigments and in photoprotection. As accessory pigments, carotenoids are involved in light harvesting and energy transfer to chlorophyll . The absBecause unfavorable conditions such as excess light energy can lead to the production of reactive oxygen species that can damage photosynthetic membranes and proteins, light absorption and energy transfer are highly regulated by numerous processes. The essential photoprotective roles of leaf carotenoids include: scavenging of reactive oxygen species, quenching of dangerous triplet states of chlorophyll and participation in thermal dissipation of excess light energy . CarotenThe carotenoid biosynthetic pathway was postulated more than three decades ago based on standard biochemical analyses using labelled precursors, specific inhibitors, and mutant characterization . Genes eArabidposis thaliana reduced leaf necrosis and lipid peroxidation and increased tolerance to high light and high temperatures  in 20092 stage,  and expr760 and S500 are the canopy reflectance at 760 nm and 500 nm wavebands, ‘k’ is the reference spectrum constant that represents the mean of the S500/S760 ratio of all genotypes at a given sampling, 1.171 is the intercept, and 2 is the factor applied to convert concentration (μg mL-1) to content (μg cm2).Canopy spectral reflectance was measured using an ASD FieldSpec, FR spectroradiometer  between 54 and 57 DAP in 2009 and 58 and 61 DAP in 2010 as previously described , and coiDescriptive statistics and Pearson correlation analysis were conducted using PROC MEAN and PROC CORR procedures of SAS Version 9.3 . To derive phenotypes for genome-wide association mapping, best linear unbiased predictions (BLUPs) values were determined to reduce error variance and shrink the phenotypic values towards the mean . For eack) ranging from 1 to 10. The correct estimation of k was provided by joining the log probability of data [LnP(D)] from the STRUCTURE output and an ad hoc statistic Δk . In. In58].  windows . Separatk = 8) and used along with kinship matrix (K) for association mapping.Association mapping was conducted based on the BLUP values using a mixed linear model (MLM) with Q-matrix and K-matrix. The Q and K matrices were used as corrections for population structure and/or genetic relatedness , 60–63. qFDR < 0.05 were considered significant. All markers that satisfied the multiple testing threshold (qFDR < 0.05) had–log10 P values ≥ 3.2, which is greater than the threshold  used in other published reports for soybean  and the other as lipoxygenase  and 15 (tCaro), and one of the loci on chromosome 10 (iCaro) . The proCanopy spectral reflectance characteristics can be assessed rapidly and non-destructively and are used for numerous purposes. In this study, two methods (tCaro and iCaro) were used to determine canopy carotenoid content based on the same canopy spectral reflectance measurements. Genome-wide association analysis using these two methods resulted in the identification of nine putative loci for iCaro and five putative loci for tCaro, including three loci that were identified for both . FourteeGenome-wide association mapping using a mixed linear model (MLM+Q+K) resulted in the identification of 28 SNPs putatively associated with soybean leaf and canopy carotenoid contents. These SNPs likely represented 14 putative loci associated with three different measures of carotenoid content of 332 soybean genotypes. The fact that these putative loci were identified based on data from two distinct growing seasons provides added confidence in their accuracy and reliability. Candidate loci identified based on canopy spectral reflectance characteristics  indicate that markers for canopy carotenoid contents can be identified and that high-throughput phenotyping based on canopy spectral reflectance can provide useful phenotypes for association mapping.S1 Fig(PDF)Click here for additional data file.S2 Figk = 8 indicates the optimum number of subgroups possible in the panel.The plateau of the graph at (PPTX)Click here for additional data file.S3 FigThe y-axis is the subgroup membership, and the x-axis is the individual genotypes in each sub population (G1–G8). All 31,253 markers with MAF ≥5% was used for analysis.(PPTX)Click here for additional data file.S4 Fig(PPTX)Click here for additional data file.S1 TableThe three methods were extractable carotenoid contents (eCaro), wavelet transformed spectral reflectance carotenoid contents (tCaro), and spectral index carotenoid contents (iCaro).(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The GSH/GSSG redox potential has become a fashionable electrochemical parameter believed to be a major driving force of redox reactions regulating biological events  of such compounds are usually not determined directly, but estimated by means of the Nernst equation from concentration changes after equilibration with other redox couples of seemingly known standard potential -phosphorylation, albeit thermodynamically favorable, is not controlled by the ΔG for ATP hydrolysis or Atkinson's highly quoted “energy charge”  that regulates cytoskeletal organization and thereby axonal outgrowth/repulsion. The biological activity of CRMP2 depends on posttranslational modifications. Redox regulation of CRMP2 during development of the zebrafish brain requires activity of the vertebrate-specific Grx2 (Bräutigam et al., 2O2. However, the bimolecular rate constants for the spontaneous reactions of low molecular mass thiols with hydroperoxides hardly reach 30 M−1s−1 (Winterbourn and Metodiewa, 7 and 108 M−1 s−1, respectively (Trujillo et al., The intention of this article was to underscore the priority of enzyme catalysis vs. thermodynamic or electrochemical parameters in GSH-dependent redox events. Although any kind of kinetically competitive reaction may interfere with a slow equilibration between redox couples, enzymatic ones are the most likely candidates. For example, thiols, in particular GSH, easily reduce HAll authors jointly wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lung cancers account for the majority of brain metastases which pose major therapeutic challenges. Biomarkers prognosticating for the development of brain metastases in patients with non-small cell lung cancers (NSCLC) may improve personalized care. Six serum proteomic biomarkers were previously investigated at Memorial Sloan Kettering but their associations with brain metastases were unknown.epidermal growth factor receptor (EGFR) mutation status were evaluated for association with the baseline presence of brain metastases using logistic regression and multivariable analysis. For patients without brain metastases at baseline, the cumulative incidence of subsequent brain metastases were compared according to baseline biomarkers and clinical factors using Gray’s test.Serum NSE, CYFRA 21–1, ProGRP, SCC-Ag, TIMP1, and HE4 by ELISA-based proteomic assays were prospectively collected from consecutive patients with stage IV NSCLC. Pre-treatment serum biomarker levels as well as age, histology, and EGFR mutation (p = 0.2), but this was not statistically significant possibly due to small sample size.A total of 118 patients were enrolled, 31  had brain metastases at baseline and a further 26  developed brain metastases subsequently. Pre-treatment serum biomarker levels were available in 104 patients. There was no significant association between the six serum biomarkers and the baseline presence or subsequent development of brain metastases. Age younger than 65 years was the only clinical factor significantly associated with brain metastasis at baseline  by multivariable analysis. A trend toward increased cumulative incidence of subsequent brain metastases was observed in patients with Serum NSE, CYFRA 21–1, Pro-GRP, SCC-Ag, TIMP1, and HE4 are not significantly associated with brain metastases. Our methods taking into account follow-up time may be applied to independent datasets to identify a patient cohort with a higher biologic propensity for developing brain metastases. Such information may be useful for the study of agents targeting the development of brain metastases. Desppatients –9. The dpatients , 11.British Journal of Cancer by Jacot et al [EGFR mutation status, associate with the development of brain metastases, taking into account survival and follow-up time.A previous study published in the ot et al  have fouot et al . Our groot et al . In furtEGFR mutation status were evaluated for association with the baseline presence and subsequent development of brain metastases.This research was approved by the Memorial Sloan Kettering Cancer Center (MSK) Institutional Review Board. We conducted a prospective study at MSK with the primary objective of assessing the prognostic value of serum-based biomarkers  . ConsecuFujirebio Diagnostics AB, Sweden) was used with two monoclonal antibodies directed against the αγ form of the glycolytic enzyme enolase . The CYFRA 21–1 EIA  was used with two monoclonal antibodies (MAb) specific for cytokeratin 19 in serum. The CanAg ProGRP EIA  non-competitive assay was used. The CanAg SCC EIA non-competitive immunoassay  was performed using the direct sandwich technique. The quantitative sandwich enzyme immunoassay was used to assess Human TIMP1 . The HE4 EIA  was used with two mouse monoclonal antibodies (2H5 and 3D8) directed against two epitopes in the C-WFDC domain of HE4.Samples were collected, stored at -80°C, processed and analyzed at a MSK Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. We performed serum biomarker analysis using validated commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kits. The CanAg NSE EIA non-competitive immunoassay  was added to each well. For TIMP1, after washing an enzyme-linked polyclonal antibody specific for TIMP1 was added to the microplate. After washing, a substrate solution was added to each well.The levels of biomarkers were dichotomized at the upper limit of normal based on previously published data: NSE (20 ng/ml), CYFRA 21–1 (3.3 ng/ml), Pro-GRP (50 pg/ml), SCC-Ag (2.5 ng/ml), TIMP1 (58.9 μg/L), and HE4 (83 pmol/L) –19. DataEGFR mutation have prognostic value for brain metastasis, we first analyzed their association with presence or absence of brain metastasis at stage IV diagnosis and then with development of subsequent brain metastasis among patients who did not have baseline brain metastasis. Association of factors with presence of brain metastasis was evaluated using logistic regression. Univariate analysis was performed on all variables, and if no significant association was observed, then no further multivariable analysis was required. If a factor was found to be significantly associated with brain metastases on univariate analysis, a multivariable logistic regression model was fitted to evaluate the association adjusted for clinically relevant covariates.To investigate whether these serum biomarkers  and clinical factors including age, histology and cmprsk package in R version 3.1.1 (http://www.R-project.org). All other statistical analysis was performed using SAS 9.4 Next, we captured the dates of the subsequent development of brain metastases in patients who did not have baseline brain metastasis. A time-to-event approach with competing risk methodology was used to analyze this outcome as it takes into account differences in follow-up time and the numerous deaths in this metastatic population that preclude observing a brain metastasis . We usedTable 1). The median age was 64 (range 36–85), 68 were women and 50 were men. By histologic subtyping, 99 had adenocarcinoma, 13 had squamous cell carcinoma, and 6 had NSCLC not otherwise specified. Baseline pre-treatment serum biomarkers were available for 104 patients. Attrition was due to either missed pre-treatment collection or lost samples. EGFR mutation testing was performed on 44 patients as testing was by clinical selection per standard of care at the time. Of those tested, 17 patients had EGFR mutation, of whom 11 (65%) had brain metastases either at baseline or subsequently.A total of 118 patients with stage IV NSCLC who were treatment naïve were enrolled, 57 (48%) of whom developed brain metastases, detected either at diagnosis  or subsequently during their course of treatment  (. Median follow-up among survivors in this cohort was 7 years (range 3.6–9 years). The number of patients with baseline biomarkers high vs. normal in relation to brain metastases are detailed in Table 2.Of the 104 patients who had pre-treatment baseline serum biomarkers collected, there was an even distribution of patients with brain metastases at baseline , as compared to patients who subsequently developed brain metastases  Table 3).For each serum biomarker tested at baseline , univariate analysis of individual biomarkers did not yield any association with the presence of brain metastasis at baseline thus no further multivariable analysis was performed . There was a trend toward higher cumulative incidence of brain metastases among patients with high baseline HE4 compared to those with normal levels of HE4 but this was not statistically significant (P = 0.07). No further multivariable analysis was performed since no factors were significantly associated with the cumulative incidence of subsequent brain metastasis. Statistical comparisons of subsequent brain metastases based on TIMP1 and SCC-Ag levels were not possible due to too few patients or events (Table 2).In patients without brain metastasis at baseline, there were no significant differences between patients with high vs. normal pre-treatment biomarker levels in relation to the incidence of subsequent brain metastases across all time points  . However, among patients without baseline brain metastases, there was no significant difference between the age groups in the incidence of subsequent brain metastases (P = 0.5) .Compared to patients aged 65 years or older, patients younger than 65 years had significantly more brain metastases at baseline  Table 3). In patients without brain metastasis at baseline, there was no significant difference between patients with adenocarcinoma and squamous cell carcinoma histology in relation to their cumulative incidence of subsequent brain metastases across all time points (P = 0.51) .With regard to histology, there were no significant differences between patients with adenocarcinoma and squamous cell carcinoma histology in relation to the baseline presence of brain metastasis   had brain metastases by the end of the study, including 4 at baseline and 7 subsequently. In comparison, only 11 of 27 patients (41%) tested negative for EGFR mutation had brain metastases by the end of the study, including 5 at baseline and 6 subsequently (Table 1). The OR associating EGFR mutation with baseline brain metastases was 1.35 (P = 0.69) (Table 2). In patients without brain metastases at diagnosis of stage IV NSCLC (n = 35), there was a trend toward higher cumulative incidence of brain metastases among patients tested positive for EGFR mutation compared to those tested negative. (P = 0.22) .Of the 44 patients tested for et al [et al [et al [et al [Our study did not find a significant association between any of 6 pre-treatment serum biomarkers and the baseline presence or the subsequent development of brain metastases in patients with stage IV NSCLC. Our study refuted the hypothesis by Jacot et al  that serl [et al  examinedl [et al  also shol [et al  did confl [et al  that basTo date, no serum biomarker for brain metastases has been validated in patients with NSCLC. Lee et al  found thThe observation from this study that patients with age younger than 65 years are significantly associated with brain metastases at baseline is consistent with previously published reports , 23. A lEGFR mutation at the time of this study, we were not able to conclusively solve the controversy regarding the association between EGFR mutation and the development of brain metastases. However, our methods were substantially different to previous studies in attempting to address this question [EGFR mutations within this selected group without accounting for survival and time to development of brain metastases [EGFR mutant lung cancers, it has never been shown whether this is due to a biologic propensity or simply a result of the longer survival of these patients due to EGFR targeted therapy [Due to the limited number of patients who had undergone question –9. Many tastases , 5, 7. W therapy –26. In oEGFR mutation testing have prevented any firm exploratory statistical analysis. Attrition due to missed collections or lost samples highlights the practical challenges in designing a biomarker study. Despite these limitations and negative results, there was no evidence to suggest that the serum biomarkers studied can be used clinically to prognosticate for the development of brain metastases. While there was a trend toward an association between high baseline HE4 and increased subsequent development of brain metastases (p = 0.07), such a biomarker cannot be recommended for clinical use without independent validation of a highly significant association. Given serum HE4 was recently shown to be associated with poor prognosis in patients with NSCLC [There are several limitations in this study. The analysis was done retrospectively which may have introduced bias. Furthermore, the sample size was relatively small and the limited number of th NSCLC , furtherEGFR mutation or other biomarkers and the development of brain metastases over time, independent datasets may be analyzed using the same methods.The main strength of this study is our methods in determining the association of biomarkers with brain metastases both uniformly at baseline and subsequently, and accounting for time and patient survival. In order to confirm the association between It is worth pointing out that while non-invasive prognostic biomarkers for the development of brain metastases are important for improving personalized therapies of patients, such biomarkers must be shown to be highly sensitive and specific in order to be clinically useful. Any positive association found by exploratory analyses must be independently validated before clinical application . Using sIn conclusion, this independent biomarker study found that the 6 pre-treatment serum biomarkers including NSE were not associated with the baseline presence or subsequent development of brain metastases in patients with metastatic lung cancers. Our methods may be applied to independent datasets to identify a patient cohort with a higher biologic propensity for developing brain metastases taking into account difference in follow-up time. Such information may be useful for the study of agents targeting the development of brain metastases.


PIK3CA) resulted in profoundly increased migration where extracellular multiscale directed migration cues and intrinsic signaling synergistically conspire to greatly outperform normal cells or any extracellular guidance cues in isolation.To investigate complex biophysical relationships driving directed cell migration, we developed a biomimetic platform that allows perturbation of microscale geometric constraints with concomitant nanoscale contact guidance architectures. This permits us to elucidate the influence, and parse out the relative contribution, of multiscale features, and define how these physical inputs are jointly processed with oncogenic signaling. We demonstrate that collective cell migration is profoundly enhanced by the addition of contract guidance cues when not otherwise constrained. However, while nanoscale cues promoted migration in all cases, microscale directed migration cues are dominant as the geometric constraint narrows, a behavior that is well explained by stochastic diffusion anisotropy modeling. Further, oncogene activation (i.e. mutant  Directional cell migration is essential for many normal and pathologic processes and is commonly regulated by gradients in chemical or physical factors and micro- and nano-structural patterns of the extracellular matrix (ECM)1235689521113141565PIK3CA mutations with features of transformation, e.g.17PIK3CA mutations1920613PIK3CA, the p110α catalytic subunit of PI3K, frequently mutated in a number of different human cancers, including those of the breast2324The effects of either ECM structure or specific oncogene activity on tumor cell motility are frequently investigated separately. However, the combinatory effect of these intra- and extra-cellular factors on the tumor cell motility and invasion has not been studied extensively and is not yet well understood. For instance, little is known regarding how specific oncogenic mutations, such as those of the phosphatidylinositol 3 kinase (PI3K) pathway that frequently occur in human breast cancer, influence how a cell interacts with the fundamental ECM architectures of the tumor microenvironment (TME). Interestingly, although studies have implicated Cell patterning on a defined substrate is a critical challenge so that fundamental questions about cell-cell and/or cell-substrate interactions and the systematic mechanisms driving their behavior in response to the local environment can be elucidated26272829303133343740433245464849To this end, we develop an engineered biomimetic cell culture platform that allows for the integrative study of complex stimuli, including micro and nanotopographic cues, and selective ECM architecture, to elucidate the roles of critical extracellular and intracellular factors and their interrelationship in the regulation of cell migration. We perform capillary force lithography and plasma lithography patterning on PDMS substrates with different geometries to guide and promote cell migration. Our results reveal that human breast epithelial cells and their genetic derivatives collectively sense and respond to the engineered microenvironment by integrating the microstructural and mechanical properties of the substrate during cell migration. Furthermore, we demonstrate that the biomimetic approach to modulate substrate-mediated cell migration may reveal essential factors that are responsible in phenotypic expression of oncogenic mutations. The results of this study signifies that topographic cues play an essential role in oncogenic activation and tumor cell metastatic behavior. Further investigations on the role of multiscale cues would be beneficial in understanding the underlying mechanisms of tumor metastasis and assist possible therapeutics development.Three different lithographic techniques were applied to create spatial cellular patterns on nanogrooved elastomeric substrates. Namely, the engineered biomimetic cell culture platform was composed of three major parts; a nanogrooved elastomeric substrate fabricated by UV-assisted capillary force lithography and PDMS molding, a microchannel patterned PDMS stamp fabricated by soft-lithography, and selectively modified ECM patterns prepared by plasma lithographic process. The large area nanogroove pattern on a silicon master (2 inches by 2 inches) was successfully transferred to the thin PUA template fabricated by UV-assisted capillary force lithography. The SEM image shown in PDMS microstamp-assisted plasma lithographic techniques were used to create spatial surface patterns for MCF-10A culture on the nanogroove patterned elastomeric substrates , step 3.2 for all experimental conditions  by tracking individual cell positions within each image.Our previous studies demonstrate that substrate nanotopography allows cells to align along the nanogroove direction and promotes cellular attachment45bstrates ,C or on bstrates ,D. SeconPIK3CA cells % faster (p < 0.001) along nanogrooved substrates than on flat substrates suggesting that encountering topographic nanogrooves results in altered motility dynamics to enhance migration speed. Moreover, oncogenic PIK3CA knockin cells migrate on average 53 (±2)% faster (p < 0.001) on both flat and nanogrooved substrates than their wild type counterparts, demonstrating that oncogenic mutations of PI3K enhance cell migration, consistent with the concept that PIK3CA may promote breast cancer metastasis. Additionally, directional persistence of both cell types  to measure trajectories including migration direction and distance . As expected, as the channel width becomes much larger than the migration persistence length the model predicts classic random 2D migration behavior for cell undergoing a random walk  bottom. PIK3CA MCF-10A lines. Selectively patterned cells migrated only on the micropatterned ECM with no remarkable difference in migration speed between cells in the middle and at the edge of the patterns consistent with earlier experiments. The nanotopography dimensions designed to provide contact guidance on the biomimetic cell culture platform were 800 nm ridges, 800 nm grooves with a height of 600 nm  to measure trajectories including migration direction and distance  shifted from 5.6 μm/hr and 8.6 μm/hr respectively, to >20 μm/hr and >40 μm/hr at 30 μm ECM pattern widths. Yet, cell migration along the ECM patterns on both substrates was up to 5.6 times faster due to the synergistic effect of substrate nanotopography and surface patterning compared to conditions that lack the nanotopography and ECM micropatterns. Furthermore, migration speed of the oncogenic PIK3CA knockin cells on both flat and nanogrooved substrates were 97% faster than their wild type cells on average  is represented asIn our experiments, with straight-edge pattern cells  that enhance migration may be conceptualized in this context as a convective driving force  additive to the diffusive flux behaviorwhere for the 2D problem this would reduce and account for anisotropy in the x and y directions. This is analogous to previous work describing directional dependent random walkIn this context the convective driving force would account for fundamental changes in the propulsion force resulting from alterations in the mechanics of cell migration resulting from contact guidance architectures. Furthermore, the ratio of these terms may have implications for characterizing the dominant cues through a dimensionless parameter, as would be the case for dominant micron scale directed migration cues in narrow channels e.g.  and 5, bPIK3CA expression resulted in significantly increased cell migration versus parental MCF-10A cells under baseline (i.e. wide and flat) conditions. In addition, exposure to nanoscale contact guidance cues significantly promoted migration of mutant cells. What is perhaps surprising is that parental MCF-10A cells showed only modestly increased migration with the addition of nanoscale contact guidance cues in the context of narrow patterns microscale geometric constrains. In contrast, oncogenic cells display a remarkable synergy where the additive benefits of microscale, nanoscale, and PI3K signaling are realized. As a result, these cells vastly outperformed wild type cells under any of the directed migration cues, suggesting that oncogenic breast epithelial cells are more heavily influenced by multiscale directed migration cues in the environment. These observations suggest that as the pattern width diminishes, microscale directed migration cues become more dominant, appearing to exceed the influence of nanotopography induced contact guidance for collective cell migration in this context. Indeed these results suggest that the activation of a single oncogene may confer a distinct advantage for migration through a given ECM architecture and this response may correlate the metastatic potential of carcinoma cells. Further, these finding support the conclusion that oncogene activation in this context resulted in a fundamental change in adhesion-mediated signaling and the dynamic cytoskeletal remodeling underlying sustained locomotion through persistent protrusion of leading edge lamellipodia over time. Yet, the exact processes by which these oncogenic cells display vastly increased persistence, as well increased speed, to profoundly increase migration remain to be elucidated. Certainly, clarifying the interrelationships between the multiscale directed migration cues presented here and the underlying intrinsic cell structure and signal transduction in a quantitative manner will add considerably to our understanding of the fundamental mechanisms regulating normal and cancerous cell migration.PI3K pathway signaling regulates numerous critical behaviors required of transformed cells, including growth, survival and invasion192324To prepare topographically nanogroove-patterned  elastomeric substrate, a mold made of polyurethane acrylate (PUA) was fabricated from a silicon master patterned via e-beam lithography  including photoresist development , deep reactive ion etching (STS ICP Etcher), and ashing process (BMR ICP PR Asher) as previously described5128The process of making cellular patterns on the substrate is composed of two different lithographic techniques. Photolithographic and plasma lithographic techniques were used to fabricate a microchannel-patterned PDMS stamp and create selective hydrophilic micropatterns, respectively. Thus creating a hydrophilic and hydrophobic array allows for cell adhesion on alternating lines . First, 66Next, the surface of the substrates were modified using plasma lithographic techniques35PIK3CA knockin cells . Culture procedures for the MCF-10A line and its derivatives has been described previously1920PIK3CA knockin cell line were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (1:1) (Life Science) supplemented with 5% horse serum, 20 ng/mL EGF, 10 g/mL insulin, 0.5 g/mL hydrocortisone, and 0.1 g/mL cholera toxin. All supplements were purchased from Sigma-Aldrich, unless otherwise noted. To generate the PIK3CA knockin line, targeting vectors were designed to introduce a single oncogenic mutation within PIK3CA. Vector transduction, colony selection, clone screening, and Cre recombinase removal of the neomycin resistance gene have been described previously682 to 1800 cells/mm2, and then cells were allowed to adhere to the flat or nanogrooved elastomeric substrates. Prior to the start of imaging and data acquisition, cells were additionally cultured with a flat stencil or a microstamp to create a dense cell monolayer as described above.All studies in this article were performed using stable lines of human-derived MCF-10A breast epithelial cells and MCF-10A mutant After initial cellular patterns were created, as shown in 5455t) the particle undergoes a displacement (δ) along a randomly chosen direction. Here the trajectory direction vector is limited so the x component is in the positive direction to account for constraints imposed by cells behind the leading edge in collective migration. Likewise, the particle displacement is constrained by restricting movement outside of the defined boundary. Similar, adjacent cells in a cell sheet can be viewed as a lateral constraint. At each time step, the displacement was evaluated to determine if it resulted in the particle crossing the boundary. If the particle crossed the boundary the displacement beyond the boundary was rejected. If the particle did not cross the boundary the next random walk displacement was implemented. Following 100,000 steps/particle averaged over 1,000 particles the mean displacement in the x direction was determined for varying widths of the migration pattern.To implement the diffusion anisotropy model presented here, particles (representing cells) perform a random walk within geometrically constrained two-dimensional space (varying constraint in the y direction). Following random walk behavior a particle moves stepwise in a random direction. That is, at each time step .