This was studied in serum-free organ cultures of microdissected human scalp HFs and skin, i.e. excluding pituitary, neural and vascular inputs. Prolactin expression was confirmed at the gene and protein level in human truncal skin, where its expression significantly increased (p = 0.049) during organ culture. There was, however, no evidence of PRL secretion into the culture medium as measured by ELISA. PRL immunoreactivity (IR) in female human epidermis was decreased by substance P (p = 0.009), while neither the classical pituitary PRL inhibitor, dopamine, nor corticotropin-releasing hormone significantly modulated PRL IR in HFs or skin respectively. Interferon (IFN) γ increased PRL IR in the epithelium of human HFs (p = 0.044) while tumour necrosis factor (TNF) α decreased both PRL and PRLR IR. This study identifies substance P, TNFα and IFNγ as novel modulators of PRL and PRLR expression in human skin, and suggests that intracutaneous PRL expression is not under dopaminergic control. Given the importance of PRL in human hair growth regulation and its possible role in the pathogenesis of several common skin diseases, targeting intracutaneous PRL production via these newly identified regulatory pathways may point towards novel therapeutic options for inflammatory dermatoses.Human scalp skin and hair follicles (HFs) are extra-pituitary sources of prolactin (PRL). However, the intracutaneous regulation of PRL remains poorly understood. Therefore we investigated whether well-recognized regulators of pituitary PRL expression, which also impact on human skin physiology and pathology, regulate expression of PRL and its receptor (PRLR)  Whilst prolactin (PRL) is appreciated for its role in the modulation of hair growth, both in human and other mammalian species in vitroet al.et al failed to identify PRL gene expression in both normal and pathological skin et al could not identify PRL gene expression in involved skin in psoriasis using reverse transcriptase polymerase chain reaction However, in human skin, the published literature has only confirmed scalp skin and scalp hair follicles (HFs) as cutaneous sources of extra-pituitary PRL production Furthermore, although the regulation of pituitary PRL synthesis and release has been extensively studied in vitro regulation of PRL and PRLR in human skin and offers unparalleled accessibility and clinical utility Given (i) the lack of any universal PRL stimulatory/inhibitory factor in vitro. Moreover, we asked whether selected regulators of pituitary PRL synthesis and/or secretion and pro-inflammatory cytokines alter epidermal and follicular PRL expression. Finally, we examined whether there are any differences between the regulation of PRLR expression in the skin and the HF.Therefore we determined whether healthy corporal human skin expresses PRL and PRLR expression at the gene and protein level, and establish whether there are any time-dependent changes in cutaneous PRL and PRLR expression in organ culture Human skin was obtained as by-products of cosmetic surgery, after written informed consent was provided. Full ethical approval was obtained from the University of Lübeck ethics committee, according to the Helsinki Declaration. For the determination of PRL and PRLR immunoreactivity (IR), immunohistochemistry was performed as described previously )  method was used. The application of antibody diluent alone served as the negative control. AEC  was again applied as the chromogen, prior to counterstaining with haematoxylin and mounting with Faramount .For PRLR immunohistochemistry, PRLR antibody Immunoreactivity for PRL and PRLR in the HF and sebaceous gland served as internal positive controls, based on previously published expression patterns In order to investigate the effect of organ culture itself on PRL and PRLR IR, 4 mm punch biopsies were obtained from redundant corporal skin, by-products of cosmetic surgery,  and placed in serum free culture medium as described previously Microdissected female anagen VI HFs were isolated from redundant scalp skin as described To enable a comparison between the effects of PRL on PRLR protein expression in the skin and HF, 7 day organ culture with PRL (400 ng/ml) was used. For skin organ culture, skin from 3♀ subjects (aged 42–63 years), was compared with HFs from 4♀ subjects (aged 49–68 years). For the remaining HF studies, the following substances were used, dopamine 75 IU/ml IFNγ does not induce catagen, while higher doses would have greatly reduced the percentage of anagen HFs. For qRT-PCR analyses, skin and/or HFs were treated for either 24–48 hours or 7 days (see figure legends for details).In order to determine whether PRLR-mediated signaling involved the canonical JAK2/STAT5 pathway, phosphorylated STAT5 levels were examined using immunohistochemistry, employing phospo-STAT5 (Tyr694) rabbit monoclonal antibody to detect STAT5a and b proteins when phosphorylated at Tyr 694 . After fixation in acetone and blocking of endogenous peroxidase, blocking solution (1% horse serum in 3% BSA-TBS-Tween) was applied for 60 minutes, followed by overnight incubation with the primary antibody at 4°C. Horse anti-rabbit biotinylated secondary antibody  was then applied for 30 minutes at room temperature, followed by the Vectastain Elite ABC®  detection technique as described above, with 3,3' diaminobenzidine (DAB) used as the chromogen.Specific mRNA transcripts of were analyzed by qRT-PCR as described (40), using TaqMan primers and probes . As internal housekeeping gene controls for the qRT-PCR experiments, transcripts of glyceraldehyde 3-phosphate dehydrogenase , β-actin , and cyclophilin A  were determined. The amount of the aforementioned transcripts was normalised to those of the most stable control gene using the ΔΔCT method. All experiments were performed in triplicate. The expression of the target genes was normalized to that of the relevant control group and independent data from 2–3 different donors were pooled, and expressed as fold changes. Statistical analysis was performed by paired or unpaired two-sample Student’s t-test.To determine whether there was any evidence of PRL secretion into the culture medium, ELISA was performed on conditioned culture media from two subjects from day one and day five. This media was from skin (♀ 31 years) and HF organ culture (♀ 52 years) respectively. ELISA was performed as described previously Given that PRL and PRLR are prominently expressed in the outer root sheath (ORS) compartment of human HFs, human ORS keratinocyte cell culture was performed to verify whether PRL and PRLR transcripts are also expressed in isolated HF keratinocytes. Moreover, we checked the expression of PRL and PRLR after treatment with TNFα (5 ng/ml for 6 hours) and tested whether this treatment influenced expression of STAT5, a key molecule in PRLR mediated signaling. ORS keratinocytes were obtained from human HFs (2♀ subjects aged 25–26 years) after digestion with trypsin Standardised reference areas in the ORS were measured for PRL and PRLR IR using National Institute of Health Image J software  Statistical analysis was performed using GraphPad Prism, Version 5 . p<0.05 was considered as statistically significant and results were expressed as the mean +/− standard error of the mean (SEM). To combine the results from the independent quantitative (immuno-) histomorphometry experiments, the results from the treatment groups were normalized to the control group (set at 100) and the results were pooled. Significance was determined as *p<0.05, **p<0.01, ***p<0.001.To determine whether PRL protein was expressed in corporal skin, PRL immunohistochemistry was performed. Prolactin specific staining was present in each subject, in a cytoplasmic distribution in the basal epidermal layer . This waNext, we determined the expression of PRLR in human corporal by immunohistochemistry. PRLR protein expression was most notable in the basal epidermal layer, again in a cytoplasmic distribution  in each Given that substance P is a recognized potential regulator of pituitary PRL secretion As a central hormone in the systemic stress response, which may also regulate pituitary PRL secretion in situ was significantly increased by IFNγ treatment  significantly decreased PRLR IR in the ORS of human HFs  after 7 Given that PRL and PRLR IR was prominent in the ORS, we checked PRL and PRLR gene expression in cultured ORS keratinocytes, which expressed PRL and PRLR at the gene level. As in organ-cultured intact human HFs, TNFα 5 ng/ml significantly decreased PRL gene expression in isolated, cultured ORS keratinocytes . In contIn order to determine whether the PRLRs detected in human skin are functional, we also examined whether PRL treatment activated epidermal STAT5 phosphorylation. STAT5 is the main mediator of PRLR signalling Finally, we assessed whether PRL impacts on the intracutaneous expression of its own receptor and whether there are any differences in the regulation of PRLR by its ligand between defined skin compartments. As expected, prominent PRLR IR was seen in the ORS and did not change significantly after PRL treatment on the protein level , Nor didin vitro, using a well established immunohistomorphological method for quantifying protein expression, measuring the intensity of protein IR in situet alin vivo, for example in response to skin trauma or psychological stress.This study demonstrates that contrary to a previous report While we found no evidence that PRL is actively secreted by the skin and/or HFs, PRL secretion into the culture medium cannot be excluded, since it could well have been below the detection limit of the employed ELISA, and may have been detectable with the more sensitive Nb2 cell proliferation bioassay in situ in human truncal skin and HFs. In contrast, dopamine, the classical negative regulator of pituitary PRL secretion, did not exert a significant regulatory effect on PRL expression in human HFs. We deliberately used dopamine itself rather than specific dopamine receptor agonists, for example cabergoline or bromocriptine, as there is no evidence at present that human HFs, or even skin, express the dopamine receptor 2 to which these agents bind. To data, only dopamine receptor 1 transcripts have been demonstrated in human skin and HFs Importantly, the study identifies the key stress mediator and neurogenic inflammation-associated neuropeptide, substance P, and the pro-inflammatory cytokines, IFNγ and TNFα as regulators of PRL protein expression Our group has consistently identified PRL and PRLR expression in female corporal skin from several locations, in a distribution pattern strikingly similar to that seen in scalp skin Given that the expression of the pituitary hormone pro-opiomelanocortin (POMC) in murine skin is reportedly up to 10,000 times lower than that in the pituitary To confirm the activity of PRLR-mediated signaling, STAT5 phosphorylation was also investigated at the protein level. In all three subjects tested, PRL treatment increased STAT5 phosphorylation at day 7. This provides preliminary evidence that cutaneous PRLR-mediated signaling can be transduced via the canonical Jak2/STAT5 pathway. However, the extent to which other recognised signaling pathways/proteins may be involved, for example the Ras/Raf/MAPK, and Src kinases or PI3K/Akt pathways, needs to be clarified in future studies. The data also provides indirect evidence that the long form of the PRLR is expressed in the skin as short PRLR isoforms are unable to activate Jak/STAT pathway.The observed effect of IFNγ on PRL expression did not seem to be related to changes in PRLR expression, since the latter was unchanged. PRL is an important immunomodulator, possibly with overall pro-inflammatory effects in response to the immunosuppressive effects of stress In addition to stimulators of PRL and/or PRLR expression we have also identified inhibitors. Substance P decreased PRL IR in the epidermis, whilst TNFα decreased PRL and PRLR IR in the HF. Given that substance P is a key mediator of neurogenic skin inflammation in situ depends on the presence of as yet unidentified HF mesenchyme-derived factors. The down-regulation of follicular PRLR expression by TNFα is also intriguing in view of the importance of TNFα in both inflammatory dermatoses such as psoriasis TNFα treatment actually increased PRLR transcription in cultured ORS keratinocytes, in contrast to its effect on PRLR protein expression in the ORS of the intact HF. This raises the question of whether the regulation of PRLR expression in human HF epithelium It is now appreciated that PRL is an important regulator of human hair growth Figure S1CRH does not alter PRL protein expression in human skin. PRL IR in the control skin (A) was not significantly different to that in (B) CRH treated skin. Positive and negative controls are shown in (C) and (D). Negative control was via omission of the primary antibody and the outer root sheath of the hair follicle and sebaceous gland served as internal positive controls. Quantitative IR measurement, the epidermis, is shown in (A) and evaluation in (E). Results were pooled from 3 ♀ subjects, aged 45–57 years.(TIF)Click here for additional data file.Figure S2Thyroid hormones have no significant effect on PRL and PRLR expression in human skin. (A) PRL IR in the epidermis of control skin was not significantly different to that in skin treated with (B) triiodothyronine (T3) or (C) thyroxine (T4) for 24 hours as demonstrated by quantitative measurement of PRL IR (D). Results pooled from 5♀ subjects aged 45–71 years. Similarly, PRLR IR in the epidermis of control skin (E) was not significantly different in (F) T3 or (G) T4 treated skin as quantified in (H). Results pooled from 3♀ subjects aged 56–71 years. Indeed, neither 24 hours of treatment with T3 (I) or T4 (J) significantly affected PRLR gene expression. Results pooled from 2♀ subjects aged 46 and 60 years.(TIF)Click here for additional data file.Figure S3Dopamine does not alter PRL protein expression in human hair follicles. PRL IR in control hair follicles (A) was not significantly different to that in Dopamine (1000 nM) treated hair follicles (B). Negative control (C). Quantitative IR measurement revealed no significant differences across the range of Dopamine concentrations tested (D) 1000 nM  and (E) 10–100 nM in 2♀ subjects aged 47–64, 15–21 HFs per group in total). Areas measured by image J are shown in (A).(TIF)Click here for additional data file.Figure S4Dopamine does not regulate PRLR expression at the gene or protein level in human hair follicles. PRL IR in the control hair follicles (A) was unchanged compared with that after Dopamine (B) 10 nM, (C) 100 nM or (D) 1000 nM treatment. (E) Negative control. Quantitative IR measurement revealed no significant differences across the range of Dopmaine concentrations tested (F) 1000 nM  and (G) 10–100 nM in 2♀ subjects aged 47–64, 14–18 HFs per group in total). (H) Dopamine 1000 nM exerted no significant effect on steady PRLR gene expression after 48 hours (56 ♀ subject).(TIF)Click here for additional data file.Figure S5IFNγ has no effect on PRLR immunoreactivity in human hair follicles at the gene or protein level. (A) PRLR IR in the outer root sheath of control hair follicles (black arrows) was unchanged by treatment with (B) IFNγ 75 IU/ml. (C) Negative control. (D) Quantitative analysis showed no significant difference in PRLR. Results were pooled from 3♀ subjects, aged 44–68 years, 13–16 HFs in total from three patients. **Arbitrary units could not be normalised due to low number of anagen hair follicles. Results were pooled from the same subjects as in (TIF)Click here for additional data file.Figure S6No evidence of compartment specific regulation of PRLR expression in human skin. PRLR IR is not significantly reduced by PRL 400 ng on the protein level in the outer root sheath of HFs after serum-free organ culture (A–D). Results pooled from 4 ♀ subjects (aged 49–68 years). 23 HFs per group in total. Moreover, epidermal PRLR IR was also unchanged after organ-culture (E–H). Results pooled from 3 ♀ subjects described in (TIF)Click here for additional data file.Table S1The differential regulation of pituitary and extrapituitary PRL synthesis and release. There is, at times, a bewildering array of seemingly contradictory regulatory effects on PRL and PRLR expression between depending on the site of PRL production. Some of these are due to methodological factors. Abbreviations: T3:triiodothyronine, T4:thyroxine.(DOC)Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that cannot be slowed substantially using any currently-available clinical tools. Through decades of studying sporadic and familial ALS (SALS and FALS), researchers are coming to understand ALS as a complex syndrome with diverse genetic and environmental etiologies. It is know appreciated that motor neuron degeneration in ALS requires active (gain of function) and passive (loss of function) events to occur in non-neuronal cells, especially astrocytes and microglia. These neuroinflammatory processes produce paracrine factors that detrimentally affect motor neurons, precipitating protein aggregation and compromising cytoskeletal integrity. The result is a loss of neuronal homeostasis and progressive die-back of motor axons culminating in death of the afflicted motor neurons. This review will discuss experimental therapeutics that have been tested in murine ALS models, with an emphasis on those that have progressed to human clinical trials. Reasons will be considered for the frequent failure of preclinical successes to translate into positive clinical outcomes. Finally, this review will explore current trends in experimental therapeutics for ALS with emphasis on the emerging interest in axon guidance signaling pathways as novel targets for pharmacological support of neural cytoskeletal structure and function in order to slow ALS.  Amyotrophic lateral sclerosis  is one member of a family of anterior  horn diseases that cause progressive, irreversible degeneration and ultimately death of spinal motor neurons and their cortical efferents . Other ahttp://alsod.iop.kcl.ac.uk/, [ALS is also distinguished from other motor neuron diseases by its frustrating lack of definable genetic causes and generally enigmatic etiology. Approximately, one fifth of ALS cases are hereditary, but even within this subset there are currently thirteen confirmed Mendelian mutations encoding proteins in disparate pathways that appear at first glance to be minimally interconnected  . It mayAmongst the remaining majority of sporadic ALS (SALS) cases, there are few confirmed genetic or environmental risk factors. To date, several independent genomewide association studies have been conducted in an effort to identify genetic contributors to SALS but these have resulted in short, nonoverlapping lists of candidates , 5–7. ThThe comparative lack of understanding of ALS etiology has understandably hindered effective therapy development. Nonetheless, there has, for decades, been consistent global research effort to find new targets and therapies for slowing ALS. This passionate effort is driven in part by the severity of ALS clinical presentation, the invariably fatal prognosis, and a growing appreciation that ALS shares many biochemical and cell biological commonalities with other much more prevalent neurological diseases. Thus, understanding the essential neurobiology of ALS could open new vistas for treating neurodegeneration in general. The identification of FALS genes and production of robust animal models (particularly of superoxide dismutase-1 (SOD1) transgenic mice in the early 1990s) have massively accelerated the pace of ALS research and therapy development efforts –11. To dThis paper will discuss the history of ALS therapy development efforts from the perspective of therapy targets, review the outcomes of major clinical trials, and suggest possible pathways for future preclinical and clinical investigation. Emphasis will be placed on discussion of past therapy development efforts that culminated in human trials, and a specific discussion will be undertaken of probably reasons why successful preclinical strategies have often failed to translate into positive outcomes during human clinical trials.G93A and SOD1G85R transgenic mouse models were introduced in the mid-1990s. The involvement of SOD1 implied, at the time, a likely redox component to disease mapping directly to toxic SOD1 gains-of-function [2+ influx leading to downstream neurotoxicity, including loss of mitochondrial functionality and concomitant oxidative stress. It was therefore not surprising that some of the first experimental treatments applied to these transgenic models involved administering antioxidants such as vitamin E , alone or in combination with the NMDA (glutamate) receptor antagonists [The central feature of ALS is death of the motor neurons. Thus, the earliest strategies to combat ALS focused on intrinsic neuron death pathways, and to this day there is much active research in this area. The key questions are why do the motor neurons die and what pathways might be pharmacologically manipulated for therapeutic benefit? Even prior to the identification of SOD1 mutations in FALS, it was appreciated that human ALS nervous system tissue exhibited protein aggregation, oxidative stress, and loss of cellular homeostasis related to defective glutamate transport . This cyfunction , 13. MorG93A mice, whereas vitamin E reportedly delayed onset of symptoms and slowed disease progression without prolonging survival [These early studies appeared quite successful: the glutamate receptor antagonists riluzole and gabapentin both were found to slow disease progression and prolong survival in SOD1survival . Moreovesurvival  and weresurvival . CurrentUnfortunately, the early enthusiasm that mutant SOD1 transgenic mice would prove good predictors of clinical drug efficacy proved unfounded. First, the efficacy of riluzole, gabapentin, and vitamin E in murine ALS models proved very poorly reproducible . Moreove2+ influx; rather, the neuroprotective effects seemed mediated downstream from the glutamate-gated receptors and possibly map to action in the mitochondria [P < 0.07) toward increased lifespan in the ALS group receiving dexpramipexole [The concept of anti-excitotoxins for ALS therapy is still a subject of active research and has yielded one very encouraging recent clinical result. The benzothiazole compound R-pramipexole (dexpramipexole or KNS-760704) emerged from Parkinson's disease research as a drug that could rescue dopaminergic neurons from glutamate excitotoxicity, but not by blocking Cachondria . Subsequchondria . No othemipexole . FurtherAlthough the fatal risk in ALS is death of the motor neurons, it has become abundantly clear that this motor neuron death is not cell-autonomous but requires active contributions from surrounding nonneuronal cells . The evidence for this is twofold. First, transgenic expression of mutant SOD1 restricted to neurons using a neurofilament light-chain promoter fails to produce motor neuron disease . Second Understanding the reasons for this could uncover feasible targets for pharmacological exploitation. As noted above, part of the glial involvement is passive, via loss-of-function(s); for instance, loss of EAAT2 expression in astrocytes likely exacerbates excitotoxic stress. A significant component of glial involvement in ALS is likely active, mediating motor neuron damage through active production of toxins or inappropriate release of paracrine factors. It has long been noted that microglial activation and proliferation occur in human and murine ALS , 24–26, α (TNFα) is elevated in human ALS blood serum [G93A mice, TNFα message and protein are both elevated in spinal cord tissue, indicating endogenous production in CNS cells [α is amongst the earliest and most aggressively upregulated gene products in SOD1G93A mouse spinal cord tissue, becoming elevated before the appearance of obvious behavioral anomalies [G93A mice indicate that the cells exist in a metastable condition that is prone to fulminant activation. Such neonatal SOD1G93A astrocytes hyperexpress endogenous TNFα as well as its principle receptor TNFRI in response to application of recombinant TNFα ligand or interferon-gamma (IFNγ) [G93A astroglia produce constitutively more prostaglandin-E2 (PGE2) and induce the leukotriene-producing arachidonate 5-lipoxygenase (5LOX) enzyme in response to inflammogens, to a much greater degree than do nontransgenic astrocytes or astrocytes overexpressing wild-type human SOD1 [G93A microglia have been shown to superexpress TNFα in response to lipopolysaccharide (toll-like receptor ligand) stimulation in vitro [G93A glia is their enhanced production of reactive oxygen and nitrogen species and enhanced cytotoxicity [In particular, several studies have found that tumor necrosis factor-od serum , 31. In NS cells –34. Morenomalies , 34. Inta (IFNγ) . These sman SOD1 . Similarin vitro . A princtoxicity , 36.α and other inflammatory cytokines with toxic glial activation in ALS has encouraged a number of preclinical and clinical trials of therapeutics selected to either suppress TNFα production or otherwise blunt the neurotoxic activation of microglia. For instance, thalidomide derivatives which suppresses TNFα message translation in addition to other actions are amongst the more effective drugs tested in SOD1G93A mice [G93A mice [The association of TNF93A mice , 37, 38.93A mice , 40. Dos93A mice . It remaα and other inflammogens in vitro [α signaling inside microglia [2 and slowed progression of murine FALS [A surprisingly large number of compounds antagonize microglial activation by TNFin vitro , 41. Theicroglia . Unfortuine FALS , failed ine FALS . Even moine FALS .α pathways in ALS has waned somewhat, due largely to reports that crossing SOD1G93A mice with TNFα-knockout mice did not significantly affect disease parameters [α is a pleiotropic cytokine that has both protective and toxic effects depending on the milieu in which it is expressed and whether one is speaking of soluble versus membrane-bound variants [α constitutively would affect both aspects of cytokine function, possibly canceling out each, with little net effect on the ALS mouse. Also, one must consider the possible compensatory biology that would come to play during development of a complete TNFα knockout mouse. In any event, TNFα may be a useful model stimulus with which to probe both negative and positive aspects of neuroinflammation in ALS mice; the role of increased TNFα presence in human ALS remains subject to debate and further research.In more recent years, interest in TNFrameters . There avariants ; knockinα-stimulated microglial activation in vitro, exceeding minocycline potency by 12-fold [G93A mice when administered orally beginning at symptomatic stages of disease [in vitro in MN-1 cells and in vivo in spinal cord synaptosomes [G93A mice but then lost efficacy with repeated administration apparently due to induced pharmacoresistance through upregulation of P-glycoprotein [Interest continues to identify safe anti-neuroinflammatory agents that might have some merit to test in ALS clinical trials. It is encouraging that polyphenols or similar compounds such as the 5LOX inhibitor nordihydroguaiaretic acid (NDGA) protect against murine ALS  and can  12-fold  extends  disease  and alsoptosomes . Interesoprotein . NDGA dioprotein , in contoprotein , suggestG93A mice, motor end plates can be visualized along with their impinging axons [α-motor neuron death, the axons disappear from the motor end plate. This is followed by demonstrable shrinkage and loss of axons at the level of ventral routes and only finally by death of the attached cell bodies in the ventral horns [Most ALS preclinical and clinical strategies thus far have fallen into two broad categories: preventing death of the motor neuron soma by pharmacologically targeting the neuron; or supporting motor neuron health indirectly by targeting the biology of ambient glia. Of course, minocycline garnered enthusiasm because of its apparent multiple targets of action both inside neurons and at the glial cell. Another tactic is gaining momentum in preclinical studies, which are beginning to reconceptualize ALS fundamentally as a cytoskeletal disease. This perspective is based on the fact that motor neuron cell death only happens near the end of an extensive clinical disease and thus likely is a final consequence of other neuronal processes. Indeed, ALS can be thought of as a progressive distal axonopathy , 51 beging axons , 51. Begal horns , 51. Theal horns , 51. ThuProgress is being made toward both goals of identifying triggers and identifying driving processes for ALS axonopathy . As for β (GSK3β) and cyclin-dependent kinase-5 (Cdk5) [β and Cdk5 act on a variety of microtubule-associated proteins including the collapsin response mediator protein (CRMP) system. CRMPs, particularly the CRMP2 isoform, function to stabilize both actin microfilaments and microtubules. Phosphorylation, expression downregulation, or sequestration into axonal aggregates can prohibit proper CRMP2 function and trigger severe axon retraction  is upregulated in terminal Schwann cells near the fast-fatigable fibers that are earliest to denervate in murine ALS . To date5 (Cdk5) , 54. GSK●NO) [●NO-induced death [There are at least two additional pieces of evidence besides Verrhaagen's finding to suggest that the Sema3A-CRMP2-cytoskeleton signaling pathway is involved at least in murine ALS and potentially amenable to pharmacological intervention. First, Pettman and colleagues recently reported that a variant of CRMP4 is induced in cultured motor neurons by exposure to nitric oxide (●NO) . Forced ed death . Thus, eα-stimulated microglial ●NO production in culture which may or may not arise from the drug's apparent action upon CRMP2 [G93A mice beginning at symptomatic disease significantly slowed disease progression and extended lifespan [Second, our group serendipitously discovered that a molecule called lanthionine ketimine (LK) binds CRMP2 to alter CRMP2 : tubulin interaction . A cell-on CRMP2 . Adminislifespan . We are G93A mice by >10%, restored axonal transport deficits, and reduced motor neuron death [Other researchers have begun attempting more direct microtubule stabilization strategies in ALS mice, with some success. Notable is a 2007 study by Fanara et al. who reported that the microtubule stabilizing agent noscapine extended lifespan of SOD1on death . This isAt this point, it is worth reconsidering the current list of generally accepted ALS genetic risk factors and Mendelian heritance factors . At firs G93A spinal cord at latter stages of disease [α, β-crystallin is also demonstrably hypercarbonylated in this mouse [All of the targets mentioned thus far in this paper—excitotoxicity, neuroinflammation, glial activation, and protein cytoskeletal dysfunction—are not mutually exclusive but likely interact with one another quite dynamically and may be mechanistically united either through sharing common origins in protein aggregation disorders or common mechanisms of enhanced oxidative stress (reviewed in ). For in disease . The prois mouse . Thus, mThe past two decades have witnessed a tremendous growth in knowledge about ALS. We now have a much more complete understanding of the genetic causes underlying FALS, though much less is known about risk factors for SALS. ALS transgenic models including mutant SOD1 transgenic mice but more recently including other transgenic constructs have provided powerful tools for exploring the basic biology of motor neuron disease and testing experimental therapeutics. This last point, however, is a pivot about which optimism for ALS therapy development has begun to vacillate. Numerous therapeutic strategies have produced small to moderate lifespan and functional effects in SOD1 mutant mouse studies , buAs others have discussed with great clarity , ALS mouAs for lessons regarding cellular targets for promising drug candidates, the only limited success to be achieved thus far in ALS clinical trials has stemmed from riluzole and tentatively, from dexpramipexole: two drugs that are thought to target either glutamate receptor activation or downstream consequences of glutamate excitotoxicity. Thus, priority might be given to drug candidates that have effects in cell or animal models that speak to excitotoxic pathways. On the other hand, not every antiexcitotoxin tested in human ALS or even in ALS mice can produce a benefit as exemplified by the failure of the NMDA receptor antagonist memantine . From whContinued effort to exploit particular neuroinflammatory pathways for ALS therapy seems warranted at this time, though researchers need to be cognizant that drug efficacy may vary from study to study in a capricious manner that is influenced in mice by pharmacotolerance in some cases . FinallFinally, it must be seriously considered that one or even two drugs will not treat ALS due to the multifactorial nature of the pathology. Though difficult to design and implement, trials of “cocktail” therapies consisting of multiple, rationally selected drug candidates may be worth attempting in future ALS mouse and human studies.In summary, ALS therapy development efforts have been characterized by cycles of enthusiasm and deep disappointment, but scientific progress has been continual and may be nearing new milestones for clinical success if the initial clinical data regarding dexpramipexole effects on ALSFRS-R can be corroborated . Rather


Interestingly, these observations compare well with what has been described for native development. We propose that our cultivation system can mimic various aspects of endothelial cell – osteogenic precursor interactions in vivo, and could find utility as a model for studies of heterotypic cellular interactions that couple blood vessel formation with osteogenesis.Tissue engineering provides unique opportunities for regenerating diseased or damaged tissues using cells obtained from tissue biopsies. Tissue engineered grafts can also be used as high fidelity models to probe cellular and molecular interactions underlying developmental processes. In this study, we co-cultured human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs) under various environmental conditions to elicit synergistic interactions leading to the colocalized development of capillary-like and bone-like tissues. Cells were encapsulated at the 1∶1 ratio in fibrin gel to screen compositions of endothelial growth medium (EGM) and osteogenic medium (OM). It was determined that, to form both tissues, co-cultures should first be supplied with EGM followed by a 1∶1 cocktail of the two media types containing bone morphogenetic protein-2. Subsequent studies of HUVECs and MSCs cultured in decellularized, trabecular bone scaffolds for 6 weeks assessed the effects on tissue construct of both temporal variations in growth-factor availability and addition of fresh cells. The resulting grafts were implanted subcutaneously into nude mice to determine the phenotype stability and functionality of engineered vessels. Two important findings resulted from these studies: ( Vhl gene, the result was higher vascularity in long bones with complementary increases in bone volumes in vivo environment.In native bone, synergistic interactions between osteoblasts/osteogenic precursors and endothelial cells enable coordinated development of vasculature and mineralized tissue. In the process of intramembranous ossification during craniofacial bone growth, this cell coupling results in close spatial relationships between the two tissues in newly forming bone, with the vascular network serving as a ‘template’ for bone mineral deposition in vivo, it has been decidedly difficult to simultaneously form capillary-like networks and mineralized deposits in vitro, within a single culture environment. Whereas beneficial effects of osteogenic precursors on vascular network formation have been demonstrated The need to vascularize tissue engineered bone grafts, during culture and following implantation, has led to studies between endothelial cells and osteoblasts/osteo-progenitors in vivo cultivation evidence of mineral deposition was revealed. Several other groups have shown that implanting biomaterial constructs with a mixture of mesenchymal and vascular or hematopoietic progenitor cells enabled the development of vascularized tissues in vivoin vitro cultivation models are required to elucidate the mechanistic interactions of both cell populations during the formation of vascularized bone.Recently, one group circumvented the difficulty of inducing adequate vascular development and osteogenic differentiation by seeding mesenchymal stem cells (MSCs) into polymer scaffolds and inducing osteogenesis prior to seeding MSCs and human umbilical vein endothelial cells (HUVECs) into a gel substrate in vitro model of bone vascularization. HUVECs and human MSCs were cultured in decellularized trabecular bone constructs using fibrin as a cell carrier to provide an environment conducive to the formation of capillary-like networks. Coordinated development of the two tissue compartments was evaluated over a two-stage procedure (6 weeks in vitro culture followed by a 2 week sub-cutaneous implantation), to establish an alternative model for engineering bone-like constructs containing vascular networks  were obtained from Peprotech . Endothelial Growth Medium-2 (EGM) was obtained from Lonza . Phosphate buffered saline (PBS) and proteinase K were obtained from Fisher Scientific . All other substances were of analytical or pharmaceutical grade and obtained from Sigma-Aldrich.Fetal bovine serum (FBS), Dulbecco's Modified Eagle Medium (DMEM), Penicillin–Streptomycin (Pen–Strep), HEPES and trypsin/EDTA were obtained from Invitrogen . Ascorbic acid-2-phosphate, dexamethasone, sodium-β-glycerophosphate, Triton X-100, fibrinogen and thrombin were obtained from Sigma-Aldrich . Basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2  were isolated from the mononuclear fraction of a bone marrow aspirate obtained from a commercial source  based on their attachment to tissue culture plastics Fresh umbilical veins were obtained from the neonatal unit at Columbia University following an approved IRB protocol (IRBAAAC4839). To isolate HUVECs, the vein was flushed with HEPES buffer to remove residual blood and treated with trypsin for 15 minutes to remove the endothelial cells. Cells were flushed from the vein with HEPES and collected in centrifuge tubes. The suspension was centrifuged at 300 g for 5 minutes and the supernatant was discarded. Cells were resuspended in EGM and cultured in tissue culture flasks. Non-adherent cells were washed out after 1 day. When the HUVECs grew to confluence, they were trypsinized, counted and cryopreserved.th passage. HUVECs used in preliminary screening studies were suspended in serum-free medium at 1×106 cells/mL and then incubated with 10 ml/mL of Vybrant DiI (Molecular Probes) for 30 minutes at 37°C. The cells were washed of excess dye three times prior to mixing with hMSCs in a 1∶1 ratio, and encapsulation in fibrin hydrogel in the wells of a 96-well plate and cultured for 4 weeks in vitro. A 1∶1 ratio was chosen in pilot studies (data not shown) as it provided robust and stable vascular networks while enabling bone formation. The hydrogels were divided into five groups depending on the various growth medium provided the constructs. These included osteogenic medium (OM) containing low glucose DMEM, 10% FBS, 1% Pen–Strep supplemented with osteogenic factors: 100 nM dexamethasone, 10 mM sodium-β-glycerophosphate, and 50 µg/mL ascorbic acid-2-phosphate. The other groups were provided EGM or a cocktail of OM and EGM in a 1∶1 ratio. The temporal sequence was investigated by providing the cells EGM only for 2 weeks followed by cocktail for the subsequent two weeks or cocktail medium supplemented with 10 ng/ml of BMP-2. The development of vascular structures was imaged with a Zeiss 510 confocal microscope using an image stack 200 µm thick with 10 µm spacing between slices.In this study, HUVECs were expanded in EGM to the 4Bone plugs (4 mm diameter×2 mm high) were prepared as in our previous studies 6 cells/mL was seeded into blot-dried scaffolds, divided into five groups, and incubated overnight to allow cell attachment as shown in 6 cells/mL. Thrombin was added to crosslink the gel, giving a final fibrin concentration of 4 mg/mL. Before crosslinking occurred, 20 µl of cell/gel suspension was pipetted into blot-dried scaffolds to allow uniform cell seeding throughout the scaffolds. Before the gels became fully cross-linked, they were aspirated under a light vacuum so that the fibrin gel coated the walls of the scaffolds, but did not fill the pore spaces. In Group 5, this same step was performed on MSC-seeded scaffolds after 4 weeks of osteogenic culture.To induce bone formation, 20 µl suspension of MSCs at a density of 10×10Group 1 (control for osteogenic conditions) utilized osteogenic medium (OM) throughout the culture, containing low glucose DMEM, 10% FBS, 1% Pen–Strep supplemented with osteogenic factors: 100 nM dexamethasone, 10 mM sodium-β-glycerophosphate, and 50 µg/mL ascorbic acid-2-phosphate, supplemented with BMP-2 (at 10 ng/mL) for 6 weeks. Group 2 (control for vasculogenic conditions) utilized endothelial growth medium (EGM) throughout the culture. Group 3 utilized EGM for 2 weeks and then a cocktail medium composed by EGM and OM at 1∶1 ratio (EGM|cocktail) for 4 weeks. Group 4 was designed exactly as Group 3 except that osteo-induced MSCs were added into scaffold pore spaces at the 2-week time point (EGM|cocktail+MSC). In Group 5, MSCs only were seeded into the scaffolds and cultured in osteogenic medium for 4 weeks, at which point HUVECs and MSCs in fibrin were added to the constructs and cultured in cocktail medium for an additional 2 weeks (OM|cocktail).Five experimental groups were established to study vascularized bone constructs, including three main groups and two controls Fig. 1. in vitro culture, constructs of all groups were evaluated for 2 weeks in a subcutaneous implantation nude mouse model in vivo. NOD SCID mice  were anaesthetized by a subcutaneous injection of ketamine (80–100 mg/kg) and xylazine (5–10 mg/kg). Analgesia was provided with buprenorphine (0.05–0.1 mg/kg). Constructs were implanted into separate subcutaneous dorsal pockets  according to an approved Columbia University Institutional Animal Care and Use Committee (IACUC) protocol. Implants were retrieved after 2 weeks for analysis of blood vessel anastomosis and bone growth.After 6 weeks of Constructs were washed in PBS, cut in half, placed into micro-centrifuge tubes and incubated overnight at 56°C in 1 mL of digestion buffer  containing 0.1 mg/mL proteinase K. The supernatants were collected and diluted 10 times to bring into the linear range of the Picogreen assay. A standard curve was prepared from a solution of bacteriophage λ DNA obtained from Molecular Probes. Picogreen dye  was added to the samples in 96-well plates (100 µL dye into each 100 µL sample) and read in a fluorescent plate reader .Constructs were cut in half, washed in PBS, incubated with calcein AM (indicating live cells) and ethidium homodimer-1 (indicating dead cells) according to manufacturer's protocol (LIVE/DEAD (R) Viability/Cytotoxicity Kit, Molecular Probes) and imaged on a confocal microscope. Optical slices were taken from the surface at 10 µm intervals, up to the depth of 160 µm and presented as a vertical projection.Constructs were washed in PBS and fixed in 10% formalin for 1 day. Samples were then decalcified for 2 days with Immunocal solution, dehydrated in graded ethanol washes, embedded in paraffin, sectioned to 5 µm and mounted on glass slides. Sections were deparaffinized with Citrisolv, rehydrated with a graded series of ethanol washes, blocked with normal serum, and stained with primary antibodies followed by the secondary antibody and development with a biotin/avidin system. Immunohistochemistry was performed for anti-human collagen I (Abcam ab6308), bone sialoprotein II (BSP) (Chemicon AB1854), CD31 (Millipore 04-1074) and von Willebrand factor (vWF) (Sigma-Aldrich F3520). Counterstaining was performed with hematoxylin. The serum, secondary antibody and developing reagents were obtained from Vector Laboratories and included in the Vector Elite ABC kit  (PK6200) and DAB/Ni Substrate kit (SK-4100). Negative controls were performed by omitting the primary antibody incubation step.in vitro culture. As a control, unseeded scaffolds were remained in OM throughout culturing period and analyzed at this time point. A modification of a previously developed protocol was used μCT was performed on all five cell-seeded groups after 6 weeks of in vitro developed capillary networks with host vasculature was evaluated by immunolocalization of human CD31 (Millipore 04-1074) as described above. Counterstaining with hematoxylin was performed to detect mouse red blood cells within human capillary lumen. New bone formation was evaluated on undecalcified sections processed according to hard tissue histology methods: constructs were fixed in 10% formalin for 1 day and dehydrated with sequential washes in 70% ethanol (2 days), 100% ethanol (2 days with twice daily solution changes), and toluene (2 days with once daily solution change). Constructs were then washed in activated methyl methacrylate (MMA) with daily changes of MMA solution for four days at 4°C, and then placed at 32°C until the MMA cured. Plastic-embedded sections were sectioned to 8 µm on a Leica hard tissue microtome. Staining for the new osteoid formation was done using the traditional Goldner's Masson trichrome stain.After 2 weeks of subcutaneous implantation in NOD SCID mice, samples were harvested, washed in PBS and macroscopic photographs were taken with the stereomicroscope. Samples were cut in half, and used for soft and hard tissue histology. Anastomosis of Statistical analysis was performed using the GraphPad Prism 4.0c software. A one-way analysis of variance (ANOVA) with Tukey's multiple comparison post-hoc test was used to verify statistically significant differences among groups. p<0.05 were considered statistically significant. Data is presented as mean±SD.in vitro only in the presence of MSCs . Robust networks were observed in confocal microscopy within two weeks of culture in constructs cultured in endothelial growth media ( group) and remained stable throughout the 6 weeks of cultivation. When OM was used at the beginning of the culture period ( group), the cells did not form networks. Instead, significant amounts of ‘debris’ from the diI-stained HUVECs were observed by confocal . Cells in the cocktail medium (EGM+OM 1∶1 ratio) formed vascular networks, as evidenced via confocal imaging of the pre-stained HUVECs, however high amount of cell debris was observed ( group).We confirmed that capillary networks formed from HUVECs are stable  group).Sequential induction was investigated in Group 3 and Group 4 constructs, using EGM first, since HUVECs did not survive well if OM was used first. In both groups, vascular networks that formed within the first two weeks were maintained for the duration of culture. However, supplementation of cocktail medium (EGM+OM 1∶1 ratio) with BMP-2 (10 ng/mL) significantly decreased the amount of cell debris, suggesting that BMP-2 may help maintain the viability of the endothelial cell population  as compared to the osteogenic conditions applied throughout culture (Group 1). Notably, the new mineral deposition was observed mostly in the close spatial proximity to vascular networks, particularly in the Fig. S3). The DNA content of constructs revealed that OM enhanced cell proliferation considerably more than EGM (p<0.05). This was clearly a specific cellular response to factors in the OM medium since EGM|cocktail also had more cells than EGM only. Not surprisingly, Group 4 (EGM|cocktail+MSC) and Group 5 (OM|cocktail) had the most cells  and EGM  were compared to Groups 3, 4 and 5 where vascular supplements were applied in sequence with osteogenic supplements. Anti-human CD31 and vWF staining demonstrated formation of vascular structures in all groups except for the OM (Group 1). Vascular structures were clearly most developed in EGM|cocktail (Group 3) and EGM|cocktail+MSC (Group 4). While there were CD31 and vWF positive cells in the OM|cocktail groups, the vascular networks did not show signs of maturity as evidenced by the sizes of lumens in the structures , which are indicative of osteogenic differentiation, with incorporation of both collagen and BSP into the new tissue matrix. In contrast, the osteogenic markers in the EGM group were confined to individual cells. In the EGM|cocktail+MSC group, high expression of BSP was observed in spatial proximity to lumenal structures  suggesting that the perfused microvasculature might be of human origin . Although there were positively stained cells in the OM|cocktail group, these vessels were not well developed and there was no evidence of perfusion. Non-decalcified grafts harvested from in vivo studies were stained with Goldner's Masson Trichrome to visualize osteoids, which were observed to a higher extent in the OM and EGM|cocktail+MSC groups. Clearly, the least osteoid formation was observed in the EGM group.Macroscopic photographs taken with the stereomicroscope indicated in-growth of large, well-developed blood vessels into each of the constructs Fig. 5. de novo bone formation by undifferentiated hMSCs over a period of several weeks. The bone formation was augmented when osteo-induced MSCs were added to the bone scaffolds at the same time as the osteogenic supplements. Moreover, several studies have shown that BMP stimulate VEGF expression in osteoblasts, and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells In initial hydrogel studies, we found that providing osteogenic medium to co-cultures of HUVECs and hMSCs was detrimental to vascular formation. As a result, subsequent studies induced robust vascular development through the application of EGM for two weeks. Early studies demonstrated that the capillary-like structures were formed by HUVECs within a couple days and were only stable in the presence of hMSCs. This suggests that the hMSCs may be playing a pericyte-like role in this system Figure 6in vitro model of bone vascularization. To this end, we systematically studied cultivation conditions that give rise to both functional vascular structures and robust osteogenesis using HUVEC-MSC co-cultures in conjunction with previously developed tissue engineering and bioreactor methods The major goal of this study was to test the hypothesis that the sequential application of growth factors, to first induce the formation of stable vasculature and then initiate osteogenic differentiation, could provide a biologically-inspired in vitro co-culture with mature endothelial cells in vivo with mature endothelial cells Fig. S1).In our model, the MSCs have two major roles: as pericytes stabilizing vascular networks and as osteogenic progenitors forming mineralized bone matrix. Several published studies show that bone marrow-derived MSCs adopt pericyte-like locations during in vitro culture. Mineralization of the tissue matrix was evident in the groups containing vascular networks, although to limited extent as cells were exposed to osteo-inductive medium for only two weeks. Thus, a longer osteo-induction period was adopted during scaffold studies.The ability of MSCs to respond with spatial and temporal specificity to differentiation cues was key in gel studies where all cells were seeded at the beginning of culture and no cells could be introduced at later stages of culture . From these preliminary studies, several key outcomes were identified that determined the selection of experimental groups for the subsequent scaffold study: (i) a sequential-induction approach enabled vascular-osteogenic outcomes within the same culture space, with the need for endothelial growth factors to be provided prior to osteogenic induction, and (ii) the role of BMPs as a vascular promoter was confirmed in vitro co-culture system and enabled the evaluation of concurrent supplementation of growth factors and fresh MSCs responding to these growth factors. We also investigated the hypothesis of MSCs losing their ability to respond to osteogenic factors during the two-week exposure to vascular supplements, by comparing Group 4  with the Group 3 . Data suggests that fresh MSCs enhance bone formation, as verified by increased bone volume fraction (EGM|cocktail). This suggests that MSCs may act initially as pericytes in vivoTrabecular bone scaffolds also provided great versatility of the fraction , osteoidfraction , and spafraction . HoweverOM|cocktail group) was moderately successful: we observed reasonable osteogenic development as evidenced by deposition of bone protein, osteoid formation and the BV/TV ratio. However, two-week exposure to vascular growth factors was not sufficient to develop robust vascular structures, as evidenced by the sizes of vessel lumens that were smaller than those observed in Groups 3 and 4. In these two groups, not only lumen were evident after in vitro cultivation, they also anastomosed well during in vivo implantation, which is consistent with the earlier reports that well-developed vascular networks are necessary for functional anastomosis in vitro cultivation By adding HUVECs at the latter stages of culture, it was possible to evaluate osteogenesis that has been induced in the scaffolds prior to providing vascular cells and growth factors, an approach similar to that taken by Tsigkou et al In future studies, it might be relevant to distinguish whether HUVECs and MSCs are equally stimulated in this group and to decipher the role of cell proliferation in tissue development. Also, despite promising results, our studies suggest that alternative cocktails (e.g. EGM|OM or increased vascular growth factor concentration in OM|cocktail group) could be tested to synergistically improve mineralization with vascular network formation. Likewise, further studies are needed to optimize the temporal stages of culture, as the two-week period of vascular induction was selected based on the robust network structures observed for fibrin gel studies.bona fide osteogenic differentiation within single tissue grafts during in vitro cultivation. Notably, functional vascular networks and viable osteoid formation were detected post-implantation. The most successful approach involved induction of vasculogenesis prior to osteogenesis, with the addition of osteo-induced MSC further improving tissue outcomes. Finally, while the focus of our study was to evaluate ‘functional’ outcomes such as vessel formation and matrix mineralization, significantly more work is needed to elucidate the underlying mechanisms that regulate heterotypic interactions and understand the interactive roles of multiple growth factors.Overall, by providing temporal variation of the culture environment, we were able to demonstrate the ability to derive vascular networks and induce In summary, it has long been accepted that vascularization is essential for the advancement of tissue-engineered bone grafts to clinical application Figure S1Gel cultivation.A: HUVECs only at 3 weeks in EGM-2. Co-culture of HUVECs with MSCs (1∶1) enables formation of stable micro-vasculature networks at 3 weeks in EGM-2 (B) that last up to 12 weeks (C) during in vitro culture. D: von Willebrand Factor (vWF) staining of lumen.(JPG)Click here for additional data file.Figure S2Gel screening studies. HUVECs (pre-stained with Di-I) and MSCs were encapsulated in fibrin hydrogels (1∶1 ratio) and cultured in small wells for 4 weeks to determine cellular responses to different medium conditions. Confocal images were used to evaluate vascular network formation and provide a read-out on HUVEC viability (cellular debris). Scale bar = 50 µm. Bottom row: von Kossa staining of mineral deposition within the gel regions. Mineral is shown as black/dark brown stains within the gels.(JPG)Click here for additional data file.Figure S3H&E staining of constructs at day 1. Cells are uniformly distributed throughout the scaffold upon seeding. Cells are located predominantly on the wall surfaces of scaffolds but grow into pore spaces subsequently.(JPG)Click here for additional data file.Figure S4EGM|cocktail+MSC group stained with anti-human CD31 mAb. Human origin lumen, with red blood cells inside (stained with hematoxylin) are pointed with yellow arrows.(JPG)Click here for additional data file.


Plutella xylostella (diamondback moth) backcross that segregated for resistance to the insecticide spinosad. Sequencing of 24 individuals was performed on a single Illumina GAIIx lane (51 base paired-end reads). Taking advantage of the lack of crossing over in homologous chromosomes in female Lepidoptera, 3,177 maternally inherited RAD alleles were assigned to the 31 chromosomes, enabling identification of the spinosad resistance and W/Z sex chromosomes. Paired-end reads for each RAD allele were assembled into contigs and compared to the genome of Bombyx mori (n = 28) using BLAST, revealing 28 homologous matches plus 3 expected fusion/breakage events which account for the difference in chromosome number. A genome-wide linkage map (1292 cM) was inferred with 2,878 segregating RAD alleles inherited from the backcross father, producing chromosome and location specific sequenced RAD markers. Here we have used RAD sequencing to construct a genetic linkage map de novo for an organism that has no previous genome data. Comparative analysis of P. xyloxtella linkage groups with B. mori chromosomes shows for the first time, genetic synteny appears common beyond the Macrolepidoptera. RAD sequencing is a powerful system capable of rapidly generating chromosome specific data for non-model organisms.Restriction-site associated DNA (RAD) sequencing is a powerful new method for targeted sequencing across the genomes of many individuals. This approach has broad potential for genetic analysis of non-model organisms including genotype-phenotype association mapping, phylogeography, population genetics and scaffolding genome assemblies through linkage mapping. We constructed a RAD library using genomic DNA from a  Discovering genes that control morphological, behavioural and physiological phenotypes is critical for understanding adaptive evolution, for plant and animal breeding and for tracking the evolutionary responses of natural populations, such as insecticide resistance in crop pests. Commonly, traits controlled by single, major Mendelian genes are isolated using genetic linkage maps created from crossing experiments. A genome-wide analysis is followed by finer scale mapping with a larger number of recombinant individuals to narrow the region of interest, and finally targeted sequencing of genome libraries. Linkage maps have been constructed for scores of organisms, generally to identify a genome region controlling a trait of interest, such as skeletal armour morphology in stickleback fish The restriction site associated DNA (RAD) sequencing method Plutella xylostella, the diamondback moth, is a worldwide pest of cruciferous crops including cabbage and broccoli P. xylostella chromosomes, (iii) compare chromosomal synteny with the reference lepidopteran genome from the silkworm Bombyx mori, (iv) identify RAD loci likely to encode transcribed genes and (v) create a genome-wide linkage map with sequenced RAD loci. Instead of mapping the RAD tags directly to a reference genome, we constructed contigs using paired-end sequences adjacent to the RAD tags and used these for gene identification and interspecific comparisons. The methods described have great potential for creating genomic scaffolds to assist in genome assembly and for identifying thousands of sequence variants to aid in detection of major as well as minor quantitative traits.P. xylostella strain Pearl-Sel 1 female. Genotyping of known markers confirmed all 12 of 12 spinosad survivors and 7 of 10 untreated controls were homozygous for the resistance mutation. A single RAD library was constructed from DNA of the two backcross parents and 22 backcross progeny . We estimated that there should be ∼6500 Sbf1 RAD loci in the P. xylostella genome, and thus generated raw Illumina reads to a depth of 65x per locus per individual based on this estimate. This was achieved in a single Illumina GAIIx lane, producing 10,217,074 51-base paired-end reads. Each forward sequence contained a 5 base MID that allowed reads to be assigned to a specific individual, followed by 6 bases of the SbfI restriction enzyme footprint (TGCAGG), leaving 40 locus-specific bases per read.Resistance to the insecticide spinosad is recessive and controlled by a single Mendelian locus in the The sequence reads were split into groups by MID, resulting in one group for each of the 24 individual moths. The average number of sequence reads per individual was 425,000, although there was high variability amongst the 24 individuals (16K-842K) . Two of The forward sequence for each RAD allele begins at a defined position; the SbfI cut site. Due to size shearing of the library, the paired-end reverse sequence read for individual RAD alleles was located a variable distance from the cut site, and could be grouped and clustered into RAD contigs using VelvetOptimiser. The length distribution of RAD contigs produced was bimodal, with average length  = 333 bp, median length  = 211 bp . RAD conP. xylostella (2n = 62) backcross progeny therefore inherit one unrecombined chromosome of each maternal pair. Crossing over does occur during spermatogenesis in males, enabling the 31 paternally inherited chromosomes to be used for linkage mapping and determining genetic distances between markers Crossing over between homologous chromosomes does not occur in female Lepidoptera. Hence all genes and markers on the same maternally inherited chromosome are completely linked 1 mother was heterozygous. The presence/absence of candidate RAD alleles in a set of progeny individuals can be represented as a binary number string of 1  or 0  per individual. For 20 progeny, more than 1 million combinations are mathematically achievable (220). However, we expect to find 62 patterns representing segregation of the 31 maternal chromosome pairs. From 4,826 candidate maternally derived RAD alleles, 3,177 were grouped into exactly 31 pairs of different binary patterns, each representing a distinct chromosome containing 49–190 sequenced markers  across the 20 backcross progeny, indicating that the FBombyx mori (432 Mb) using an expect value cut-off of <1E-10. RAD contigs with hits to more than one chromosome were removed from the analysis. From each P. xylostella linkage group, the number of significant hits to each B. mori chromosome were counted to identify the most likely chromosomal homologue. Due to differences in chromosome number, it was expected that multiple P. xylostella chromosomes would collapse into a single B. mori chromosome. The 31 linkage groups collapsed into the 28 chromosomes of B. mori, and have been numbered according to B. mori chromosome nomenclature with three exceptions: B. mori chromosome (BmChr) 11 represents a fusion of P. xylostella linkage group (PxLG) 11 and 29; BmChr 23 fuses PxLG 23 and 30; and BmChr 24 fuses PxLG 24 and 31 , two of which have previously been identified as sex-linked genes in Lepidoptera  there were three or fewer base differences in the 40 base forward sequence and b) each paternal RAD allele matched either i) a single maternally derived RAD allele or ii) multiple RAD alleles on the same chromosome. Markers with multiple matches to multiple chromosomes (or no chromosome matches) were not assigned to a chromosome.The experimental cross analysed here was intended to associate RAD alleles with chromosomes, and in particular, identify sequenced markers on the chromosome encoding spinosad resistance. Nonetheless, despite the small number of individuals sampled we were able to construct a linkage map of the Once a subset of the paternal candidate RAD alleles had been assigned to chromosomes, the segregation patterns of all paternal markers were analyzed. Candidate RAD alleles that showed a segregation pattern identical to that of a previously chromosome-assigned marker were also assigned to that chromosome. Candidate RAD alleles displaying the inverse of these segregation patterns were also included. Finally paternally derived candidate RAD alleles with segregation patterns that differed by only one genotype from a chromosome-assigned marker were linked to that chromosome.To minimize the likelihood of erroneous markers contributing to the linkage map, a robust criterion was established: each locus had to contain three or more distinct RAD alleles. Segregation patterns with less than three RAD alleles often had questionable support and so were discarded. Valid loci containing one or two RAD alleles could be manually identified, however a criterion of at least three markers per loci was sufficient for our purposes.B. mori genome using TBLASTX, with 636 (89%) having at least one hit to their assigned chromosome. Linkage groups were assembled independently (JoinMap 3.0), producing a map length of 1292 cM (P. xylostella haploid genome size to be 339.4 (+/−1.1) Mb for homogametic males and 338.7 (+/−1.1) Mb for heterogametic females. This implies a physical map distance of 262.7 kb/cM in males.Of the 4,042 paternally inherited and segregating candidate RAD alleles, 2,878 met this criterion. This set further collapsed into 285 distinct segregation patterns, or markers, containing between 3 and 71 RAD alleles at each position. Of these 2,878 RAD alleles, the associated RAD contigs of 718 hit the  1292 cM . Using mRAD contigs were compared to the UniRef90 protein database using BLASTX to predict the number of chromosome linked RAD markers that are likely to be parts of protein-coding genes. Using an expect value significance cut-off of 1E-10, 282 (8.8%) of the 3,177 maternally derived RAD contigs and 231 (8.0%) of the 2,878 paternally derived RAD contigs are predicted to derive from protein-coding genes. As a proportion of markers were allelic between the cross parents, there were hits to 365 unique proteins .P. xylostella we used paired-end sequences adjacent to the RAD tags for interspecific comparisons to the genome sequence of the model lepidopteran Bombyx mori. Here, using a single lane of Illumina GAIIx sequencing, we have identified over 8,000 candidate loci with about 14,000 alleles. The number of loci identified is larger than the 6,652 expected from a simple Poisson model of SbfI site frequency in the 339Mb P. xylostella genome, likely reflecting the inadequacy of this model in a complex, repeat-rich, eukaryote genome. These RAD-derived molecular markers have been assigned to chromosomal locations in the genome of the insect pest P. xylostella.Understanding the molecular control of novel phenotypes in complex organisms is a central goal of modern evolutionary biology. Identifying the general region of a genome containing an adaptive locus is often the first critical phase in this process that ultimately aims to pinpoint the causal sequence under selection. However, technical limitations have made this a considerable challenge in most species. Restriction-site associated DNA (RAD) sequencing presents a powerful approach to dramatically improve molecular marker density across genomes and considerably accelerate the identification of genomic regions linked to traits of interest. This technique has evolved from pioneering studies utilizing microarray hybridization techniques to score presence/absence of marker regions adjacent to restriction sites in the genome Due to a lack of crossing over in female Lepidoptera, we were able to confidently establish a set of 3,177 maternally inherited, chromosome-specific RAD markers identified by their segregation pattern among backcross progeny. Each of the 31 chromosomes contained 49–190 sequenced markers and, overall, 8% of the contigs associated with mapped RAD alleles showed homology to predicted proteins. From just a limited number of backcross progeny, we accurately mapped a Mendelian spinosad insecticide resistance trait to chromosome 5. The resistance chromosome produced the greatest number of maternally derived RAD markers (190), although it is unclear whether this is due to the chromosome size or physical linkage to the resistance locus. The spinosad resistant Pearl strain was previously crossed to susceptible strain Geneva88 to select for survival on an artificial diet and then reselected for spinosad resistance to produce the mapping strain, Pearl-Sel. The effect of this laboratory selection prior to the Pearl-Sel x Geneva88 backcross presented here meant that many polymorphisms were shared by cross parents, except those at or perhaps near to the resistance locus itself. This may have increased the number of markers that could be mapped on the resistance chromosome.B. moriP. xylostella linkage groups and B. mori chromosomes, including the three putative fusion or breakage events. This high degree of synteny conservation at the level of whole chromosomes is consistent with previous analyses within the Macrolepidoptera, and suggests that the conservation seen previously between butterflies and B. moriThe maternally derived paired-end data allow a comprehensive genome wide comparison of synteny with the sequenced genome of Heliconius melpomene (1616 cM) B. mori (between 1305 cM and 3229 cM) Bicyclus anynana (between 1352 cM –1642 cM) B. anynana, Van't Hof (2008) calculated total linkage distances of 1354 cM and 1873 cM from the same data set using two different mapping programs.Linkage maps have been produced using RAD sequence data in barley The methods used for this study were suitable for our purposes but may not be appropriate for RAD studies in other species with different aims. We have provided information on the effect of using different numbers of mismatches and fragment count thresholds for our data  and S4 aBicyclus anynana was constructed based on EST sequences, but required costly prior identification of SNPs before mapping could be carried out Plutella xylostella based on RAD alleles, large numbers of which can be readily identified as orthologues of known genes in reference genomes. Presence/absence patterns of RAD alleles furnish the basic data for linkage map construction, SNP variation within the 40-base RAD alleles enables integration of paternally- and maternally-informative linkage maps, and longer sequences adjacent to the RAD alleles provide enough information for cross-species comparisons. This will provide a wealth of genome sequence data for future gene-finding and genomic studies in this economically important pest species. Furthermore, our dataset demonstrates the feasibility of this approach for future genomic studies of any non-model organism.The strength of RAD-based linkage maps is the volume of accountable genomic sequence. Traditional linkage maps based on anonymous markers are useful in determining rates of crossing over, or focusing on a specific locus. However, the anonymity of these markers limits the utility of such maps for further studies, and they provide no information on comparative genomics. More recently, a genetic linkage map for In what follows, a read is the raw sequence determined from the P1 adapter, containing the captured restriction site overhang, obtained on an Illumina sequencing instrument. The sequence read from the P2 adapter at the other end of the DNA fragment is called the paired-end read. A RAD locus is a region downstream of a restriction site. Each RAD locus contains one or more RAD alleles, depending on whether the locus is homozygous or heterozygous within and across individuals. RAD loci and RAD alleles are initially described as candidates to indicate that further error correction  will take place downstream during processing. A RAD contig is an assembly of the paired-ends for a single RAD allele into a long contiguous sequence. Each allele will have a read count  and a fragment count . A segregation pattern is a pattern of presence/absence of a particular RAD allele across all individuals in the sample. A RAD marker is a segregation pattern of alleles at a locus used in the construction of the linkage map.1 female to a Pearl-Sel male. Approximately 60 backcross progeny were treated with a discriminating dose of spinosad (10 ppm) and, as resistance is recessive, survivors were inferred to be homozygous for the spinosad resistance allele. Some backcross progeny were reared without exposure to spinosad as controls and were either homozygous or heterozygous for the resistance allele The spinosad-resistant strain Pearl was collected from Hawaii in 2001. A resistant derivative, Pearl-Sel, was isolated after crossing Pearl to a sensitive line (Geneva88) to facilitate laboratory adaptation. The reference spinosad-sensitive strain, Geneva88, was collected in 1988 and reared in the laboratory without exposure to insecticide. Pearl-Sel shows >1000 fold resistance to spinosad compared to Geneva88 et al. (2008). DNA samples from the backcross father, F1 mother (400 ng DNA each), 10 untreated controls and 12 bioassay survivors (200 ng each) were used to produce a single Illumina sequencing library.After sexing, single adult moths were homogenised in DNA isolation buffer and genomic DNA purified using three phenol extractions followed by a single chloroform extraction Genomic DNA from each of the 24 library individuals was digested separately for 30 minutes at 37°C, in 50 µL reactions with 15 units of SbfI (NEB). The enzyme was heat inactivated at 65°C for 20 minutes. A different P1 adapter, each with a unique 5 bp molecular identifying sequence (MID) , was theFragment ends were repaired using the Quick Blunting Kit (NEB) , incubated at room temperature for 30 minutes, purified with MinElute Reaction Cleanup Kit (Qiagen) and eluted in 16 µL water. dATP overhangs were added to the DNA using 15 µL of purified library template, dATP (1 µL 100 mM), 15 units Klenow exo- (NEB) and 2 µL NEB buffer 2. The reaction was incubated at 37°C for 30 minutes, then purified with a MinElute column and eluted in 21 µL of water.http://www.ebi.ac.uk/ena/data/view/ERP000449).Paired-end P2 adapter, containing T overhangs, was prepared by combining HPLC purified oligonucleotides P2-PE-FOR and P2-PE-REV  in 1X anRaw Illumina reads from the P1 adapter were separated into pools according to the MID assigned to each individual sample in the RAD library. The 5 base MID and 6 base partial restriction site were removed from each 51 base read, leaving 40 bases of downstream sequence. For each individual, candidate RAD loci were identified and candidate RAD alleles called within loci as follows.Identical reads were collapsed into unique sequences, with median quality values calculated for each unique sequence, and singleton reads discarded. Uniques less than a threshold distance apart were grouped together, with each group of uniques treated as a candidate RAD locus. Uniques were separated into different groups if they were more than a distance of 7 bases apart, allowing for sequence diversity and sequencing error between uniques within a group. This is likely to be an overestimate of sequence diversity in a real biological dataset, as it does not take repeat content into account, but a more liberal approach to clustering is preferred because there are many criteria by which clusters can be corrected at later stages. In fact, increasing the number of mismatches above three for this dataset has a minor effect on the number of candidate loci and alleles present in more than one individual in the final marker set see . The disEach group of unique sequences was considered to be a candidate RAD locus. The set of uniques will include not only real RAD alleles but also sequencing errors. To correct these errors, for each possible base at each position in the locus, the mean quality score was calculated across all uniques. Bases that appeared in only one unique were rejected as likely errors. Uniques were then clustered together into candidate RAD alleles, merging uniques that were identical except for bases whose average qualities fell below a chosen quality threshold .Candidate RAD loci and RAD alleles were defined based on the read containing the restriction site overhang, ignoring the paired-end read. Once RAD alleles were defined, the paired-end reads were grouped together by RAD allele. For each RAD allele, duplicate paired-end reads were collapsed into unique sequences, with the number of unique sequences taken to represent the number of DNA fragments in the original sample. These fragment counts were used instead of read counts in the following analysis.m as a standard , based on the curves shown in i. The scale factor for individual i was calculated as iSF = (mmin/imin + mmax/imax)/2. The number of fragments for each candidate allele for individual i was then multiplied by iSF before thresholds were applied. RAD alleles with less than 3 fragments or only appearing in a single individual were discarded , using the ‘max’ optimisation function, which aims to generate the longest contig possible. The RAD contig produced does not overlap the associated RAD allele since the smallest fragment formed by shearing (approx 300 bp) is longer than the length of the read plus the paired-end read.For each RAD allele, paired-end reads for that RAD allele were pooled together across all individuals and assembled using VelvetOptimiser version 2.1.4 http://www.radseq.info).A general version of this pipeline has been implemented in the open source toolkit RADtools . Due to the small number of progeny analyzed  some chromosomes contained multiple linkage groups, or RAD markers that did not group together. In these cases, raw genotypes were manually analyzed to assess likely order. A maximum of 5/20 genotype differences (representing 5 crossing over events) were considered, with distance approximation of 1 event ∼ 5cM. Markers with more than 5 recombinants were not grouped together and are presented as distinct linkage groups for a particular chromosome.P. xylostella was estimated by comparing red fluorescence from propidium iodide-stained nuclei isolated from brain tissue of single adults to that produced by co-prepared nuclei from the head of a single Drosophila melanogaster adult (1C = 175 Mb) using flow cytometry The genome size of The dataset for this study has been submitted to the Sequence Read Archive under accession number ERP000449.Figure S1Normalisation of fragment counts. Numbers of unique paired-end fragments per candidate allele are plotted against number of candidate alleles, for the mother, father and one backcross individual, control 07. For example, the mother has 149 candidate alleles with 6 fragments each and 259 candidate alleles with 27 fragments each. Both the axes are truncated, obscuring a large peak of alleles with three fragments or less (likely to be sequencing errors) and a long tail of alleles with eighty fragments or more (expected to be repeat clusters). Each curve shows a trough (marked with a circle) and a peak (marked with an arrowhead) which represent mini and maxi respectively for each individual i (see Methods).(PDF)Click here for additional data file.Table S1Uniref90 hits to P. xylostella chromosomes. RAD allele sequences are listed with the best Uniref90 hit to their RAD contig. E-values are for TBLASTX. Alleles are listed as Mother (M) or Father (F) derived.(XLS)Click here for additional data file.Table S2Dataset for linkage map construction. 2,878 paternally derived RAD alleles were collapsed into 285 discrete RAD markers and used for linkage map construction (truction . The for(XLS)Click here for additional data file.Table S3Effect of grouping uniques into loci with different numbers of mismatches. Reads for each individual were processed with the RADtools pipeline, allowing different numbers of mismatches between uniques when clustering them into candidate loci. The resulting candidate loci for each individual were then clustered together using the RADmarkers tool, with no thresholding of read or tag count and no mismatches allowed between sequences, only clustering together loci with identical alleles across individuals. After clustering, the loci present in only one individual were removed, as they are likely to be sequencing error. The numbers of loci and alleles fall sharply when using three mismatches instead of one mismatch, but only show small decreases as more than three mismatches are used.(PDF)Click here for additional data file.Table S4Effects of normalisation, varying fragment count threshold and number of mismatches allowed during clustering with RADtools. Loci with normalised fragment counts (A) and raw fragment counts (B) were clustered, allowing 0-6 mismatches and accepting only alleles with fragment counts equal to or above thresholds 1-6 . All loci appearing in one individual only were discarded.(PDF)Click here for additional data file.


Transcatheter arterial chemoembolization (TACE) is an effective treatment for hepatocellular carcinoma (HCC) that would occasionally lead to energy malnutrition through therapeutic hypoxic stress. We aimed to clarify the correlation between the energy malnutrition after TACE and low tolerability for hypoxia of non-tumoral liver before TACE.We performed a prospective cohort study involving 100 HCC patients who underwent TACE at Kobe University Hospital. Indirect calorimetry was performed before and 7 days after TACE, and non-protein respiratory quotient (npRQ) as an indicator of the energy malnutrition was measured. Blood biochemical examinations were also performed before TACE. As an indicator of hypoxic marker, des-γ-carboxy prothrombin (DCP) was measured before TACE. The correlation between npRQ ratio (7 days after/before TACE) and DCP (before TACE) was statistically examined. Spearman’s correlation coefficient test showed that npRQ ratio (Day 7/Day 0) was significantly related to DCP (Day 0) . On the other hand, npRQ ratio (Day 7/Day 0) was not related to alpha fetoprotein (Day 0) .The npRQ reduction after TACE was related to a high value of DCP before TACE. The energy malnutrition after TACE would originate from low tolerability for hypoxia of non-tumoral liver. The HCC patients with a high value of DCP before TACE would clinically have a high risk of the energy malnutrition after TACE. Transcatheter arterial chemoembolization (TACE) is an effective treatment for hepatocellular carcinoma (HCC) that can deteriorate liver function. The long-term deterioration of liver function after TACE can originate from the progression of energy malnutrition  who underwent TACE for HCC. All patients were Japanese and had liver cirrhosis. Overall, 58 patients were classified into Child’s grade A and 42 were classified into grade B. The etiology of cirrhosis was as follows: hepatitis B in 12 patients, hepatitis C in 71, alcoholic liver dysfunction in 28, primary biliary cirrhosis in 2, autoimmune hepatitis in 2, non-alcoholic fatty liver disease in 5, and unknown in 1, although each category overlaps with others. Blood biochemical examinations were performed before TACE and indirect calorimetry was performed before and 7 days after TACE. The therapeutic volume was calculated from the distribution of Lipiodol deposits on a liver CT scan after TACE. The relationship between two variables was investigated by Spearman’s correlation coefficient test. Logarithmic transformation was performed only when variables displayed skewed distributions.Spearman’s correlation coefficient test showed that npRQ ratio (Day 7/Day 0) was significantly related to des-γ-carboxy prothrombin (DCP) (Day 0) (Figure In general, DCP is an HCC tumor marker (Liebman et al. In conclusion, npRQ reduction after TACE was related to a high value of DCP before TACE. The energy malnutrition after TACE would originate from low tolerability for hypoxia of non-tumoral liver. The HCC patients with a high value of DCP before TACE would clinically have a high risk of the energy malnutrition after TACE.